Regenerative Medicine - Stem Cell Applications PDF

Regenerative Medicine NB ARTICLES !!! Article questions (stem cell applications) https://prod-files-secure.s3.us-west-2.amazonaws.com/db640146-473b-4d4b-9e45-299899b34c36/30b28c11-2e 63-4711-9abe-f59eddb62a8b/READING_MATERIAL_WEEK__2_Stem_cells_overview.pdf Possible TEST question from thi...

Regenerative Medicine NB ARTICLES !!! Article questions (stem cell applications) https://prod-files-secure.s3.us-west-2.amazonaws.com/db640146-473b-4d4b-9e45-299899b34c36/30b28c11-2e 63-4711-9abe-f59eddb62a8b/READING_MATERIAL_WEEK__2_Stem_cells_overview.pdf Possible TEST question from this article : stem cell applications Outline Transitional Medicine Regenerative Medicine (subtopic of translational) Stem cells Stem Cell niche/environment NB Cancer cells vs stem cells !!NB Stem cell populations → both in bone marrow Stem cells classification (NB) Potency - stemness Stem cell types (difference NB) Applications for stem cell therapy Trachea application Umbilical cord derived stem cells Mesenchymal stem cell markers !!! Successful animal stem cell studies (NB for test) Successful human stem cell studies (NB) How To Monitor Stem Cell Therapy? → research techniques Stem cell isolation protocol (Exam Q) 5 marks Other stem cell sources Adipose tissue Menstrual-Derived Mesenchymal Stem Cells Perinatal Tissue Induced Pluripotent Stem Cells (iPSCs) Satellite Cells → stem cells for muscle What are Satellite cells? Myocyte after exercise Where are satellite cells found? Satellite → myotubes process (myogenesis) Gene expression identifies stem cell and progenitor cell populations in adult skeletal muscle MyoD is expressed in somites and developing muscle during embryogenesis Satellite cells have a specialized niche in adult skeletal muscle Satellite cell response to myotrauma Muscle repair is characterized by discrete stages of regeneration Satellite cell regulation Inflammatory Processes and Myocyte Regeneration Aging and Stem cells Regenerative vs translational medicine (different things) Transitional Medicine Translational = from research to clinic; from lab to bedside Translational biology take things in lab that work well and translate into clinic (patients) Translational Medicine as an interdisciplinary branch of the biomedical field supported by three main pillars: Bench-side, -- lab Bedside, - patient clinic Community – population as whole … referring to the triad of laboratory discoveries, clinical application and their interaction with/effect on patients and the population. A field of biomedical research →Focus on bridging gap between laboratory research and clinical practice Goal is to accelerate the application of scientific discoveries into real world medical treatments Bench to bedside, clinical trials, Interdisciplinary collaboration, personalized medicine, combat disease on a global scale Regenerative Medicine (subtopic of translational) Regenerative medicine Definition: a branch of translational research in cell and tissue engineering and molecular biology Deals with the process of replacing, engineering, or regenerating human cells, tissues, or organs to restore or establish normal function This field holds the promise of engineering damaged tissues and organs by stimulating the body's own repair mechanisms to functionally heal previously irreparable tissues or organs “Regenerate own cells” Not prosthetics/ diets/supplementations Manipulate and take advantage of your own healing processes in body Anti-aging medicine → not traditional regenerative medicine Stem cells -> also in adults (very NB) serve important functions ( liver inflamed, replace cells) stem cell ability diminishes as you get older Includes: Growing tissues and organs in the lab, modifying them, training them, and reintroducing them back into the patient Examples (traditional) ie : stem cell therapy, gene therapy, tissue engineering, cell training Stem cell Therapy stem cell therapy: take stem cells out of body (bone marrow = main source) , isolate them, transplant them Disease on cellular level/organ level → can depoly derivatives ( proteins and molecules that are released by exosomes ie vesicles) of stem cells Can put in petri dish → differentiate, extract derivatives or stem cell populations Promote cell rejuvenation CAR T-cell therapy Different to stem cell therapy take out T-cells and plant back in individual Engineering own tissue and bodys healing mechanism Train (t cells) leukocytes to attack cancer cells train the T cells that are already inadequate to attack cancer be reactive to antigen that they are normally not activated towards to could be certain population that will not be able to react to antigen Negate need of organ transplants If the regenerated organ would be derived from the patient's own tissue or cells, this would potentially solve the problem of the shortage of organs available for donation, and the problem of organ transplant rejection Convalescent plasma as treatment (NB) [5-10 mark question] - ipad drawing convalescent = recover Exam Q: http://samj.org.za/index.php/samj/article/view/13019 most infective disease can apply recover well and in good health isolate specific antibodies from donor and give concentrated plasma to pateint ( need to make sure glucose levels and etc are good) Convalescent plasma – antibodies present in, even though patient not having active infection COVID application Convalescent plasma therapy investigated in severe coronavirus disease 2019 (COVID-19) Can produce hyperimmune immunoglobulin (antibody) solution from this Relevance of giving plasma: (NB) antibodies neutralize SARS covid can prevent patient B from getting acutely hospitalized potential therapies for reducing the morbidity and mortality burdens of the disease, particularly among those who are severely or critically ill clinical improvement in all participants, and no participant died after 37 days of follow-up Four of the participants experienced undesirable effects: 1 had moderate fever (39ºC), and 3 had anaphylactic shock after receiving convalescent plasma. Article info: (SAMJ) https://prod-files-secure.s3.us-west-2.amazonaws.com/db640146-473b-4d4b-9e45-299899b34c36/1c ab630e-79b0-424e-84e1-33512732d9f8/13019-58338-1-PB.pdf SA clinical trial: SANBS Starting on 4 May 2020, the SANBS began taking appointments from volunteers who wish to book appointments to donate convalescent plasma. What does the SANBS hope to achieve with the clinical trial? Currently, there is not enough scientific evidence to prove whether COVID-19 convalescent plasma is a safe and effective treatment for patients with the virus. Well-designed clinical trials will help provide the information necessary to prove whether convalescent plasma is a safe and effective treatment. The results from the clinical trial will inform future decisions on the wider availability of convalescent plasma. It will be an important contribution to research on a global scale that could help patients in South Africa and around the world. Examples: Nontraditional Note: While treatments like hyaluronic acid injections and PRP facials might not be as complex or obvious as stem cell or gene therapies, they still fit within regenerative medicine technically They aim to restore, rejuvenate, or enhance the body's natural functions, particularly in the context of skin health and appearance Traditionally, stem cell therapy, gene therapy, tissue engineering, cell training, etc… Anti-aging medicine Hyaluronic Acid (HA) → ECM what is it Naturally occurring linear polysaccharide composed of repeating disaccharide units of D-glucuronic acid and N-acetyl-D-glucosamine linked by β-1-3 and β-1-4 glycosidic bonds Holds water extremely well, hydrates tissues can supplement exogenously body produces endogenously → HA is a primary component of the extracellular matrix (endogenous molecule) It is an important structural element in the skin Present in high concentration in the synovial joint fluids, vitreous humor of the eyes, hyaline cartilage, intervertebral disc nucleus pulposus, and umbilical cord and connective tissues maintains moisture, primary constituent of ECM ( epithelial barrier – is just ECM, connective tissue) ECM loses function and structure, if can replace with hyaluronic acid What is it used for used for supplementation of impaired synovial fluid in arthritic patients aesthetic medicine such as dermal fillers soft tissue surgery such as vocal fold augmentation as scaffold for tissue regenerative applications Hydrogels (wound dressings, drug delivery systems, etc.) → very expensive hydrogels are specific