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STEM CELL AND REGENERATIVE BIOLOGY

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STEM CELL AND REGENERATIVE BIOLOGY

 

Ayesha Tungekar 

 

 

 

STEM CELL NICHE: ROLE IN STEM CELL HOMEOSTASIS AND REGENERATIVE MEDICINE

 

1-INTRODUCTION:

The stem cell niche is the microenvironment that supports stem cells facilitated by soluble factors, and adhesion molecules that lead to subsequent signal transduction. The stem cell niche instructs the stem cells to proliferate, migrate and differentiate. Their function is to repair and regenerate the tissue and also maintain tissue homeostasis. The stem cells consist of extracellular matrix and cell adhesion molecules that play a role in the attachment of stem cells to the niche. The stem cell niche is well-studied in hematopoietic stem cells, hair follicles, germline stem cells, and intestinal stem cells. It is known that aging causes the niche to weaken this is due to reduced expression of factors responsible for maintaining stem cell self-renewal and also reduced expression of CAM(cell adhesion molecules). Drosophila and mice serve as excellent models to study stem-cell-niche interactions.

Stem cell niche

Stem cell niche

Image 1:- Stem cell niche (Mooney et al., 2015)

 

2-CELL ADHESION IN STEM CELL NICHE:

 

Cadherin molecules lead to cell-cell interactions. The cadherin molecules consist of the intra- cellular domain and extracellular domain. The intracellular domains of cadherin can connect cadherins to the cytoskeletal network and thus forms stable adherent junctions was found that E.cadherin in Drosophila ovary accumulates at the junction between the germline stem cells and their niche cells. E-cadherin removal showed that the Germline stem cell niche departs from

their niche thus highlighting the fact that E-cadherins are crucial for self-renewal and GSC maintenance.

Gap junctions (intracellular protein channels) function n connecting the two adjacent cells. Gap junctions play a role in the direct transfer of small molecules between the cells and also cause electrical and chemical communication between the cells. Connexin 43  facilitates the transfer of reactive oxygen species into stromal cells and thus prevents HSC senescence.

 

3-HEMATOPOIETIC STEM CELL NICHE

 

Hematopoietic stem cells residing in bone borrow can give rise to all mature blood types and tissue. Blood  production occurs due to the proliferation, and differentiation of HSCs in bone marrow

Haematopoietic stem cell niche

Image 2: Haematopoietic stem cell niche(Mendelson & Frenette, 2014)

Pre-vascular cells in the HSC niche:

Myeloma-associated cell adhesion molecules (MACM), CD51, and nestin all account for bone marrow MPSC (mesenchymal stem and progenitor cells). These cell express genes that encode XML-12 and SCF (Stem Cell Factor). The pre-vascular cells having CAR i.e., CXCL-12 abundant reticular play a role in HSC self-renewal, its proliferation, and trafficking. CXCL-12 is a niche factor responsible for maintaining HSC differentiation, and dormancy and inducing multilineage reconstitution. Deletion of this CXCL-12have shown deletion of this CXCL-12 receptor in Mx1-Cre mice showed a reduction in HSC levels in the bone marrow. In the Prx1-Cre deletion strain deletion of CXCL-12 revealed its importance in repopulating HSCs and maintaining quiescence. However, it was noted that  CXCL-2 DOES

NOT PLAY ROLE in maintaining HSC and leads to movement of HSCs to blood when CXCL-12 were deleted from car cells using osx-care. (Mendelson & Frenette, 2014)

Notch-pathway:

Expression of the Notch pathway in early hematopoiesis plays role in cell differentiation decisions. Imaging techniques have revealed that the is a homogeneous distribution found in homeostatic HSCs whereas HSCs that are transplanted are found in the trabecular bone region. Transplanted HSC regions are confined to nearby vascular structures within five hrs of transplantation.

Conditions like anemia, chemotherapy, irradiation, and hypoxia can affect HSC homeostasis and can disrupt regeneration capability. Chemotherapy and irradiation are responsible for causing blood cell death. In a study, it was found that The apoptosis caused can be mitigated if activated protein C(APC) or thrombomodulin is administered in mice after 24hrs of lethal irradiation.

HSC in regenerative medicine:

Identification and understanding of markers will help to improve the knowledge and understanding of the maintenance and regulation of hematopoietic stem cells. Generating engraftable hematopoietic stem cells from pluripotent stem cells may throw light on developmental cues in understanding the relation or connection between HSC self-renewal and quiescence.

