stem cell research for diabetes type 2

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“When my son was diagnosed [with Type 1], I knew nothing about diabetes. I changed my research focus, thinking, as any parent would, ‘What am I going to do about this?’” says Douglas Melton.

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Breakthrough within reach for diabetes scientist and patients nearest to his heart

Harvard Correspondent

100 years after discovery of insulin, replacement therapy represents ‘a new kind of medicine,’ says Stem Cell Institute co-director Douglas Melton, whose children inspired his research

When Vertex Pharmaceuticals announced last month that its investigational stem-cell-derived replacement therapy was, in conjunction with immunosuppressive therapy, helping the first patient in a Phase 1/2 clinical trial robustly reproduce his or her own fully differentiated pancreatic islet cells, the cells that produce insulin, the news was hailed as a potential breakthrough for the treatment of Type 1 diabetes. For Harvard Stem Cell Institute Co-Director and Xander University Professor Douglas Melton, whose lab pioneered the science behind the therapy, the trial marked the most recent turning point in a decades-long effort to understand and treat the disease. In a conversation with the Gazette, Melton discussed the science behind the advance, the challenges ahead, and the personal side of his research. The interview was edited for clarity and length.

Douglas Melton

GAZETTE: What is the significance of the Vertex trial?

MELTON: The first major change in the treatment of Type 1 diabetes was probably the discovery of insulin in 1920. Now it’s 100 years later and if this works, it’s going to change the medical treatment for people with diabetes. Instead of injecting insulin, patients will get cells that will be their own insulin factories. It’s a new kind of medicine.

GAZETTE: Would you walk us through the approach?

MELTON: Nearly two decades ago we had the idea that we could use embryonic stem cells to make functional pancreatic islets for diabetics. When we first started, we had to try to figure out how the islets in a person’s pancreas replenished. Blood, for example, is replenished routinely by a blood stem cell. So, if you go give blood at a blood drive, your body makes more blood. But we showed in mice that that is not true for the pancreatic islets. Once they’re removed or killed, the adult body has no capacity to make new ones.

So the first important “a-ha” moment was to demonstrate that there was no capacity in an adult to make new islets. That moved us to another source of new material: stem cells. The next important thing, after we overcame the political issues surrounding the use of embryonic stem cells, was to ask: Can we direct the differentiation of stem cells and make them become beta cells? That problem took much longer than I expected — I told my wife it would take five years, but it took closer to 15. The project benefited enormously from undergraduates, graduate students, and postdocs. None of them were here for 15 years of course, but they all worked on different steps.

GAZETTE: What role did the Harvard Stem Cell Institute play?

MELTON: This work absolutely could not have been done using conventional support from the National Institutes of Health. First of all, NIH grants came with severe restrictions and secondly, a long-term project like this doesn’t easily map to the initial grant support they give for a one- to three-year project. I am forever grateful and feel fortunate to have been at a private institution where philanthropy, through the HSCI, wasn’t just helpful, it made all the difference.

I am exceptionally grateful as well to former Harvard President Larry Summers and Steve Hyman, director of the Stanley Center for Psychiatric Research at the Broad Institute, who supported the creation of the HSCI, which was formed specifically with the idea to explore the potential of pluripotency stem cells for discovering questions about how development works, how cells are made in our body, and hopefully for finding new treatments or cures for disease. This may be one of the first examples where it’s come to fruition. At the time, the use of embryonic stem cells was quite controversial, and Steve and Larry said that this was precisely the kind of science they wanted to support.

GAZETTE: You were fundamental in starting the Department of Stem Cell and Regenerative Biology. Can you tell us about that?

MELTON: David Scadden and I helped start the department, which lives in two Schools: Harvard Medical School and the Faculty of Arts and Science. This speaks to the unusual formation and intention of the department. I’ve talked a lot about diabetes and islets, but think about all the other tissues and diseases that people suffer from. There are faculty and students in the department working on the heart, nerves, muscle, brain, and other tissues — on all aspects of how the development of a cell and a tissue affects who we are and the course of disease. The department is an exciting one because it’s exploring experimental questions such as: How do you regenerate a limb? The department was founded with the idea that not only should you ask and answer questions about nature, but that one can do so with the intention that the results lead to new treatments for disease. It is a kind of applied biology department.

GAZETTE: This pancreatic islet work was patented by Harvard and then licensed to your biotech company, Semma, which was acquired by Vertex. Can you explain how this reflects your personal connection to the research?

MELTON: Semma is named for my two children, Sam and Emma. Both are now adults, and both have Type 1 diabetes. My son was 6 months old when he was diagnosed. And that’s when I changed my research plan. And my daughter, who’s four years older than my son, became diabetic about 10 years later, when she was 14.

When my son was diagnosed, I knew nothing about diabetes and had been working on how frogs develop. I changed my research focus, thinking, as any parent would, “What am I going to do about this?” Again, I come back to the flexibility of Harvard. Nobody said, “Why are you changing your research plan?”

GAZETTE: What’s next?

MELTON: The stem-cell-derived replacement therapy cells that have been put into this first patient were provided with a class of drugs called immunosuppressants, which depress the patient’s immune system. They have to do this because these cells were not taken from that patient, and so they are not recognized as “self.” Without immunosuppressants, they would be rejected. We want to find a way to make cells by genetic engineering that are not recognized as foreign.

I think this is a solvable problem. Why? When a woman has a baby, that baby has two sets of genes. It has genes from the egg, from the mother, which would be recognized as “self,” but it also has genes from the father, which would be “non-self.” Why does the mother’s body not reject the fetus? If we can figure that out, it will help inform our thinking about what genes to change in our stem cell-derived islets so that they could go into any person. This would be relevant not just to diabetes, but to any cells you wanted to transplant for liver or even heart transplants. It could mean no longer having to worry about immunosuppression.

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GIOSTAR Announces FDA Clearance of the IND for Starting PHASE-2 Clinical Trial for Developing Specific Stem Cell Therapy for Type II Diabetes.

SAN DIEGO--(BUSINESS WIRE)-- Global Institute of Stem Cell Therapy and Research, Inc. known as GIOSTAR , a San Diego, California based Global organization at the forefront of stem cell research over two decades, developing a novel cellular therapy pipeline to improve the standard of care for treating Type II diabetes patients, today announced that the United States Food and Drug Administration (FDA) has cleared its investigational new drug (IND) application to start a Phase–2 clinical trial for DT2-SCT. The Company’s novel approach using autologous mesenchymal stem cells to alleviate the disease-caused damage in diabetics offers a new hope to address the sufferings in diabetes patients without much side-effects.

Diabetes is not just a disease but a global health crisis. It is estimated to affect more than one billion people worldwide. The financial burden on the global healthcare system to treat diabetes is expected to reach more than one trillion dollars annually. GIOSTAR CEO, President, and Cofounder Mr. Deven Patel stated, “Upon a successful completion of the clinical trials, GIOSTAR intends to make this treatment affordable to masses and poised to capture significant global market share due to GIOSTAR’s existing global infrastructure of hospitals and research centers.”

According to the Chairman and Cofounder of GIOSTAR, Dr. Anand Srivastava , “DT2-SCT is a cellular therapy for Type II diabetics which uses autologous stem cells, isolated from the visceral tissues of the recipients, developed to target systemic ill-effects caused by diabetes-induced pathology in patients. We are pleased to reach this milestone following extensive research and development.”

GIOSTAR expects to complete the Phase-2 clinical trial using the DT2-SCT in Type II diabetics within 12 to 18 months. GIOSTAR anticipates enrolling participants for the study at few sites across the United States.

“The diabetes is now considered as a lifestyle disease. In many cases, even lifestyle changes are not enough to eliminate the risk of developing diabetes due to certain genetic risk factors,” stated Patel . “Our innovative and noninvasive stem cell-based therapeutics may offer better treatment option for diabetic patients.”

About: ( GIOSTAR )

GIOSTAR is a global stem cell research organization committed to the discovery, development, and commercialization of stem cell based treatments to make a meaningful difference in the lives of people impacted by difficult-to-treat degenerative and other diseases. The company also aims to develop stem cell-based therapies for arthritis, long COVID complications , cancer vaccines and the fairly uncommon area of making red blood cells from stem cells. GIOSTAR red blood cell technology is ready to scale and preparing its IND for US FDA. The leadership of GIOSTAR aims to make these stem cell treatments available to the masses at affordable prices.

Investor & Media Inquiries: Mr. Deven Patel CEO, President and Cofounder Global Institute Of Stem Cell Therapy And Research [email protected] www.giostar.com

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Smart insulin and stem cell transplants: Research Highlights October 2024

We take a look at some of the exciting diabetes research developments announced in October, and what the findings could mean for people living with or affected by diabetes. 

In this month's article:

• A step forward in making insulin 'smarter'
• World-first stem cell transplant helps person with type 1 diabetes produce insulin
• Regenerating cells with electrical impulses in people with type 2 diabetes
• New driver of diabetic retinopathy discovered

A step forward in making insulin ‘smarter’ 

A new type of ‘smart’ insulin has shown promise in early-stage research. In animals, the insulin was found to only turn on when blood sugar levels are high and stay off when levels are low. 

Millions of people with all types of diabetes in the UK use insulin to manage their blood sugar levels. This requires a careful balancing act and it’s difficult to get right. So, scientists are trying to develop cleverer insulins that can sense blood sugar levels and respond in the right way. This would make life for people with diabetes dramatically simpler.  

In new research published in Nature , an international research team tested a new type of ‘smart’ insulin they’ve developed, called NNC2215. It has a special molecular switch.

When blood sugars are low, a molecule called glucoside locks NNC2215 in the ‘off’ position so it won’t lower blood sugars further. When blood sugar levels rise, glucose (sugar) in the blood replaces the glucoside, switching NNC2215 ‘on’ so it will bring blood sugars down. 

In studies with rats and pigs, researchers found NNC2215’s switch worked as they’d hoped. It was able to turn on and off in response to changing blood sugar levels. And it was as effective as regular insulin at lowering high blood sugar, without causing it to drop too low.  

NNC2215 hasn’t been tested in humans yet, and there will be many more steps before it could be. But these findings are encouraging.  

Dr Elizabeth Robertson, our Director of Research, said: 

“This research represents a significant step forward in the global effort to develop the next generation of ‘smart’ insulins. The hope is that these will ease the constant challenge of managing blood sugar highs and lows, and improve the physical and mental health of millions of people worldwide with diabetes who rely on insulin therapy. "We’re excited to part of this endeavour through our Type 1 Diabetes Grand Challenge programme .” 

World-first stem cell transplant helps person with type 1 produce insulin 

A new type of stem cell treatment, created from a person’s own cells, is showing early promise in allowing people with type 1 diabetes to make their own insulin.  

Stem cells are like shape-shifters - they can turn into almost any type of cell the body needs. In the quest for new treatments and a cure for type 1 diabetes, scientists have been working to turn stem cells into insulin-making beta cells.

The hope is these stem cells-turned-beta cells could replace those that have been destroyed by the immune system in people with type 1 diabetes. 

Until now, most clinical trials of stem cell therapies in people with type 1 diabetes have used donor stem cells. Because these stem cells are foreign, trial participants need to take immunosuppressant drugs to prevent their immune system from recognising and destroying them. These drugs come with significant side effects.  

In a new study, published in Cell , a 25-year-old woman with type 1 diabetes became the first person in the world to receive a stem cell transplant created from her own cells. Scientists hope that this approach could reduce or eliminate the need for immunosuppressants.  

Researchers at Peking University, Beijing, extracted cells from the participant and used chemicals in the lab to make what are known as chemically induced pluripotent stem cells. They then reprogrammed the stem cells to become beta cells, and injected around 1.5 million into the woman’s stomach. 

Two-and-a-half months after this procedure, the woman’s new beta cells were producing enough insulin for her to stop insulin injections. A year later, she was still producing her own insulin, and her blood sugars were in range for 98% of the time. The researchers also didn’t see any major safety concerns. 

This is an important and exciting development in stem cell therapies for type 1 diabetes. But the participant in this trial was already taking immunosuppressants because of a prior liver transplant. This means we can’t be sure if the new type of stem cell procedure alone was the reason she didn’t reject the transplanted cells, or if the immunosuppressants played a role.  

So far, two other participants have also had the stem cell procedure, and the researchers are set to report their results soon. There will then need to be more studies, with more people to check if and how well the treatment works. 

To speed up progress and get us to effective beta cell therapies quicker, we’re investing in cutting-edge research through the Type 1 Diabetes Grand Challenge .

Regenerating cells with electrical impulses in people with type 2 diabetes 

Some people with type 2 diabetes were able to stop taking insulin after receiving a new procedure, which uses electrical impulses to improve the body’s response to insulin, alongside the blood sugar lowering medication  semaglutide .  

Researchers at the Amsterdam University Medical Centre ran the first trial of a new treatment, called Recellularisation via Electroporation Therapy (ReCET). It aims to improve the body’s sensitivity to its own insulin using pulsed electrical fields that target cells in the gut involved in blood sugar control.

The idea is to regenerate these cells, making them better at using insulin and bringing down blood sugar levels.  

In the trial, 14 people living with type 2 diabetes had the ReCET procedure, which is done under sedation. They then began taking an existing type 2 diabetes medication: semaglutide, a GLP-1 agonist . This medication helps the body to produce more insulin. 

Participants were followed up at six, 12 and 24 months. The study was set up to look at the safety of the ReCET procedure and promisingly there weren’t any serious safety concerns reported.  

Over the two years, 12 of the 14 people (86%) no longer needed to take insulin to keep their blood sugars stayed in a target range.  

This is exciting. But we need to keep in mind this was an early safety study, and the research wasn’t designed to look at the treatment’s effectiveness. As participants were taking a GLP-1 medication it also isn’t clear if the ReCET procedure gave any additional benefit.

Next, it will be important to compare ReCET with other existing type 2 treatments in head-to-head trials, including against GLP1-s alone. 

We’ll also need to see if any benefits last longer term and which groups of people with type 2 it could hold potential to help. The team are already working on more studies, so answers will be coming.  

Dr Celine Busch, lead author of the study, said:

“These findings are very encouraging, suggesting that ReCET is a safe and feasible procedure that, when combined with semaglutide, can effectively eliminate the need for insulin therapy.” 

These results were presented at United European Gastroenterology Week (UEGW) 2024 in Vienna. 

New driver of diabetic retinopathy discovered 

With our funding, researchers at Queen’s University Belfast have uncovered a new factor that could help explain why diabetic retinopathy progresses and causes vision problems. The research shows a protein, called Pentraxin 3 (PTX3), might be driving harmful inflammation in the eyes of people with diabetes.  

Diabetic retinopathy  is a common complication of diabetes caused by high blood sugar levels damaging blood vessels in the eye. Inflammation can make this eye damage worse, but scientists haven’t been sure what causes it. 

Recent evidence has shown that PTX3 is made in the eye, so our researchers led by Professor Reinhold Medina delved into its role in retinopathy for the first time. They compared two groups of mice with diabetes: one group who had PTX3 in the retina and a second group who didn’t make the protein. The researchers monitored the mice over nine months.   

The researchers found that the mice without PTX3 showed less eye damage. These mice had fewer signs of inflammation in the retina and better vision, compared to the PTX3 mice. In the PTX3 mice, they saw that the protein activated certain cells that cause swelling.  

The team then looked at samples from people with diabetic retinopathy and found that PTX3 levels were higher in people with more severe eye problems. These results suggest that PTX3 is involved in retinopathy progression by fuelling inflammation in the eye. 

This knowledge opens the door to developing new treatments that target and block PTX3.  

Dr Lucy Chambers, our Head of Research Communications, said:

“Eye problems are a frightening and too frequent complication of diabetes. By advancing our understanding of the biological factors contributing to eye damage, this research could take us closer to new and better treatments that help more people with diabetes avoid devastating harm to their sight.” 

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© The British Diabetic Association operating as Diabetes UK, a   charity registered in England and Wales (no. 215199) and in Scotland (no. SC039136). A company limited by guarantee registered in England and Wales with (no.00339181) and registered office at Wells Lawrence House, 126 Back Church Lane London E1 1FH

Towards Standardized Stem Cell Therapy in Type 2 Diabetes Mellitus: A Systematic Review

Affiliations.

• 1 Faculty of Medicine, Department of Histology, Universitas Indonesia, Jakarta, Indonesia.
• 2 Stem Cell and Tissue Engineering Research Center, Indonesian Medical Education and Research Institute (IMERI), Faculty of Medicine Universitas Indonesia, Jakarta, Indonesia.
• 3 Stem Cell Medical Technology Integrated Service Unit, Cipto Mangunkusumo Central Hospital - Faculty of Medicine Universitas Indonesia, Jakarta, Indonesia.
• 4 Tissue Engineering Program, Life Sciences Institute, National University of Singapore, Singapore.
• PMID: 29732994
• DOI: 10.2174/1574888X13666180502143657

Objective: To compile and analyze the published studies on cell therapy for type 2 diabetes mellitus (T2DM) to obtain a better insight into management of T2DM that involved stem cell therapy.

Methods: We searched all published studies in Pubmed/Medline, and Cochrane library, using keywords: ‘stem cell’ AND ‘therapy’ AND ‘diabetes type 2’. Inclusion criteria: original articles on the use of stem cells in humans with T2DM. Exclusion criteria: articles in the non-English literature, studies on T2DM complications that did not assess both adverse events and any of the common diabetes study outcomes. Data collection: type of study, number of cases, and all data that were related to outcome and adverse events. Data were analyzed descriptively to conclude the possible cause of adverse reactions, and which protocols gave a satisfactory outcome.

Results: We collected 25 original articles, out of which 17 studies did not have controls and were classified as case reports, while there were 8 studies that were controlled clinical trials. Most studies used autologous bone marrow mononuclear cells (BM-MNCs) or autologous or allogeneic mesenchymal stem cells (MSCs) from various sources. Adverse events were mild and mostly intervention related. Efficacy of autologous BM-MNCs that were given via interventional route was comparable to Wharton jelly or umbilical cord MSCs that were given via intravenous (IV), Intra muscular (IM), or subcutaneous (SC) route.

Conclusion: Further controlled studies that compare BM-MNCs to BM-MSCs or WJ-MSCs or UCSCs are recommended to prove their comparable efficacy. In addition, studies that compare various routes of administration (IV, IM or SC) versus the more invasive interventional routes are needed.

Keywords: Type 2 diabetes mellitus; Wharton’s jelly; bone marrow; mesenchymal stem cells; mononuclear cells; umbilical cord..

Copyright© Bentham Science Publishers; For any queries, please email at [email protected].

Publication types

• Systematic Review
• Bone Marrow Cells / cytology*
• Cell Differentiation / physiology
• Cell- and Tissue-Based Therapy* / methods
• Diabetes Mellitus, Type 2 / therapy*
• Mesenchymal Stem Cell Transplantation* / methods
• Mesenchymal Stem Cells / cytology*

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Stem cells: past, present, and future

Wojciech zakrzewski, maciej dobrzyński, maria szymonowicz, zbigniew rybak.

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Corresponding author.

Collection date 2019.

Open Access This article is distributed under the terms of the Creative Commons Attribution 4.0 International License ( http://creativecommons.org/licenses/by/4.0/ ), which permits unrestricted use, distribution, and reproduction in any medium, provided you give appropriate credit to the original author(s) and the source, provide a link to the Creative Commons license, and indicate if changes were made. The Creative Commons Public Domain Dedication waiver ( http://creativecommons.org/publicdomain/zero/1.0/ ) applies to the data made available in this article, unless otherwise stated.

In recent years, stem cell therapy has become a very promising and advanced scientific research topic. The development of treatment methods has evoked great expectations. This paper is a review focused on the discovery of different stem cells and the potential therapies based on these cells. The genesis of stem cells is followed by laboratory steps of controlled stem cell culturing and derivation. Quality control and teratoma formation assays are important procedures in assessing the properties of the stem cells tested. Derivation methods and the utilization of culturing media are crucial to set proper environmental conditions for controlled differentiation. Among many types of stem tissue applications, the use of graphene scaffolds and the potential of extracellular vesicle-based therapies require attention due to their versatility. The review is summarized by challenges that stem cell therapy must overcome to be accepted worldwide. A wide variety of possibilities makes this cutting edge therapy a turning point in modern medicine, providing hope for untreatable diseases.

Keywords: Stem cells, Differentiation, Pluripotency, Induced pluripotent stem cell (iPSC), Teratoma formation assay, Stem cell derivation, Growth media, Tissue banks, Tissue transplantation

Stem cell classification

Stem cells are unspecialized cells of the human body. They are able to differentiate into any cell of an organism and have the ability of self-renewal. Stem cells exist both in embryos and adult cells. There are several steps of specialization. Developmental potency is reduced with each step, which means that a unipotent stem cell is not able to differentiate into as many types of cells as a pluripotent one. This chapter will focus on stem cell classification to make it easier for the reader to comprehend the following chapters.

Totipotent stem cells are able to divide and differentiate into cells of the whole organism. Totipotency has the highest differentiation potential and allows cells to form both embryo and extra-embryonic structures. One example of a totipotent cell is a zygote, which is formed after a sperm fertilizes an egg. These cells can later develop either into any of the three germ layers or form a placenta. After approximately 4 days, the blastocyst’s inner cell mass becomes pluripotent. This structure is the source of pluripotent cells.

