Stem Cell Biology

Stem Cells Overview

• Stem cells possess the unique ability to self-renew and differentiate into specialized tissues and organs.
• Most cells in the body self-renew, but stem cells can generate differentiated tissues and organs.
• Stem cells maintain tissue homeostasis by balancing self-renewal, differentiation, and mature cell death.
• Symmetric division in stem cells leads to two new stem cells, replenishing the population.
• Asymmetric division results in one stem cell and one cell committed to differentiation.
• Homeostatic equilibrium is maintained in normal tissue through balanced stem cell replication, differentiation, and mature cell death.
• Cell numbers are regulated by altering stem cell input, apoptosis, proliferation, and differentiation rates.

Types of Stem Cells

• Embryonic stem cells from the inner mass of the blastocyst generate any cell in the body.
• Embryonic stem cells are totipotent, possessing limitless renewal capacity.
• Adult stem cells replace damaged cells and maintain cell populations within specific tissues.
• Adult stem cells have limited regenerative capability and reside in specialized tissue microenvironments called niches.
• Stem cell differentiation potential varies depending on source and developmental stage.
• Totipotent stem cells can generate all cell types, including extraembryonic tissues like the placenta.
• Pluripotent stem cells can differentiate into any cell type of the body, but not extraembryonic structures.
• Pluripotent cells give rise to cells from the ectoderm, mesoderm, and endoderm.
• The ectoderm forms skin, the nervous system, and sensory organs.
• The mesoderm forms muscles, bones, blood, kidneys, and gonads.
• The endoderm forms the digestive tract, liver, lungs, and endocrine glands.

Differentiation of Stem Cells

• Embryonic stem cells transition from totipotent to pluripotent, then to multipotent.
• Pluripotent stem cells divide asymmetrically to maintain a stable pool while generating more restricted cells.
• Embryonic stem cells in vitro differentiate into cells from all three germ layers.
• Adult stem cells typically produce tissue-specific cells.
• Hematopoietic stem cells from bone marrow replenish marrow after chemotherapy or replace defective blood cells.
• Mesenchymal stem cells from bone marrow and fat differentiate into chondrocytes, osteocytes, myocytes, and adipocytes.

Challenges and Solutions in Regenerative Medicine

• Regenerative medicine seeks to use stem cells to restore tissues with low intrinsic regenerative capacity.
• Challenges include delivering stem cells to the damage site and functionally integrating them into the tissue.
• Immunologic rejection is triggered by human leukocyte antigen (HLA) molecules on stem cells.
• Induced pluripotent stem cells (iPSCs) are generated from a patient's somatic cells to avoid immunologic rejection.
• iPSCs express the same HLA markers as the patient's cells and can differentiate into cells from all three germ layers.
• iPSCs are a potential tool for gene editing or genetic correction.

Stem Cell Summary

• Embryonic stem cells are totipotent and generate all cell types in the body.
• Adult stem cells are multipotent with limited regenerative potential.
• iPSCs have the potential to create personalized regenerative medicine approaches.
• Asymmetric division balances stem cell self-renewal with differentiated cell production for tissue homeostasis.
• Restoring functionality in tissues with limited regenerative capacity requires proper integration of transplanted cells.
• Environmental factors influence stem cell division, affecting tissue regeneration.
• Mechanical properties of the stem cell niche can affect tissue regeneration.

Cell Cycle Overview

• The cell cycle is a series of events for cell proliferation, crucial for organism development and tissue homeostasis.
• Tissue homeostasis replaces dead or damaged cells through stem cells.
• Accurate DNA replication and component division are essential for proper cell function.
• The cell cycle includes G (gap) phases, S (synthesis) phase, and M (mitosis) phase.
• Cells in G0 are not actively cycling, including highly specialized permanent cells like neurons and cardiac myocytes.
• Stable cells, like hepatocytes, are quiescent in G0 but can re-enter the cell cycle.
• Labile tissues, such as gastrointestinal and epidermis cells, cycle continuously.
• G1-S and G2-M checkpoints prevent aberrant cells from progressing through the cycle.
• Cancer cells evade checkpoints, leading to uncontrolled division.