to field of study → medical hydrogel made up of mostly water, it maintains gel-like structure application: Wounds - tissue repair and regeneration if can dress wounds with hydrogel , has HA and healing factors → promote better healing ( inhibits scar formation) Have immobile HA , enable a portion of it to be soluable so it can dissolve into wound and interact with wound, stimulate fibrobalst activation, collagen formation etc → stimulate healing graded/free HA released into the wound promotes cell proliferation, cell migration and angiogenesis A HA-rich wound matrix facilitates wound repair inside: use ceramics (give hydrogel its structure) application: drug delivery (article) HA especially is important for drug delivery benefits in anti-aging and regenerative medicine PRP facials - vampire facial → Example to manipulate biological functioning to aid What can impair this procedure? Systemic inflammation Smoking Use plasma and platelets → regeneration Must Use a Healthy Person’s Blood to Extract and Use Platelets micro-needle face and inject own platelets back into holes Why specifically platelets? after platelet plug – clot – stop blood loss – need to regenerate damaged epithelial cells – fibroblasts ( produce ECM components) , SM cells ( get from stem cells and progenitor cells ( intermediate cells between stem cells and final differentiated cells ) platelets: release important growth factors (epithelial GF , vascular endothelial growth factor, TGF , EGF – remodel ECM , stimulate fibroblast to produce ECM components) When get older – lose ECM components collagen loss – platelets stimulate fibroblasts to create more collagen take out platelets and stimulate them to release compounds (PDGF , VEGF etc) to stimulate fibroblasts Results aren’t as promising as hypothesis – evidence isn’t that strong Estrogen replacement therapy (hormone therapy) Not technically regenerative medicine Alleviating symptoms associated with hormone deficiency But, hormone replacement therapy can be used for dysfunctional tissues, organs? Hypercoagulation as a potential side effect Thrombo-embolic stroke (not hemorrhagic) Why use estrogen – after menopause, estrogen levels decrease -> promotes symptoms of menopause Can hinder symptoms by supplement exogenous estrogens Estrogen – steroid hormone (can ingest) Problem – estrogen – prothrombotic – stroke = danger hemorragic – vessel ruptures (caused by annurism) thrombo stroke – clot blocks Estrogen effects on RBC: causes RBC to undergo apoptosis - EROPTOSIS changes RBC High estrogen implicates clot network – no gaps , hard for cells to move through and hard to degrade Increase clot persistence – increase chance of having stroke /mycocardial inafrction estrogen activates platelets Can show in test why estrigen is dangerous as supplement Receoptors on platelets – that can bind to lipid soluable hormones – etsrogen binds – leads to activation Platelets need to be in actiavtion or rest when actaivted – release contents ( clot formation and healing promote) Diets and supplements (not regenerative) Stem cells NB Q: 3 characteristics of stem cells and expand on each for 2 marks Definition: Clonogenic should be a clonogenic cell ( ability of a single cell to grow into a colony of cells) clonogenic cell survival assay determines the ability of a cell to proliferate indefinitely, thereby retaining its reproductive ability to form a large colony or a clone. This cell is then said to be clonogenic Differentiation Capable of generating at least 1 differentiated cell type Self-Renewing self-renewing (proliferate and self renew) Remarkable ability to develop into many cell types, ability to rejuvenate Internal repair system, dividing to replenish lost, defective, sick cells Unspecialized Cells, capable of renewal, even after long periods of inactivity This is the case with adult stem cells that live in an environment called the stem cell niche under certain physiologic or experimental conditions, they can be induced to become tissue- or organ-specific cells with special functions stem cell niche is very important for mainetenance - Possible question: Changes in niche can induce pathology Can induce them to differentiate in vitro – force by extracellular communication (antagonists /agonists) -. for regnerative purposes ie stroke : glial cells/neurons Stem Cell niche/environment NB All about stem cell niche they reside in – determines what happens to stem cells if die out/ differentiate / stay stem cells Article questions: stem cell vs cancer cell https://prod-files-secure.s3.us-west-2.amazonaws.com/db640146-473b-4d4b-9e45-299899b34c36/ef0afbd7- 05f7-4935-ab52-0e1642634fb6/READING_MATERIAL_WEEK_1_AND_2_Stem_cell_cancer_niche.pdf The stem cell niche in adult somatic tissues plays an essential role in maintaining stem cells or preventing tumorigenesis by providing primarily inhibitory signals for both proliferation and differentiation Loss of the niche can lead to loss of stem cells, indicating the reliance of stem cells on niche signalling Therefore, cancer stem cells may arise from an intrinsic mutation, leading to self-sufficient cell proliferation, and/or may also involve deregulation or alteration of the niche by dominant proliferation-promoting signals. Furthermore, the molecular machinery used by normal stem cells for homing to or mobilizing from the niche may be "hijacked" by cancer stem cells for invasion and metastasis Stem cell niche definition: Stem Cell Niche (from Google) the in vivo microenvironment where stem cells both reside and receive stimuli that determine their fate Therefore, the niche should not be considered simply a physical location for stem cells, rather as the place where extrinsic signals interact and integrate to influence stem cell behavior Cancer cells vs stem cells !!NB Cancer niche – cancer can modulate ( very good at surviving) Normal stem cells in adult somatic tissues and cancer stem cells share the common features of self- renewal and slow cycling. Mutations in stem/progenitor cells most likely undergo uncontrolled proliferation (i.e. lead to malignancy) – hemopoietic stem cells NB relevance for blood cells ( why terminal RBC cannot be cancerous) Q in exam ( use article for question to supplement) One of the differences between normal stem cells and cancer stem cells is their degree of dependence on the stem cell niche, a specialized microenvironment in which stem cells reside (cancer cells modulate their extracellular environment) Stem cell populations → both in bone marrow 2 populations (in bone marrow) – know differences between populations Possible questions Hemopoietic (HSC) Mesenchymal (MSC) pluripotent ?? Different synonyms: Mesenchymal stem cells OR mesenchymal stromal cells OR bone marrow stem cells (differentiate into white & red blood cells, and OR bone marrow stroma cells – synonyms platelets) – all blood cells multipotent → connective tissue ( relevant for location ie bone) can differentiate into osteoblasts, osteoclasts, adipocytes and chondrocytes also glial and muscle, hepatic cells a population of non- Found in the endosteal region and near blood hematopoietic cells described in the bone vessels in the bone marrow marrow, adipose tissue, liver, amniotic fluid, embryonic placenta, umbilical cord blood, and Different populations in bone marrow stem cells: other tissues Long-term hematopoietic stem cells BM-MSc – bone marrow mesenchymal stem cells Short-term hematopoietic stem cells Multi-potent progenitor cells Bone marrow stem cells go between bone marrow itself and blood Bone marrow transplant can be used for treatment of blood diseases (leukemia) ie in leukemia, antigens and replace stem cells → not exhibit cancer Leukemia NB Found virtually in all tissues Leukemia cancer of leukocytes(Can also have Play a role in homeostasis and repair during cancer of myeloid lineage) disease NB: RBC and thrombocytes: Mesenchymal cells: regenerative medicine RBC and platelets lack a nucleus, mitochondria and Why? ribosomes Easily culture in vivo → RBC and platelet cannot become cancer High proliferation rates You get cancer of progenitor cells & stem cells that Can become many cell types: e.g. Osteoblasts, form blood cells and platelets – the differentiated Chondrocytes, Adipocytes, Hepatocytes, Neurons cells are then defective RBC /leukocyte and glial cells need nucleus and ribosome and mitochondria to be a cancer potential cell Note: Where do RBC get energy from – diffusion ( nitric oxide) deform and force a vessel to dilate sometime ( release nitric oxide ) Easy to deal with and manipulate ( by agonists – Treatment: bone marrow transplant NB signaling molecules that lead to cell differentiated into to) Now we know we can get failures: presence of immune regulatory T-cells (why???) An agonist is a ligand that can stimulate (agonize) the GPCR to activate intracellular Regulator T cells – regulate activity of other T signaling and trigger a biological response cells High yield - in clinic and lab When have a transplant – want to inhibit T regs ( rejection) T regulator cells NB ( know immune section) suppress immune responses : both innate and adaptive Stem cells classification (NB)  NB: cell types that are multipotent https://prod-files-secure.s3.us-west-2.amazonaws.com/db640146-473b-4d4b-9e45-299899b34c36/e21252d8 -a9d1-4573-b0d1-15a284e6efa6/F344_Additional_background_terminology_for__stem_cells_ENGLISH.pdf Stem cell: are special human cells that are able to develop into many different cell types. (Clonogenic, differentiation, self-renewal) Progenitor cells: are descendants of stem cells that then further differentiate to create specialized cell types. They are more specialized than stem cells intermediate between stem cells and terminal cell (ie hemopoietic stem cell) There are many types of progenitor cells throughout the human body Each progenitor cell is only capable of differentiating into cells that belong to the same tissue or organ When stem cells differentiate – always progenitor cell in lineage the progenitor cells (common) Potency - stemness potency → how many different types of cells a SC can differentiate into Once form progenitors ( cant reverse) When differentiated – potency is decreased but the degree of senescence Is possible to reverse in vitro neuron cells to stem cells Aging – accumulative defects Senescence – problem at cellular level Telomeres – protective on chromosomes (shorten with every division) – contribute to senescence Overt senesces - last divide for cell Application in regenerative medicine: Telomere Shortening During Ageing (cellular senescence) → NB for regenerative medicine Telomeres form the ends of human chromosomes A telomere is a region of repetitive nucleotide sequences at each end of a chromosome, which protects the end of the chromosome from deterioration or from fusion with neighboring chromosomes However, as a downside, there is growing evidence indicating that telomere shortening also limits stem cell function, regeneration, and organ maintenance during ageing Moreover, telomere shortening during ageing and disease is associated with increasing cancer risk Stem cell types (difference NB) Adult stem cell Embyronic stem cell have less as get older Can proliferate/renew indefinitely children have more than adults Pluripotent Adult Stem Cells (mesenchymal stem cells, Due to their ability to maintain their telomeres hemopoietic stem cells, myeloid stem cells, intact satellite stem cells, epidermal stem cells) This is in contrast to normal cells where Children and babies – have lots of adult stem telomeres shorten during successive cell cells, more active , more responsive divisions Number, activity and responsiveness between Ethical issues with obtaining embryonic stem cells stem cells in adults / children - scratch out embryo cells adults – decreases in size, responsiveness (ability to respond to extracellular signalling) Adults – have less embryonic stem cells / not any ( not to say we don’t have any embryonic or that stem cells can replace cells) Babies and children have MORE adult stems cells and population is more active big advantage for IPSCs IPSC induced pluripotent stem cells take terminally diffrentiated cell and differentiate it back to the progenitor cell or stem cell (in vitro) adv: dont have to do invasive surgery to get to bone marrow example: Take muscle tissue cells and force to revert back (have matched cells – don’t need donation and not invasive surgery to obtain mesenchymal cells from bone marrow) Applications for stem cell therapy Trachea application In June 2008, at the Hospital clinic de Barcelona, Professor Macchiatiniand his team, of the University of Barcelona, performed the first tissue engineered trachea /bronchus area transplantation. Adult stem cells were extracted from the patient's bone marrow, grown into a large population, and matured into cartilage cells, or chondrocytes, using an adaptive method originally devised for treating osteoarthritis. The team then seeded the newly grown chondrocytes, as well as epithelial cells, into a decellularised (free of donor cells) tracheal segment that was donated from a 51-year-old transplant donor who had died of cerebral hemorrhage. decellurised: Remove cells from organ – connective tissue is left (won't elicit immune response) Repopulate with cells → use the cells obtained from person who is sick (mesenchymal stem cells) – differentiate them to chondrocytes In vitro – stimulate and differentiate and upkeep environment they are in chondrocytes → can further form connective tissue of trachea Investigate which cells have populated trachea – ie epithelial cells and goblet cells After four days of seeding, the graft was used to replace the patient's left main bronchus. After one month, a biopsy elicited local bleeding, indicating that the blood vessels had already grown back successfully. Application: Can transplant lab-grown organ into body Umbilical cord derived stem cells UCB stem cells are considered between ESCs and adult stem cells ethical considerations: (NB) using umbilical cord vs embryo Umbilical cord – not harming any organism If take from embryo – invasive why store umbilical cord: Routine procedure to store chord blood Future disease Multilineage potential Why use umbilical cord? These stem cells have neuronal, bone, adipose tissue markers HSCc and MSCs – 2 types in umbilical cord From umbilical cord - Don’t have antigens that would typically induce immune rejection response Better to use to cellularsise decellularised organ and use for stem cells They have immunoregulatory properties: they can suppress lymphocyte proliferation and decrease pro- inflammatory cytokine levels Very anti-inflammatory Models that has been developed using these stem cells: cerebral ischemia; hepatic cirrhosis; pulmonary fibrosis = decreases inflammation Immune compatibility – less likely to cause immune rejection than adult stem cells (not fully developed, lack certain markers that might be/can be viewed as a foreign antigen) Haemopoietic potential (blood disorders) Mesenchymal potential Less ethical issues than embryonic stem cells; ease of collection Question: Explain ethical benefit of using umbilical stem cells Non-controversial source: Since umbilical stem cells are harvested from a byproduct of birth, it avoids the ethical dilemmas tied to embryonic stem cell research, which involves the destruction of embryos. No harm: Collecting umbilical stem cells does not require any medical procedure that could harm the donor. Consent: Parents can give informed consent for the donation or storage of umbilical cord blood, making the process ethically transparent. Wide availability: The umbilical cord is typically discarded after birth, so using it for stem cell collection makes use of an otherwise wasted resource, increasing accessibility and reducing wastage. Mesenchymal stem cell markers !!! NB for Exam (10mark questions) 5 marks per protein NB look at assignment what is needed to define a MSC? Pleiotropic – one protein, one gene, multiple functions Presence of CD73, CD90 and CD105 on the cell membranes Use of flow cytometry and fluorescent image stream analyses Fluorescent markers – conjugated to antibodies Confirm Mesenchymal cell: Develop antibodies for stem cell markers – can conjugate to Fluorencnce If cells are positive for markers – it can confirm cells Note: Hemopoietic stem cells – may have different markers CD73 – also shared for hemopoietic stem cells CD73 CD73, a membrane-bound nucleotidase Also known as a glycosyl phosphatidylinositol (GPI)-linked, membrane-bound glycoprotein which hydrolyzes extracellular nucleoside monophosphates into bioactive nucleoside intermediates. Surface-bound CD73 metabolizes adenosine 5′-monophosphate (AMP) to adenosine, which when released can activate one of four types of, seven transmembrane spanning adenosine receptors (which are G-protein coupled receptors) or can be internalized through dipyridamole-sensitive carriers Other functions ( know) CD73, also known as ecto-5'-nucleotidase, plays several important roles in cellular processes. Adenosine Production: CD73 catalyzes the conversion of extracellular ATP (adenosine triphosphate) and ADP (adenosine diphosphate) into adenosine. This is a crucial step in the extracellular nucleotide metabolism pathway. Adenosine, produced by CD73, is a key molecule involved in regulating various physiological and pathological processes. Regulation of Immune Responses: Adenosine has significant immunomodulatory effects. It can inhibit T- cell proliferation, reduce the activity of natural killer (NK) cells, and suppress the production of pro- inflammatory cytokines. By producing adenosine, CD73 helps modulate immune responses, often promoting an anti-inflammatory or immunosuppressive environment. Tissue Repair and Regeneration: In the context of tissue injury and repair, adenosine generated by CD73 can contribute to tissue regeneration and reduce damage by modulating inflammation and promoting cell survival. Endothelial Function: CD73 is involved in regulating endothelial cell function and vascular biology. By generating adenosine, CD73 helps in maintaining endothelial cell integrity and modulating vascular responses. Cell Migration and Adhesion: CD73 can influence cell migration and adhesion through its effects on the extracellular matrix and cell signaling pathways, partly mediated by the adenosine it produces. Cancer: In the tumor microenvironment, CD73's role in producing adenosine can contribute to immune evasion by tumors. Elevated CD73 activity in cancers can help tumors avoid immune surveillance and promote a more favorable environment for tumor growth. CD90 CD90 is a 25-35 kD GPI-anchored protein Cell adhesion molecule It is heavily N glycosylated, glycophosphatidyinositol(GPI) anchored conserved cell surface protein with a single V-like immunoglobulin domain, originally discovered as a thymocyte antigen (Thymocytes are hematopoietic progenitor cells present in the thymus) Thymocytes (when immature) – matures in thymus to T cells It is also known as Thy-1 Thy-1 is present in the outer leaflet of lipid rafts in the cell membrane not an immunoglobulin ( antibody) CD105 Also known as Endoglin(ENG) It is a type I membrane glycoprotein located on cell surfaces and is part of the TGF beta receptor complex – represses immune responses What is TGF beta → tumour supressor Cell growth: It tells cells when to grow or stop growing. Wound healing: TGF-beta helps tissues repair themselves when injured. Immune system: It controls immune responses, making sure they don't become too strong and cause damage. Tissue development: It helps with the formation of organs and tissues during development. Summary 1. CD90 (Thy-1) Function: Cell adhesion: CD90 plays a key role in mediating cell-cell and cell-matrix interactions, influencing cellular adhesion and migration. Stem cell marker: CD90 is widely used as a marker for mesenchymal stem cells (MSCs), hematopoietic stem cells, and other progenitor cells. Immune regulation: It modulates T-cell activity, as well as influencing inflammation and immune responses in tissue injury. Neuronal differentiation: CD90 is involved in neural cell development and plays a role in neuron survival and axonal growth. 2. CD73 (5'-nucleotidase) Function: Enzymatic activity: CD73 is an enzyme that catalyzes the conversion of AMP (adenosine monophosphate) to adenosine, which plays a crucial role in regulating extracellular levels of adenosine. Immunosuppression: Through its role in adenosine production, CD73 is involved in immune suppression and inflammation regulation, particularly in the tumor microenvironment. Tissue repair: CD73-generated adenosine contributes to tissue repair and protection by limiting inflammatory responses and promoting vascular growth and function. Stem cell marker: CD73 is also a marker for mesenchymal stem cells (MSCs) and contributes to their immunomodulatory functions. 3. CD105 (Endoglin) Function: Angiogenesis: CD105 is a co-receptor for transforming growth factor-beta (TGF-β) and plays a critical role in endothelial cell function and blood vessel formation (angiogenesis). Vascular integrity: It is crucial for maintaining vascular integrity and is upregulated in proliferating endothelial cells during angiogenesis, such as in tumor development. Stem cell marker: CD105 is a marker for MSCs and is often used in regenerative medicine for its involvement in promoting tissue repair and vascular development. TGF-β signaling modulation: CD105 regulates TGF-β signaling pathways, influencing processes such as fibrosis, inflammation, and stem cell differentiation. Summary of Differences: CD90 is primarily involved in cell adhesion, immune modulation, and neuronal differentiation. CD73 acts as an enzyme involved in adenosine production, playing roles in immunosuppression and tissue repair. CD105 is crucial for angiogenesis and TGF-β signaling, contributing to vascular development and stem cell function. Successful animal stem cell studies (NB for test) Prepare for an application based answer Like trachea example ( don’t need to know case but application) Need to apply stem cell therapy to a question Use this as examples NB application for stroke case etc To treat: Intervertebral disc regeneration link to helpful article implantation of mesenchymal stem cells faces multiple challenges, such as the durability and survival of MSCs upon implantation, uncertain pathways to discogenic differentiation, and the adverse impact of a harsh microenvironment on cell survival MSCs are expected to promote intervertebral disc tissue regeneration and repair by differentiating into beneficial cells such as nucleus pulposus cells (NPCs) The potential of stem cell therapy in disc degeneration is to repopulate the disc with viable cells capable of producing the ECM and restoring damaged tissue. Myocardial infarction A myocardial infarction (MI), or heart attack, occurs when blood flow to part of the heart is blocked, leading to tissue death due to lack of oxygen. Since heart tissue (cardiomyocytes) has limited regenerative capacity, damage from an MI can lead to heart failure. SCT: The goal of regenerative medicine for MI is to regenerate damaged heart tissue and restore heart function. Techniques: link Stem Cell Therapy: Various types of stem cells, including cardiac stem cells, MSCs, and induced pluripotent stem cells (iPSCs), have been used to repair damaged heart tissue. They may differentiate into cardiomyocytes or secrete factors that promote healing. Autologous cell transplantation uses stem cells to regenerate myocardium. This process involves implantation of bone marrow stem cells into the infarcted area of heart in an effort to restore the viability of the tissue. Skeletal Myoblasts MSC Studies have shown the ability of MSCs to engraft into host tissue, rapidly differentiate into vascular endothelial and cardiomyocyte-like cells, recruit endogenous stem cells, and improve LV function post- MI challenges: hostile enviro of ischemic/infarcted cardiac tissue Other studies have suggested that perhaps the hypoxic environment of infarcted tissue induces expression and release of growth factors that support angiogenesis, trigger proliferation and migration of cardiac progenitors, and promote MSC differentiation into cardiomyocytes. Factors such as vascular endothelial growth factor (VEGF), hepatocyte growth factor (HGF), and insulin-like growth factor (IGF) have been implicated in the upregulation of cardiomyocyte genes to direct differentiation and promote other reparative processes embryonic stem cells Embryonic stem cells (ESCs) are a population of pluripotent cells that arise from the inner cell mass of the blastocyst during embryonic development in mammals. They can give rise to any/all adult cell types, and thus have the potential to regenerate lost myocardium Exosome Therapy: Exosomes (small vesicles released by cells) from stem cells have been shown to help repair heart tissue by delivering regenerative signals to damaged areas.