Pluripotent stem cells (PSCs) form cells of all germ layers but not extraembryonic structures, such as the placenta. Embryonic stem cells (ESCs) are an example. ESCs are derived from the inner cell mass of preimplantation embryos. Another example is induced pluripotent stem cells (iPSCs) derived from the epiblast layer of implanted embryos. Their pluripotency is a continuum, starting from completely pluripotent cells such as ESCs and iPSCs and ending on representatives with less potency—multi-, oligo- or unipotent cells. One of the methods to assess their activity and spectrum is the teratoma formation assay. iPSCs are artificially generated from somatic cells, and they function similarly to PSCs. Their culturing and utilization are very promising for present and future regenerative medicine.

Multipotent stem cells have a narrower spectrum of differentiation than PSCs, but they can specialize in discrete cells of specific cell lineages. One example is a haematopoietic stem cell, which can develop into several types of blood cells. After differentiation, a haematopoietic stem cell becomes an oligopotent cell. Its differentiation abilities are then restricted to cells of its lineage. However, some multipotent cells are capable of conversion into unrelated cell types, which suggests naming them pluripotent cells.

Oligopotent stem cells can differentiate into several cell types. A myeloid stem cell is an example that can divide into white blood cells but not red blood cells.

Unipotent stem cells are characterized by the narrowest differentiation capabilities and a special property of dividing repeatedly. Their latter feature makes them a promising candidate for therapeutic use in regenerative medicine. These cells are only able to form one cell type, e.g. dermatocytes.

Stem cell biology

A blastocyst is formed after the fusion of sperm and ovum fertilization. Its inner wall is lined with short-lived stem cells, namely, embryonic stem cells. Blastocysts are composed of two distinct cell types: the inner cell mass (ICM), which develops into epiblasts and induces the development of a foetus, and the trophectoderm (TE). Blastocysts are responsible for the regulation of the ICM microenvironment. The TE continues to develop and forms the extraembryonic support structures needed for the successful origin of the embryo, such as the placenta. As the TE begins to form a specialized support structure, the ICM cells remain undifferentiated, fully pluripotent and proliferative [ 1 ]. The pluripotency of stem cells allows them to form any cell of the organism. Human embryonic stem cells (hESCs) are derived from the ICM. During the process of embryogenesis, cells form aggregations called germ layers: endoderm, mesoderm and ectoderm (Fig.  1 ), each eventually giving rise to differentiated cells and tissues of the foetus and, later on, the adult organism [ 2 ]. After hESCs differentiate into one of the germ layers, they become multipotent stem cells, whose potency is limited to only the cells of the germ layer. This process is short in human development. After that, pluripotent stem cells occur all over the organism as undifferentiated cells, and their key abilities are proliferation by the formation of the next generation of stem cells and differentiation into specialized cells under certain physiological conditions.

Oocyte development and formation of stem cells: the blastocoel, which is formed from oocytes, consists of embryonic stem cells that later differentiate into mesodermal, ectodermal, or endodermal cells. Blastocoel develops into the gastrula

Signals that influence the stem cell specialization process can be divided into external, such as physical contact between cells or chemical secretion by surrounding tissue, and internal, which are signals controlled by genes in DNA.

Stem cells also act as internal repair systems of the body. The replenishment and formation of new cells are unlimited as long as an organism is alive. Stem cell activity depends on the organ in which they are in; for example, in bone marrow, their division is constant, although in organs such as the pancreas, division only occurs under special physiological conditions.

Stem cell functional division

Whole-body development.

During division, the presence of different stem cells depends on organism development. Somatic stem cell ESCs can be distinguished. Although the derivation of ESCs without separation from the TE is possible, such a combination has growth limits. Because proliferating actions are limited, co-culture of these is usually avoided.

ESCs are derived from the inner cell mass of the blastocyst, which is a stage of pre-implantation embryo ca. 4 days after fertilization. After that, these cells are placed in a culture dish filled with culture medium. Passage is an inefficient but popular process of sub-culturing cells to other dishes. These cells can be described as pluripotent because they are able to eventually differentiate into every cell type in the organism. Since the beginning of their studies, there have been ethical restrictions connected to the medical use of ESCs in therapies. Most embryonic stem cells are developed from eggs that have been fertilized in an in vitro clinic, not from eggs fertilized in vivo.

Somatic or adult stem cells are undifferentiated and found among differentiated cells in the whole body after development. The function of these cells is to enable the healing, growth, and replacement of cells that are lost each day. These cells have a restricted range of differentiation options. Among many types, there are the following:

Mesenchymal stem cells are present in many tissues. In bone marrow, these cells differentiate mainly into the bone, cartilage, and fat cells. As stem cells, they are an exception because they act pluripotently and can specialize in the cells of any germ layer.

Neural cells give rise to nerve cells and their supporting cells—oligodendrocytes and astrocytes.

Haematopoietic stem cells form all kinds of blood cells: red, white, and platelets.

Skin stem cells form, for example, keratinocytes, which form a protective layer of skin.

The proliferation time of somatic stem cells is longer than that of ESCs. It is possible to reprogram adult stem cells back to their pluripotent state. This can be performed by transferring the adult nucleus into the cytoplasm of an oocyte or by fusion with the pluripotent cell. The same technique was used during cloning of the famous Dolly sheep.

hESCs are involved in whole-body development. They can differentiate into pluripotent, totipotent, multipotent, and unipotent cells (Fig.  2 ) [ 2 ].

Changes in the potency of stem cells in human body development. Potency ranges from pluripotent cells of the blastocyst to unipotent cells of a specific tissue in a human body such as the skin, CNS, or bone marrow. Reversed pluripotency can be achieved by the formation of induced pluripotent stem cells using either octamer-binding transcription factor (Oct4), sex-determining region Y (Sox2), Kruppel-like factor 4 (Klf4), or the Myc gene

Pluripotent cells can be named totipotent if they can additionally form extraembryonic tissues of the embryo. Multipotent cells are restricted in differentiating to each cell type of given tissue. When tissue contains only one lineage of cells, stem cells that form them are called either called oligo- or unipotent.

iPSC quality control and recognition by morphological differences

The comparability of stem cell lines from different individuals is needed for iPSC lines to be used in therapeutics [ 3 ]. Among critical quality procedures, the following can be distinguished:

Short tandem repeat analysis—This is the comparison of specific loci on the DNA of the samples. It is used in measuring an exact number of repeating units. One unit consists of 2 to 13 nucleotides repeating many times on the DNA strand. A polymerase chain reaction is used to check the lengths of short tandem repeats. The genotyping procedure of source tissue, cells, and iPSC seed and master cell banks is recommended.

Identity analysis—The unintentional switching of lines, resulting in other stem cell line contamination, requires rigorous assay for cell line identification.

Residual vector testing—An appearance of reprogramming vectors integrated into the host genome is hazardous, and testing their presence is a mandatory procedure. It is a commonly used procedure for generating high-quality iPSC lines. An acceptable threshold in high-quality research-grade iPSC line collections is ≤ 1 plasmid copies per 100 cells. During the procedure, 2 different regions, common to all plasmids, should be used as specific targets, such as EBNA and CAG sequences [ 3 ]. To accurately represent the test reactions, a standard curve needs to be prepared in a carrier of gDNA from a well-characterized hPSC line. For calculations of plasmid copies per cell, it is crucial to incorporate internal reference gDNA sequences to allow the quantification of, for example, ribonuclease P (RNaseP) or human telomerase reverse transcriptase (hTERT).

Karyotype—A long-term culture of hESCs can accumulate culture-driven mutations [ 4 ]. Because of that, it is crucial to pay additional attention to genomic integrity. Karyotype tests can be performed by resuscitating representative aliquots and culturing them for 48–72 h before harvesting cells for karyotypic analysis. If abnormalities are found within the first 20 karyotypes, the analysis must be repeated on a fresh sample. When this situation is repeated, the line is evaluated as abnormal. Repeated abnormalities must be recorded. Although karyology is a crucial procedure in stem cell quality control, the single nucleotide polymorphism (SNP) array, discussed later, has approximately 50 times higher resolution.

Viral testing—When assessing the quality of stem cells, all tests for harmful human adventitious agents must be performed (e.g. hepatitis C or human immunodeficiency virus). This procedure must be performed in the case of non-xeno-free culture agents.

Bacteriology—Bacterial or fungal sterility tests can be divided into culture- or broth-based tests. All the procedures must be recommended by pharmacopoeia for the jurisdiction in which the work is performed.

Single nucleotide polymorphism arrays—This procedure is a type of DNA microarray that detects population polymorphisms by enabling the detection of subchromosomal changes and the copy-neutral loss of heterozygosity, as well as an indication of cellular transformation. The SNP assay consists of three components. The first is labelling fragmented nucleic acid sequences with fluorescent dyes. The second is an array that contains immobilized allele-specific oligonucleotide (ASO) probes. The last component detects, records, and eventually interprets the signal.

Flow cytometry—This is a technique that utilizes light to count and profile cells in a heterogeneous fluid mixture. It allows researchers to accurately and rapidly collect data from heterogeneous fluid mixtures with live cells. Cells are passed through a narrow channel one by one. During light illumination, sensors detect light emitted or refracted from the cells. The last step is data analysis, compilation and integration into a comprehensive picture of the sample.

Phenotypic pluripotency assays—Recognizing undifferentiated cells is crucial in successful stem cell therapy. Among other characteristics, stem cells appear to have a distinct morphology with a high nucleus to cytoplasm ratio and a prominent nucleolus. Cells appear to be flat with defined borders, in contrast to differentiating colonies, which appear as loosely located cells with rough borders [ 5 ]. It is important that images of ideal and poor quality colonies for each cell line are kept in laboratories, so whenever there is doubt about the quality of culture, it can always be checked according to the representative image. Embryoid body formation or directed differentiation of monolayer cultures to produce cell types representative of all three embryonic germ layers must be performed. It is important to note that colonies cultured under different conditions may have different morphologies [ 6 ].

Histone modification and DNA methylation—Quality control can be achieved by using epigenetic analysis tools such as histone modification or DNA methylation. When stem cells differentiate, the methylation process silences pluripotency genes, which reduces differentiation potential, although other genes may undergo demethylation to become expressed [ 7 ]. It is important to emphasize that stem cell identity, together with its morphological characteristics, is also related to its epigenetic profile [ 8 , 9 ]. According to Brindley [ 10 ], there is a relationship between epigenetic changes, pluripotency, and cell expansion conditions, which emphasizes that unmethylated regions appear to be serum-dependent.

hESC derivation and media

hESCs can be derived using a variety of methods, from classic culturing to laser-assisted methodologies or microsurgery [ 11 ]. hESC differentiation must be specified to avoid teratoma formation (see Fig.  3 ).

Spontaneous differentiation of hESCs causes the formation of a heterogeneous cell population. There is a different result, however, when commitment signals (in forms of soluble factors and culture conditions) are applied and enable the selection of progenitor cells

hESCs spontaneously differentiate into embryonic bodies (EBs) [ 12 ]. EBs can be studied instead of embryos or animals to predict their effects on early human development. There are many different methods for acquiring EBs, such as bioreactor culture [ 13 ], hanging drop culture [ 12 ], or microwell technology [ 14 , 15 ]. These methods allow specific precursors to form in vitro [ 16 ].

The essential part of these culturing procedures is a separation of inner cell mass to culture future hESCs (Fig.  4 ) [ 17 ]. Rosowski et al. [ 18 ] emphasizes that particular attention must be taken in controlling spontaneous differentiation. When the colony reaches the appropriate size, cells must be separated. The occurrence of pluripotent cells lasts for 1–2 days. Because the classical utilization of hESCs caused ethical concerns about gastrulas used during procedures, Chung et al. [ 19 ] found out that it is also possible to obtain hESCs from four cell embryos, leaving a higher probability of embryo survival. Additionally, Zhang et al. [ 20 ] used only in vitro fertilization growth-arrested cells.

Culturing of pluripotent stem cells in vitro. Three days after fertilization, totipotent cells are formed. Blastocysts with ICM are formed on the sixth day after fertilization. Pluripotent stem cells from ICM can then be successfully transmitted on a dish

Cell passaging is used to form smaller clusters of cells on a new culture surface [ 21 ]. There are four important passaging procedures.

Enzymatic dissociation is a cutting action of enzymes on proteins and adhesion domains that bind the colony. It is a gentler method than the manual passage. It is crucial to not leave hESCs alone after passaging. Solitary cells are more sensitive and can easily undergo cell death; collagenase type IV is an example [ 22 , 23 ].

Manual passage , on the other hand, focuses on using cell scratchers. The selection of certain cells is not necessary. This should be done in the early stages of cell line derivation [ 24 ].

Trypsin utilization allows a healthy, automated hESC passage. Good Manufacturing Practice (GMP)-grade recombinant trypsin is widely available in this procedure [ 24 ]. However, there is a risk of decreasing the pluripotency and viability of stem cells [ 25 ]. Trypsin utilization can be halted with an inhibitor of the protein rho-associated protein kinase (ROCK) [ 26 ].

Ethylenediaminetetraacetic acid ( EDTA ) indirectly suppresses cell-to-cell connections by chelating divalent cations. Their suppression promotes cell dissociation [ 27 ].

Stem cells require a mixture of growth factors and nutrients to differentiate and develop. The medium should be changed each day.

Traditional culture methods used for hESCs are mouse embryonic fibroblasts (MEFs) as a feeder layer and bovine serum [ 28 ] as a medium. Martin et al. [ 29 ] demonstrated that hESCs cultured in the presence of animal products express the non-human sialic acid, N -glycolylneuraminic acid (NeuGc). Feeder layers prevent uncontrolled proliferation with factors such as leukaemia inhibitory factor (LIF) [ 30 ].

First feeder layer-free culture can be supplemented with serum replacement, combined with laminin [ 31 ]. This causes stable karyotypes of stem cells and pluripotency lasting for over a year.

Initial culturing media can be serum (e.g. foetal calf serum FCS), artificial replacement such as synthetic serum substitute (SSS), knockout serum replacement (KOSR), or StemPro [ 32 ]. The simplest culture medium contains only eight essential elements: DMEM/F12 medium, selenium, NaHCO 3, l -ascorbic acid, transferrin, insulin, TGFβ1, and FGF2 [ 33 ]. It is not yet fully known whether culture systems developed for hESCs can be allowed without adaptation in iPSC cultures.

Turning point in stem cell therapy

The turning point in stem cell therapy appeared in 2006, when scientists Shinya Yamanaka, together with Kazutoshi Takahashi, discovered that it is possible to reprogram multipotent adult stem cells to the pluripotent state. This process avoided endangering the foetus’ life in the process. Retrovirus-mediated transduction of mouse fibroblasts with four transcription factors (Oct-3/4, Sox2, KLF4, and c-Myc) [ 34 ] that are mainly expressed in embryonic stem cells could induce the fibroblasts to become pluripotent (Fig.  5 ) [ 35 ]. This new form of stem cells was named iPSCs. One year later, the experiment also succeeded with human cells [ 36 ]. After this success, the method opened a new field in stem cell research with a generation of iPSC lines that can be customized and biocompatible with the patient. Recently, studies have focused on reducing carcinogenesis and improving the conduction system.

Retroviral-mediated transduction induces pluripotency in isolated patient somatic cells. Target cells lose their role as somatic cells and, once again, become pluripotent and can differentiate into any cell type of human body

The turning point was influenced by former discoveries that happened in 1962 and 1987.

The former discovery was about scientist John Gurdon successfully cloning frogs by transferring a nucleus from a frog’s somatic cells into an oocyte. This caused a complete reversion of somatic cell development [ 37 ]. The results of his experiment became an immense discovery since it was previously believed that cell differentiation is a one-way street only, but his experiment suggested the opposite and demonstrated that it is even possible for a somatic cell to again acquire pluripotency [ 38 ].

The latter was a discovery made by Davis R.L. that focused on fibroblast DNA subtraction. Three genes were found that originally appeared in myoblasts. The enforced expression of only one of the genes, named myogenic differentiation 1 (Myod1), caused the conversion of fibroblasts into myoblasts, showing that reprogramming cells is possible, and it can even be used to transform cells from one lineage to another [ 39 ].

Although pluripotency can occur naturally only in embryonic stem cells, it is possible to induce terminally differentiated cells to become pluripotent again. The process of direct reprogramming converts differentiated somatic cells into iPSC lines that can form all cell types of an organism. Reprogramming focuses on the expression of oncogenes such as Myc and Klf4 (Kruppel-like factor 4). This process is enhanced by a downregulation of genes promoting genome stability, such as p53. Additionally, cell reprogramming involves histone alteration. All these processes can cause potential mutagenic risk and later lead to an increased number of mutations. Quinlan et al. [ 40 ] checked fully pluripotent mouse iPSCs using whole genome DNA sequencing and structural variation (SV) detection algorithms. Based on those studies, it was confirmed that although there were single mutations in the non-genetic region, there were non-retrotransposon insertions. This led to the conclusion that current reprogramming methods can produce fully pluripotent iPSCs without severe genomic alterations.

During the course of development from pluripotent hESCs to differentiated somatic cells, crucial changes appear in the epigenetic structure of these cells. There is a restriction or permission of the transcription of genes relevant to each cell type. When somatic cells are being reprogrammed using transcription factors, all the epigenetic architecture has to be reconditioned to achieve iPSCs with pluripotency [ 41 ]. However, cells of each tissue undergo specific somatic genomic methylation. This influences transcription, which can further cause alterations in induced pluripotency [ 42 ].

Source of iPSCs

Because pluripotent cells can propagate indefinitely and differentiate into any kind of cell, they can be an unlimited source, either for replacing lost or diseased tissues. iPSCs bypass the need for embryos in stem cell therapy. Because they are made from the patient’s own cells, they are autologous and no longer generate any risk of immune rejection.

At first, fibroblasts were used as a source of iPSCs. Because a biopsy was needed to achieve these types of cells, the technique underwent further research. Researchers investigated whether more accessible cells could be used in the method. Further, other cells were used in the process: peripheral blood cells, keratinocytes, and renal epithelial cells found in urine. An alternative strategy to stem cell transplantation can be stimulating a patient’s endogenous stem cells to divide or differentiate, occurring naturally when skin wounds are healing. In 2008, pancreatic exocrine cells were shown to be reprogrammed to functional, insulin-producing beta cells [ 43 ].

The best stem cell source appears to be the fibroblasts, which is more tempting in the case of logistics since its stimulation can be fast and better controlled [ 44 ].

Teratoma formation assay

The self-renewal and differentiation capabilities of iPSCs have gained significant interest and attention in regenerative medicine sciences. To study their abilities, a quality-control assay is needed, of which one of the most important is the teratoma formation assay. Teratomas are benign tumours. Teratomas are capable of rapid growth in vivo and are characteristic because of their ability to develop into tissues of all three germ layers simultaneously. Because of the high pluripotency of teratomas, this formation assay is considered an assessment of iPSC’s abilities [ 45 ].

Teratoma formation rate, for instance, was observed to be elevated in human iPSCs compared to that in hESCs [ 46 ]. This difference may be connected to different differentiation methods and cell origins. Most commonly, the teratoma assay involves an injection of examined iPSCs subcutaneously or under the testis or kidney capsule in mice, which are immune-deficient [ 47 ]. After injection, an immature but recognizable tissue can be observed, such as the kidney tubules, bone, cartilage, or neuroepithelium [ 30 ]. The injection site may have an impact on the efficiency of teratoma formation [ 48 ].

There are three groups of markers used in this assay to differentiate the cells of germ layers. For endodermal tissue, there is insulin/C-peptide and alpha-1 antitrypsin [ 49 ]. For the mesoderm, derivatives can be used, e.g. cartilage matrix protein for the bone and alcian blue for the cartilage. As ectodermal markers, class III B botulin or keratin can be used for keratinocytes.

Teratoma formation assays are considered the gold standard for demonstrating the pluripotency of human iPSCs, demonstrating their possibilities under physiological conditions. Due to their actual tissue formation, they could be used for the characterization of many cell lineages [ 50 ].

Directed differentiation

To be useful in therapy, stem cells must be converted into desired cell types as necessary or else the whole regenerative medicine process will be pointless. Differentiation of ESCs is crucial because undifferentiated ESCs can cause teratoma formation in vivo. Understanding and using signalling pathways for differentiation is an important method in successful regenerative medicine. In directed differentiation, it is likely to mimic signals that are received by cells when they undergo successive stages of development [ 51 ]. The extracellular microenvironment plays a significant role in controlling cell behaviour. By manipulating the culture conditions, it is possible to restrict specific differentiation pathways and generate cultures that are enriched in certain precursors in vitro. However, achieving a similar effect in vivo is challenging. It is crucial to develop culture conditions that will allow the promotion of homogenous and enhanced differentiation of ESCs into functional and desired tissues.

Regarding the self-renewal of embryonic stem cells, Hwang et al. [ 52 ] noted that the ideal culture method for hESC-based cell and tissue therapy would be a defined culture free of either the feeder layer or animal components. This is because cell and tissue therapy requires the maintenance of large quantities of undifferentiated hESCs, which does not make feeder cells suitable for such tasks.