Therapeutic delivery of cytokines like IFN-γ, and FGF, and delivery of APC will help in increasing multilineage hematopoietic reconstitution.(Mendelson & Frenette, 2014)

 

4-GERMLINE STEM CELL NICHE

 

C.ELEGANS AS AN EXCELLENT MODEL FOR STUDYING GERMLINE STEM CELL NICHE:

Germline stem cell niche helps to control stem cell behavior and C.elegans can be used as a useful model to understand this. C.elegans have a male and a hermaphrodite. In the case of males, one gonad arm is present and thus produces one sperm.  In the case of hermaphrodites, there are two identical arms and each one produces sperm and then an oocyte. The oocytes move to the uterus where they are fertilized. In C.elegans germline stem cells are located at the distal ends of gonads. The germline stem cell niche is controlled by a single DTC (distal tip cell).

GLP-1/notch signaling helps to maintain homeostasis between cell proliferation and differentiation and removal of either DTC or GLP-1 signaling will lead to an exit from mitosis and differentiation. GLP-1/notch signaling is known to activate fbf-2 in germ cells and this fbf-2 along with fbf-1 will help to inhibit the expression of genes like  GLD-1,2,3  thereby controlling germline stem cell self-renewal. GSCs enter meiosis if there is a high level of gold-1 and mitotic division is seen when there is a low level of gold-1. GSCs’ self-renewal is controlled BY PUF-8 which belongs to the PUF family. This PUF-8 controls the self-renewal of GSCs by enhancing mitotic proliferation.

 

GSCs in drosophila ovary:

 

The adult Drosophila ovary has a well-characterized stem cell niche. About 15 ovarioles are present in the adult Drosophila ovary which contains a specialized structure called germarium. Three types of stem cells i.e., escort stem cells, germline stem cells, and follicle stem cells are seen at each anterior tip of the germarium. Terminal filaments(TF) located anterior to the cap cell connects with the IGS (inner germarium sheath) cells. In adult drosophila the germline stem cells are connected to the niche cells via adheres junctions.DE- cadherins provide anchorage to GSCs in their niche and. Escort stem cells interact with each other to form a germline stem cell niche.

howing female Drosophila germanium

Image3: showing female Drosophila germanium(Ram Singh, 2012)

 

GSCs in Drosophila testis:

 

At the tip of the Drosophila testis, there is a germinal proliferation center that consists of germline stem cells and cyst progenitor cells which are attached to hub cells. The GSCs divided mitotically and orient the mitotic spindle in a manner that is perpendicular to hub cells whereas

the cyst progenitor cells also called cyst stem cells (CSC) has the property of self-renewal and produces daughter cell that will differentiate into SCC (somatic cyst cells). The SSCs will now grow and form a thin layer around the spermatogonial cyst. The JAK/STAT signaling controls the early differentiated germ cell cyst to differentiate back into germline stem cells and also play role in controlling  GSCs/cyst stem cells’ self-renewal and proliferation.DER/RAF/MAPK signaling is used to maintain differentiation.

Image4: Showing drosophila testicular niche (Ram Singh, 2012)

 

Spermatogonia stem cell (SSC)niche in mouse testis:

 

Spermatogenesis is maintained by undifferentiated spermatogonial stem cells in the mouse. The reason why the research on the mouse spermatogonial stem cell has gained importance is because of the simple tubular structure, and the ability of spermatogonial stem cells to undergo transplantation. The genetics and transplantation findings have revealed that niche is critical for the maintenance of spermatogonia stem cells. These stem cells lie close to the basement membrane of the seminiferous tubule. Leydig cells Sertoli cells are a functional niche for SSC. Sertoli cells express ERM(Ets-related molecule) which is necessary for the self-renewal of spermatogonial stem cells. Active JAK0STAT and PI3K/Akt pathway signaling and suppression of TGF-β signaling are necessary for the self-renewal of SSC.Rac1  controls the proliferation of SSCs.Markers expressed by SSCs are CD9, STRA-8, and Thy-1  and express receptors like GDNF receptor, and c-Ret play tyrosine kinase receptor. (Ram Singh, 2012)

 

5-INTESTINAL STEM CELL NICHE:

Illustration of intestinal stem cell niche

Image5: Illustration of intestinal stem cell niche

Source: https://www.researchgate.net/publication/314195000

The Transient amplifying cells undergo division and form about 300 cells per crypt per day. Each crypt consists of four to six intestinal stem cells. These transient amplifying cells undergo differentiation to produce mucosa, enteroendocrine, enterocyte, and Paneth cells. ISC is located at the +4 position from the bottom of the crypt.  The +4 position stem cells express m Tert and f Bmi1. Some studies also found that crypt-based columnar cells(CBC) also stem cells. In the study carried out it was found that Lgr 5 expresses crypt-based columnar cells. These crypt-based columnar cells are rapidly dividing. The Lgr5 are multipotent and thus have the capability to differentiate into all cell types in the intestine. CBC(Lgr5+) receive niche support from their specialized progeny. These CBCs express a high level of Sox-9,β-catenin, and Lgr5. The +4 cells are quiescent and CBCs are active. The Paneth cells act as a stem cell niche and these Paneth cells are known to regulate ISC maintenance and proliferation. Various signaling events control ISC proliferation and maintenance. Wnt signaling regulates the upward and downward migration of differentiated epithelial cells present in the intestine. Further proliferation in the intestine is inhibited by Hedgehog signaling which negatively regulates the Wnt signalling pathway. The size of the ISC niche is maintained by Lrig1. Lrig1 is known to be highly expressed in ISCs. The +4 cells express hope (homeobox protein) which can generate crypt-based columnar cells.Lgr5+ is known to maintain tissue homeostasis and its complete loss is compensated by BMI-1-expressing stem cells. (Ram Singh, 2012)

 

6-EPIDERMAL STEM CELL NICHE:

 

The skin consists of the epidermis, hair follicles, and sebaceous glands. Hair follicle stem cells are located in the bulge region and can regenerate the hair and sebaceous gland.HFSCs located in the bulge region are multipotent and can differentiate into all hair follicle cell lineages. At the base of the bulge region hair germ- small clusters of cells are present which resemble HFSCs. In the bulge, niche melanocyte resides. These melanocytes stem cells have the potential to produce and transfer pigment cells to the matrix The stem cell at the bulge region have a repairing role and thus can repair the damaged hair follicle. In a study, it has been observed that the stem cells at the bulge region can repopulate the sebaceous gland by traveling upwards to the isthmus. (Ram Singh, 2012)

 

7-NEURAL STEM CELL NICHE:

 

Two niches have been identified in the adult mammalian brain., these regions are the subventricular and subgranular zone. Neural stem cells possess the ability to differentiate into several interneuron subtypes. These Neural stem cells  are known to generate several neurons for the olfactory (Ram Singh, 2012)

 

8- STEM CELL NICHE AGING:

 

Adult stem cells play the role in establishing homeostasis and are also known to repair damaged or injured cells. But due to aging various intrinsic and extrinsic factors are affected which impairs normal stem cell tissue behavior.  Ageing-related studies carried out in Drosophila testis revealed that several changes like reduction in cell adhesion molecules and self-renewal signal were seen in the stem cell niche and this is in turn responsible for the reduction of decline in GSC number. In female drosophila, the GSC’s proliferative ability was reduced because of the decrease in expression of E-cadherin at the stemcell niche junctions. The hematopoietic stem cells and neural stem cells become less active with age. Hair begins to grey as a result of loss of melanocyte stem cell self-renewal. (Ram Singh, 2012)

 

Image

stem cell and niche interaction in normal

6:  Showing  stem cell and niche interaction in normal and aging cells(Ram Singh, 2012)

Gut stem cell aging is driven by the mTORC1 pathway:

In a study carried out by using mice that were 17-month-olds and 3.5 months old it was found that MTORC1expression in mice regulates villus cell aging. MTORC1 IS hyperactivated IN Intestinal stem cells and TA which causes aging of the villus by inhibiting intestinal stem cell or progenitor cell proliferation by amplifying the MMK6-p38-p53 stress response pathway. It was found that MAP-K ablation can prevent mTORC-1-driven villus cell aging. (He et al., 2020) .

 

9-STEM CELL IN REGENERATIVE MEDICINE:

 

The niche is a dynamic micro-environment that can adapt to physiological or diseased conditions. To target the stem cell niche for therapeutics it is necessary to understand cell-to-cell contact, electrical stimuli, cell to matrix contact. The surface containing the Synthetic peptide  Arginine-glycine-Aspartate can be used as a motif for cell attachment and thus can be used for cell expansion. The advantage that synthetic peptides offer is that it is animal component-free and it is potentially scalable.