(not fully understood) Encephalitis Encephalitis is an inflammation of the brain tissue, usually caused by viral infections, autoimmune disorders, or bacterial infections. It can lead to brain damage, neurological deficits, and even death if untreated In cases where encephalitis leads to brain damage, regenerative approaches aim to repair or replace the lost neurons and brain tissue. UC-MSC → link When the immune system responds to injury or infection in the brain, by-products like fluid, dead cells, and viral remnants accumulate, affecting neuron function. This buildup increases pressure in the brain, potentially leading to reduced consciousness. Glial cells, which support neurons, absorb the excess fluids and harmful chemicals to protect neurons. However, when overwhelmed, glial cells die, releasing the toxins back into the brain, which further damages neurons. The high levels of these toxic substances can destroy weakened neurons by damaging their membranes or overstimulating them until they die. Stroke Stroke occurs when blood flow to a part of the brain is interrupted, either by a blockage (ischemic stroke) or a burst blood vessel (hemorrhagic stroke). This results in brain damage and loss of function, depending on the affected area. Stem Cell Therapy: NSCs, iPSCs, and MSCs are being used to regenerate neurons and repair damaged brain tissue. These stem cells can differentiate into neuronal cells and promote the formation of new blood vessels (angiogenesis). anti-inflammatory too can regenerate glial cells prevent cells from dying - apoptosis link !! Successful human stem cell studies (NB) Cartilage defects: collagen gel or HA-hydrogel infused by bone marrow mesenchymal stem cells link better link Background: Cartilage defects often occur due to injury or degenerative diseases like osteoarthritis, which lead to pain and limited mobility. Cartilage has poor regenerative capacity, making it a prime candidate for regenerative therapies. Intervention: Collagen Gel/HA-Hydrogel: These biomaterials act as scaffolds that support the growth and regeneration of cartilage tissue. Collagen gel and hyaluronic acid (HA)-hydrogels are both used to mimic the extracellular matrix environment, promoting cell attachment, proliferation, and differentiation. Bone Marrow MSCs: These stem cells have the ability to differentiate into chondrocytes (cartilage-producing cells). When infused into the collagen or HA-based hydrogels, MSCs can help regenerate damaged cartilage. Mechanism: MSCs secrete growth factors that stimulate tissue regeneration and reduce inflammation. The collagen or HA-hydrogel scaffold provides mechanical stability and helps the cells remain in place, allowing for localized healing. Liver regeneration with bone marrow mesenchymal stem cells into the hepatic artery and other stem cells (find out) HSC MSC https://prod-files-secure.s3.us-west-2.amazonaws.com/db640146-473b-4d4b-9e45-299899b34c36/26339 d37-8e57-4415-b60a-f559cf369c2c/liver.pdf Research HA and othjer stem cell therapies Keywords for Research: Hyaluronic acid (HA) and stem cell therapy. Mesenchymal stem cells (MSCs) in cartilage repair. HA-hydrogel in regenerative medicine. Biomaterials in tissue engineering and stem cells. How To Monitor Stem Cell Therapy? → research techniques Fluorescence (marker binding), MRI or transmission electron microscopy (superparamagnetic iron oxide nanocomposites that are internalized by the stem cells) – can tract nanoparticle Can track stem cells with nanoparticles – see if stem cell population is growing or been rejected etc MRI very good for clinical because microscopy cant do in humans Gold nanotracers monitored Gold – non reactive with biological tissue and silver NBBBB → link In vivo monitoring of Au NT labeled-MSCs using combined ultrasound and photoacoustic imaging In photoacoustic imaging, non-ionizing laser pulses are delivered into biological tissues (when radiofrequency pulses are used, the technology is referred to as thermoacoustic spectroscopy) Monitor particles in reallife ( location and velocity ) – use tracers Instead of MRI Spectroscopy and ultrasound Better analysis to get down to cellular level – MRI is great technique in vivo in clinic tract stem cell activity ( need to make sure they are differentiating into correct population and that they have not been rejected) Stem Cells monitored by ultrasound and photo-acoustic methods combine with AU NT labeled Stem cell isolation protocol (Exam Q) 5 marks NB watch umbilical cord video How to Isolate Cells Directly from Whole Blood - EasySep™ Direct Protocol, EasyEights Magnet https://youtu.be/YfnGz51Hpcc Isolation and Characterization of Mesenchymal Stromal Cells from Human Umbilical Cord and Fetal Placenta https://www.jove.com/v/55224/isolation-characterization-mesenchymal-stromal-cells-from-human Can stem cell therapy be harmful? https://www.fda.gov/consumers/consumer-updates/fda-warns-about-stem-cell-therapies Other stem cell sources Adipose tissue Remember, not just a storage site for lipids But a highly active endocrine organ, important for metabolism, inflammation, and tissue repair In adipose tissue, we can also find: Adipose Mesenchymal Stem Cells AKA: Adipose-derived adult stem cells Adipose-derived adult stromal cells Adipose-derived stromal cells Adipose mesenchymal cells Adipose-derived stromal/stem cells In a sample of adipose tissue we will find adipocytes, preadipocytes, mesenchymal stem cells, leukocytes, fibroblasts, endothelial cells, smooth muscle cells…. Preadipocytes are progenitor cells committed to the adipocyte lineage (not yet mature adip’s) Obtained via liposuction or excision, and stem cells are systematically isolated from samples Adipose stem cells are derived from the stromal vascular fraction (SVF) SVF – a heterogeneous mixture of cells High proliferative and angiogenic capacity, relevant to regenerative practices Obtaining SVF: https://www.ncbi.nlm.nih.gov/pmc/articles/PMC6391981/ Obtain fat tissue via liposuction or excision Mince tissue Collagenase exposure Filter solution through a 100um filter Centrifuge to obtain pellet (SVF) Remove supernatant (contains some adipocytes) Resuspend pellet.. (further processing, i.e. cell culture, flow cytometry, differentiation)  Markers Adipose derived stem cells should be + for CD73, CD90, CD105, and – for CD45 → because MSC Menstrual-Derived Mesenchymal Stem Cells NB 3 characteristics of stem cells → clonogenicity, self-renewal and differentiation Human endometrium: 2 layers (stem cells found in both layers) Functional layer – always undergo reorganisation (shed during menstruation) Basal layer– mostly loose conjunctive tissue (remains unchanged) Stem cells from this tissue obtained through: Hysterectomy OR Collection of menstrual blood (in vessels containing heparin and antibiotics) Easy adhesion to plastic → MSC have this property when grown in lab Three kinds of stem cells exist in the human endometrium: epithelial stem cells, mesenchymal stem cells and endothelial stem cells, which are identified based on the expression of specific surface markers and on their differentiation potential Advantage: Multipotent and minimally non-invasive and can transfer (or become) to: Cardiomyocytes Respiratory epithelium Neural cells Myocytes Endothelial cells Pancreatic cells Osteocytes Hepatocytes Adipocytes Perinatal Tissue https://doi.org/10.3390/genes12010006 Placenta, amniotic fluid, umbilical cord, Wharton’s Jelly Wharton’s Jelly - largely made up of mucopolysaccharides. It acts as a mucous connective tissue containing some fibroblasts and macrophages (insulates/protects umbilical cord) Mesenchymal and *haematopoietic stem cells Induced Pluripotent Stem Cells (iPSCs) Somatic cells can be re-programmed to revert to the embryonic-like pluripotent stage Through ectopic expression of transcription factors Adipocyte stem cells can generate IPSCs Advantages of iPSCs Ethical considerations Personalized medicine (patient’s own tissue) Unlimited supply (adult stem cell population decreases) Disease modelling (patient’s own genetic mutations/genotype) Pluripotent (not just multipotent) Lower risk of tumor formation (than embryonic stem cells) Ease of collection Applications Fractured bones, tissue injuries Joint/cartilage tissue in rheumatoid arthritis Wound healing (MSCs – growth factors, angiogenesis, etc.) – used in diabetes for chronic ulcers/sores, burns Immunomodulation (anti-inflammatory, graft vs host disease, autoimmune) Clinical trials ongoing for myocardial infarction Neurodegeneration (as well as spinal cord injuries) Vehicles for drug delivery (affinity towards sites of injury, cancer/tumours) Research models (lab vs clinic) Eye conditions And more… Concept of obtaining stem cells from tissue – need to know that Satellite Cells → stem cells for muscle https://prod-files-secure.s3.us-west-2.amazonaws.com/db640146-473b-4d4b-9e45-299899b34c36/31f3e851-b1f 6-4e3c-85c3-24491e25e076/Genes_Dev.