Most directed differentiation protocols are formed to mimic the development of an inner cell mass during gastrulation. During this process, pluripotent stem cells differentiate into ectodermal, mesodermal, or endodermal progenitors. Mall molecules or growth factors induce the conversion of stem cells into appropriate progenitor cells, which will later give rise to the desired cell type. There is a variety of signal intensities and molecular families that may affect the establishment of germ layers in vivo, such as fibroblast growth factors (FGFs) [ 53 ]; the Wnt family [ 54 ] or superfamily of transforming growth factors—β(TGFβ); and bone morphogenic proteins (BMP) [ 55 ]. Each candidate factor must be tested on various concentrations and additionally applied to various durations because the precise concentrations and times during which developing cells in embryos are influenced during differentiation are unknown. For instance, molecular antagonists of endogenous BMP and Wnt signalling can be used for ESC formation of ectoderm [ 56 ]. However, transient Wnt and lower concentrations of the TGFβ family trigger mesodermal differentiation [ 57 ]. Regarding endoderm formation, a higher activin A concentration may be required [ 58 , 59 ].

There are numerous protocols about the methods of forming progenitors of cells of each of germ layers, such as cardiomyocytes [ 60 ], hepatocytes [ 61 ], renal cells [ 62 ], lung cells [ 63 , 64 ], motor neurons [ 65 ], intestinal cells [ 66 ], or chondrocytes [ 67 ].

Directed differentiation of either iPSCs or ESCs into, e.g. hepatocytes, could influence and develop the study of the molecular mechanisms in human liver development. In addition, it could also provide the possibility to form exogenous hepatocytes for drug toxicity testing [ 68 ].

Levels of concentration and duration of action with a specific signalling molecule can cause a variety of factors. Unfortunately, for now, a high cost of recombinant factors is likely to limit their use on a larger scale in medicine. The more promising technique focuses on the use of small molecules. These can be used for either activating or deactivating specific signalling pathways. They enhance reprogramming efficiency by creating cells that are compatible with the desired type of tissue. It is a cheaper and non-immunogenic method.

One of the successful examples of small-molecule cell therapies is antagonists and agonists of the Hedgehog pathway. They show to be very useful in motor neuron regeneration [ 69 ]. Endogenous small molecules with their function in embryonic development can also be used in in vitro methods to induce the differentiation of cells; for example, retinoic acid, which is responsible for patterning the nervous system in vivo [ 70 ], surprisingly induced retinal cell formation when the laboratory procedure involved hESCs [ 71 ].

The efficacy of differentiation factors depends on functional maturity, efficiency, and, finally, introducing produced cells to their in vivo equivalent. Topography, shear stress, and substrate rigidity are factors influencing the phenotype of future cells [ 72 ].

The control of biophysical and biochemical signals, the biophysical environment, and a proper guide of hESC differentiation are important factors in appropriately cultured stem cells.

Stem cell utilization and their manufacturing standards and culture systems

The European Medicines Agency and the Food and Drug Administration have set Good Manufacturing Practice (GMP) guidelines for safe and appropriate stem cell transplantation. In the past, protocols used for stem cell transplantation required animal-derived products [ 73 ].

The risk of introducing animal antigens or pathogens caused a restriction in their use. Due to such limitations, the technique required an obvious update [ 74 ]. Now, it is essential to use xeno-free equivalents when establishing cell lines that are derived from fresh embryos and cultured from human feeder cell lines [ 75 ]. In this method, it is crucial to replace any non-human materials with xeno-free equivalents [ 76 ].

NutriStem with LN-511, TeSR2 with human recombinant laminin (LN-511), and RegES with human foreskin fibroblasts (HFFs) are commonly used xeno-free culture systems [ 33 ]. There are many organizations and international initiatives, such as the National Stem Cell Bank, that provide stem cell lines for treatment or medical research [ 77 ].

Stem cell use in medicine

Stem cells have great potential to become one of the most important aspects of medicine. In addition to the fact that they play a large role in developing restorative medicine, their study reveals much information about the complex events that happen during human development.

The difference between a stem cell and a differentiated cell is reflected in the cells’ DNA. In the former cell, DNA is arranged loosely with working genes. When signals enter the cell and the differentiation process begins, genes that are no longer needed are shut down, but genes required for the specialized function will remain active. This process can be reversed, and it is known that such pluripotency can be achieved by interaction in gene sequences. Takahashi and Yamanaka [ 78 ] and Loh et al. [ 79 ] discovered that octamer-binding transcription factor 3 and 4 (Oct3/4), sex determining region Y (SRY)-box 2 and Nanog genes function as core transcription factors in maintaining pluripotency. Among them, Oct3/4 and Sox2 are essential for the generation of iPSCs.

Many serious medical conditions, such as birth defects or cancer, are caused by improper differentiation or cell division. Currently, several stem cell therapies are possible, among which are treatments for spinal cord injury, heart failure [ 80 ], retinal and macular degeneration [ 81 ], tendon ruptures, and diabetes type 1 [ 82 ]. Stem cell research can further help in better understanding stem cell physiology. This may result in finding new ways of treating currently incurable diseases.

Haematopoietic stem cell transplantation

Haematopoietic stem cells are important because they are by far the most thoroughly characterized tissue-specific stem cell; after all, they have been experimentally studied for more than 50 years. These stem cells appear to provide an accurate paradigm model system to study tissue-specific stem cells, and they have potential in regenerative medicine.

Multipotent haematopoietic stem cell (HSC) transplantation is currently the most popular stem cell therapy. Target cells are usually derived from the bone marrow, peripheral blood, or umbilical cord blood [ 83 ]. The procedure can be autologous (when the patient’s own cells are used), allogenic (when the stem cell comes from a donor), or syngeneic (from an identical twin). HSCs are responsible for the generation of all functional haematopoietic lineages in blood, including erythrocytes, leukocytes, and platelets. HSC transplantation solves problems that are caused by inappropriate functioning of the haematopoietic system, which includes diseases such as leukaemia and anaemia. However, when conventional sources of HSC are taken into consideration, there are some important limitations. First, there is a limited number of transplantable cells, and an efficient way of gathering them has not yet been found. There is also a problem with finding a fitting antigen-matched donor for transplantation, and viral contamination or any immunoreactions also cause a reduction in efficiency in conventional HSC transplantations. Haematopoietic transplantation should be reserved for patients with life-threatening diseases because it has a multifactorial character and can be a dangerous procedure. iPSC use is crucial in this procedure. The use of a patient’s own unspecialized somatic cells as stem cells provides the greatest immunological compatibility and significantly increases the success of the procedure.

Stem cells as a target for pharmacological testing

Stem cells can be used in new drug tests. Each experiment on living tissue can be performed safely on specific differentiated cells from pluripotent cells. If any undesirable effect appears, drug formulas can be changed until they reach a sufficient level of effectiveness. The drug can enter the pharmacological market without harming any live testers. However, to test the drugs properly, the conditions must be equal when comparing the effects of two drugs. To achieve this goal, researchers need to gain full control of the differentiation process to generate pure populations of differentiated cells.

Stem cells as an alternative for arthroplasty

One of the biggest fears of professional sportsmen is getting an injury, which most often signifies the end of their professional career. This applies especially to tendon injuries, which, due to current treatment options focusing either on conservative or surgical treatment, often do not provide acceptable outcomes. Problems with the tendons start with their regeneration capabilities. Instead of functionally regenerating after an injury, tendons merely heal by forming scar tissues that lack the functionality of healthy tissues. Factors that may cause this failed healing response include hypervascularization, deposition of calcific materials, pain, or swelling [ 84 ].

Additionally, in addition to problems with tendons, there is a high probability of acquiring a pathological condition of joints called osteoarthritis (OA) [ 85 ]. OA is common due to the avascular nature of articular cartilage and its low regenerative capabilities [ 86 ]. Although arthroplasty is currently a common procedure in treating OA, it is not ideal for younger patients because they can outlive the implant and will require several surgical procedures in the future. These are situations where stem cell therapy can help by stopping the onset of OA [ 87 ]. However, these procedures are not well developed, and the long-term maintenance of hyaline cartilage requires further research.

Osteonecrosis of the femoral hip (ONFH) is a refractory disease associated with the collapse of the femoral head and risk of hip arthroplasty in younger populations [ 88 ]. Although total hip arthroplasty (THA) is clinically successful, it is not ideal for young patients, mostly due to the limited lifetime of the prosthesis. An increasing number of clinical studies have evaluated the therapeutic effect of stem cells on ONFH. Most of the authors demonstrated positive outcomes, with reduced pain, improved function, or avoidance of THA [ 89 – 91 ].

Rejuvenation by cell programming

Ageing is a reversible epigenetic process. The first cell rejuvenation study was published in 2011 [ 92 ]. Cells from aged individuals have different transcriptional signatures, high levels of oxidative stress, dysfunctional mitochondria, and shorter telomeres than in young cells [ 93 ]. There is a hypothesis that when human or mouse adult somatic cells are reprogrammed to iPSCs, their epigenetic age is virtually reset to zero [ 94 ]. This was based on an epigenetic model, which explains that at the time of fertilization, all marks of parenteral ageing are erased from the zygote’s genome and its ageing clock is reset to zero [ 95 ].

In their study, Ocampo et al. [ 96 ] used Oct4, Sox2, Klf4, and C-myc genes (OSKM genes) and affected pancreas and skeletal muscle cells, which have poor regenerative capacity. Their procedure revealed that these genes can also be used for effective regenerative treatment [ 97 ]. The main challenge of their method was the need to employ an approach that does not use transgenic animals and does not require an indefinitely long application. The first clinical approach would be preventive, focused on stopping or slowing the ageing rate. Later, progressive rejuvenation of old individuals can be attempted. In the future, this method may raise some ethical issues, such as overpopulation, leading to lower availability of food and energy.

For now, it is important to learn how to implement cell reprogramming technology in non-transgenic elder animals and humans to erase marks of ageing without removing the epigenetic marks of cell identity.

Cell-based therapies

Stem cells can be induced to become a specific cell type that is required to repair damaged or destroyed tissues (Fig.  6 ). Currently, when the need for transplantable tissues and organs outweighs the possible supply, stem cells appear to be a perfect solution for the problem. The most common conditions that benefit from such therapy are macular degenerations [ 98 ], strokes [ 99 ], osteoarthritis [ 89 , 90 ], neurodegenerative diseases, and diabetes [ 100 ]. Due to this technique, it can become possible to generate healthy heart muscle cells and later transplant them to patients with heart disease.

Stem cell experiments on animals. These experiments are one of the many procedures that proved stem cells to be a crucial factor in future regenerative medicine

In the case of type 1 diabetes, insulin-producing cells in the pancreas are destroyed due to an autoimmunological reaction. As an alternative to transplantation therapy, it can be possible to induce stem cells to differentiate into insulin-producing cells [ 101 ].

Stem cells and tissue banks

iPS cells with their theoretically unlimited propagation and differentiation abilities are attractive for the present and future sciences. They can be stored in a tissue bank to be an essential source of human tissue used for medical examination. The problem with conventional differentiated tissue cells held in the laboratory is that their propagation features diminish after time. This does not occur in iPSCs.

The umbilical cord is known to be rich in mesenchymal stem cells. Due to its cryopreservation immediately after birth, its stem cells can be successfully stored and used in therapies to prevent the future life-threatening diseases of a given patient.

Stem cells of human exfoliated deciduous teeth (SHED) found in exfoliated deciduous teeth has the ability to develop into more types of body tissues than other stem cells [ 102 ] (Table  1 ). Techniques of their collection, isolation, and storage are simple and non-invasive. Among the advantages of banking, SHED cells are:

Guaranteed donor-match autologous transplant that causes no immune reaction and rejection of cells [ 103 ]

Simple and painless for both child and parent

Less than one third of the cost of cord blood storage

Not subject to the same ethical concerns as embryonic stem cells [ 104 ]

In contrast to cord blood stem cells, SHED cells are able to regenerate into solid tissues such as connective, neural, dental, or bone tissue [ 105 , 106 ]

SHED can be useful for close relatives of the donor

Types of stem cells in human exfoliated deciduous teeth

Fertility diseases

In 2011, two researchers, Katsuhiko Hayashi et al. [ 107 ], showed in an experiment on mice that it is possible to form sperm from iPSCs. They succeeded in delivering healthy and fertile pups in infertile mice. The experiment was also successful for female mice, where iPSCs formed fully functional eggs .

Young adults at risk of losing their spermatogonial stem cells (SSC), mostly cancer patients, are the main target group that can benefit from testicular tissue cryopreservation and autotransplantation. Effective freezing methods for adult and pre-pubertal testicular tissue are available [ 108 ].

Qiuwan et al. [ 109 ] provided important evidence that human amniotic epithelial cell (hAEC) transplantation could effectively improve ovarian function by inhibiting cell apoptosis and reducing inflammation in injured ovarian tissue of mice, and it could be a promising strategy for the management of premature ovarian failure or insufficiency in female cancer survivors.

For now, reaching successful infertility treatments in humans appears to be only a matter of time, but there are several challenges to overcome. First, the process needs to have high efficiency; second, the chances of forming tumours instead of eggs or sperm must be maximally reduced. The last barrier is how to mature human sperm and eggs in the lab without transplanting them to in vivo conditions, which could cause either a tumour risk or an invasive procedure.

Therapy for incurable neurodegenerative diseases

Thanks to stem cell therapy, it is possible not only to delay the progression of incurable neurodegenerative diseases such as Parkinson’s disease, Alzheimer’s disease (AD), and Huntington disease, but also, most importantly, to remove the source of the problem. In neuroscience, the discovery of neural stem cells (NSCs) has nullified the previous idea that adult CNS were not capable of neurogenesis [ 110 , 111 ]. Neural stem cells are capable of improving cognitive function in preclinical rodent models of AD [ 112 – 114 ]. Awe et al. [ 115 ] clinically derived relevant human iPSCs from skin punch biopsies to develop a neural stem cell-based approach for treating AD. Neuronal degeneration in Parkinson’s disease (PD) is focal, and dopaminergic neurons can be efficiently generated from hESCs. PD is an ideal disease for iPSC-based cell therapy [ 116 ]. However, this therapy is still in an experimental phase ( https://www.ncbi.nlm.nih.gov/pmc/articles/PMC4539501 /). Brain tissue from aborted foetuses was used on patients with Parkinson’s disease [ 117 ]. Although the results were not uniform, they showed that therapies with pure stem cells are an important and achievable therapy.

Stem cell use in dentistry

Teeth represent a very challenging material for regenerative medicine. They are difficult to recreate because of their function in aspects such as articulation, mastication, or aesthetics due to their complicated structure. Currently, there is a chance for stem cells to become more widely used than synthetic materials. Teeth have a large advantage of being the most natural and non-invasive source of stem cells.

For now, without the use of stem cells, the most common periodontological treatments are either growth factors, grafts, or surgery. For example, there are stem cells in periodontal ligament [ 118 , 119 ], which are capable of differentiating into osteoblasts or cementoblasts, and their functions were also assessed in neural cells [ 120 ]. Tissue engineering is a successful method for treating periodontal diseases. Stem cells of the root apical areas are able to recreate periodontal ligament. One of the possible methods of tissue engineering in periodontology is gene therapy performed using adenoviruses-containing growth factors [ 121 ].

As a result of animal studies, dentin regeneration is an effective process that results in the formation of dentin bridges [ 122 ].

Enamel is more difficult to regenerate than dentin. After the differentiation of ameloblastoma cells into the enamel, the former is destroyed, and reparation is impossible. Medical studies have succeeded in differentiating bone marrow stem cells into ameloblastoma [ 123 ].

Healthy dental tissue has a high amount of regular stem cells, although this number is reduced when tissue is either traumatized or inflamed [ 124 ]. There are several dental stem cell groups that can be isolated (Fig.  7 ).

Localization of stem cells in dental tissues. Dental pulp stem cells (DPSCs) and human deciduous teeth stem cells (SHED) are located in the dental pulp. Periodontal ligaments stem cells are located in the periodontal ligament. Apical papilla consists of stem cells from the apical papilla (SCAP)

Dental pulp stem cell (DPSC)

These were the first dental stem cells isolated from the human dental pulp, which were [ 125 ] located inside dental pulp (Table  2 ). They have osteogenic and chondrogenic potential. Mesenchymal stem cells (MSCs) of the dental pulp, when isolated, appear highly clonogenic; they can be isolated from adult tissue (e.g. bone marrow, adipose tissue) and foetal (e.g. umbilical cord) [ 126 ] tissue, and they are able to differentiate densely [ 127 ]. MSCs differentiate into odontoblast-like cells and osteoblasts to form dentin and bone. Their best source locations are the third molars [ 125 ]. DPSCs are the most useful dental source of tissue engineering due to their easy surgical accessibility, cryopreservation possibility, increased production of dentin tissues compared to non-dental stem cells, and their anti-inflammatory abilities. These cells have the potential to be a source for maxillofacial and orthopaedic reconstructions or reconstructions even beyond the oral cavity. DPSCs are able to generate all structures of the developed tooth [ 128 ]. In particular, beneficial results in the use of DPSCs may be achieved when combined with other new therapies, such as periodontal tissue photobiomodulation (laser stimulation), which is an efficient technique in the stimulation of proliferation and differentiation into distinct cell types [ 129 ]. DPSCs can be induced to form neural cells to help treat neurological deficits.

Detailed information about the differentiation of DPSCs and the studies connected to them [ 176 ]

Stem cells of human exfoliated deciduous teeth (SHED) have a faster rate of proliferation than DPSCs and differentiate into an even greater number of cells, e.g. other mesenchymal and non-mesenchymal stem cell derivatives, such as neural cells [ 130 ]. These cells possess one major disadvantage: they form a non-complete dentin/pulp-like complex in vivo. SHED do not undergo the same ethical concerns as embryonic stem cells. Both DPSCs and SHED are able to form bone-like tissues in vivo [ 131 ] and can be used for periodontal, dentin, or pulp regeneration. DPSCs and SHED can be used in treating, for example, neural deficits [ 132 ]. DPSCs alone were tested and successfully applied for alveolar bone and mandible reconstruction [ 133 ].

Periodontal ligament stem cells (PDLSCs)

These cells are used in periodontal ligament or cementum tissue regeneration. They can differentiate into mesenchymal cell lineages to produce collagen-forming cells, adipocytes, cementum tissue, Sharpey’s fibres, and osteoblast-like cells in vitro. PDLSCs exist both on the root and alveolar bone surfaces; however, on the latter, these cells have better differentiation abilities than on the former [ 134 ]. PDLSCs have become the first treatment for periodontal regeneration therapy because of their safety and efficiency [ 135 , 136 ].

Stem cells from apical papilla (SCAP)

These cells are mesenchymal structures located within immature roots. They are isolated from human immature permanent apical papilla. SCAP are the source of odontoblasts and cause apexogenesis. These stem cells can be induced in vitro to form odontoblast-like cells, neuron-like cells, or adipocytes. SCAP have a higher capacity of proliferation than DPSCs, which makes them a better choice for tissue regeneration [ 137 , 138 ].

Dental follicle stem cells (DFCs)

These cells are loose connective tissues surrounding the developing tooth germ. DFCs contain cells that can differentiate into cementoblasts, osteoblasts, and periodontal ligament cells [ 139 , 140 ]. Additionally, these cells proliferate after even more than 30 passages [ 141 ]. DFCs are most commonly extracted from the sac of a third molar. When DFCs are combined with a treated dentin matrix, they can form a root-like tissue with a pulp-dentin complex and eventually form tooth roots [ 141 ]. When DFC sheets are induced by Hertwig’s epithelial root sheath cells, they can produce periodontal tissue; thus, DFCs represent a very promising material for tooth regeneration [ 142 ].

Pulp regeneration in endodontics

Dental pulp stem cells can differentiate into odontoblasts. There are few methods that enable the regeneration of the pulp.

The first is an ex vivo method. Proper stem cells are grown on a scaffold before they are implanted into the root channel [ 143 ].

The second is an in vivo method. This method focuses on injecting stem cells into disinfected root channels after the opening of the in vivo apex. Additionally, the use of a scaffold is necessary to prevent the movement of cells towards other tissues. For now, only pulp-like structures have been created successfully.

Methods of placing stem cells into the root channel constitute are either soft scaffolding [ 144 ] or the application of stem cells in apexogenesis or apexification. Immature teeth are the best source [ 145 ]. Nerve and blood vessel network regeneration are extremely vital to keep pulp tissue healthy.

The potential of dental stem cells is mainly regarding the regeneration of damaged dentin and pulp or the repair of any perforations; in the future, it appears to be even possible to generate the whole tooth. Such an immense success would lead to the gradual replacement of implant treatments. Mandibulary and maxillary defects can be one of the most complicated dental problems for stem cells to address.

Acquiring non-dental tissue cells by dental stem cell differentiation

In 2013, it was reported that it is possible to grow teeth from stem cells obtained extra-orally, e.g. from urine [ 146 ]. Pluripotent stem cells derived from human urine were induced and generated tooth-like structures. The physical properties of the structures were similar to natural ones except for hardness [ 127 ]. Nonetheless, it appears to be a very promising technique because it is non-invasive and relatively low-cost, and somatic cells can be used instead of embryonic cells. More importantly, stem cells derived from urine did not form any tumours, and the use of autologous cells reduces the chances of rejection [ 147 ].