Regulation of the Cell Cycle

• Cyclins bind to cyclin-dependent kinases (CDKs) to form active complexes.
• Cyclin A, B, D, and E have specific roles in the cell cycle.
• Cyclin synthesis increases kinase activity of CDK binding partners, promoting phosphorylation.
• G1-S checkpoint monitors DNA integrity before replication, regulated by cyclin D-CDK4/6 and cyclin A-CDK2 complexes.
• Phosphorylation of retinoblastoma protein (Rb) regulates the G1 to S phase transition
• G2-M checkpoint ensures accurate DNA replication, regulated by cyclin B-CDK1 complex.
• DNA defects trigger repair, delay division, or induce senescence or apoptosis.
• Cyclin-dependent kinase inhibitors (CKIs) inhibit cyclin-CDK complexes, halting progression.
• p21, p27, and p57 are broad inhibitors, while p15, p16, p18, and p19 are specific to CDK4 and CDK6.

Links to Disease

• Cancer cells bypass checkpoints, leading to uncontrolled proliferation.
• Disabling the ability to degraded cyclins leads to unregulated cell growth.
• Elevated Cyclin D levels promote the G1-S phase, indicating increased cell proliferation of cancerous growth.
• Inhibiting CDK4 and CDK6 prevents the phosphorylation of Rb protein, which is essential for initiating the cell cycle and DNA replication.

Stem Cells & Chromatin Modifications (ChX)

• Chromatin modifications are chemical changes to DNA or histone proteins that control which genes are expressed.
• Chromatin modifications influence hematopoietic stem cell (HSC) fate by affecting DNA packing.
• Open chromatin allows expression of self-renewal genes, maintaining the HSC pool.
• H3K4 methylation activates genes for self-renewal.
• Closed chromatin silences self-renewal genes, activating differentiation pathways.
• H3K27 methylation represses genes that maintain the stem cell state, promoting differentiation.
• Chemotherapy destroys rapidly dividing cells, including blood cells.
• HSCs must regenerate the blood system after chemotherapy, requiring precise chromatin regulation for self-renewal and differentiation.
• Inhibited p53 leads to proliferation of cells with damaged DNA

Cell & Tissue Regeneration

• Tissue repair restores tissue architecture and function after injury of epithelium, parenchyma and connective tissue.
• If there is a small area damaged, the area can resolve on its own
• Large Tissue damage with pus accumulation can cause fibrosis and scarring
• Persistent injuries can transform acute inflammation into chronic inflammation with lymphocytes and macrophages.
• Regeneration returns tissue to normal structure and function; repair involves fibrosis and scarring

Proliferative Capacity:

• Labile cells continually divide, like epithelial and bone marrow cells.
• Stable cells are quiescent but can divide following tissue injury, like hepatocytes.
• Permanent cells are non-proliferative, such as cardiac and brain cells.
• Proliferation involves growth factors, extracellular matrix integrity, and tissue stem cells.
• Key players include vascular endothelial cells for angiogenesis, fibroblasts for collagen, and tissue stem cells.
• Environmental factors influence stem cell division, affecting tissue regeneration.

Liver Regeneration:

• The liver regenerates after partial hepatectomy if structural tissue remains.
• Major damage leads to fibrosis and scarring, for example hepatitis.
• Liver regeneration occurs via hepatocyte proliferation or progenitor cell repopulation.
• The Priming phase involves Kupffer cells secreting Interleukin-6, stimulated by TNF-alpha.
• Transforming Growth Factor Alpha (TGF-α) is associated with cell proliferation for increased cell numbers.
• Hepatocytes in G0 enter the cell cycle triggered by growth factors.
• The Termination phase involves cells stopping proliferation and becoming quiescent.
• Axolotls and invertebrates regenerate limbs; mammals regenerate liver.
• Small damage to a healthy liver can cause resolution via regeneration; larger areas cause repair with fibrosis.