-2006-Shi-1692-708.pdf What are Satellite cells? stem cells for the muscle AKA Muscle stem cells Satellite stem cells Etc. you’ll see various synonyms in the literature Myocyte = Muscle cell/myofiber/muscle fiber Myoblasts = satellite cells committed to the myocyte lineage (progenitor) Already in process of differentiation Myotube = myocyte ( myotube = fused myoblasts) Does satellite cells conform to the definition of a perfect stem cell? Remember our definition of a perfect stem cell Clonogenic, Differentiation, Self-Renewal In the lab researchers can generate colonies of differentiation-competent progeny in vitro We can do this in the lab by isolating mononucleated cells from adult skeletal muscle = enzymatic degradation (why using enzymes in this step) NB Enzymes are used in this step to break down the connective tissue and extracellular matrix (the material surrounding the cells) that hold the muscle fibers together. Plated and form colonies Once the cells are isolated, they are placed on a dish (plated) in a controlled environment, where they can grow and form clusters or colonies of cells. A proportion of the cells will retain capacity to differentiate into myogenic clones myogenic clones - satellite cells (self renewal) Some cells form fibroblastic type cells A portion of them may become fibroblastic-type cells, which are cells that are more like fibroblasts (cells that produce the connective tissue). These cells don't contribute to muscle formation but instead form the structural framework of tissues. Myocyte after exercise Myocytes are damaged in response to exercise Satellite cells repair tissue Continuous exercise leads to increase in myocyte size and number, but also adaptive changes on the cellular level: increased mitochondria, increased enzymes and activity, enhanced metabolism, greater contractile capacity myocytes are terminally differentiated, therefore no proliferation means: once they become myocytes , they cannot change into a different type of cell or divide to produce new myocytes When myocytes are damaged ( from exercise/toxins) → stimulate/activate satellite cells How do the myocytes activate them: inflammation, cytokines, etc ie intracellular chemicals(kept inside myocyte) , not meant to go extracellular, ie muscle cells burst open when exercise = chemicals intracellular are released into extracellular Cytokines and calcium, growth factors – all contained in cells Stimulate satellite cells (in quiescent state) Toxin induced damage → can also produce and regenration response my satellite cells if myoctes are damaged Damage myocytes– satellite cells regenerate them , repopulate damaged myocyte population Where are satellite cells found? Each myofibre is associated with a number satellite cells, beneath the basal lamina in close proximity to the sarcolemma satellite cell in close proximity to myocyte basal lamina → supports and wraps a single myofibre Muscle regeneration is dependent on the persistence of a reserve population of muscle stem cells – satellite cells without reserve population → Will not undergo hypertrophy or increase contractile capacity The niche in which satellite cells reside is regulated, hence dysregulation → results in over or under generation of myoctes = defective tissue Note: resident macrophages reside in muscle tissue (monocytes in blood and mature in tissue) Satellite → myotubes process (myogenesis) When under no stress/exercise, satellite cells are mitotically quiescent but become activated to divide in response to signals released following muscle damage or increased workload Satellite maturation – dependent on signal Replaces old, damaged, defective post-mitotic (muscle cells are post mitotic), differentiated cells (myocytes) → terminally differentiated Satellite cells → myoblasts → myocyte Process (NB markers at each step) Satellite cells are mononucleated Myocytes (myotubes) are multinucleated Multiple nuclei result from the fusion of myoblasts to form a myocyte nuclei shifted to the side of cell so that contractile components tke up bulk of cell Pax7 → differentiating transcription factor Quiescent satellite cells express Pax7 protein transcription factor → a protein that helps control which genes are turned on or off in a cell. It acts like a switch by attaching to specific parts of DNA and telling the cell to start or stop making certain proteins. vitale for transforming satellite cell into myocyte Pax7 is a transcription factor –that enables transcription of other genes Pax (paired box) transcription factors function as key regulators of myogenesis (during embryogenesi) important in maintenance of satellite cell NB MyoD → diffrenetiating transcription factor (what triggers) Quiescent satellite cell → negative for MyoD (dont express protein) Another important transcription factor One of the Myogenic Regulatory Factors (MRFs), drives myogenesis (commitment) Expressed by myoblasts (committed into turning into myocyte) ; Myf5 is semi-synonymous(another protein) (*knockout of MyoD) Knocked out (not expressed) and stimulated exercise, and measured satellite cells capacity to regenerate – still myocytes but defective, not strong in terms of contractibility Help determine purpose and function of myoD Satellite cells can express Pax7 and Myf5 (Myf5 indicates some activation) hence satellite cells that express Myf5 and Pax7 → early activation Damaged mycoytes relase agonsists and bind to satellite cells and activate them Trigger for MyoD transcription & Expression? The release of pro-inflammatory cytokines, neurotrophic factors, growth factors, or oxygen tension (which mediates the hypoxia-inducible gene program such as Hif1α, Hif2α, NO, VEGF, etc.) collectively orchestrates and modulates the status of the satellite cell pool Oxygen deficit – we are not measuring levels, satellite cells respond to cellular processes that result from low oxygen environment eg HIF-1 also during exercise, muscle cells accumulate byproducts Satellite cells respond to Growth factors – FGF2, IGF-1, HGF FGF2 - fibroblast growth factor 2 This growth factor helps stimulate the growth and division of many types of cells, especially in the development of tissues like blood vessels, skin, and muscles. It’s important for wound healing and tissue repair. IGF-1 manages effects of GH promotes bone and tissue growth HGF promote cell proliferation, resisting apoptosis, and inducing cell motility or invasion Changes to ECM – laminars and collagen Crushed muscle fibers themselves release growth factors, other cellular contents Notch Signalling Pathway (we’ll cover this later/soon) Why does it need to activate new genes and produce new proteins? chemical signalling , stimulate process that will transform cell need to reorganize cellular structure to become myocyte Transcribe genes to produce proteins to create myocytes Earliest marker of myogenic commitment Expressed at extremely low and essentially undetectable levels in quiescent satellite cells BUT expression of MyoD is activated in response to exercise or muscle tissue damage The effect of MyoD on satellite cells is dose-dependent; high MyoD expression represses cell renewal, promotes terminal differentiation and can induce apoptosis. Myf5 Myf5 is semi-synonymous(another protein) to myoD myogenic stem cell pool ( MSC /satellite cells) myogenic progenitor cells ( MPC or those characterized by expression of MyoD,Myf5) Transcription factors govern molecular cascades that ultimately regulate the fate of cell populations Gene expression identifies stem cell and progenitor cell populations in adult skeletal muscle In response to an injury, the quiescent satellite cell is activated by factors (including Fgfs, Hgf, activated Notch or NICD, Tnf-α) and up-regulates MyoD expression and this can happen within 2 h of injury. When satellite cells activated - *Self-Renewal – replenish pool of satellite cells – stem cells MyoD is expressed in somites and developing muscle during embryogenesis somites → morphological region in embryo → expect lots of mesodermal tissue to be derived from epithelial spheres = somites MyoD is expressed in somites and developing muscle during embryogenesis. (A) Whole-mount image of the MyoD promoter (containing the core enhancer and distal regulatory region)-GFP transgenic embryo at E11.5. Note expression in somites and limb muscles. (B) In situ hybridization of a parasagittal section of a E13.5 mouse embryo using a 35S-labeled MyoD riboprobe. Note expression in back, intercostal, tongue, and limb muscle groups. MyoD expressed in somites and established skeletal muscle during embryogenesis studies suggest that myogenic satellite cells population originate principally from the somite somite → blocks of mesodermal cells located on either side of the neural tube and the notochord Mesodermal cells are cells derived from the mesoderm, one of the three primary germ layers formed during embryonic development. The mesoderm is situated between the ectoderm (outer