Use of graphene in stem cell therapy

Over recent years, graphene and its derivatives have been increasingly used as scaffold materials to mediate stem cell growth and differentiation [ 148 ]. Both graphene and graphene oxide (GO) represent high in-plane stiffness [ 149 ]. Because graphene has carbon and aromatic network, it works either covalently or non-covalently with biomolecules; in addition to its superior mechanical properties, graphene offers versatile chemistry. Graphene exhibits biocompatibility with cells and their proper adhesion. It also tested positively for enhancing the proliferation or differentiation of stem cells [ 148 ]. After positive experiments, graphene revealed great potential as a scaffold and guide for specific lineages of stem cell differentiation [ 150 ]. Graphene has been successfully used in the transplantation of hMSCs and their guided differentiation to specific cells. The acceleration skills of graphene differentiation and division were also investigated. It was discovered that graphene can serve as a platform with increased adhesion for both growth factors and differentiation chemicals. It was also discovered that π-π binding was responsible for increased adhesion and played a crucial role in inducing hMSC differentiation [ 150 ].

Therapeutic potential of extracellular vesicle-based therapies

Extracellular vesicles (EVs) can be released by virtually every cell of an organism, including stem cells [ 151 ], and are involved in intercellular communication through the delivery of their mRNAs, lipids, and proteins. As Oh et al. [ 152 ] prove, stem cells, together with their paracrine factors—exosomes—can become potential therapeutics in the treatment of, e.g. skin ageing. Exosomes are small membrane vesicles secreted by most cells (30–120 nm in diameter) [ 153 ]. When endosomes fuse with the plasma membrane, they become exosomes that have messenger RNAs (mRNAs) and microRNAs (miRNAs), some classes of non-coding RNAs (IncRNAs) and several proteins that originate from the host cell [ 154 ]. IncRNAs can bind to specific loci and create epigenetic regulators, which leads to the formation of epigenetic modifications in recipient cells. Because of this feature, exosomes are believed to be implicated in cell-to-cell communication and the progression of diseases such as cancer [ 155 ]. Recently, many studies have also shown the therapeutic use of exosomes derived from stem cells, e.g. skin damage and renal or lung injuries [ 156 ].

In skin ageing, the most important factor is exposure to UV light, called “photoageing” [ 157 ], which causes extrinsic skin damage, characterized by dryness, roughness, irregular pigmentation, lesions, and skin cancers. In intrinsic skin ageing, on the other hand, the loss of elasticity is a characteristic feature. The skin dermis consists of fibroblasts, which are responsible for the synthesis of crucial skin elements, such as procollagen or elastic fibres. These elements form either basic framework extracellular matrix constituents of the skin dermis or play a major role in tissue elasticity. Fibroblast efficiency and abundance decrease with ageing [ 158 ]. Stem cells can promote the proliferation of dermal fibroblasts by secreting cytokines such as platelet-derived growth factor (PDGF), transforming growth factor β (TGF-β), and basic fibroblast growth factor. Huh et al. [ 159 ] mentioned that a medium of human amniotic fluid-derived stem cells (hAFSC) positively affected skin regeneration after longwave UV-induced (UVA, 315–400 nm) photoageing by increasing the proliferation and migration of dermal fibroblasts. It was discovered that, in addition to the induction of fibroblast physiology, hAFSC transplantation also improved diseases in cases of renal pathology, various cancers, or stroke [ 160 , 161 ].

Oh [ 162 ] also presented another option for the treatment of skin wounds, either caused by physical damage or due to diabetic ulcers. Induced pluripotent stem cell-conditioned medium (iPSC-CM) without any animal-derived components induced dermal fibroblast proliferation and migration.

Natural cutaneous wound healing is divided into three steps: haemostasis/inflammation, proliferation, and remodelling. During the crucial step of proliferation, fibroblasts migrate and increase in number, indicating that it is a critical step in skin repair, and factors such as iPSC-CM that impact it can improve the whole cutaneous wound healing process. Paracrine actions performed by iPSCs are also important for this therapeutic effect [ 163 ]. These actions result in the secretion of cytokines such as TGF-β, interleukin (IL)-6, IL-8, monocyte chemotactic protein-1 (MCP-1), vascular endothelial growth factor (VEGF), platelet-derived growth factor-AA (PDGF-AA), and basic fibroblast growth factor (bFGF). Bae et al. [ 164 ] mentioned that TGF-β induced the migration of keratinocytes. It was also demonstrated that iPSC factors can enhance skin wound healing in vivo and in vitro when Zhou et al. [ 165 ] enhanced wound healing, even after carbon dioxide laser resurfacing in an in vivo study.

Peng et al. [ 166 ] investigated the effects of EVs derived from hESCs on in vitro cultured retinal glial, progenitor Müller cells, which are known to differentiate into retinal neurons. EVs appear heterogeneous in size and can be internalized by cultured Müller cells, and their proteins are involved in the induction and maintenance of stem cell pluripotency. These stem cell-derived vesicles were responsible for the neuronal trans-differentiation of cultured Müller cells exposed to them. However, the research article points out that the procedure was accomplished only on in vitro acquired retina.

Challenges concerning stem cell therapy

Although stem cells appear to be an ideal solution for medicine, there are still many obstacles that need to be overcome in the future. One of the first problems is ethical concern.

The most common pluripotent stem cells are ESCs. Therapies concerning their use at the beginning were, and still are, the source of ethical conflicts. The reason behind it started when, in 1998, scientists discovered the possibility of removing ESCs from human embryos. Stem cell therapy appeared to be very effective in treating many, even previously incurable, diseases. The problem was that when scientists isolated ESCs in the lab, the embryo, which had potential for becoming a human, was destroyed (Fig.  8 ). Because of this, scientists, seeing a large potential in this treatment method, focused their efforts on making it possible to isolate stem cells without endangering their source—the embryo.

Use of inner cell mass pluripotent stem cells and their stimulation to differentiate into desired cell types

For now, while hESCs still remain an ethically debatable source of cells, they are potentially powerful tools to be used for therapeutic applications of tissue regeneration. Because of the complexity of stem cell control systems, there is still much to be learned through observations in vitro. For stem cells to become a popular and widely accessible procedure, tumour risk must be assessed. The second problem is to achieve successful immunological tolerance between stem cells and the patient’s body. For now, one of the best ideas is to use the patient’s own cells and devolve them into their pluripotent stage of development.

New cells need to have the ability to fully replace lost or malfunctioning natural cells. Additionally, there is a concern about the possibility of obtaining stem cells without the risk of morbidity or pain for either the patient or the donor. Uncontrolled proliferation and differentiation of cells after implementation must also be assessed before its use in a wide variety of regenerative procedures on living patients [ 167 ].

One of the arguments that limit the use of iPSCs is their infamous role in tumourigenicity. There is a risk that the expression of oncogenes may increase when cells are being reprogrammed. In 2008, a technique was discovered that allowed scientists to remove oncogenes after a cell achieved pluripotency, although it is not efficient yet and takes a longer amount of time. The process of reprogramming may be enhanced by deletion of the tumour suppressor gene p53, but this gene also acts as a key regulator of cancer, which makes it impossible to remove in order to avoid more mutations in the reprogrammed cell. The low efficiency of the process is another problem, which is progressively becoming reduced with each year. At first, the rate of somatic cell reprogramming in Yamanaka’s study was up to 0.1%. The use of transcription factors creates a risk of genomic insertion and further mutation of the target cell genome. For now, the only ethically acceptable operation is an injection of hESCs into mouse embryos in the case of pluripotency evaluation [ 168 ].

Stem cell obstacles in the future

Pioneering scientific and medical advances always have to be carefully policed in order to make sure they are both ethical and safe. Because stem cell therapy already has a large impact on many aspects of life, it should not be treated differently.

Currently, there are several challenges concerning stem cells. First, the most important one is about fully understanding the mechanism by which stem cells function first in animal models. This step cannot be avoided. For the widespread, global acceptance of the procedure, fear of the unknown is the greatest challenge to overcome.

The efficiency of stem cell-directed differentiation must be improved to make stem cells more reliable and trustworthy for a regular patient. The scale of the procedure is another challenge. Future stem cell therapies may be a significant obstacle. Transplanting new, fully functional organs made by stem cell therapy would require the creation of millions of working and biologically accurate cooperating cells. Bringing such complicated procedures into general, widespread regenerative medicine will require interdisciplinary and international collaboration.

The identification and proper isolation of stem cells from a patient’s tissues is another challenge. Immunological rejection is a major barrier to successful stem cell transplantation. With certain types of stem cells and procedures, the immune system may recognize transplanted cells as foreign bodies, triggering an immune reaction resulting in transplant or cell rejection.

One of the ideas that can make stem cells a “failsafe” is about implementing a self-destruct option if they become dangerous. Further development and versatility of stem cells may cause reduction of treatment costs for people suffering from currently incurable diseases. When facing certain organ failure, instead of undergoing extraordinarily expensive drug treatment, the patient would be able to utilize stem cell therapy. The effect of a successful operation would be immediate, and the patient would avoid chronic pharmacological treatment and its inevitable side effects.

Although these challenges facing stem cell science can be overwhelming, the field is making great advances each day. Stem cell therapy is already available for treating several diseases and conditions. Their impact on future medicine appears to be significant.

After several decades of experiments, stem cell therapy is becoming a magnificent game changer for medicine. With each experiment, the capabilities of stem cells are growing, although there are still many obstacles to overcome. Regardless, the influence of stem cells in regenerative medicine and transplantology is immense. Currently, untreatable neurodegenerative diseases have the possibility of becoming treatable with stem cell therapy. Induced pluripotency enables the use of a patient’s own cells. Tissue banks are becoming increasingly popular, as they gather cells that are the source of regenerative medicine in a struggle against present and future diseases. With stem cell therapy and all its regenerative benefits, we are better able to prolong human life than at any time in history.

Acknowledgements

Not applicable.

This work is supported by Wrocław Medical University in Poland.

Availability of data and materials

Please contact author for data requests.

Abbreviations

Basic fibroblast growth factor

Bone morphogenic proteins

Dental follicle stem cells

Dental pulp stem cells

Embryonic bodies

Embryonic stem cells

Fibroblast growth factors

Good Manufacturing Practice

Graphene oxide

Human amniotic fluid-derived stem cells

Human embryonic stem cells

Human foreskin fibroblasts

Inner cell mass

Non-coding RNA

Induced pluripotent stem cells

In vitro fertilization

Knockout serum replacement

Leukaemia inhibitory factor

Monocyte chemotactic protein-1

Fibroblasts

Messenger RNA

Mesenchymal stem cells of dental pulp

Myogenic differentiation

Osteoarthritis

Octamer-binding transcription factor 3 and 4

Platelet-derived growth factor

Platelet-derived growth factor-AA

Periodontal ligament stem cells

Rho-associated protein kinase

Stem cells from apical papilla

Stem cells of human exfoliated deciduous teeth

Synthetic Serum Substitute

Trophectoderm

Vascular endothelial growth factor

Transforming growth factors

Authors’ contributions

WZ is the principal author and was responsible for the first draft of the manuscript. WZ and ZR were responsible for the concept of the review. MS, MD, and ZR were responsible for revising the article and for data acquisition. All authors read and approved the final manuscript.

Ethics approval and consent to participate

Consent for publication, competing interests.

The authors declare that they have no competing interests.

Publisher’s Note

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Contributor Information

Wojciech Zakrzewski, Email: [email protected].

Maciej Dobrzyński, Email: [email protected].

Maria Szymonowicz, Email: [email protected].

Zbigniew Rybak, Email: [email protected].

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• Published: 11 November 2024

Autologous stem-cell derived islets — the ultimate frontier in diabetes mellitus?

• A. M. James Shapiro   ORCID: orcid.org/0000-0002-6215-0990 1  

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A patient with longstanding type 1 diabetes mellitus has achieved insulin independence for at least 1 year after transplantation of autologous stem cell islets. These cells were differentiated from inducible pluripotent stem cells from adipose tissue and were transplanted into the rectus sheath of the abdominal wall.

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Shapiro, A.M.J. Autologous stem-cell derived islets — the ultimate frontier in diabetes mellitus?. Nat Rev Endocrinol (2024). https://doi.org/10.1038/s41574-024-01064-x

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Emerging insights into epigenetics and hematopoietic stem cell trafficking in age-related hematological malignancies

• Yang Xinyi   ORCID: orcid.org/0009-0009-2320-0834 1 ,
• Reshetov Igor Vladimirovich 1 ,
• Narasimha M. Beeraka 3 , 4 , 5 , 9 ,
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Hematopoiesis within the bone marrow (BM) is a complex and tightly regulated process predominantly influenced by immune factors. Aging, diabetes, and obesity are significant contributors to BM niche damage, which can alter hematopoiesis and lead to the development of clonal hematopoiesis of intermediate potential (CHIP). Genetic/epigenetic alterations during aging could influence BM niche reorganization for hematopoiesis or clonal hematopoiesis. CHIP is driven by mutations in genes such as Tet2, Dnmt3a, Asxl1, and Jak2, which are associated with age-related hematological malignancies.

This literature review aims to provide an updated exploration of the functional aspects of BM niche cells within the hematopoietic microenvironment in the context of age-related hematological malignancies. The review specifically focuses on how immunological stressors modulate different signaling pathways that impact hematopoiesis. Methods: An extensive review of recent studies was conducted, examining the roles of various BM niche cells in hematopoietic stem cell (HSC) trafficking and the development of age-related hematological malignancies. Emphasis was placed on understanding the influence of immunological stressors on these processes.

Recent findings reveal a significant microheterogeneity and temporal stochasticity of niche cells across the BM during hematopoiesis. These studies demonstrate that niche cells, including mesenchymal stem cells, osteoblasts, and endothelial cells, exhibit dynamic interactions with HSCs, significantly influenced by the BM microenvironment as the age increases. Immunosurveillance plays a crucial role in maintaining hematopoietic homeostasis, with alterations in immune signaling pathways contributing to the onset of hematological malignancies. Novel insights into the interaction between niche cells and HSCs under stress/aging conditions highlight the importance of niche plasticity and adaptability.

The involvement of age-induced genetic/epigenetic alterations in BM niche cells and immunological stressors in hematopoiesis is crucial for understanding the development of age-related hematological malignancies. This comprehensive review provides new insights into the complex interplay between niche cells and HSCs, emphasizing the potential for novel therapeutic approaches that target niche cell functionality and resilience to improve hematopoietic outcomes in the context of aging and metabolic disorders.

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This review introduces novel concepts regarding the plasticity and adaptability of BM niche cells in response to immunological stressors and epigenetics. It proposes that targeted therapeutic strategies aimed at enhancing niche cell resilience could mitigate the adverse effects of aging, diabetes, and obesity on hematopoiesis and clonal hematopoiesis. Additionally, the review suggests that understanding the precise temporal and spatial dynamics of niche-HSC interactions and epigenetics influence may lead to innovative treatments for age-related hematological malignancies.

Graphical abstract

Introduction

During the developmental periods, mesenchymal stem cells (MSCs) and other stem cells exhibit microheterogeneity, specific series of temporal events [ 1 ]. The significant intricate potential of these cells in tissue repair, and cell replacement makes remarkable development of stem cell-based treatments, which require substantial preclinical and clinical studies. However, the unproven efficacy of these cells to ameliorate patient maladies successfully is questionable in terms of science and medical applications.

The bone marrow is a complex and vital component of the human body. It has a diverse and dynamic environment consisting of characteristic cells that work in unison to produce blood and immune cells. The bone marrow houses hematopoietic stem cells (HSCs) and multipotent progenitors (MPPs). Bone marrow niche is perpetually dynamic, where billions of cells are produced to replace transient cells. These are periodically cleared and create an endless supply of blood factors and cells necessary for oxygenation and homeostatic regulation. Bone marrow possesses a unique “demand-adapted” hematopoiesis which is triggered during injury or infection, where there is a surge in production and levels of high-demand cells. This sheds light on the inflammatory response pathway of bone marrow [ 2 ]. The bone marrow (BM) microenvironment is highly vascularized and composed of several niche components such as stromal and nonstromal cells. Niche components inside the BM could act directly on the hematopoietic stem cells (HSCs). For instance, the perivascular cells are confined to the BM and exemplified by the expression of Nestin + mesenchymal stem cells (MSCs). These cells can be categorized into Nestin-GFP bright and Nestin-GFP dim in Nestin-GFP transgenic mice. The Nestin-GFP dim cells are confined to the sinusoids whereas the Nestin-GFP bright cells are confined to the arterioles [ 3 , 4 ]. Stromal cells are categorized into NG2 + cells CXCL-12 abundant reticular cells [ 5 ], and leptin receptor cells [ 6 , 7 , 8 ]. Stem cells confined to the BM could undergo self-renewal and generate a progeny of lineages such as myeloid and lymphoid cells. Mainly, HSCs maintain contact with the osteoblasts in the sinusoidal endothelium in the BM microenvironment. Several anchoring cells are evident in the BM to foster HSCs’ survival, retention, and proliferation. Specifically, endothelial cells (ECs) and MSCs are supporting cells that can promote the survival of HSCs and subsequently foster BM regeneration. Furthermore, sympathetic and parasympathetic innervations of the autonomic system onto the BM could affect the hematopoietic system.

Aging [ 9 ], diabetes [ 10 , 11 ], obesity-induced proinflammatory states [ 12 ], and the formation of fatty BM [ 12 ] are a few potential reasons for clonal hematopoiesis of intermediate potential (CHIP)-associated pathological conditions [ 12 ]. In this review, we have discussed the updated mechanistic role of several niche cells in the BM in the promotion of HSCs proliferation and differentiation upon aging-induced genetic/epigenetic changes, and metabolic diseases in order to target age-related haematological malignancies. We discussed the role of several biological and pharmacological molecules in modulating the HSPCs trafficking in both hematopoiesis during aging-related hematological malignancies by modulating several signaling pathways.

Literature search

We conducted a comprehensive literature review by gathering data from various databases, including Pubmed, Medline, eMedicine, Scopus, Google Scholar, the National Library of Medicine (NLM), and ReleMed. We focused on published reports and articles spanning from 1950 to 2023, exploring studies related to the implications of niche cells within the bone marrow (BM) on hematopoiesis, the influence of neural signaling on hematopoiesis during hematological malignancies while addressing immunological stressors, and their potential clinical implications. Key search terms such as “aging,” “obesity,” “diabetes,” “hematopoiesis,” “BM niche cells,” “epigenetics/genetics”, “clonal hematopoiesis,” “clonal hematopoiesis of indeterminate potential (CHIP),” “immune stressors,” and “hematological malignancies” were utilized to ensure effective literature collection. Relevant articles focusing on the implications of BM niche cells on hematopoiesis during hematological malignancies, particularly in the context of overcoming immunological stressors, were identified for analysis.

BM microenvironment

The bone marrow microenvironment is composed of several different kinds of cell types including hematopoietic stem cell progenitors, osteoblasts, immune cells, osteoclasts, and perivascular cells [ 13 , 14 ]. Primitive hematopoietic cells undergo trafficking to the specific vascular regions of the bone marrow where CXCL12 and E-selectin are abundant. Different kinds of MSCs can regulate the HSCs’ survival in the specialized bone niches. However, the significant diversity and the lineage relationships are yet to be explored vividly for MSCs in the BM. The majority of them are confined across the perivascular space arterioles, and sinusoids and generate several significant niche factors such as SCF and CXCL-12. Consequently, these factors are identified by the leptin receptor [ 15 , 16 ], Nestin [ 17 ], or NG2 (Cspg4) [ 3 ], these were explored by studying the Lepr-cre and Nes-GFP reporter non-inducible mouse lines respectively [ 17 , 18 ]. Bone marrow damage is evident in chemotherapy or radiotherapy after irradiation. Irradiation could damage sinusoids across the bone marrow [ 13 , 19 , 20 ] and the arteriolar blood vessels are preserved in the endosteum. However, it is yet to be examined the role of these factors such as SCF and CXCL-12 in producing either distinct or overlapping cell lineages during hematopoiesis across BM.

• Hematopoiesis

HSCs are multipotent primitive cells that can differentiate into different kinds of blood cells such as myeloid-lineage and lymphoid-lineage cells [ 21 ]. These cells can be abundant in peripheral blood, and bone marrow. These lineages are significantly generated through the self-renewing multipotent HSCs. A vivid understating of the self-renewal and differentiation mechanisms could have significant clinical implications pertinent to disease type and severity. The minimal amount of this HSC population can foster hematopoiesis [ 22 ]. Murine models : Previous studies described the long-term dormancy and quiescence of the HSCs is mediated by several genetic and epigenetic factors as described in the studies related to p21cip1/waf1 by using p21-/- mice [ 23 , 24 , 25 ]. HSCs confined to the bone marrow are significantly involved in the generation of all kinds of blood cells. Albeit HSCs undergo division infrequently, it is suggested that the HSC pool turns over randomly depending on the internal cues and they often undergo division through entering/exiting the cell cycle.

Previous studies have delineated the flow cytometry-based label-retaining assays using BrdU and histone H2B-GFP to observe a specific population of dormant HSCs in murine models within linSca1 + cKit + CD150 + CD48CD34 population [ 26 ]. In addition, computational models described that the dormant HSCs undergo division every 145 days and 5 times in their lifetime [ 26 ]. Stimulation through G-CSF activity or bone marrow injury could induce self-renewal HSCs suggesting their efficacy to switch from dormancy to self-renewal at the time of hematopoietic stress [ 26 ]. As soon as they re-established the homeostasis, they can return to dormancy suggesting that HSCs are not stochastically undergoing the cell cycle but undergo a reversible switch from dormancy to self-renewal at the time of hematopoietic stress [ 26 ].