Tissue Regeneration & Repair:

• Key components are blood vessels (angiogenesis) and fibroblasts (collagen).
• Cells such as fibroblast produced structural proteins such as collagen and elastin,.
• Proteins secreted from the cells include collagen and elastin as well as proteoglycans.
• Tissue regeneration and repair includes a tissue injury, regeneration and remodeling
• Tissue Injury is comprised of hemostasis and inflammation phases where you will need to plug up the bleed.
• Neutrophils and macrophages are recruited to reduce bacteria from entering wounds.
• The Regeneration Phase will see stem cell proliferation and differentiation
• In the connective tissue, you are laying down the connective tissue for scaffolding as well as increased angiogenesis
• The end point is remodeling which has a conversion of collagen type III within the granulation tissue, into a deposition of collage type I
• Macrophages and fibroblasts are your keyplayers during fibroblast proliferation.
• Inhibited TGF-β prevents the degradation of newly synthesized matrix components

Regulation and Timelines

• Cytokines, such as your macrophage secretions influence these regenerative properties.
• Fibroblasts start laying down collagen with angiogenic properties.
• Peri-endothelial cell recruitment increases angiogenic properties as well as suppression of endothelial proliferation
• One Day, One Week, One Month timeline for tissue repair stages
• Inflammation starts with an injury leading to a bloodclot
• At day one of injury, materials accumulate which includes angiogenesis properties
• Towards the first month, an increased fibroblastic proliferation activity replaces collagen type three

Wound outcomes:

• If regeneration is not possible it leaves a scar that may or may not be closed.
• Regenerative properties show an end product, which may not show evidence of damage after an event.
• Restorative properties lead to and increased amount of collagen can lead to fibrosis to repair the damage.
• Damaged tissue needs collagen deposition to lead to the increased amount of collagen to lead to fibrosis to repair the damage.

Factors Affecting Tissue Repair:

• Infections can hinder the regenerative process by prolonging the inflammatory phase.
• Diabetes will slow and compromise wound healing due to hyperglycemia, which can promote bacteria and decrease blood flow.
• Nutritional Status: Protein, acute phase proteins, clotting factors, albumin, vitamin C
• Protein deficiencies and hydroxylation of proline reduce appropriate processes.
• Anti-Inflammatory effects through corticoids/glucocorticoids suppress TGF beta formation and tissue production.
• Mechanical Factors causing movement and poor perfusion reduce would healing properties
• Foreign Bodies such as peritoneum synovial cavities, increase exudate, an accumulation of proteinaceous material.
• Increased exudate can be absorbed and digested
• Poor response is caused by loss of tissue or loss of integrity.

Clinical Correlations:

• Prolonged pneumonia infections may be caused by hyperglycemia and would result in prolonged inflammation.
• This may cause damages to the immune system's response, which would increase healing time
• Damages to the stem calls are repaired through fibroblast proliferation and collagen synthesis.
• Fibroblast proliferation, collagen synthesis replace damaged tissue with scar tissue.
• Elevated Cyclin D levels promote the G1-S phase, indicating increased cell proliferation of cancerous growth.
• Inhibiting CDK4 and CDK6 prevents the phosphorylation of Rb protein, which is essential for initiating the cell cycle and DNA replication.

Types of Wound Coverage

• Healing by First Intention:
• The primary goals are to promote clean/uninfected areas as surgical incisions, leading to surgical sutures which have low areas to remold Healing by Second Intention:
• Promotes severe and deeper injuries which require time to heal through a secondary manner.

Abnormal Tissue Coverage

• Loss of the structural integrity and collagen will lead to less stable injuries
• Hypertrophic scars: Raised margins with thermal burns in the dermis
• Keloids are excessive growths with an exuberant presence that don't regress and can be easily form in specific individuals.
• Trichrome Stains are used to enhance properties for the collagen fibers and what the tissue would like. Contractures: An extreme exaggeration that causes deformities to the woulds to deform the surrounding tissue.

Chronic Wounds

• Venous Leg Ulcers are found on the medial malleolus
• Arterial Ulcers are found on the pressure points on the toes.
• Pressure sores are caused by prolonged pressure causing a risk of infection on a patient.
• In the absence of an intact extracellular matrix, the body cannot regenerate tissue and will create scar tissue.