layer) and endoderm (inner layer) and gives rise to a variety of tissues and organs in the body. Satellite cells have a specialized niche in adult skeletal muscle The satellite cells have a specialized niche in adult skeletal muscle. (A) Electron micrograph of adult skeletal muscle demonstrating the myocyte nucleus (MC) and the satellite cell nucleus (SC). Note that the satellite cell is characterized by its size (i.e., small), high nuclear to cytoplasm ratio, relative absence of cytoplasmic organelles, and increased nuclear heterochromatin representing a quiescent state of the cell. (B) Schematic of A emphasizing that the satellite cell lies between the basal lamina (black arrowhead or green line) and the sarcolemma (white arrowhead or red line). Satellite cell response to myotrauma Skeletal muscle trauma or injury may be minor (e.g., resistance training) or may be more extensive (e.g., toxin injection (in lab), Duchenne muscular dystrophy) Some of the satellite cells will re-esta blish a quiescent satellite cell pool th rough a process of self-renewal. Satellite cells will migrate to the dama ged region and, depending on the severit y of the injury, fuse to the existing my ofiber or align and fuse to produce a ne chemotaxis: tell cell where to go w myofiber. movement of an organism or entity in response to In the regenerated myofiber, the newly f a chemical stimulus used satellite cell nuclei will initiall y be centralized but will later migrate to assume a more peripheral location. Muscle repair is characterized by discrete stages of regeneration (A)Day 1 to day 14 (B)After injury (C)Regeneration by stem/progenitor cells results in the formation of small, centronucleated (slightly basophilic) myofibers between 5 and 7 d post-injury (arrowhead marks the centronucleus of the newly regenerated myofiber). Hematoxylin and eosin-stained transverse section of Centrally nucleus of myocyte – indicates that the satellite post-injured skeletal muscle reveals restoration of the cells have been activated ( have a central nucleus; only in cellular architecture within ∼2 wk of injury. The nucleus mycotes have peripheral nuclei) occupies a peripheral position in fully mature myofibers. 1. Stages of Muscle Regeneration (Panel A): Activation of Satellite Cells: These cells are activated within 2 hours of muscle injury. Proliferative Phase: Satellite cells rapidly proliferate, with peak cellular proliferation occurring between 2–3 days after injury. These proliferating cells are known as transit amplifying cells. Differentiation: This stage is marked by the development of centronucleated myofibers. Maturation: Myofibers continue maturing after their formation, restoring muscle tissue. 2. Post-Injury Muscle Damage and Repair (Panel B): Following an injection of cardiotoxin, which causes myonecrosis (muscle cell death), about 70%–90% of the muscle is destroyed. Stem or progenitor cells regenerate the muscle, forming small, centronucleated myofibers (as seen 5–7 days after injury, with the nucleus centrally located in the myofibers). 3. Restoration of Muscle Structure (Panel C): A Hematoxylin and Eosin-stained transverse section of the muscle shows the cellular architecture of the muscle being restored within approximately 2 weeks post-injury. Fully mature myofibers are characterized by nuclei positioned at the periphery of the cells. Satellite cell regulation Damaged muscle fibres release growth factors that stimulate satellite cells FGF2 → fibroblast growth factor HGF → hepatocyte growth factor (why in muscle cells→ just name ) The hepatocyte growth factor (HGF) is named for its role in liver cells, but it's not exclusive to the liver. While it was initially identified for its effects on hepatocytes (liver cells), HGF has broader biological functions and can be found in various tissues and cells throughout the body IGF → insulin growth factor TGF-β → Transforming growth factor beta multifactoral cytokine maintains quiescnet state , tumour supressor protein PDGF (more commonly release by non-muscle cells, fibroblasts and platelets) And non-growth factors (cytokines, oxygen-deficit inducible factors, etc. → stimulate myogenesis autocrine factors → maintain quescient state cell secretes , acts on surface receptors of that same cell Motor neuron → remember what happens at neuromuscular junction Inflammatory Processes and Myocyte Regeneration https://www.youtube.com/watch?v=vLbdXaFZNcs myocytes are damaged in response to exercise Inflammtory cells recruite 1. Neutrophils → invade araea , release reactive chemicals , cause more mycoyte damage but clear space for new satellite cells to proflierate and differentiate (in this context neutrophil damage is beneficial) a. NETS: Neutrophil Extracellular Traps, trap and neytralize pathogens, facilitate destrcution b. defensins: disrupting pathogen membranes c. ROS: damage pathogen membranes d. MPO: myeloperoxidase → causes ROS , it is an enzyme that degrades the damaged muscle fibers to create space for satellite cells 2. monocytes recruited → M1 macrophages (proinflammatory) a. Damaged myocytes – release chemokines that attract other leukocytes b. FAP and satellite cells activated and profliferate c. FAP also promote expansion of satellite cells i. FAP → Fibro-adipogenic progenitors (FAPs) play a crucial role in skeletal muscle regeneration, as they generate a favorable niche that allows satellite cells to perform efficient muscle regeneration. 3. within 48hrs of damage → neutrophils decrease at site of injiury 4. FAP decrease because of M1 macrophage presence 5. M1 macrophages decrease, giving rise to M2 macrophages a. M2 macrophges - antinflammatory i. suppprt satellite actiavtion and diffreentiation ii. support remaining FAP NOTE: muscel regenartion depends on tiemly change between M1 and M2 macrophages → If M2 expand too early, excess matrix deposition is observed  as a result of FAP survival and deposition also wont be able to clear as much damage NOTE: M1 and M2 are diffrenet macrophage phenotypes tissue engineering/manipulation (not important - only 3 mark Q) → NB : NOT a treatment for muscular dystrophy https://www.sciencedirect.com/science/article/pii/S0958166917300320 Two general approaches of skeletal muscle tissue engineering. (a)Muscle cells and other supporting cells are combined with biomaterials in vitro, followed by transplantation either after extended culture to promote muscle formation, or immediately. (b) Biomaterials, either alone or combined with cytokines, growth factors, or cells secreting paracrine signals, are delivered to the body to induce regeneration by host muscle cells. Tissue-engineered skeletal muscle holds promise as a source of graft tissue for repair of volumetric muscle loss and as a model system for pharmaceutical testing In particular, hepatocyte and fibroblast growth factors are used to accelerate satellite cell activation and proliferation, followed by addition of insulin-like growth factor as a potent inducer of differentiation, are proven methods for increased myogenesis in engineered muscle Biomaterials can improve muscle regeneration by presenting chemical and physical cues to muscle cells that mimic the natural cascade of regeneration. Howeveer in vitro myocytes do not have the same contractability as in vivo myocytes anchoring of myocytes is also not as effective Dilemmas with Engineered Muscle? Engineered muscle does not produced forces comparable to native muscle (reduced contractile capacity), limiting its potential for repair and for use as an in vitro model for pharmaceutical testing Dexamethasone (DEX)(anti-inflammatory) , a glucocorticoid that stimulates myoblast differentiation and fusion into myotubes, as a solution to tissue engineered three-dimensional skeletal muscle units Addition of DEX to isolated muscle satellite cells in culture can improve functional and structural characteristics of tissue engineered skeletal muscle when administered at optimal doses and timings. Addition of DEX before induction of differentiation improved myogenic proliferation of muscle satellite cells, which subsequently led to increased myogenic differentiation and myotube fusion. could possible get in vitro muscle cells to function like in vivo Aging and Stem cells Aging Extrinsic and intrinsic changes in aging refer to two broad categories of factors that affect the aging process of stem cells and tissue regeneration, as seen in your image. Extrinsic changes with aging These are external factors that influence stem cells and tissue regeneration, often arising from the surrounding environment or systemic changes