B1. Methods of Stem Cells Identification: Stem cells live with other stem cells so we need some methods to identify them from the other normal and differentiated cells. A few methods utilized for the identification of stem cells are discussed below:

The mechanisms of stem cell action are complex and multifaceted, and they can vary depending on the specific type of stem cell and the context in which they are applied. However, there are several general mechanisms through which stem cells exert their effects. Here are some key mechanisms of stem cell action:

Differentiation: Proliferation and differentiation of tissue stem cells to replace damaged cells: Cells divide by symmetric or asymmetric cell kinetics programs. Symmetric division expands the stem cell pool, whereas asymmetric division maintains a constant number. Stem cell divides to produce transiently-amplifying progenitor cells, which differentiate into functional and terminally-differentiated cells (Figure 6). Ultimately, depending on the environmental cues, stem cells expand to increase inside the tissue and maintain tissue homeostasis (Figure 7).

Figure 7: Symmetric and asymmetric division of tissue stem cell resulting in two daughter cells of similar or dissimilar types.

Figure 8: Expansion of stem and progenitor cells which ultimately produce terminally differentiated cells (R. Choudhary, 2014).

Paracrine signaling – The paracrine action of stem cells has emerged as one of the mechanisms of stem cell-mediated therapeutic applications. Soluble factors released from stem cells may act on damaged neighbor cells mediated through microvesicles (M.V.s). M.V.s are loaded with proteins, peptides, miRNAs, bioactive lipids, and other second messenger molecules are transferred to injured cells that reprogram damaged cells to restore functionality or maybe even regain stem cell-like phenotype (Figure 8).

Figure 9: Paracrine signaling. Microvesicles released from stem cells repairs injured cells and restore their functions. It has also been postulated that microvesicles released by the damaged cells may induce differentiation of stem cells and thus maintain cell turnover.

Immunomodulation: Stem cells possess immunomodulatory properties, meaning they can influence the immune response. They can suppress immune reactions, reduce inflammation, and promote tissue healing by interacting with immune cells and modulating their activity. Stem cells can interact with various immune cells, such as T cells, B cells, macrophages, and dendritic cells, through direct cell-to-cell contact or through the secretion of soluble factors or transfer cell organelles or molecules through tunneling nanotubes (TNT). Interaction of molecules with immune cells results in (i) Suppression of inappropriate or excessive immune responses, (ii) Reduction of inflammation, (iii) polarization of immun cells like development of regulatory T-cells or Tregs, and others like promotion of tissue healing.

Tunneling nanotubes (TNT) formation: TNT are considered a novel cargo route between the cell through which subcellular organelles and secreted factors (secretomes) are transferred (Figure 9). For example, hemopoietic progenitor cells are connected via F-actin-dependent TNT, and the stem cell marker CD133 is transferred between hemopoietic cells. In another study, TNT-mediated mitochondria transfer was observed between bone marrow-derived mesenchymal stem cells and pulposus cells of the brain (Yang et al., 2022).

Figure 10: Exchange of cell organelles and biologically active lipids and peptides between stem cells and neighboring cells through tunneling nanotubes.

Cell replcement: By differentiating into the appropriate cell types, stem cells can integrate into the damaged tissue and restore its structure and function. When stem cells are introduced into a damaged tissue or organ, they can integrate into the existing cellular infrastructure and undergo differentiation based on the surrounding signals and cues. For example, MSCs differentiate into various cell types such as osteoblasts (bone cells), chondrocytes (cartilage cells), and adipocytes (fat cells) dependig upon the signal of diffeentiotion. Stem cells can also integrate into the damaged tissue and restore its structure and function. In a study conducted using human ESCs, stem cells fused with fibroblasts resulted in hybrid cells, which have the typical characteristics of ESCs. The result established that ESCs could reprogram other cells to an embryonic-like stateClick or tap here to enter text.. Later, a fusion of mesenchymal stem cells with tumor cells was observed, and mechanistic molecular regulation of fusion events was elucidated (Zhang et al., 2021).

Figure 11: The fusion of stem cells with neighboring cells for restoring the function of damaged cells has also been a postulate for the mechanisms of stem cell action.