Several transplantation studies described that the HSCs can live longer when compared to the donor from which they specifically isolated [ 27 ]. Similar to stem cells, telomerase in the HSCs is significantly involved in the stability of chromosomes at the time of cell division [ 28 ]. Furthermore, the HSCs of aged donors are significantly different from young donors [ 29 , 30 ]. For instance, HSCs of aged donors exhibit significantly a rapid cell cycle, altered cell surface phenotypic immune markers, and generate a higher number of myeloid cells, and these HSCs are associated with minimal efficacy in homing to bone marrow when compared to younger individuals [ 31 , 32 , 33 , 34 , 35 , 36 ]. During aged conditions, the HSCs exhibit comparatively a minimal self-renewal capacity when compared to their younger counterparts due to the DNA damage in HSCs [ 37 , 38 ]. However, future studies are required to explore the age-related changes in the behavior of HSCs. Different subsets of HSCs exhibit distinct characteristics such as self-renewal capacities, repopulation kinetics, and differentiation capacities [ 39 , 40 , 41 , 42 ]. The clonal analysis described the HSC compartment in aged individuals and observed the three kinds of HSC subsets reported in younger mice [ 39 , 43 , 44 ]. Normally, the generation of myeloid and lymphoid cells is in a similar ratio to the HSCs self-renewal process in the blood of unmanipulated mice. Myeloid-biased HSCs could produce a minimal number of subsequent generations of lymphoid progeny whereas the lymphoid-biased HSCs could produce a minimal number of myeloid cells [ 39 ]. A minimal number of prethymic T-cell precursors as well as B-cell precursors are produced by the myeloid-biased HSCs. Significantly a slow response of lymphoid progeny has been observed to the IL-7 lymphokine at the time of minimal lymphopoiesis. Myeloid-biased HSCs from young murine models suggested a delay in the onset of repopulation after transplantation but could be conducive to profoundly longer peripheral hematopoiesis when compared to the other kinds of HSCs [ 39 , 45 ].

MSCs derived from the bone marrow are considered rare hematopoietic cells with self-renewal capacity to undergo differentiation into bone, cartilage, and fat. These cells could surround the arterioles and form wrapping loosely around the sinusoidal vessels. Furthermore, along with HSCs, the MSCs can form the CFC-Fs or mesenchymal spheres in in vitro conditions. During the transplantation of MSCs, they could be attributed to the ectopic formation of the hematopoietic niche consequently we can observe BM stromal cells and active haematopoiesis [ 46 ].

Murine models

Human bone marrow-derived CD146 + mesenchymal stem and progenitor cells (MSPCs) upon transplantation through subcutaneous route into the murine models have induced the generation of heterotopic hematopoietic niche associated with host-derived HSCs as well as donor-derived perivascular CD146 + MSPCs [ 47 ]. In addition, the transplantation of CD105 + CD15 + MSPCs obtained from the bone tissue of fetal mice into the renal capsule of adult murine models could foster the formation of ectopic hematopoietic marrow [ 48 ]. Thus, the intricate role of MSPCs requires substantial studies in the irradiated murine models to explore their ability to organize the HSC niche [ 49 ]. MSCs confined to the perivascular region constituted a significant fraction of CD146 in humans [ 47 ] and CXCL12-GFP, Nestin-GFP, leptin receptor, Prx-1-Cre, inducible Mx-1Cre in the murine models could produce the osteoblast cells and foster the generation of several factors to induce the maintenance of HSCs [ 50 ]. The CXCL12 abundant reticular (CAR) cells are confined to the sinusoids and they were co-localized with HSCs across bone marrow [ 51 ]. For instance, irradiating the mice with CXCL-12 expressing BM cells resulted in the depletion of HSCs consequently causing defects in both adipogenic as well as osteogenic efficacy of BM [ 5 ]. Furthermore, the human CD146 + skeletal stem cells can undergo localization adjacent to sinusoids across BM and are involved in fostering substantial amounts of HSC niche factors including SCF, and CXCL-12 [ 47 ]. BM stromal cells express the fibroblast activation protein (FAP) and also a phenotypic expression of CXCL-12, SCF, and Sca-1 is abundant in these cells, and involved in modulating the hematopoiesis [ 52 , 53 ]. For instance, the ablation of BM stromal cells expressing FAP + could cause depression in the BM followed by the eventual occurrence of anemia, hypocellularity, and decline in the osteogenic cells [ 54 , 55 ]. Future studies are warranted to explore these mechanisms in the BM microenvironment.

Endothelial cells (ECs)

The endothelial cells are confined to the perivascular HSC niche [ 56 ] and evidence described the deletion of gp130 cytokine receptors in ECs that could cause hypocellularity of BM and the decline in overall HSCs [ 57 ]. Loss of VEGF receptor-2 functions in the murine models by irradiation caused the impairment of regeneration of sinusoidal ECs consequently blocking the LSK stem/progenitor cells and spleen colony-forming cells [ 20 ]. The ECs could promote the maintenance of HSC in the culture conditions [ 58 , 59 ]. E-selectin expressed by ECs is involved in the HSCs maintenance in BM and for instance, blocking the activity of E-selectin could cause a higher quiescence of HSCs and make them more resistant to irradiated ablation [ 60 ]. These studies described the crucial functions of ECs in the maintenance of HSC niche but future studies required to explore the role of ECs in regulating the HSCs maintenance in the in vivo conditions. It is crucial to explore the activity of ECs involvement in modulating the vascular permeability in NPY-treated mouse models by examining the HSPC egress.

T reg cell’s role in hematopoiesis

T Regulatory cells (Foxp3 + regulatory T cells) play a crucial role in maintaining the immune system and self-tolerance. These specialized cells, characterized by the expression of transcription factor Foxp3, have been extensively studied for their ability to regulate immune response. However, a gray area in research has been the role of T cells in hematopoiesis and post-transplant reconstitution [ 61 ]. In recent times, several experiments have been undertaken to explore the impact of Treg cells on B-cell lymphopoiesis ( Fig.  1 ) and the function of the BM microenvironment [ 61 ]. Researchers have analyzed several T-cell-depleted mice models to understand the relation between T cells and HSC [ 61 ]. In T-cell-depleted mice models, it was evident that the B cell lymphopoiesis was downregulated in the bone marrow, however, the B cell population was replenished in a compensatory manner by the transfer of affected HSCs or bone marrow cells into T-reg competent recipients [ 61 ] (Fig.  2 ). Intriguingly, B-cell reconstitution was not impaired in both syngeneic and allogeneic transplantation models with Treg-depleted mice as recipients [ 61 ].

To further understand the underlying mechanisms, researchers investigated the production of interleukin-7 (IL-7), a growth factor crucial for B-cell lymphopoiesis. It was revealed that the T cell governs the physiological production of IL-7 which is majorly assisted by a subgroup of ICAM1 + perivascular stromal cells [ 61 ]. In the event of T cell depletion, IL-7 production by these stromal cells decreased, suggesting a strong physiological influence of T cells on the differentiation of B cells. There exists a crucial, intricate relationship between T reg cells, B cell differentiation, and the production of essential growth factors. IL-7 can modulate cell fate decisions by increasing the expression profiles of BACH2, EBF1, and PAX5 that concomitantly together conferring to the specification or commitment of B cell progenitors. Previous studies described the significant role of HSC transplantation in patients with T-B + SCID and subsequently concluded the functional role of IL-7 receptor signaling in leukemogenesis [ 62 ]. Understanding these mechanisms can help to gain valuable insights into T cell biology, physiology, and its implications for clinical applications for modulating the immunological homeostasis among normal individuals and individuals with BM transplants [ 61 ].

The impact of Treg cells on immune reconstitution after transplantation is of particular interest. Development of tolerance by T cells is observed in several transplantation models [ 63 ]. Co-infusion of donor Treg cells has been shown to ameliorate graft failure in the allogeneic transplants performed in mice models with induced T cell depletion. Treg cells can provide an immune niche to HSCs, helping them to evade host immunity and favoring their survival. Impaired downstream signaling of inflammatory cytokines such as IFN or TNF can confer to dysregulation of HSCs in the BM [ 64 , 65 ]. These mediators may be crucial in T cell-depleted transplant models. Interestingly, the functional HSC population remained relatively stable in the T-cell-depleted mice model despite the transition of phenotypic HSC into the cell cycle and expansion phase model. However, as a result of T cell depletion, the differentiation of B cells was observed. This indicates that the BM microenvironment aids the B cell differentiation, which was extremely vulnerable to T cell depletion-mediated immune cell activation. This evidence corroborates the role of T cells in regulating the secretion of inflammatory cytokines (which in turn governs HSC quiescence) and BM physiology and function. The HSC and bone marrow environment have susceptibility to host immune cells. The absence of host Treg cells led to increased host-versus-graft BM alloreactivity, resulting in bone marrow aplasia and decreased survival rates. Furthermore, studies suggest that transplant failure is often observed as a result of T cell depletion and has evident manifestations when those mice are subjected to allogenic transplants.

The HSC niche is composed of various essential components, including a cytokine pool which plays a crucial role in HSC progenitor differentiation into lymphoid cell lineages [ 66 ]. Perivascular stromal cells, a crucial component of the HSC niche, secrete growth factors such as CXCL12 and IL-7 for B cell differentiation. In vivo T cell-depleted mice models successfully restored donor B-cell reconstitution when administered external IL-7, highlighting that T cells govern IL-7 production. Subsequently, IL-7 has also been used to restore immune response post-transplant in preclinical studies and clinical trials [ 67 , 68 ].

( A ). Bone marrow adipocyte lineages and their role in the hematopoietic stem cells and their role on the osteoblasts, and osteocytes across the bone marrow. ( B ). Heterogeneity of macrophages inside the bone marrow and their role in the hematopoiesis by modulating the functional aspects of niche cells. HSCs dormancy and their maintenance have relied on the macrophages. For instance, the MSCs are modulated by the signals induced by the macrophages. In addition, the macrophages are associated with erythroid progenitors, and factors such as alpha-SMA, DARC, and LXR are expressed by macrophages which could significantly modulate the hematopoiesis by altering HSC function

Administration of IL-7 to syngeneic transplanted mice with adequate T cell levels showed no improvement in the immune reconstitution when compared to their untreated counterparts which proves the satisfactory levels of IL-7 observed in the model and low levels of IL-7 observed only in T cell-depleted mice models [ 61 ].

Osteoblast role in hematopoiesis

Recent studies have shed light on the individual contributions of the different hematopoietic lineages, and the evidence thus far suggests that osteoblasts and vasculature are particularly important players in this intricate multiple lineage system [ 69 , 70 , 71 ]. CD146 + subendothelial cells have been identified as skeletal progenitors that possess an innate ability to restore and reorganize the hematopoietic niche by creating a conducive environment for transplantation in humans [ 47 ]. Studies show that the external endosteal surface has abundant vasculature, whose walls exist in close contact with osteoblasts [ 13 , 19 ].

The trabecular bone lacks endosteal and perivascular/vascular niches, although osteoblasts and vascular cells have different functions. Several reports described the role of osteoblasts within the BM, especially their contribution to maintaining and stabilizing the HSC niche when compared to the osteoblast precursors in different stages which could aid the development of B lymphocytes, which is one of the well-elucidated specific hematopoietic lineages. These studies could provide significant implications for therapeutic interventions, such as BM transplantation and regenerative medicine [ 72 ]. Anatomic evidence suggests a longstanding supportive role of osteoblasts in the hematopoiesis within the bone marrow [ 73 , 74 ]. Osteoblasts do not directly influence HSC function and their maintenance; however, they do govern B cell lineage progenitor activity. This is evident in experiments where osteoblast ablation in adult mice led to the destruction of common lymphoid progenitors (CLPs) whereas B-cell lymphopoiesis was enhanced in cultures enriched for osteoblasts. Cultures enriched for osteoblasts support B lymphopoiesis and ablation of osteoblasts in adult mice acutely depletes common lymphoid progenitors (CLPs) [ 75 , 76 ]. Gs-alpha knockout osteoblasts are essential for PTH receptor signaling and dramatically reduce pro and pre-B cells which are restored with exogenous IL-7 [ 77 ]. Nearly 30% of IL7R + Lineage-bone marrow cells enriched for early lymphoid progenitors are recruited instantaneously towards bone lining cells at the endosteum [ 15 ] (Fig.  1 ).

Macrophages

Macrophages localized in the bone marrow niche are major cell populations that facilitate hematopoiesis by modulating the role of downstream HSCs and MPPs. Macrophages possess an exceptional sensing capacity towards inflammatory markers and they can adjust to the dynamic environment while reducing and/or promoting inflammation. They have a significant impact on HSC function. Therefore, macrophages have a profound impact on hematopoiesis and can be a significant therapeutic target where macrophages are involved in disease pathogenesis. CXCL12 is a chemokine factor that enhances the proliferation and maintenance of B lineage progenitors [ 78 , 79 ] and CLPs [ 80 , 81 ]. One of the fundamental processes that macrophages are involved in is the clearance of apoptotic cells through a process known as efferocytosis. This process is essential for the efficient removal of dead or damaged cells. Macrophages facilitate efferocytosis by efficient disposal of neutrophils and activation of the LXR-dependant mechanism. This substantiates the influence of macrophage physiology and function on the homeostatic regulation of blood progenitors. Any dysfunction in circadian rhythms due to aging comorbid with obesity and lifestyle and environmental stress can have profound consequences on the mobilization of HSC and HSPC, which has to be explored through substantial future studies. Inflammatory signaling facilitates HSC and HSPC mobilization into circulation simultaneously with the production and release of granulocytes [ 82 , 83 , 84 ]. The driving factor for mobilization and granulopoiesis is the G-CSF, a growth factor that facilitates proliferation and differentiation [ 85 , 86 , 87 ]. The consequence of this event is the downregulation of CXCL12, G-CSF receptor signaling is often not necessary in hematopoietic progenitors themselves [ 88 ] ( Fig.  1 ) , but macrophages are typically required for the G-CSF-dependent HSC mobilization [ 89 ]. G-CSF can induce a dramatic reduction of macrophage levels in the bone marrow but the levels remain relatively unaffected in other tissues like the spleen [ 90 , 91 ]. This demonstrates that localized reduction of the macrophage population inside the bone marrow could foster the mobilization of HSC. This was consistently observed in a study involving “clodronate-loaded liposome” mediated depletion of macrophages which resulted in HSC mobilization and concomitantly promoted the G-CSF-driven HSC mobilization [ 92 ].

The resulting mobilization under the above circumstances accounts for a high number of HSCs in the circulation which suppresses CXCL12, and various HSC retention signals [ 92 , 93 ], some of which may drive the HSC/HSPC pool to the dormancy [ 94 ]. Furthermore, macrophages are a diverse population of cells that play crucial roles in tissue homeostasis, inflammation, and immune responses during aging, and obesity. Along with the mobilization of HSCs, macrophages are required for the HSCs bone marrow engraftment post-transplant [ 94 ].

Bone marrow macrophages exhibited radioresistance after irradiation and this resistance persisted for 30 days post-radiation. These tolerant macrophages were typically significant for HSC engraftment and reconstitution post-radiation, which is evident by the ‘reduction of HSC engraftment’ by CD169 expressing macrophage depletion mediated by diphtheria toxin receptor (DTR) [ 94 ]. This substantiates the persistence of macrophages post-lethal radiation across BM, as these macrophages can undergo slow clearance or proliferation and also possess self-renewal capacity potentiated by M-CSF. Exogenous M-CSF facilitates the expansion of the host macrophage pool which is demonstrated to have a protective effect on native macrophages and reduction of graft vs. host disease during transplantation [ 94 ].

In an independent study, HSC retention and mobilization in bone was potentiated by the depletion of macrophages which consequently resulted in stable hematopoietic chimerism [ 95 ]. However, another study provided contrasting evidence that the reduction in HSC engraftment due to macrophage depletion consequently when combined with radiation has led to the development of a proinflammatory environment which resulted in impairment of engraftment [ 96 ]. Clod-lip-induced macrophage depletion is not the sole factor for inflammatory response. This inconsistency highlights the important gaps in the interpretation of macrophage depletion studies since they only allow for in vivo modulation of inflammation and immune cells. Clod-lip-mediated macrophage depletion has been widely studied and the underlying mechanism is well established. It essentially involves apoptosis of the phagocytic cell (engulfing liposome) which is triggered by the release of the bisphosphonate intracellularly [ 97 ]. However, its deletion in osteoblastic cells led to a reduction in CLPs and other premature lymphoid progenitors in the BM without affecting HSCs [ 15 ]. Hence some premature lymphoid progenitors rely on the osteoblast microenvironment rather than the HSC-specific niche. Other lineage-specific microenvironments include the erythroid niche where macrophages are crucial for erythropoietic cell maturation [ 98 ]. An in-depth analysis of the erythroid niche is necessary to determine characteristic cellular components to further elucidate the interlink between this niche and existing HSC lymphoid progenitor niches. In vivo, experiments involving induced and conditional alteration or deletion of niche-specific factors and analyzing the manifestations and effects on stem/progenitor cell maintenance provides insights on the stem cell specific-niche and specific progenitor belonging to haematopoietic system, restricted by the precision of available Cre alleles. Thus, gaining an understanding of the cellular and functional differences between these niches is important for advancing our knowledge of haematopoiesis and immune cell development. It opens up new avenues for research into how the bone microenvironment influences the fate and function of different types of blood cells. This knowledge could have implications for developing new strategies to manipulate immune cell production and function, potentially leading to novel therapies for immune-related disorders.

Perivascular cells

Perivascular cells are a crucial niche for modulating the role of adult HSCs. These perivascular cells coexist in an interactive environment with HSCs in adult BM and serve as precursors to mesenchymal stem/stromal cells (MSCs) that foster differentiation into various cell kinds [ 99 ], including osteoblasts, adipocytes, and chondrocytes. Pericytes are typically located on the abluminal side of blood vessels; they also maintain significant association with endothelial cells (ECs) and express markers including CD146, NG2, PDGFRβ, α-SMA, Nes, and LepR [ 99 ].

However, pericytes are not a homogenous population. Different subsets exhibit distinct immunophenotypes and mesenchymal features, including varying capacities to support hematopoiesis. Both arteriolar and sinusoidal pericytes play crucial roles in regulating HSC behavior, maintenance, and HSC quiescence, and trafficking inside the adult BM via paracrine signaling [ 99 ]. Various studies have described hematopoietic-supportive pericyte subpopulations with the aid of diverse markers through murine experiments. Arteriolar pericytes, are marked by upregulated α-SMA and NG2 levels, and they are associated with the maintenance of HSC quiescence and long-term repopulating capacity. Sinusoidal pericytes, often expressing CD146 and PDGFRβ, are implicated in the regulation of HSC mobilization and homing [ 99 ]. For instance, Nestin-expressing pericytes have been shown to secrete high levels of CXCL12, a critical chemokine for HSC retention in the BM niche. Similarly, LepR-positive pericytes have been identified as key regulators of HSC maintenance through their support of the sinusoidal microenvironment. These findings suggest that specific pericyte subpopulations create distinct niche environments that differentially influence HSC function [ 99 ]. Detailed molecular profiling of pericyte subpopulations will enhance our understanding of their specific roles in HSC regulation. Single-cell RNA sequencing could provide insights into the gene expression profiles and signaling pathways active in different pericyte subsets [ 99 ]. Investigating the functional impact of pericyte-HSC interactions using advanced in vivo models and genetic manipulation techniques will clarify the mechanisms through which pericytes influence HSC behavior. Conditional knockout models targeting specific pericyte markers or signaling pathways could elucidate their roles in HSC maintenance and mobilization. Leveraging the knowledge of pericyte subpopulations to engineer BM niches in vitro could have significant implications for HSC transplantation and regenerative medicine. Creating bioengineered niches that mimic the in vivo perivascular environment may improve the expansion and functional maintenance of HSCs ex vivo. Exploring the changes in pericyte function and HSC support with aging or in disease contexts, such as myelodysplastic syndromes or leukemia, will provide insights into how pericyte-HSC interactions are altered in pathological states. This could lead to targeted interventions that restore normal niche function and improve patient outcomes [ 99 ].

The rapid growth of the skeletal system during embryonic and postnatal life necessitates a coordinated interplay between cell proliferation, differentiation, mineralization, and the expansion of local vasculature [ 100 , 101 ]. Several bone-forming cells including chondrocytes and osteoblasts are implicated in this process by releasing VEGF, which stimulates angiogenesis by stimulating VEGF receptors confined on ECs [ 100 , 101 , 102 ]. Vascular ECs can generate paracrine signaling which could modulate growth as well as regeneration in multiple organs including the skeletal system [ 103 , 104 , 105 , 106 , 107 ]. For instance, osteogenesis is particularly mediated through a specialized capillary EC subtype known as type H; type H-containing capillary ECs are characterized by the expression of CD31/PECAM1 as well as Endomucin (CD31 hi Emcn hi ) markers. These cells are predominantly located in the metaphysis and endosteum of postnatal long bones [ 103 , 104 , 105 , 106 , 107 , 108 ]. Type H ECs not only promote angiogenic growth but also release molecular signals that act on osteoprogenitor cells, subsequently coupling angiogenesis with osteogenesis. Conversely, type L ECs, which exhibit lower CD31 and Emcn expression (CD31 lo Emcn lo ), together associated with sinusoidal vessel network across BM [ 103 , 104 , 105 , 106 , 107 , 108 , 109 ].