in the body. Examples from the image include: Together, these extrinsic factors lead to loss of quiescence in stem cells, causing them to activate more frequently to repair tissue damage. Over time, this increased activation depletes stem cell reserves and leads to decreased regeneration capacity. tissue damage In short, tissue damage in this context is one of the triggers that leads to stem cell activation as the body attempts to repair itself. However, with aging, the body's ability to handle this damage diminishes, leading to chronic damage and reduced regenerative capacity. changes in local and systemic inflammatory cytokine & growth factors Changes in local and systemic inflammatory cytokine and growth factors: With age, the levels and balance of inflammatory molecules and growth factors in the body shift. This can promote a chronic low- level inflammatory state, leading to reduced tissue repair and increased tissue damage. altered matrix remodelling and mechanotransduction Altered matrix remodeling and mechanotransduction: The extracellular matrix (ECM), which provides structural support to tissues, changes with age, affecting the signals that cells receive from their surroundings. Mechanotransduction refers to how cells sense and respond to mechanical cues, and aging impairs this ability. signalling molecules Intrinsic changes These are internal changes within the stem cells themselves, such as genetic and molecular alterations that occur as part of the aging process. Examples from the image include: Intrinsic changes contribute to decreased self-renewal and increased differentiation of stem cells, meaning they are less capable of maintaining a population of quiescent stem cells and more likely to become differentiated, specialized cells, thus depleting the stem cell pool. changes in transcription factor and epigenetic signatures (epigenetic modifications & transcription regulation very Nb in stem cells) Changes in transcription factors and epigenetic signatures: Aging affects the regulation of genes (via transcription factors) and alters the epigenome (chemical changes that control gene expression without changing the DNA sequence). These modifications influence how cells renew and differentiate. altered activation of signal transduction and cell cycle regulators Altered activation of signal transduction and cell cycle regulators: As stem cells age, there are changes in the pathways that control how they respond to external signals and how they regulate their cell cycle. These alterations can disrupt their ability to maintain quiescence or renew effectively. Extra: telomerase Telomerase is involved in intrinsic aging by influencing telomere length and cellular replicative capacity. Reduced telomerase activity in adult stem cells leads 9due to aging) to telomere shortening, which contributes to the intrinsic aging of cells. This results in a gradual decline in the self-renewal capacity of stem cells, one of the hallmarks of aging. autophagy is defective DNA damage chaperone proteins cant degrade small proteins properly Aged stem cell niche (dysregulated signaling) contributes to loss of quiescence and impaired regenerative function Pathways → NB look on ipad Dysregulated in ageing/aged muscle Crucial for the regulation stem cell function, including their maintenance and maintenance of quiescence, self- renewal, and differentiation Notch and Wnt → all stem cells have pathways Notch Function Satellite cells, which are stem cells in muscle tissue, remain quiescent (in a resting state) until there is injury. When injury occurs, Notch ligands are released, activating the satellite cells and triggering their proliferation. This proliferation helps expand the satellite cell pool so there are enough cells to regenerate and repair damaged muscle tissue during the process of myogenesis (muscle formation). The role of Notch signaling is to balance both the activation and replication of these cells, ensuring the muscle has enough satellite cells for repair and growth after injury. What is it? Notch pathway mediates cell-to-cell interactions (membrane-bound) Maintenance of quiescence helps maintain the undifferentiated state of stem cells by inhibiting differentiation Notch receptor spans the membrane, with an intracellular domain → Transmembrane protein has Intracellular, extracellular domain Ligands for Notch Ligands for Notch include: (not all exert same functions) Delta (1, 3, and 4) Jagged – activation and expansion of satellite cells in prep for myogenesis Serrate Lag 2 Ligands are membrane bound (cell-cell interactions) → expressed by myocytes Function: Ligand proteins binding to the extracellular domain induce proteolytic cleavage and release of the intracellular domain, which enters the nucleus to modify gene expression. Y-secretase cleaves NICD → leads to transcription of Hay and Hes genes → maintenance of quiescence Notch in the nucleus (don’t need to know transcription factor names) Within the nucleus, the active NICD interacts with transcriptional repressors of the CSL family (CBF1/RBP-J, Suppressor of Hairless (Su[H]), and Lag-1) and converts them to transcriptional activators (NICD-CSL). With involvement of other proteins, the CSL-NICD complex regulates target genes such as Hey and Hes genes imprtant in maintain quiescence Hes prevents differentiation During muscle injury Damaged myocytes express notch ligands (delta often associated with quiescence, jagged often associated with activation and proliferation) Jagged ligands from exercising muscle Notch activation therefore in the presence of resting myocytes prompts quiescence Notch activation in the presence of damaged myocytes prompts activation and proliferation (NOT DIFFERENTIATION) – preparing for tissue regeneration Later in myogenesis process, niche environment changes and Notch ligands decrease, prompting differentiation of myocytes Summary: The summary of your description about satellite cell activation during exercise and Notch/Wnt signaling is: Extracellular signals change throughout myogenesis. Early on, Notch ligands are high, promoting quiescence, but later they decrease, allowing satellite cells to begin differentiation. Less Notch Intracellular Domain (NICD) moves into the nucleus, reducing transcription of quiescence genes. As Notch decreases, other factors, such as HIF (hypoxia-inducible factor) and cytokines, begin to activate satellite cells, pushing them to differentiate and repair muscle. In the resting state, satellite cells are maintained in muscle tissue, but the numbers are insufficient for large-scale muscle regeneration. Exercise causes significant damage to muscle fibers, requiring a large-scale replenishment of satellite cells. This means both proliferation and self-renewal (a stem cell trait) are needed to expand the satellite cell pool. Notch and Wnt signaling are involved in controlling stem cells, including satellite cells. Before exercise, Notch ligands help maintain quiescence. During exercise, other factors like Jagged ligands, HIF, and cytokines activate satellite cells for both activation and differentiation, replenishing the muscle's regenerative capacity. Notch & aging Decreased ligands in ageing Leading to decrease/loss of stem cell population Impaired control of quiescence Dysregulated differentiation Impaired tissue regulation Also, altered intrinsic function (impaired signal transduction, oxidative stress, etc.) And ageing-related extrinsic factors (inflammation, etc.) Wnt Function Aids in satellite cell proliferation and commitment to myocyte differentiation; myotube formation Drives myotube formation What is it & aging complex network function with Notch In aging – increased circulating Wnt3a (wingless-type MMTV integration site family member 3a) Circulating Wnt3a stimulates beta-catenin signaling = induces aberrant fibrogenic differentiation of muscle stem cells and progenitor cells AND Wnt antagonizes Notch signaling Wnt ligands are a large family of secreted glycoproteins These glycoproteins are cysteine-rich and highly hydrophobic Increase Wnt – stimulate malformed progenitor cells to form myotubes , not desired myotubes ( dysfunctionality) Beta-catenin β-catenin is a dual function protein: involved in regulation and coordination of cell-cell adhesion and proliferation How does it work The Wnt signaling cascade needs soluble Wnt ligands to interact with Frizzled receptors and low-density lipoprotein receptor-related protein co-receptors (LRP). This coordination stimulates phosphorylation of Dishevelled and inactivates GS3Kß's phosphorylation of ß catenin.