Tissue protection and anti-apoptosis: Stem cells have the ability to protect existing cells from injury and cell death. Apoptotic bodies of stem cells engulfed by the macrophage to induce cytokine production – Apoptosis is necessary for exerting the immunomodulatory functions of stem cell therapy. It has been observed recently that apoptotic mesenchymal stem cells, after intravenous injection, were found in the lungs of mice. An alveolar macrophage of the lungs engulfed apoptotic bodies of stem cells and released cytokine -IL10 through phosphatidylserine-recruited macrophage that acted on neutrophils and natural killer cells (Figure 11). Click or tap here to enter text.Investigators concluded that MSC apoptosis and phagocytosis by the macrophage are pivotal in their therapeutic efficacy (Pang et al., 2021).

Figure 12: Apoptosis, or programmed cell death, has been considered the significant step in inducing epithelial cell proliferation and hence a strategic player in tissue regeneration. Recent studies suggest apoptosis of mesenchymal stem cells in immunosuppression and required for therapeutic functions.

• Apoptosis- A process of programmed cell death mediated by intrinsic and extrinsic factors
• Asymmetric Cell Division –Cell divisions that produce two different types of cells.
• Asymmetric Self-Renewal Division – The cell division process by which a tissue stem cell produces a new tissue stem cell and a differentiated progenitor cell.
• Blastocyst – After successful fertilization, the zygote divides rapidly and makes a rapidly dividing ball of cells called the blastocyst. It contains ‘inner cell mass’ or ICM and trophoblasts that surround the inner cell mass for nourishment. The ICM is the source of embryonic stem cells (ESCs).
• Blood Cells – Various types of cells derived from hematopoietic stem cells (HSC) are called blood cells. Different blood cells derived from myeloid progenitors are megakaryocytes, erythrocytes, mast cells, and megablast. Megablasts further give rise to basophils, neutrophils, eosinophils, and monocytes (called macrophages in tissue). Other blood cells derived from lymphoid progenitors are natural killer cells, T-lymphocytes, B-lymphocytes, and plasma cells.
• Bone Marrow – The spongy tissue found inside the long bones is called bone marrow. Bone marrow is the reservoir of mesenchymal stem cells (MSCs) called bone marrow-derived MSCs. It is also a major source of HSCs, which can be mobilized by specific factors to enter the peripheral bloodstream.
• Cancer – A disease that is associated with the abnormal growth of cells or tissue that has the potential to invade other tissue. A mass of cancer cells is called a tumor. The process of oncogenesis is hypothesized to be mediated through cancer stem cells.
• Cardiovascular – Related to heart and blood vessels.
• Cell –The biological living unit of large-bodied organisms like humans. Trillions of cells multiply and change into different forms and structures that integrate to make the organs and tissues of multicellular plants and animals.
• Cell Division – The process by which cells multiply by growing in mass, duplicating their DNA, and splitting into two new cells.
• Cell Kinetics – Processes that result in changes in cell number.
• Chimera – Presence of two types of cells derived from two different individuals of the same or even from another species. A chimera is created when pluripotent stem cells of another embryo of the same or different species are added to another developing embryo.
• Chromosome – Densely packed structures that contain a cell’s nuclear DNA. Normal human cells have 46 chromosomes.
• Chromosome segregation – The process by which dividing cells place an equal number of chromosomes into each new cell produced.
• Congenital Arthritis – Joint defects that are deformed at birth
• Cytokines – Cell signaling molecules (peptides) released from various immune and non-immune cells that affect (enhance or slow down) the body's immune system.
• Differentiation – A process of changing stem cells to more specialized cell types. The production of progenitor cells by stem cells and then the formation of terminally-differentiated cells from progenitor cells are the events of tissue cell differentiation.
• ESC – Embryonic stem cells are the cells derived from the inner cell mass of the blastocyst. ESCs are pluripotent.
• Fibroblast – A type of differentiated cell often used for reprogramming cells toward the generation of induced pluripotent stem cells (iPSCs) in stem cell biology.
• Gene – The basic unit of heredity and the part of DNA that codes for proteins.
• Genetic Medicine – These genetic materials, DNA or RNA, are delivered into the body as therapeutics.
• Genome - Entire genetic material of an organism or a cell. It includes all nuclear DNA plus the mitochondrial DNA of a human cell.