Future studies are required to explore research into modulating the VEGF signaling pathway specifically in type H ECs might offer novel approaches to enhance bone healing and repair, particularly in aging populations where bone regeneration is impaired. In addition, identifying and validating biomarkers for type H and type L ECs could facilitate the monitoring of therapeutic outcomes and the development of precision medicine approaches for bone-related diseases. By addressing these future directions, it is possible to develop more effective treatments for bone diseases, enhance bone repair, and improve overall skeletal health [ 109 ].

Correlation of arterial and sinusoidal niches in promoting HSC quiescence

Cell cycle quiescence is significant to maintain HSC’s role in BM. Although several stromal cells are proposed as HSC niches, the precise spatial localization of HSCs in the quiescent stage yet remained ambiguous. A study by Yuya Kunisaki et al. 2013 described that quiescent HSCs are typically confined to small arterioles located across endosteal BM. These arterioles are positively ensheathed for NG2 + pericytes which are distinct from LepR + cells. Decline in NG2 + cells could cause enhanced HSC cycling and a decline in the repopulating HSCs on a long-term basis suggesting that arteriolar niches are crucial to maintaining HSC quiescence [ 3 ].

Structurally, arterioles and sinusoids are different; these structural differences are reflected in the altered unique transcriptional programs of ECs across arterioles and venules. For instance, arteriolar Nes peri cells and sinusoidal Nes retic cells are uniquely differentiated through their transcriptional activities; for instance, Nes peri cells exhibit a greater enrichment in genes related to cell cycle quiescence as well as for maintaining the HSC niche. Under genotoxic stress, mitotically active Nes retic sinusoidal niche cells are undergoing destruction but quiescent Nes peri arteriolar niche cells exhibit chemoresistance. This dual association of hematopoietic and mesenchymal stem cells with arterioles suggests that these vessels could coordinate hematopoietic and regeneration of stroma [ 3 , 110 ]. Further investigations have revealed that the interaction between HSCs and their niche is more complex than previously understood. HSCs in periarteriolar niches are mediated in a quiescence state through the influence of NG2 + pericytes, which secrete factors that suppress cell cycling. This is contrasted by the LEPR + perisinusoidal niche, which supports more active HSC states. The dynamics between these niches suggest a sophisticated regulatory mechanism that ensures the preservation and timely activation of HSCs in response to physiological demands. Moreover, the chemoresistance observed in quiescent Nes peri arteriolar niche cells under genotoxic stress highlights a potential therapeutic target for enhancing HSC protection during chemotherapy. Understanding the molecular signals that confer this resistance could lead to strategies to bolster HSC resilience in patients undergoing aggressive treatments. Detailed molecular studies to elucidate the specific signals and pathways involved in maintaining HSC quiescence within arteriolar niches enable the development of therapeutic interventions that can mimic the protective environment of the arteriolar niche to enhance HSC survival during treatments like chemotherapy. Investigating the interactions between different niche cells, including pericytes, ECs, and mesenchymal progenitors is essential to develop a comprehensive map of HSC regulatory networks. By addressing these, it is possible to understand HSC regulation and develop novel therapeutic approaches to enhance hematopoietic health and regeneration [ 3 ].

Bone marrow adipocytes (BMA) are metabolically active and possess abundant lipid reserves, mitochondria, and endoplasmic reticulum. The number of bone marrow adipocytes is variable during growth and development as is influenced by various internal and external factors like osteoporosis, aging, and calorie-deficit diets [ 111 ]. Current research indicates that bone marrow adipocytes have an inhibitory effect on hematopoiesis [ 111 , 112 ]. Caudal vertebrae are composed of adipocytes, a low number of HSCs, and short-term progenitors when compared to the thoracic vertebrae which contain no adipocytes. Moreover, the pharmacological and genetic downregulation of adipogenesis expedites the restoration of hematopoiesis post-irradiation and bone marrow transplant [ 112 ]. However, there is no concrete evidence to justify the role and influence of BMAs on HSC and the bone marrow niche. Recent evidence suggests that adipocytes localized in long bones aid restoration of hematopoiesis post-irradiation by providing a crucial HSC survival factor, stem cell factor (SCF) [ 113 ]. However, adipocytes localized in tail vertebrates are known to downregulate hematopoiesis [ 114 ]. Fatless A-ZIP/F1 mice models when exposed to radiation resulted in the loss of bone marrow cellularity and HSC inside the long bones, but a higher number of HSCs was observed in caudal vertebrae [ 114 ]. This stark contrast was associated with the increased vasculature in the tail vertebrae of the mice, which is absent in long bones [ 114 ]. The main requirement for restoration of hematopoiesis is an adequate vascularization of bone marrow [ 20 ]. Along with SCF, adiponectin and leptin are two other biomolecules produced by adipocytes that could enhance the proliferation of HSC [ 115 , 116 ].

The rhesus macaque model has been used to validate the relation between BMAs and hematopoiesis in primates. HSPCs reside adjacent to BMAs. Additionally, BMAT (Bone marrow adipocyte tissue)-enriched medium facilitates the HSC proliferation and differentiation ex vivo. A quantitative protein examination of the BMAT-conditioned medium was performed to understand the underlying mechanism. Out of a total of 994 BMAT-derived proteins, including TGFB1, FBLN1, IGFBP2, LGALS1, TIMP1, and C3, were reported to possess the capacity to regulate and increase the differentiation, adhesion, and mobility of HPSC [ 117 ]. Of the 994, 430 proteins have a complex framework, possess paracrine function, and have origin from microvesicles or exosomes. It is also essential to understand that BMAT contains cells apart from BMA including macrophages and granulocytes [ 117 ]. Thus, the proteins derived from the BMAT could play a regulatory role in HSC function via these immune cells. BMA’s role in leukemia is a subject of debate and is largely lineage-specific. In vitro and in vivo experiments provide evidence that BMAs inhibit T-acute lymphoblastic leukemia (ALL) proliferation in ALL conditions [ 118 ].

Further, BMAs, when cocultured with AML blasts, exhibited typically diminished apoptotic effect and higher proliferative effect in acute myeloid leukemia (AML) [ 119 ]. AML blasts enhance the lipolysis of BMAs and the resulting fatty acids are transported to AML cells where they undergo β-oxidation. Recent evidence suggests that AML reduces the adipocyte cellularity in the bone marrow and AML xenograft implying that AML selectively influences adipocyte number along with potentiating lipolysis of existing adipocytes [ 120 ]. A comprehensive analysis of the global transcriptome of BMSCs isolated from AML or healthy patients reported altered adipogenic differentiation caused by AML [ 120 ]. Furthermore, transwell assays were conducted to explore the interlink between BMA reduction and mitigated myelo-erythropoiesis in AML conditions. From this study, it is evident that BMAs can promote the maturation of myeloid and erythropoietic cells. In another study, adipogenesis was activated by GW1929, a PPARγ agonist, which was found to restore hematopoietic maturation in addition to the inhibition of leukemic growth [ 120 ].

In conclusion, BMAs are crucial for the normal maturation of myeloid and erythropoietic cells and hence a valid target for therapy to ameliorate bone marrow failure observed in AML (Fig.  1 ). BMAs are susceptible to changes in homeostatic metabolism and hence need to be researched further to accurately determine their physiology and role in the pathogenesis of diseases like hematological malignancies [ 2 , 121 ].

The fine balance between adipogenic and osteogenic differentiation of MSCs is orchestrated by crucial signaling pathways and transcription factors. Signaling pathways such as TGF-β/BMP, Wnt, Hedgehog, Notch, and FGFs play pivotal roles in this regulation. These pathways influence key transcription factors, PPARγ and C/EBPs for adipogenesis, and Runx2 and Osterix for osteogenesis-ensuring a precise differentiation balance. ( A ). The role of regulatory T cells (Tregs) in the bone marrow environment is significant. Foxp3 + Tregs can influence the activity of cytotoxic T cells and ICAM1 + perivascular cells. Activation of T cells leads to the depletion of Tregs, which in turn reduces UL-7 production by perivascular cells, affecting B cell differentiation from hematopoietic stem cells (HSCs). ( B ) HSCs within the bone marrow microenvironment are supported by the endosteal and perivascular niches, including osteoclasts, osteoblasts, and other supportive cells. Factors such as G-CSF, osteopontin, annexin-2, thrombopoietin, and angiopoietin-1 regulate HSC functions in lineage commitment. Additionally, osteoblasts, endothelial cells, LepR + perivascular cells, and Nestin + MSCs producing CXCL12 and CAR cells play crucial roles in the homing, self-renewal, and differentiation of HSCs, facilitating hematopoiesis

Primitive hematopoietic cells are confined to the endosteal surface while the progenitors are located centrally within the marrow space. Intravital microscopy experiments were conducted along with HSPCs labeled with significant markers have further confirmed the proximity of HSPCs to the endosteal osteoblasts during the HSPC engraftment conditions where a significant number of mature progenitors were positioned further from osteoblasts [ 13 , 19 ]. Furthermore, migration of hematopoiesis from fetal liver to bone marrow greatly relies on normal bone formation and turnover during embryonic development. Previous reports suggested that the mice lacking runx2, a key transcription factor for osteoblast differentiation, developed weak, demineralized, or structurally skeletons [ 122 , 123 , 124 ] which led to compensatory extramedullary [ 124 ].

Mice with defective M-CSF exhibit typically reduced levels of osteoclasts which eventually results in osteopetrosis and extramedullary hematopoiesis [ 125 ]. In vitro studies have demonstrated the supportive function of stromal cells [ 126 ] to the osteoblast lineage to foster hematopoietic cell differentiation [ 127 , 128 ]. Targeted ablation of osteoblasts was carried out in the mice presenting differentiated osteoblasts with herpes simplex thymidine kinase through ganciclovir administration [ 76 ]. This led to a reduction in bone marrow niche and conferred to the extramedullary hematopoiesis, highlighting the role of osteoblasts in supporting hematopoiesis in vivo across the BM [ 129 ]. The osteoblast ablation resulted in a downregulation of B-cell lymphopoiesis and erythropoiesis in the BM, eventually mitigating the primitive hematopoietic cell population in bone marrow [ 75 , 129 ]. Osteoblasts have also been implicated in HSC mobilization, particularly in response to G-CSF [ 130 , 131 ] (Fig.  2 ). However, the precise molecular mechanisms and the requirement for direct contact between HSCs and osteoblasts in vivo are still being investigated.

Parathyroid hormone exerts a synergistic effect through osteoblasts on B lymphopoiesis. PTH/PTH-related peptide receptor (PPR) signaling plays a role in osteoblast-mediated B-cell development. Genetic manipulation or alteration of either osteoblast-specific PPR [ 132 ] or the BMPR1a receptor [ 133 ] results in a higher number of osteoblasts and increased HSC niche [ 133 ]. However, a low level of osteoblasts was observed in biglycan knockout mice which is independent of any hematopoietic defect or decrease in HSC [ 134 ]. This indicates that osteoblast is not necessarily the sole determining factor of the HSC population [ 134 ].

Osteoblasts can regulate functions of various hematopoietic factors including angiopoietin-1 [ 135 ], osteopontin [ 136 , 137 ], thrombopoietin [ 138 ], Wnts [ 139 ], and extracellular calcium [ 140 ]. A recent study suggested that the overexpression of Notch ligand Jagged-1 (Jag1) in osteoblasts by PPR activation directly leads to the increase in the HSC population, thus implicating notch signaling as a factor for hematopoiesis. The overexpression of HSCs can be suppressed by γ-secretase inhibitors [ 132 ]. It has also been demonstrated that the deletion of Jag1 Mx1-Cre-mediated in the microenvironment yielded no phenotype [ 141 ].

The role of N-cadherin is widely debated among researchers, some who advocate its synergism and importance [ 19 , 133 ] and others who contest the same [ 134 ]. N-cadherin associated with β-catenin/Wnt-signaling has a significant role in the HSCs’ interactions with their niche [ 142 ]. Previous studies reported the role of osteoblast N-cadherin mainly cadherin-11 confined to the osteoblasts is upregulated during differentiation. Another study reported that overexpression of c-Myc and Rbm15 can mitigate N-cadherin expression and speculated that the mitigation in the expression of N-cadherin is required for the release from the stem cell niche [ 143 , 144 , 145 ].

Therefore, while the role of osteoblasts in maintaining the homeostasis of the HSC niche is validated through the studies of in vivo models, the crucial underlying mechanisms remain uncertain. The molecular mechanisms underlying the cross-talk between the osteoblast lineage of the skeletal system and perivascular/vascular cells of the hematopoietic niche is another gray area. Cell-specific ablation of factors will be crucial to addressing several of these issues and bridging the research gap [ 72 ].

In vitro cultures of HSCs are not feasible unlike other stem cells sourced from other tissues which can be readily cultured in vitro. This could hinder their therapeutic and transplantation scope, one of them being gene therapy where the transfected HSCs need to be cultured to assess the quality before transplantation. Several factors could influence HSC survival through osteoblasts and the pleiotrophin supplementation promoted HSC survival in vitro [ 156 ] and the lack thereof resulted in HSC reduction and dysregulation of haematopoiesis [ 157 ]. Pleiotrophin is produced by sinusoidal liver endothelial cells and perivascular stromal cells expressing CXCL12 to promote HSC function [ 157 ]. A key slit receptor, Robo4, is expressed by both endothelial cells and HSCs. This receptor governs the HSC localization in bone marrow microenvironment [ 158 , 159 ]. The slit2 ligand exhibits selective activity towards MSCs and some osteoblast lineage cells. This indicates that both pleiotrophin and Robo4/Slit2 are essential components of the perivascular environment. Evidence also suggests a possible positive or negative influence of Tenascin-C [ 160 ], osteopontin [ 136 , 137 ], and non-canonical Wnts on the HSC population (Tables  1 and 2 ).

Importance of the different signaling pathways involved with BM niche

As we discussed above, osteoblasts were the first cell type identified to support HSC expansion in vitro, notably via the presentation of granulocyte G-CSF [ 161 ]. These cells secrete a variety of proteins, including angiopoietin-1, CXCL12, SCF, and TPO, all of which are pivotal for promoting HSC growth [ 136 , 140 , 162 ]. Additionally, osteopontin and SDF-1α produced by osteoblasts are crucial for the mobilization and egress of HSCs [ 163 ]. The perivascular niche, encompassing ECs, perivascular stromal cells, and MSCs, plays an essential role in maintaining HSC physiology near blood vessels. MSCs, which are highly heterogeneous, express markers such as CD146, CXCL12, Nes, and LepR, all of which are vital for HSC survival [ 16 , 47 ].

Primary ECs obtained from non-hematopoietic organs have been shown to enhance HSCs repopulation in vitro [ 164 ]. HSC migration is facilitated by derived from CD31 + ECs as well as E-selectin ligand-1 (ESL-1) in HSPCs. Blocking this interaction results in HSCs becoming quiescent and exerting resistance to the irradiation [ 60 , 165 ]. Various mature cells in the bone marrow also play significant roles in modifying the niche. Trophic endosteal macrophages, for example, support both osteoblast function and the entire endosteal HSC niche. Their absence can lead to HSC egress into the bloodstream [ 93 , 166 ]. Non-myelinating Schwann cells regulate HSC pools through the modulation of TGF-β signaling [ 39 , 166 ]. Expansion of HSPCs is also regulated by the Notch stimulation [ 167 ]. Notch signaling plays a dual role: it could foster the morphology development and artery specification subsequently mediates communication among niche cells by enabling Notch receptor expression or ligands. Another critical signaling is the involvement of the canonical Wnt pathway. For instance, overexpression of β-catenin has been associated with a higher hematopoietic cascade [ 168 ]. Quiescent LT-HSCs are characterized by the Frizzled 8 expression, which antagonizes Wnt signaling, while Wnt5a maintains HSC quiescence through the inhibition of Wnt3a-mediated canonical Wnt pathway [ 133 ]. In addition to these pathways, other signaling mechanisms indirectly influence HSC development, including BMP as well as the Hedgehog pathway. BMP pathway can support the function of spindle-shaped N-cadherin + osteoblasts to control bone marrow niche size and also the fate of HSCs. Sonic Hedgehog is involved in fostering primitive HPCs differentiation and myeloid differentiation [ 169 , 170 ]. Further, in vivo studies are required for conducting comprehensive in vivo studies to better understand the dynamic interactions within the BM niche and their impact on HSC behavior under various physiological and pathological conditions. As part of regenerative medicine studies, it is crucial to explore the potential of using niche-modifying agents in regenerative medicine to enhance bone and vascular health, thereby improving outcomes in BM transplantation and other hematological therapies.

Adipo-osteogenic differentiation of MSCs and molecular signaling

MSCs undergo a 2-step differentiation involving lineage commitment and maturation, transitioning from multipotent stem cells to lineage-specific progenitors and finally into specialized cell types. Key signaling pathways regulating MSC differentiation include TGFβ/bone morphogenic protein (BMP), Wnt, Hedgehog (Hh), Notch, as well as fibroblast growth factors (FGFs) [ 171 ].

The TGFβ/BMP signaling pathway is known to have dual roles in MSC differentiation, affecting both adipogenesis and osteogenesis [ 172 ]. This pathway operates through canonical Smad-dependent and non-canonical Smad-independent mechanisms, such as the p38 MAPK pathway [ 173 ]. Activation of these pathways regulates the expression of key transcription factors like runt-related gene 2 (Runx2/Cbfa1) [ 174 ] and PPARγ, which in turn dictate MSC differentiation [ 172 ]. The cytokine composition in the MSC microenvironment is thus crucial for determining lineage commitment.

Wnt signaling can confer osteogenic differentiation but impair adipogenic differentiation [ 175 ]. For instance, Wnt3a stimulates osteogenesis via TAZ activation by PP1A-mediated dephosphorylation, and YAP/TAZ can modulate Wnt signaling-induced osteogenesis [ 176 , 177 ]. Aging-related enhancement in adipocytes is linked to reduced Wnt10b levels. Furthermore, β-catenin loss in the developing mouse uterus mesenchyme switches differentiation towards adipogenesis [ 178 , 179 , 180 ].

Notch signaling plays a complex role in MSC differentiation. Impairment of Notch pathway promotes autophagy-associated adipogenesis by modulating PTEN-PI3K/AKT/mTOR pathway [ 181 ]. Notch also suppresses osteogenesis by inhibiting Wnt/β-catenin signaling but can promote osteogenesis through BMP2 signaling cross-talk [ 173 , 182 ].

Hedgehog signaling is downregulated during adipogenesis, with decreased Gli expression. Hedgehog pathway actuation inhibits adipogenesis by repressing PPARγ and C/EBPα expression, while Gli inhibition promotes adipogenesis [ 183 ]. Conversely, Hedgehog signaling supports osteogenesis [ 184 , 185 , 186 ] and can interact with BMP signaling to enhance Smad-mediated osteogenesis, highlighting its pro-osteogenic and anti-adipogenic roles [ 171 ] (Fig.  2 ).

Impact of aging on adipo-osteogenic differentiation

Aging is associated with a shift in the differentiation balance of MSCs, leading to increased bone marrow adiposity and decreased osteogenesis [ 187 , 188 , 189 ]. A previous study identified several molecules linked to age-related osteogenic potential loss, including decreased levels of chloride intracellular channel 1 (CLIC1) as well as prohibitin, and increased levels of LIM and SH3 domain protein 1 (LASP1) and annexin V. Furthermore, aging also increases ROS- mediated oxidative stress, which play significant role in age-induced bone loss as well as differentiation balance through pathways involving FOXO, Wnt, or PPARγ [ 189 , 190 , 191 ]. For instance, PPARγ, a key transcription factor to modulate adipogenesis, inhibits osteoblast differentiation. Increased PPARγ expression in aged MSCs promotes adipogenesis and inhibits osteogenesis [ 171 ]. It is crucial to investigate the interactions between different signaling pathways (e.g., TGFβ/BMP, Wnt, Notch, Hedgehog) to better understand the regulatory networks governing MSC differentiation. Subsequently exploring how microenvironmental factors, such as cytokine composition and mechanical stimuli, influence MSC lineage commitment and differentiation is needed. This could lead to targeted therapies that modulate these factors to promote desired differentiation outcomes. By addressing these research directions, we can better harness the potential of MSCs for regenerative medicine, particularly in the context of aging and bone-related diseases [ 171 ].

BM niche regulates stem cell trafficking and age may influence the trafficking in the following signaling pathways

The bone marrow microenvironment is composed of several different kinds of cell types including hematopoietic stem cell progenitors, osteoblasts, immune cells, osteoclasts, and perivascular cells [ 13 , 14 ]. Irradiation could damage sinusoids across the bone marrow [ 13 , 19 , 20 ] and the arteriolar blood vessels are preserved in the endosteum.