A-Organoid model in studying stem cell niche:

Organoids are very small and self-organized 3D cultures of the tissue, which are derived from the cells. Organoids are considered as the mini version of the organs that are produced and developed by in vitro process. Organoids are derived from adult stem cells or embryonic or induced pluripotent stem cells(iPSCs). The 3D nature of the organoids mimics many of the anatomical and physiological features including cellular movement, and cell-cell interaction.

Organoids provide an in-vitro environment for studying and analysis of the normal and disease organoids derived from the stem cell and thus provide insights to analyze the stem-cell niche interactions.  By combining organoids and CRISPR CAS9 various diseases can be cured and provide new opportunities for researchers and scientists to produce cures for human diseases like leukemia, Parkinson’s disease, or Alzheimer’s disease. Engineered organoids can measure the properties like signaling pathway and mechanical forces by incorporating non-cellular materials such as sensors within the living tissue. (Murrow et al., 2017).

 

B-HSC expansion in-vitro

 

BM-HSC expansion in-vitro has gained a lot of importance. For treating patients with Hematological malignancies like leukemia, thrombocytopenia, and multiple myeloma HSC transplant is the only curative therapy. However, in many cases problems arises where patients do not have HLA- identical donor, and thus therapy cannot be performed on these patients. Focus is being made on cord-blood transplants as this reduce the need for HL-A identical donor and minimize the risk of chronic graft -versus host disease.HSC cells can be maintained in-vitro in absence of any growth factors and by inhibiting mTOR and facilitating activation of Wnt signaling., all these things are done to expand the HSCs cell in-vitro. Valproic acid and lithium are known to prevent the differentiation of genes and preserve the genes involved in stem cell expression. Inhibition of GSK inhibitors will promote the activation of β-catenin which will promote HSC proliferation. However, this alone is not sufficient enough to maintain self-renewal capacity also inhibition of mTOR, anti-apoptotic effect by SCF, and modulation of histone acetylation are necessary to preserve the self-renewal capacity. Small molecules like UM171, SR1 and CHIR99021 are useful in the expansion of HSCs in-vitro whereas Rapamycin, and Valproic acid is used to maintain the HSCs in-vitro. (Sugimura, 2016)

 

C-In-vitro  Bioengineering of HSC niche:

Engineering of HSC niche followed by drug screening

Image 7: Engineering of HSC niche followed by drug screening(Sugimura, 2016)

Application of organ on a chip culture can be used to activate the physiological response and mechanics of the entire organ. So Bone marrow, a part of an organ on a chip can provide useful insights and help in replicating the HSC niche. In the experiment implantation of the chip-size bone, the matrix is done inside the mice so that the native hematopoietic stem cells can migrate to the matrix and reconstitute BM.  BM- on a chip helps in understanding the consequence of an HSC niche thereby allowing drug screening for BM- regeneration. Disease modeling such as leukemia can be studied followed b developing potential development of the drug. (Sugimura, 2016)

 

D-Mesenchymal stem cells(MSCs): Use in regenerative medicine:

 

MSCs can self-renew and differentiate into tissue types like adipocytes,  osteoblasts, and chondrocytes. MSCs produce secretory factors that play a pivotal role in maintenance and tissue repair that support both autocrine and paracrine functions and also engraftment functions. (Ullah et al., 2015).

SEM  micrographs of hydrogel fibres prepared with simple extrusion and electro-stretching

MSC transplantation into Amyotrophic lateral sclerosis(ALS) disease patient

This disease causes the degeneration of muscular neurons. MSCs have the potential to differentiate into neurons. Mesenchymal stem cells can be isolated from the bone marrow of a patient suffering from ALS and injected back into the spinal cord of the same patient, followed by tracking of MSCs using MRI. The result showed no abnormal cell proliferation but mild effects like leg sensory dysesthesia were observed which was reduced within few weeks. (Ullah et al., 2015).

Parkinson’s Disease:

In Parkinson’s disease, there is a substantial loss of dopaminergic neurons. Bone marrow-derived mesenchymal cells can be effectively used in regenerative medicine.