• Germ Cells- Reproducing cells of the body, sperm cells in males and eggs in females are the germ cells.
• Hematopoiesis – Formation of blood cellular components which is derived from hematopoietic stem cells (see blood cells)
• Homeostasis – Process of self-regulation to maintain internal steady state condition for the body's optimal functioning.
• Immunohistochemistry – A method of detecting antigens in a cell of a tissue section by exploiting functions of antigen-antibody binding. Antigens of the cell are detected by an anti-antigen antibody that is labeled with an enzyme of fluorophores. Visualization of chromogen (in the case of enzyme-labeled antibody) or fluorophores indicate the presence of specific antigens in the cell.
• Immortal DNA strands – The oldest nuclear DNA molecules in the chromosomes that are non-randomly retained by asymmetrically self-renewing tissue stem cells. They were first postulated to exist by Cairns in 1975.
• iPSCs – Induced pluripotent stem cells are bioengineered ESC-like cells produced by reprogramming somatic cells with pluripotent transcription factors.
• Kinetic Stem Cell (KSC) Counting – A method for determining the specific fraction or dosage of tissue stem cells based on monitoring how the produce progenitor and terminally-arrested cells in culture.
• Label Retention – This technique was previously used to identify slow-cycling cells or tissue stem cells with non-random chromosome segregation. A label like bromodeoxyuridine (BrdU) is once incorporated into the DNA of dividing cells, including stem cells. Retention of the label by cells after successive division in the absence of the label allows the identification of label-retaining cells (LRC). If LRCs are shown independently to be actively cycling, they are defined to be tissue stem cells.
• Macrophage – A type of blood cell that has phagocytic activity. When they reach a specific tissue location, monocytes can be induced to mature into macrophages.
• Mesenchymal Stem Cells (MSCs) – Multilineage stromal cells that have the potency to self-renew and differentiate into many cell types. MSCs can be isolated from various tissues, including bone marrow, adipose tissue, placenta, umbilical cord blood, endometrium, and others.
• Microvesicles – Microvesicles (MVs) are the vesicular structures shed off by the cells through blebbing of the plasma membrane. MVs are typically in the range of 0.1-1.0 µm in diameter.
• Morphology – Refers to a cell's size, shape, and structure.
• Neonatal – Newly born
• Neuron – Also called a nerve cell- is the fundamental brain and nervous system unit. The nervous system is made up of neurons.
• Newt – A newt is a salamander- amphibian
• Non-random Chromosome Segregation – A form of chromosome segregation specific to asymmetrically self-renewing tissue stem cells. Instead of new and old chromosomes having an equal likelihood of going to either new cell, the stem cell always gets the chromosomes with the older DNA.
• Organelle – Structures within the cells surrounded by a membrane and has a specific function are called cell-organelle.
• Paracrine – A type of cellular signaling where the signal (biomolecules) produced in a cell induces changes in nearby cells.
• Perinatal – Associated with birth; referring to the gestational tissues, the placenta, umbilical cord, and amnion sac.
• Phagocytosis – A process in which cells use their plasma membrane and cytoplasmic to engulf large particles like cell debris or bacteria. The formation of an internal structure with large particles inside the cell is called a phagosome.
• Postnatal – Occurring after birth
• Prenatal - Before birth
• Progenitor Cell – The progeny of stem cells with lesser potency than stem cells but higher potency than terminally differentiated cells are called progenitor cells. They are also called “transiently-amplifying cells” due to their high proliferation capacity.
• Regeneration – The process of restoring or replacing damaged/injured cells with functional ones is regeneration. Stem cells have regeneration power to replace or repair damaged cells with new or functional cells.
• Somatic Cell- All other cells of the body except germ cells (egg and sperm) are called somatic cells.
• Tunneling Nanotubes (TNT) – A membranous elongation of cells that connect each other to mediate trafficking materials (organelles, biomolecules, bioactive peptides, ions, and other cues) across them.
• Totipotent – Stem cells that can give rise to any cell type or a complete embryo, including embryonic and extra-embryonic tissues.
• Transcription – The process of formation of RNA from the DNA.

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• https://stemcells.nih.gov/
• https://stemcells.nih.gov/info/basics/stc-basics
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