Aging is a highly intricate physiological process associated with significant alterations in tissue-specific changes due to alterations in gene expression and cell composition [ 192 ]. In the bone marrow (BM), aging leads to an expansion of the HSC pool, with HSCs exhibiting a biased differentiation toward myeloid progenitors at the expense of lymphoid ones, along with a diminished regenerative potential [ 35 , 193 ]. Mitosis analysis described that HSCs, and MPPs exhibit a quiescent nature in a steady state whereas the granulocyte-macrophage lineage-restricted progenitors (GMLPs) exhibit a higher proliferation rate [ 194 ]. Aging in HSCs is characterized by various intrinsic changes, including loss of cell polarity, heightened Wnt5a non-canonical signaling, disrupted autophagy [ 195 ], deregulation of the mitochondrial unfolded protein response, reduced mitochondrial acetylation mediated by SIRT3, and alterations in the epigenome, including increased symmetry of epigenetic division [ 196 , 197 , 198 , 199 , 200 , 201 , 202 , 203 ]. These intrinsic alterations could affect the functions of HSC function irrespective of the BM niche, a phenomenon termed “intrinsic” HSC aging, extensively reviewed elsewhere [ 204 ]. Recent investigations into middle-aged BM microenvironments have identified decreased IGF1 levels as a crucial aging-promoting factor affecting both HSCs and niche cells. Restoring IGF1 signaling has been shown to rescue Cdc42 and tubulin polarity, reduce γH2AX focus, and alleviate myeloid differentiation skewing in middle-aged LT-HSCs [ 205 ]. Interestingly, fasting-induced decrease of IGF1-dependent stimulation of PKA activity has been identified as a key factor promoting HSC self-renewal, balanced differentiation, stress resistance, and regenerative capacities after chemotherapy in aged mice [ 206 ]. This apparent discrepancy might be explained by the different downstream pathways activated by IGF1, with fasting-induced IGF1-mediated effects passing through PKA activation, while aging-associated effects activate the mTOR signaling [ 205 , 206 ]. This suggests that more research is needed to fully understand the regulation of HSC function by IGF1 during aging. Additionally, aging results in the degeneration and remodeling of various niche compartments, impacting HSC behavior and function on different levels [ 207 ].

As the age increases, the alterations occur in the vasculature, marked by the decline in CD31 high Endomucin −/low arterioles [ 147 , 151 , 208 ]. Stromal cells inside BM also change, such as the depletion of periarteriolar Osteolectin (+) cells, potentially contributing to the decline in lymphoid progenitors [ 209 ]. In contrast, sinusoids remain preserved during aging, potentially explaining the absence of age-related depletion of HSCs in most mouse strains, unlike lymphoid progenitors [ 208 ]. The bone marrow’s inflammatory milieu increases during the aging process subsequently contributing to hematopoietic changes. A higher expression of inflammatory factors, including interferons, IL-1β, IL-6, and TNFα by stromal and hematopoietic cells is evident as the age increases in the individuals fostering increased myelopoiesis [ 4 , 150 , 210 , 211 , 212 ]. Additionally, alterations in nerve fibers within the bone marrow may play a role in age-related changes in hematopoiesis [ 146 , 213 ]. Hence, our study specifically focuses on the influence of neural signaling in the hematopoiesis of aging-related hematological malignancies with subsequent need for the development of gene therapies.

Hematopoietic system during aging

With the global population aging, health implications such as cancer, neurodegenerative, and other several ailments contribute to significant public health concerns [ 214 ]. Aging pertinent to the hematopoietic system disrupts immunity and homeostasis, increasing the risk of blood malignancies due to impaired HSC function [ 35 , 215 , 216 , 217 ]. Myeloid malignancies are more common with age, whereas lymphoid malignancies are more prevalent in younger individuals [ 216 , 218 , 219 ]. Understanding age-related HSC behavior is crucial for addressing the physiology of the hematopoietic system as age increases. Aging in the hematopoietic system manifests through enhanced myelopoiesis, mitigated ability of adaptive immune functions, and decreased HSC functionality, which are essential for sustaining hematopoiesis. The aging process is characterized by alterations in different HSC subset levels, although the regulatory mechanisms, particularly given HSC heterogeneity, are not fully elucidated. A previous report by Tsu-Yi Su et al. shed light on how aging affects different HSC subset functions, marked by CD49b [ 219 ].

Shifts in lineage bias, epigenetic and transcriptional changes with aging

As per the previous reports, both lymphoid-biased as well as myeloid-biased HSC subsets show a shift towards increased myeloid subsets as they age. Additionally, gene expression and regulatory mechanisms in HSCs start to change from the juvenile stage and continue progressively, indicating intrinsic modifications in both cellular and molecular properties due to aging] [ 219 ]. Aging is linked to substantial alteration in the transcriptional as well as epigenetic changes that impact the differentiation of HSCs. Previous reports described differentiation-associated gene loci becoming hypermethylated, while self-renewal-associated loci become hypomethylated, with enhanced histone marks activity in aged HSCs [ 35 , 220 , 221 , 222 ]. The study of epigenetic changes in lineage-biased HSC subsets during aging has faced significant limitations, primarily due to difficulties in separating functionally different HSC subsets. As a result, the specific epigenetic modifications that occur in these lineage-biased HSCs have not been thoroughly investigated. Recent advancements in single-cell epigenomics and improved isolation techniques have begun to shed light on these processes. These innovations allow for a more precise dissection of the epigenetic landscape of HSCs, revealing how age-related changes contribute to lineage bias and functional decline. Understanding these mechanisms is crucial for developing targeted therapies to mitigate age-associated hematopoietic dysfunction.

Tsu-Yi Su et al. [ 219 ]identified integrin CD49b as a significant marker for differentiating functional subsets among primitive Lineage–Sca-1 + c-Kit+ (LSK) CD48–CD34–CD150hi (CD150hi) HSC compartment [ 223 ]. CD49b– HSCs are confined highly to the myeloid-biased cells, whereas CD49b + HSCs predominantly exhibit lymphoid-biased features. Despite transcriptional similarities, these subsets show different profiles of chromatin accessibility, describing epigenetic regulation of these functionally distinct lineage-biased HSCs [ 223 ].

Myeloid shift and HSC composition with aging

The increased myeloid output in aging has been attributed to the mitigated efficacy of HSCs to generate lymphoid subsets. Identification of distinct subsets of HSCs describes alterations in the clonal composition of HSCs which could drive enhanced subsets of myeloid-biased cells [ 215 , 216 ]. Numerous studies on aging HSCs have examined heterogeneous HSC compartments containing a variety of subtypes, making it difficult to draw clear conclusions about age-related changes in specific, highly enriched HSC subsets. Additionally, research on aging in mouse models often contrasts young adult mice (2–4 months old) with older mice (1.5-2 years old). It’s important to note that HSCs transition from a fetal to an adult phenotype approximately one month after birth, during a phase characterized by rapid tissue growth and high self-renewal activity [ 224 , 225 ]. This developmental stage may contribute to the higher incidence of lymphoid malignancies observed in children, highlighting the need to include the juvenile period in studies of age-related alterations in HSCs [ 218 ]. Recent studies utilizing advanced single-cell sequencing techniques and more refined isolation methods have been initiated to address these challenges, offering deeper insights into the epigenetic and functional dynamics of HSCs across different life stages. This enhanced understanding is essential for identifying therapeutic targets to counteract age-related hematopoietic disorders [ 219 ].

HSC quiescence and proliferation with age

HSCs are getting more quiescent and undergo minimal proliferation as the age increases [ 219 ]. However, the cell cycle pertinent to these aged HSCs is yet unexplored vividly and debated [ 31 , 221 , 226 ], but as per the conclusions given by Tsu-Yi Su et al. juvenile mice typically undergo symmetric self-renewal process whereas aged HSCs could induce the generation of progenitors via symmetric proliferation [ 224 ]. Hence, in the aged HSCs, the enhanced production of progenitors aligns with stemness loss and mitigated functionality of HSCs [ 31 , 215 , 216 , 217 ]. Despite the reduction in this functionality, the phenotypic HSC population is enhanced in older age and maintains a larger population size by constraining the cell cycle process. Typically, the CD49b + HSCs were observed to be less quiescent and exhibits higher proliferation rate subsequently associated with minimal engraftment efficacy concluding to explore the CD49b– and CD49b + HSCs ability undergo symmetric or asymmetric division as the age increases; this exploration presenting a significant aspect for future research to uncover HSC heterogeneity as the age of the individuals increases.

Epigenetic regulation of HSC lineage bias

Lineage bias is considered to be a heritable trait [ 227 ] suggesting epigenetics may control heterogeneity of HSCs [ 228 ]. According to past reports, the functionally distinct HSCs do have identical gene expression patterns but display different epigenetic profiles. Single-cell sequencing/ATAC sequencing described the age-induced gene expression alteration and elucidated that juvenile HSCs have dissimilar molecular identity from fetal HSCs but are transcriptionally identical to adult HSCs. For instance, the chromatic accessibility of HSCs has eventually; the chromatin accessibility typically increased in both HSC subsets, correlating with the loss of SPIB and SPI1 (PU.1) transcription factor binding sites, consistent with the published reduction in PU.1 expression with age [ 220 , 222 , 223 , 229 ]. Mitigated SPIB TFBS is vividly observed in adult HSCs and suggests that certain gene expression alterations during aging originate from the juvenile stage typically at the time of the growth restriction process into adulthood [ 230 ]. Therefore, these kinds of age-mediated alterations could benefit to identification of epigenetics-associated signatures in age-related hematological malignancies. Subsequently, future studies are required to explore whether targeting chromatin remodeling across these epigenetic regions can reverse or mitigate aging-induced modifications in HSCs.

Subset-specific epigenetic differences

Predominantly, PU.1 levels are critical for myeloid bias; however, PU.1 deficiency in vivo models has been shown to enhance myelopoiesis while blocking lymphopoiesis, thereby increasing the propensity for myeloid leukemia as age increases [ 231 , 232 , 233 ]. These findings indicate that aging and lineage-biased differentiation may involve shared transcription factors. Furthermore, several other candidate genes have been identified in controlling HSC lineage bias. Specifically, Bcr, Abl1, and Tet1 are typically involved in both myeloid and lymphoid differentiation. In contrast, the role of Kcnn1 in the hematopoietic system remains unexplored. Modulations in the expression or mitigated expression of Bcr, Abl1, and Tet1 have been linked to hematopoietic malignancies, describing a crucial understanding of regulatory functions to modulate normal blood cell lineage differentiation [ 234 , 235 , 236 , 237 , 238 ]. Additional reports are necessary to elucidate the roles of these genes in regulating HSC lineage bias [ 219 ].

HSC aging research: Comprehensive genomic analysis of aging HSCs

To explore intrinsic aging mechanisms that compromise somatic stem cell function, we [ 220 ] conducted a detailed genomic analysis including histone analysis, and transcriptome/DNA analysis comparing young and aged murine HSCs. According to this analysis, a decrease in TGF-β signaling and disruptions in genes is critical for HSC proliferation or differentiation. Furthermore, aged HSCs displayed broader H3K4me3 peaks across genes associated with HSC self-renewal, alongside enhanced methylation rate of DNA typically at transcription factor binding sites of differentiation-promoting genes and decreased methylation at genes essential for HSC maintenance. These epigenetic modifications collectively reinforce self-renewal while diminishing differentiation, mirroring phenotypic aging in HSCs [ 220 ]. Additionally, ribosomal biogenesis is considered to be targeted during aging for modulating the ribosomal protein or RNA genes [ 220 ]. Previous reports offer a valuable resource for future epigenomic studies on stem cell aging. The observed epigenetic changes align with documented functional and phenotypic alterations in HSCs, such as increased self-renewal, reduced differentiation potential, and a myeloid-biased differentiation ratio. These modifications, though not directly pathological, create a cellular environment prone to age-related diseases like myelodysplastic syndrome (MDS) and leukemia [ 220 ].

Reduced TGF-β signaling in aged HSCs

A previous report described a significant reduction in TGF-β signaling pathways in aged HSCs, corroborating previous reports on aging in cardiac and neural tissues [ 239 , 240 ]. Differential expression of genes involved in the actuation of ligand and bioavailability include MMP-2 and MMP-9 [ 241 , 242 ], as well as mitigated levels of Smad6 expression, a gene interfering with Smad2 phosphorylation. These insights describe the necessity for further investigation into TGF-β pathway role in HSC aging.

Ribosomal gene transcription alterations

A report by Deqiang Su et al. [ 220 ] revealed significant alterations in ribosomal gene transcription with aging. Despite aged HSCs not being more proliferative than their younger counterparts [ 35 , 36 ], the increase in ribosomal protein gene transcription suggests enhanced splicing and potential translation efficiency. This finding aligns with studies linking ribosome biogenesis defects to bone marrow failure syndromes and malignancies like Diamond-Blackfan anemia [ 243 ]. Given the repeated association of ribosomal biogenesis with aging in various models [ 244 , 245 , 246 , 247 ], revisiting its role in mammalian stem cell aging is imperative.

H3K4me3 modifications and aging

Increased breadth of H3K4me3 peaks in aged HSCs, particularly on genes associated with self-renewal, suggests a correlation with functional declines observed in aging HSCs. This phenomenon mirrors observations in C. elegans, where mutations in H3K4 methylation genes extend longevity [ 248 ]. H3K4me3 accumulation on genes critical for self-renewal may drive the observed functional impairments in aged HSCs [ 249 , 250 ].

Linking HSC aging to myeloid malignancies

MDS and AML prevalence increases as the age increases, which is characterized by mitigated differentiation and a myeloid-lineage bias. Aging HSCs acquire changes predisposing them to these malignancies even without mutations. For instance, genes like Dnmt3a and Ezh2, often mutated in MDS and AML, show slight but significant downregulation with age [ 251 , 252 ]. Furthermore, hypomethylation and upregulation of HSC-specific genes (e.g., Gata2, Hmga2) and hypermethylation of differentiation-related transcription factors (e.g., Pu.1) suggest a predisposition towards malignant transformation. These observations warrant further investigation into the causal relationships between epigenetic changes and hematologic malignancies [ 251 , 252 , 253 , 254 ]. Developing novel interventions to modulate HSC function based on these insights holds promise for mitigating age-associated hematopoietic dysfunctions and improving health outcomes. By integrating these detailed genomic analyses and identifying specific future research directions, this discussion offers a comprehensive perspective on the mechanisms driving HSC aging and potential therapeutic strategies [ 220 ].

Aging, Diabetes, and obesity-induced BM alterations: hematopoiesis

Transcriptional dysregulation could be one of the factors that can promote aging. Cell cycle regulators [ 221 ], upregulated myeloid signatures [ 221 , 255 ], leukemia-causing genes [ 35 ], upregulation of inflammatory signaling through NF-kB, TNF-α [ 256 , 257 ], and mitigation in the DNA repair pathways [ 36 ] are predominantly accompanied by aging. Furthermore, the epigenetic alterations concomitant with transcriptional lesions are significantly observed in HSPCs derived from elderly individuals [ 220 , 258 ]. NTN1 I is a linchpin molecule actively involved in regulating BMEC-LepR + MSC niche A study by Pradeep Ramalingam et al. 2023 described the regulatory role of NTN1 in BMEC-LepR + MSC niche interactions inside the sinusoids of BM and ameliorate the accumulation of adipocytes [ 9 ]. Future studies are warranted to explore the role of BM niche-derived signaling to modulate the role of HSCs in vascular integrity, oxygenation, and adiposity inside the aging BM with hematological malignancies.

Leukemia: Insights into epigenetic regulation and clonal hematopoiesis in aging

Epigenetic regulation plays a crucial role in fostering transcriptional functions in stem cells in aging. Numerous clinical studies have identified gene mutations that could encode epigenetic enzymes in elderly individuals with oligoclonal hematopoiesis or patients with MDS or AML. The significant genes mainly affected are DNMT3A, EZH2, TET2, and SETDB1 [ 259 , 260 , 261 ]. Although the enzymatic activities of these proteins (DNA methylation/demethylation, H3K27/H3K9 tri-methylation) are well characterized, their specific effects on stem cell function remain unclear. It is elucidated that gene mutations pertinent to DNMT3A, EZH2, TET2, and SETDB1 can induce changes in the epigenetic memory of stem cells; this leads to modulation in transcriptional alterations which in turn fosters the self-renewal ability of mutant cells. Over time, these mutations promote clonal expansion within the bone marrow. Murine model-based reports described that changes in the expression of wild-type or mutate epigenetic writes can modulate the self-renewal process of HSCs [ 259 , 260 , 261 , 262 ].

Clonal hematopoiesis is a common phenomenon in older individuals. Initial studies, which examined X-chromosome inactivation patterns in the blood cells of elderly females, suggested that blood cells in older individuals originate from fewer stem cells [ 263 , 264 , 265 ]. More recent large-scale sequencing studies have confirmed these early observations and revealed that clonal hematopoiesis in healthy individuals increases the risk of developing leukemia, thereby associating it with increased mortality [ 265 ]. This raises critical questions about the detrimental nature of clonal hematopoiesis and its underlying mechanisms.

Despite its association with disease, clonal hematopoiesis is present in many healthy elderly individuals without any clinical symptoms [ 266 , 267 , 268 ]. This suggests that while clonal hematopoiesis can be linked to disease, it may also represent a benign aging process. Mutations in epigenetic genes might be a factor to drive clonal hematopoiesis might not be directly oncogenic but rather increase HSC self-renewal, leading to clonal expansion. When leukemic transformations occur, these actively self-renewing stem cells are more likely to acquire additional mutations, contributing to disease progression. Therefore, it is important to differentiate between mutations that drive benign clonal expansion and those that initiate leukemia.

Despite the established prevalence of clonal hematopoiesis in the elderly, the reports related to clonal stem cell impact on hematopoiesis are limited. A few reports proposed, to describe ranging from clonal succession to stochastic behavior, but no consensus has been reached [ 269 , 270 ]. This uncertainty arises because clonal descent of blood cells is historically difficult to trace. Early methods involved ex vivo barcoding of purified HSCs, followed by transplantation into recipient mice [ 271 , 272 , 273 ]. More recent approaches have used in vivo DNA barcoding and fluorescent dyes for clonal marking in transgenic mice and fish [ 274 , 275 ]. Exploring the roles of candidate genes identified in our ATAC-seq analysis, such as Bcr, Abl1, and Tet1, in regulating HSC lineage bias will be crucial for understanding the regulatory mechanisms pertinent to normal blood lineage differentiation and developing strategies to address hematopoietic malignancies associated with aging.

Understanding the functional impact of epigenetic changes on mature blood cells derived from aged stem cells will provide deeper insights into the links between clonal hematopoiesis and non-hematological diseases. Moreover, reconciling the differences in clonal contribution models and determining the precise number of stem cells contributing to steady-state hematopoiesis at the time of aging will be essential. Enhanced methods for tracing clonal descent in human studies, combined with experimental data from transgenic models, will be vital in addressing these gaps. Finally, translating these findings into clinical strategies to monitor and potentially modulate clonal hematopoiesis could lead to improved management of age-related hematopoietic and cardiovascular diseases [ 217 ].

Induces the reduction in the circulating BM-derived stem/progenitor cells and causes severe BM-related complications [ 10 , 276 , 277 ]. Inflammation has a significant role in the derangement of the BM microenvironment in the mouse models of type 1 diabetes as indicated by the presence of proinflammatory cytokines, and other monocyte/macrophage stimulating factors [ 278 ]. Changes in the cytokine expression in the long-term T1D conditions are accompanied by higher levels of IGF-1, IGFBP5, and osteoprotegerin and VEGF; concomitantly mitigated gene expression of Bmi1 could induce a decline in the HSPC senescence and fosters impairment of regenerative ability of HSC niche in the sinusoids of BM [ 10 , 279 ].

Disautonomia is associated with diabetes which could affect bone marrow function and damage to the peripheral clock. These events cause damage to the HSC niche during G-CSF-induced HSC mobilization in murine models [ 280 , 281 ]. Cardiovascular diabetic autonomic neuropathy is an abnormality that can induce mitigated levels of HSPCs with the upregulated expression of src homology & collagen homology domain (p66Shc), which consequently downregulates the expression of sirtuin 1 (Sirt1) [ 282 ]. Several in vivo studies delineated the diabetes-induced defects inside the microenvironment of BM including microangiography and neuropathy subsequently impair the stem cell mobilization egress. However, these mechanisms yet should be explored during hematological malignancies comorbid with diabetes.

Comorbid conditions such as obesity, and diabetes could foster proinflammatory conditions which could can induce CHIP-associated pathologies. The relationship between obesity and CHIP was described by Santosh P et al. [ 12 ]. In this study, authors elucidated the Ca2+/calcineurin-mediated Nfat and IRG1 signaling in the ob/ob fatty bone marrow recipient mice transplanted with Tet2–/– HSC/Ps which was accompanied by the substantial levels of itaconate [ 283 ], a metabolite involved in retrieving TCA cycle and impairs succinate dehydrogenase [ 284 ]. In addition, the obesity-mediated alterations in glucose availability induce a higher acetyl-CoA which concomitantly enhances the influx of divalent calcium into the cell. Subsequently, these events could cause activation and translocation of NFAT into the nucleus and result in the activation of lysine acetyltransferase p300 to foster the site-specific regulation of H3K27ac and promote Irg1 upregulated expression and cause accumulation of itaconate. This metabolite bonds to the alpha-ketoglutarate of the TET enzyme and impairs its catalytic activity at the time of DNA demethylation [ 285 , 286 ]. Santosh P et al. concluded that the transgenic Tet2-/-ob/ob mutant mice and chimeric ob/ob FBM recipient mice transplanted with Tet-/- HSC/Ps induced higher divalent calcium levels [ 12 ] and consequently declined the 5-hmC levels in their HSC/Ps; this a vivid role of obesity mediated CHIP-associated hematological malignancies are evident and the novel therapeutic modalities like gene-corrected stem cells may deliver effective clinical outcomes in the aging-associated mutated CHIP populations of BM and future studies are warranted to explore these modalities in clinical sectors.