In a research carried out at Tubingen University, MSCs were delivered through the nose of a Parkinson’s disease rat. After about four and a half months, it was observed that the administration of MSCs increased the level of tyrosine hydroxylase and

Type1 Diabetes:

Type1 Diabetes is an autoimmune disease. In this condition, the body produces auto-antibody against the β- cells and destroys these cells. Insulin is the hormone that is necessary for controlling glucose levels in our bodies. Due to the destruction of these β-cells very low

levels of insulin are produced which is not sufficient to control blood insulin. Human BM-MSCs can differentiate into glucose-competent pancreatic endocrine cells. In research carried out by Unsal.et.al,2014 it was found that the survival rate of engrafted islet cells was enhanced when mesenchymal stem cells together with islet cells were introduced in mice treated with streptozotocin. This technique can be useful for curing type 1 diabetes patients (Ullah et al., 2015).

 

E-BIOMIMETIC NANOFIBRES FOR TISSUE REGENERATION.

 

Nanofibers facilitate stem cell survival, proliferation, and differentiation. Nanofibers provide allows the stem cells to differentiate and organize themselves. Nanofibres can be formed by natural polymers like collagen, cellulose, and keratin, or can also be derived from synthetic polymers like polyethersulfone(PES), and polylactide(PLA). However natural polymers are preferred because of the good biocompatibility advantage they offer. The biochemical properties, topography, and structures have an impact on stem cell fate and tissue regeneration.

SEM  micrographs of hydrogel fibres prepared with simple extrusion and electro-stretching

Image: SEM  micrographs of hydrogel fibers prepared with simple extrusion and electro-stretching.(Xiaowei Li et.al.,2017)

Brain tissue regeneration:

In the brain, the lesion cavity is formed close to the cortex due to stroke and traumatic injury. This can result in poor cell survival and less differentiation of engrafted stem cells. When RADA16-IKVAV peptide solution is injected into the damaged brain tissue region lesion site it quickly accumulated into a nanofibrous hydrogel at the original region that filled the lesion cavity. This technique created a suitable environment for the axons to regenerate, connected brain tissues together, reduced the formation of reactive glial cells, and increased the survival of neuronal stem cells. (Xiaowei et.al.,2017)

Liver regeneration:

The liver is composed of epithelial cells like cholangiocytes and hepatocytes. These cells work together with the mesenchymal, endothelial, and stromal cells to carry out crucial metabolic functions like metabolic, exocrine, and endocrine functions which are necessary for body homeostasis. Nanofiber scaffoldlike collagen nanofibers can be used to mediate the differentiation of hMSCs into hepatocytes. This can improve hepatocyte cell function like urea and albumin synthesis and secretion. (Xiaowei et.al.,2017)

10-CONCLUSION:

Importance is being made to study and understand the microenvironment in which the stem cells reside and maintain their specific characteristics.to develop effective therapies for stem cell regeneration. Such therapies will help in curing many diseases. By using some genomic and biochemical markers expressed by various stem cells, telomere length and cellular morphology can be studied. This can be used further for clinical and commercial applications.

 

 REFERENCES:
  • He, D., Wu, H., Xiang, J., Ruan, X., Peng, P., Ruan, Y., Chen, Y.-G., Wang, Y., Yu, Q., Zhang, H., Habib, S. L., De Pinho, R. A., Liu, H., & Li, B. (2020). Gut stem cell aging is driven by mTORC1 via a p38 MAPK-p53 pathway. Nature Communications, 11(1), 37
  • Mendelson, A., & Frenette, P. S. (2014). Hematopoietic stem cell niche maintenance during homeostasis and regeneration. Nature Medicine, 20(8), 833–846.
  • Murrow, L. M., Weber, R. J., & Gartner, Z. J. (2017). Dissecting the stem cell niche with organoid models: An engineering-based approach. Development, 144(6), 998–1007.
  • Ram Singh, S. (2012). Stem Cell Niche in Tissue Homeostasis, Aging, and Cancer. Current Medicinal Chemistry, 19(35), 5965–5974.

 

  • Sugimura, R. (2016). Bioengineering Hematopoietic Stem Cell Niche toward Regenerative Medicine. Advanced Drug Delivery Reviews, 99, 212–220.

 

 

  • Ullah, I., Subbarao, R. B., & Rho, G. J. (2015). Human mesenchymal stem cells—Current trends and future prospective. Bioscience Reports, 35(2), e00191.

 

Ayesha Tungekar

 

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