Impact of atherosclerosis and myocardial infarction (MI) on the HSC niche

Haematopoiesis, the process by which mature immune cells are produced, plays a crucial role in responding to vascular injury, including in atherosclerosis and MI. During these cardiovascular events, infiltrating monocytes release pro-inflammatory cytokines such as TNFα, IL-1β, IL-6 and reactive oxygen species, which amplify local inflammation and promote further leukocyte recruitment. These monocytes also secrete metallopeptidases that contribute to pathogenic remodeling, such as destabilizing the fibrous cap in atherosclerosis and forming infarct scars in MI [ 287 , 288 ]. In the context of MI, monocyte infiltration occurs in two phases: an initial phase dominated by classical monocytes (Ly6hi CCR2hi in mice; CD14hi CD16 − in humans) that drive inflammation and phagocytosis, followed by a phase where nonclassical monocytes (Ly6Clo CX3CR1hi in mice; CD14lo CD16hi in humans) predominate to facilitate inflammation amelioration and cardiac remodeling. Nonclassical monocytes in this second phase are primarily derived from the conversion of classical monocytes, with minimal recruitment from the periphery [ 289 , 290 , 291 , 292 ].

Neutrophils, the most abundant circulating leukocytes, play a significant role in atherogenesis by releasing granules containing pro-inflammatory mediators, proteases, and enzymes that produce reactive oxygen species. These actions promote further leukocyte recruitment and the formation of neutrophil extracellular traps, which contribute to thrombosis. During MI, neutrophils also aid in angiogenesis and macrophage polarization toward an anti-inflammatory state, promoting cardiac tissue healing [ 290 ].

Correlation of MI and Clonal Hematopoiesis of Indeterminate Potential (CHIP)

As per the previous reports, the influence of post-MI heart failure on the vasculature of bone marrow elucidated that a significant reduction in type H bone endothelial cells (ECs), typically associated with inflammation due to the release of a higher IL-1β in type H vessels preceding their loss after MI. Blocking generation of IL-1β protected bone vascular niche, suggesting inflammatory signaling as the primary cause of type H vasculature reduction post-MI. It is speculated that IL-1β activation in CD31hiEMCNhi ECs can confer to pyroptosis and subsequently damage the endothelium of H vessels subsequently leading to age-induced damage to bone endothelium which further results in damage to hematopoiesis and activity of HSCs [ 151 , 293 , 294 , 295 ].

Given that MI could foster myeloid skewing and increase the number of LT-HSCs, it is plausible that MI could cause speedy HSC expansion with CHIP-driver mutations. In reality, individuals with ischemic heart disease are associated with a greater chance of incidence of CHIP-driver mutations when compared to age-matched cohorts [ 296 , 297 ]. An exploratory analysis from the CANTOS trial revealed that patients with CHIP mutations (e.g., TET2) experienced lower mortality than those without CHIP mutations [ 298 ].

To further elucidate the mechanisms underlying the interaction between cardiovascular diseases and the HSC niche, a detailed investigation into how cytokines and DAMPs interact with the NLRP3 inflammasome and other inflammatory pathways will be essential in understanding the progression from cardiovascular injury to hematopoietic dysregulation. Research should focus on how specific HSC subsets contribute to hematopoiesis during cardiovascular disease and aging. Investigating the differential adhesion and migration properties of these subsets could reveal targets for therapeutic intervention. On the other hand, conducting longitudinal studies using advanced clonal tracking techniques will help determine the stability and contribution of various HSC clones over time, particularly in the context of cardiovascular diseases. Expanding clinical trials to test the efficacy of anti-inflammatory treatments, like canakinumab, in broader populations and different stages of cardiovascular disease could provide new insights into managing CHIP and related hematopoietic anomalies. Furthermore, comprehensive profiling of genetic and epigenetic changes in HSCs from patients with cardiovascular diseases will help identify biomarkers for early detection and potential targets for personalized therapies. By addressing these areas, we can better understand and potentially mitigate the adverse effects of cardiovascular diseases on the hematopoietic system, improving outcomes for affected individuals [ 299 ].

Bi-directional impact of clonal hematopoiesis on the aging bone marrow niche

Understanding the bi-directional influences of CHIP on the aging bone marrow niche is crucial, as this interaction may elucidate the early changes that precede myeloid disease manifestation and potentially reveal targets for early intervention.

Inflammaging and Niche remodeling

A key concept in CHIP-related research is the acceleration of inflammaging, a chronic, low-grade inflammation associated with aging, which exacerbates BM niche remodeling beyond what is observed in normal aging. Genetic subtypes of CHIP, such as DNMT3A, TET2, and JAK2 mutations [ 300 ], have been linked to enhanced inflammatory responses. CHIP carriers exhibit typically enhanced proinflammatory cytokines, such as IL-6, IL-8, TNF, IL-1β, and IL-18 [ 268 , 301 , 302 ]. Gene-specific analyses highlight that TET2 mutations are associated with enhanced IL-1β, while JAK2 as well as SF3B1 mutations correlate with higher IL-18 levels [ 268 , 301 , 302 ]. This elucidates the necessity for gene-specific studies to understand the distinct inflammatory profiles induced by different CHIP mutations.

CHIP mutations also appear to influence bone structure, particularly in DNMT3A-mutant carriers, who are prone to osteoporosis. Proinflammatory cytokines from DNMT3A-deficient macrophages, such as IL-20, enhance osteoclastogenesis, leading to increased bone resorption and reduced bone mass, thereby deteriorating the endosteal niche [ 268 , 301 , 302 ]. Similarly, Tet2 inactivation in mice results in decreased bone mass, although the effects are less severe [ 268 , 301 , 302 ]. The involvement of growth factors like TGF-β and BMPs in bone resorption and MDS progression further illustrates the complex interplay between CHIP and bone health [ 303 , 304 ]. Emerging evidence suggests that CHIP mutations may impact MSC function and differentiation capacity. For instance, DNMT3A-mutant HSPCs induce MSC senescence via IL-6 production, which may result in clonal expansion and myeloid skewing [ 305 , 306 , 307 ]. Another condition including sympathetic neuropathy induced through mutant cells could also impair MSCs’ HSC-supportive function, highlighting a potential mechanism for CHIP-induced niche remodeling [ 305 , 306 , 307 ]. CHIP mutations may also promote vascular niche remodeling. Tet2-deficient immune cells, particularly myeloid cells, have been shown to enhance angiogenesis through increased S100A8/A9 secretion in a lung cancer model [ 308 ]. This pro-angiogenic and inflammatory environment likely increases BM vascularization and perivascular permeability, activating transcriptional activities pertinent to inflammation inside stromal cells as well as endothelial cells in BM during aging [ 267 , 309 ].

Selective advantage and clonal expansion

Aging and adipocyte-enriched BM is characterized by prolonged TNF signaling, which confers a selective advantage to DNMT3A- and TET2-mutant clones, facilitating their expansion [ 12 , 310 ]. In this case, IL-1R pathway is reported in driving Tet2+/− CHIP progression with aging [ 311 , 312 ]. Additionally, TET2 mutations may impair natural killer (NK) cell-mediated immune surveillance, further promoting the persistence of malignant clones [ 313 ].

The interplay between CHIP and the aging BM niche involves a continuous bi-directional inflammatory loop. Mutant HSPCs not only adapt to but also drive the remodeling of both the endosteal and vascular niches, creating a microenvironment that favors clonal expansion. This intricate relationship between intrinsic HSPC mutations and extrinsic niche factors elucidates the complexity of CHIP and its progression to hematologic malignancies [ 314 ]. Further studies should explore the molecular pathways through which CHIP mutations induce niche remodeling and inflammaging for identifying potential therapeutic targets to modulate the BM niche and inhibit CHIP progression. Developing strategies for early intervention in CHIP carriers is essential to prevent the transition to overt myeloid malignancies. In addition, it is advantageous to conduct gene-specific analyses to better understand the distinct inflammatory and remodeling responses induced by different CHIP mutations. By elucidating these mechanisms, we can develop targeted therapies to mitigate the adverse effects of CHIP on the aging BM niche and improve outcomes for individuals at risk of hematologic diseases [ 314 ].

Conclusions and future acknowledgments

Aging, obesity, and diabetes can cause significant alterations in the bone marrow. The factors concomitantly generated by these conditions can influence signaling across the BM, subsequently causing changes in hematopoiesis. These alterations can disrupt the normal function and regulation of HSCs and HSPCs, leading to an increased risk of hematological disorders and malignancies. Future studies are warranted to explore the following areas: Influence of autonomic signaling: Investigate how autonomic signaling affects the regulation of hematopoiesis, particularly in the context of aging, obesity, and diabetes. Understanding these mechanisms could reveal novel therapeutic targets to maintain healthy hematopoiesis. Age-related changes in hematopoiesis: Delve deeper into how aging-induced epigenetic influences hematopoietic processes at the cellular and molecular levels. Identifying specific age-related changes could help in developing strategies to counteract the adverse effects of aging on the hematopoietic system. Obesity-related clonal hematopoiesis: Study the impact of obesity on clonal hematopoiesis and its role in promoting hematological malignancies. Research in this area could lead to interventions that mitigate the risk of cancer in obese individuals by targeting the pathways involved in clonal expansion and mutation accumulation. Addressing these areas in future research could significantly advance our understanding of the complex interplay between systemic conditions like aging, obesity, diabetes, and hematopoietic regulation. This knowledge would be instrumental in developing novel therapeutic strategies to prevent and treat hematological malignancies associated with these conditions.

Abbreviations

Bone marrow

Tet methylcytosine dioxygenase 2

DNA (cytosine-5)-methyltransferase 3 A

ASXL transcriptional regulator 1

Janus kinase 2

C-X-C motif chemokine ligand 12

Stem cell factor

Bone marrow fibroblastoid colony-forming cells

Vascular endothelial growth factor

hematopoietic stem and progenitor cells

forkhead box P3

Intercellular adhesion molecule-1

B-cell-specific transcription repressor

early B cell factor-1

Paired Box (PAX) transcription factor 5

severe combined immunodeficiency

tumor necrosis factor

Runt-related transcription factor-2

Macrophage colony-stimulating factor

Multipotent long-term HSCs

Cell Division Cycle 42

Slit guidance ligand 2

insulin-like growth factor-1

Specificity protein-1

chemokine receptor 4

Beta-3-adrenergic receptors

Suppressor of Mothers against Decapentaplegic

Granulocyte-macrophage colony-stimulating factor

macrophage inhibitory protein-1 alpha

Sympathetic nervous system

Dipeptidyl peptidase 4

metalloproteinase

Bone Marrow endothelial cells

Insulin-like growth factor binding protein

Acute myeloid Leukaemia

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Our sincere thanks to department staff of Human Anatomy and Histology, Sechenov University, Moscow, Russia.

This study was supported by Herman Wells Pediatric Research Center, Indianapolis, USA.

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Yang Xinyi (YX), Reshetov Igor Vladimirovich (RIV), Narasimha M Beeraka (NMB), Allaka Satyavathi (AS), Dinisha Kamble (DK), Vladimir N Nikolenko (VNN), Allaka Naga Lakshmi (ANL), Basappa Basappa (BB), Padmanabha Reddy Y (PRY), Ruitai Fan (RF), Junqi Liu (JL) designed the concept, and YX (Primary author) NMB (Main corresponding author) and RF wrote the manuscript; NMB, YX proofread, edited, and analyzed the content of the article. All authors reviewed the manuscript and approved it before submission.

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Xinyi, Y., Vladimirovich, R.I., Beeraka, N.M. et al. Emerging insights into epigenetics and hematopoietic stem cell trafficking in age-related hematological malignancies. Stem Cell Res Ther 15 , 401 (2024). https://doi.org/10.1186/s13287-024-04008-4

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Published on: Wednesday, 13 November 2024 05:23 PM

SAN DIEGO, CA, USA I November 13, 2024 I Global Institute of Stem Cell Therapy and Research, Inc. known as GIOSTAR , a San Diego, California based Global organization at the forefront of stem cell research over two decades, developing a novel cellular therapy pipeline to improve the standard of care for treating Type II diabetes patients, today announced that the United States Food and Drug Administration (FDA) has cleared its investigational new drug (IND) application to start a Phase–2 clinical trial for DT2-SCT. The Company’s novel approach using autologous mesenchymal stem cells to alleviate the disease-caused damage in diabetics offers a new hope to address the sufferings in diabetes patients without much side-effects.

Diabetes is not just a disease but a global health crisis. It is estimated to affect more than one billion people worldwide. The financial burden on the global healthcare system to treat diabetes is expected to reach more than one trillion dollars annually. GIOSTAR CEO, President, and Cofounder Mr. Deven Patel stated, “Upon a successful completion of the clinical trials, GIOSTAR intends to make this treatment affordable to masses and poised to capture significant global market share due to GIOSTAR’s existing global infrastructure of hospitals and research centers.”

According to the Chairman and Cofounder of GIOSTAR, Dr. Anand Srivastava , “DT2-SCT is a cellular therapy for Type II diabetics which uses autologous stem cells, isolated from the visceral tissues of the recipients, developed to target systemic ill-effects caused by diabetes-induced pathology in patients. We are pleased to reach this milestone following extensive research and development.”

GIOSTAR expects to complete the Phase-2 clinical trial using the DT2-SCT in Type II diabetics within 12 to 18 months. GIOSTAR anticipates enrolling participants for the study at few sites across the United States.

“The diabetes is now considered as a lifestyle disease. In many cases, even lifestyle changes are not enough to eliminate the risk of developing diabetes due to certain genetic risk factors,” stated Patel . “Our innovative and noninvasive stem cell-based therapeutics may offer better treatment option for diabetic patients.”

About: ( GIOSTAR )

GIOSTAR is a global stem cell research organization committed to the discovery, development, and commercialization of stem cell based treatments to make a meaningful difference in the lives of people impacted by difficult-to-treat degenerative and other diseases. The company also aims to develop stem cell-based therapies for arthritis, long COVID complications , cancer vaccines and the fairly uncommon area of making red blood cells from stem cells. GIOSTAR red blood cell technology is ready to scale and preparing its IND for US FDA. The leadership of GIOSTAR aims to make these stem cell treatments available to the masses at affordable prices.

SOURCE: GIOSTAR

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Over 800 million adults have diabetes globally, many untreated: Study

In 2022, there were around 828 million people aged 18 years and older with type 1 and type 2 diabetes worldwide, the study found.

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Nanotherapy offers new hope for the treatment of Type 1 diabetes

Individuals living with Type 1 diabetes must carefully follow prescribed insulin regimens every day, receiving injections of the hormone via syringe, insulin pump or some other device. And without viable long-term treatments, this course of treatment is a lifelong sentence.

Pancreatic islets control insulin production when blood sugar levels change, and in Type 1 diabetes, the body’s immune system attacks and destroys such insulin-producing cells. Islet transplantation has emerged over the past few decades as a potential cure for Type 1 diabetes. With healthy transplanted islets, Type 1 diabetes patients may no longer need insulin injections, but transplantation efforts have faced setbacks as the immune system continues to eventually reject new islets. Current immunosuppressive drugs offer inadequate protection for transplanted cells and tissues and are plagued by undesirable side effects.

Now a team of researchers at Northwestern University led by SQI members Evan Scott and Guillermo Ameer has discovered a technique to help make immunomodulation more effective. The method uses nanocarriers to re-engineer the commonly used immunosuppressant rapamycin. Using these rapamycin-loaded nanocarriers, the researchers generated a new form of immunosuppression capable of targeting specific cells related to the transplant without suppressing wider immune responses. The  paper was published  Jan. 17 in the journal Nature Nanotechnology .

Specifying the body’s attack

Ameer has been working on improving the outcomes of islet transplantation by providing islets with an engineered environment, using biomaterials to optimize their survival and function. However, problems associated with traditional systemic immunosuppression remain a barrier to the clinical management of patients and must also be addressed to truly have an impact on their care, said Ameer, the Daniel Hale Williams Professor of Biomedical Engineering at Northwestern’s  McCormick School of Engineering and Professor of Surgery at Feinberg School of Medicine . Ameer also serves as the director of the  Center for Advanced Regenerative Engineering (CARE) .

“This was an opportunity to partner with Evan Scott, a leader in immunoengineering, and engage in a convergence research collaboration that was well executed with tremendous attention to detail by Jacqueline Burke, a National Science Foundation Graduate Research Fellow,” Ameer said.

Rapamycin is well-studied and commonly used to suppress immune responses during other types of treatment and transplants, notable for its wide range of effects on many cell types throughout the body. Typically delivered orally, rapamycin’s dosage must be carefully monitored to prevent toxic effects. Yet, at lower doses it has poor effectiveness in cases such as islet transplantation.

Scott, also a member of CARE, said he wanted to see how the drug could be enhanced by putting it in a nanoparticle and “controlling where it goes within the body.”

“To avoid the broad effects of rapamycin during treatment, the drug is typically given at low dosages and via specific routes of administration, mainly orally,” said Scott, the Kay Davis Professor and an associate professor of biomedical engineering at McCormick and microbiology-immunology at Feinberg. “But in the case of a transplant, you have to give enough rapamycin to systemically suppress T cells, which can have significant side effects like hair loss, mouth sores and an overall weakened immune system.”

Following a transplant, immune cells, called T cells, will reject newly introduced foreign cells and tissues. Immunosuppressants are used to inhibit this effect but can also impact the body’s ability to fight other infections by shutting down T cells across the body. But the team formulated the nanocarrier and drug mixture to have a more specific effect. Instead of directly modulating T cells — the most common therapeutic target of rapamycin — the nanoparticle would be designed to target and modify antigen presenting cells (APCs) that allow for more targeted, controlled immunosuppression.

Using nanoparticles also enabled the team to deliver rapamycin through a subcutaneous injection, which they discovered uses a different metabolic pathway to avoid extensive drug loss that occurs in the liver following oral administration. This route of administration requires significantly less rapamycin to be effective — about half the standard dose.

“We wondered, can rapamycin be re-engineered to avoid non-specific suppression of T cells and instead stimulate a tolerogenic pathway by delivering the drug to different types of immune cells?” Scott said. “By changing the cell types that are targeted, we actually changed the way that immunosuppression was achieved.”

A ‘pipe dream’ come true in diabetes research

The team tested the hypothesis on mice, introducing diabetes to the population before treating them with a combination of islet transplantation and rapamycin, delivered via the standard Rapamune® oral regimen and their nanocarrier formulation. Beginning the day before transplantation, mice were given injections of the altered drug and continued injections every three days for two weeks.

The team observed minimal side effects in the mice and found the diabetes was eradicated for the length of their 100-day trial; but the treatment should last the transplant’s lifespan. The team also demonstrated the population of mice treated with the nano-delivered drug had a “robust immune response” compared to mice given standard treatments of the drug.

The concept of enhancing and controlling side effects of drugs via nanodelivery is not a new one, Scott said. “But here we’re not enhancing an effect, we are changing it – by repurposing the biochemical pathway of a drug, in this case mTOR inhibition by rapamycin, we are generating a totally different cellular response.”

The team’s discovery could have far-reaching implications. “This approach can be applied to other transplanted tissues and organs, opening up new research areas and options for patients,” Ameer said. “We are now working on taking these very exciting results one step closer to clinical use.”

Jacqueline Burke, the first author on the study and a National Science Foundation Graduate Research Fellow and researcher working with Scott and Ameer at CARE, said she could hardly believe her readings when she saw the mice’s blood sugar plummet from highly diabetic levels to an even number. She kept double-checking to make sure it wasn’t a fluke, but saw the number sustained over the course of months.

Research hits close to home

For Burke, a doctoral candidate studying biomedical engineering, the research hits closer to home. Burke is one such individual for whom daily shots are a well-known part of her life. She was diagnosed with Type 1 diabetes when she was nine, and for a long time knew she wanted to somehow contribute to the field.

“At my past program, I worked on wound healing for diabetic foot ulcers, which are a complication of Type 1 diabetes,” Burke said. “As someone who’s 26, I never really want to get there, so I felt like a better strategy would be to focus on how we can treat diabetes now in a more succinct way that mimics the natural occurrences of the pancreas in a non-diabetic person.”

The all-Northwestern research team has been working on experiments and publishing studies on islet transplantation for three years, and both Burke and Scott say the work they just published could have been broken into two or three papers. What they’ve published now, though, they consider a breakthrough and say it could have major implications on the future of diabetes research.

Scott has begun the process of patenting the method and collaborating with industrial partners to ultimately move it into the clinical trials stage. Commercializing his work would address the  remaining issues  that have arisen for new technologies like  Vertex’s stem-cell derived pancreatic islets  for diabetes treatment.

The paper, “Subcutaneous nanotherapy repurposes the immunosuppressive mechanism of rapamycin to enhance allogeneic islet graft viability,” was supported by the National Science Foundation (Award no. 1453576), the National Institute of Health Director’s New Innovator Award (NHLBI 1DP2HL132390-01), and the Center for Advanced Regenerative Engineering.

This article first appeared on Northwestern Now .