Stem Cells Lecture Notes PDF

Stem cells Lecturer: Dr. Michelle Kuzma Adapted from: Dept. Head, Dr. Danuta Mielżyńska-Švach Molecular biology, 2023/2024 Lecture 10  https://biochem118.stanford.edu/Stem%20Cell/annurev%20Stem%20Cell%20Niche.pdf  https://www.nature.com/articles/s41423-023-01034-9  Chapter 20 ...

Stem cells Lecturer: Dr. Michelle Kuzma Adapted from: Dept. Head, Dr. Danuta Mielżyńska-Švach Molecular biology, 2023/2024 Lecture 10  https://biochem118.stanford.edu/Stem%20Cell/annurev%20Stem%20Cell%20Niche.pdf  https://www.nature.com/articles/s41423-023-01034-9  Chapter 20  Stem cells Stem cells occur in the human body starting from the embryonic stage through fetal life and into adulthood Stem cells are cells that have not yet differentiated into a specific cell type All stem cells display two properties,:  the capacity for an unlimited number of divisions  The ability to differentiate into a particular type of body cell  Stem cells Morphological features of a stem cell:  large nucleus  narrow cytoplasmic rim, sparse area Types of division:  symmetric  asymmetric Other stem cell features:  ability to divide after long periods of inactivity ("sleeping" state in cell cycle)  increased resistance to radiotherapy and cytostasis  Stem cell division Asymmetric division (self-renewal process):  one daughter cell retains the properties of stem cell ("mother cell")  the other daughter cell is partially differentiated ("precursor cell") Symmetrical divisions:  both daughter cells retain properties of stem cells  both daughter cells are partially differentiated Stem cell division Stem cell division  Stem cells Most often, the term "stem cells" refers only to cells that are capable of differentiating into all cell types (pluripotent) that comprise:  tissues of endodermal origin  tissues of ectodermal origin  tissues of mesodermal origin  sex cells  Stem cell classification Stem cells are divided up according to their:  potential for differentiation  source of origin They are sub-divided in accordance to their differentiation potential (potency) into:  totipotent or omnipotent (full-potential)  pluripotent (multi-potential)  multipotent (few-potential)  unipotent (one-potential)  Stem cell classification Stem cells are sub-divided based on their source of origin:  embryonic  fetal and extra-embryonic tissue  somatic cells of adults Totipotent (omnipotent) stem cells Totipotent (omnipotent) cells are stem cells that can give rise to:  all types of cells that make up an organism, including sex cells  extra-embryonic structures, such as the fetal membrane or placenta Totipotent cells include:  the zygote – the cell created as result of fertilization  blastula cells – cluster of cells up to the stage of several blastomeres  Pluripotent stem cells Pluripotent cells have the ability to form all types of cells in the body, except for sex cells and extra-embryonic structures Pluripotent cells form the blastula that forms in the first week of embryo development The blastula gives rise to the three germ layers from which all tissues in the human body will develop Pluripotent stem cells cannot transform back into totipotent/omnipotent cells  Multipotent stem cells Multipotent cells can develop into all cell types, but only within a given germ layer, i.e.,  the ectoderm (e.g., sensory epithelium, nervous system)  the endoderm (e.g., respiratory system, pancreas and liver)  the mesoderm (e.g., dermis, skeletal and cardiac muscles)  Unipotent cells Unipotent cells are cells that are capable of differentiating into only one type of specialized cell Unipotent cells usually comprise a reservoir for renewal and repair of tissues in adult body  Types of stem cells Multipotent  Stem cell types Based on their origin, stem cells are sub-divided into:  embryonic stem cells (ESC)  fetal stem cells (FSC)  somatic, adult or tissue stem cells (SSC, ASC or TSC)  induced pluripotent stem cells (iPSC)  Embryonic stem cells Embryonic stem cells (ESC) are  embryonic cells at the 4 - 8 cell stage (morula composed of blastomeres)  cells of the blastocyst embryonic node (16-cell stage/blastocyst) Embryonic stem cells have number of specific genes that are used as stem cell markers (e.g., transcription factors OCT3/4, NANOG, SOX2)  Embryonic stem cells Only embryonic stem cells (ESCs) derived from blastomeres have totipotent properties They can differentiate into any type of cell in body, including germ cells and those that form extra-embryonic structures (i.e., fetal membranes, placenta, etc.) Zona pellucida  Embryonic stem cells Embryonic stem cells (ESC) derived from blastocysts:  exhibit only pluripotent properties  Have the greatest capacity for differentiation (plasticity) They can differentiate into three germ layers and then into tissue and organ cells They can give rise to cells of all 210 tissues that make up the human body  Fetal stem cells Fetal stem cells (FSC) are found in:  fetal tissues (blood, liver, bone marrow, pancreas, kidneys, spleen)  extra-embryonic tissues (placenta, chorion, amnion, amniotic fluid, umbilical cord blood) Fetal stem cells are considered a transitional form between embryonic stem cells and differentiated somatic cells  Somatic stem cells Somatic stem cells (SSCs) occur in many tissues of adult organisms Somatic stem cells can be:  multipotent  unipotent SSCs that are multipotent can give rise to several different types of cells with similar properties and of embryonic origin SSCs with unipotent properties are characterized by the ability to only differentiate into one cell type Types of stem cells  Somatic stem cells SCCs are located in specific areas called stem-cell niches located between differentiated cells It is assumed that these are cells that did not participate in organogenesis during embryogenesis, but rather remained in a quiescent state Their function is to:  replace old and worn-out cells with new ones  repair damaged tissue  Stem-cell niches Stem-cell niches contain:  framework of the niche (microenvironment) - proteins and proteoglycans of the extracellular matrix  stem cells  mature, differentiated cells of given tissue  immune cells  signaling substances produced by surrounding cells and tissues  oxygen, nutrients, as well as, hormonal (blood) and neuronal (nerve) signals Stem cell niches  Somatic stem cells The number of somatic stem cells present in adult organisms decreases with age Somatic stem cells present in epithelial tissue and bone marrow divide regularly Somatic stem cells located in skeletal muscle or the pancreas divide less frequently or only when the organ or tissue is damaged  Somatic stem cells Somatic stem cells:  usually are metabolically inactive  may not undergo cell division for a long time They are most often activated as result of injury to the tissue (organ) in which they occur or development of disease Activated stem cells:  begin cell division  transform into specialized cells necessary for tissue regeneration  SCC organ niches Organ niches of somatic stem cells in humans are present in organs and tissues, such as:  bone marrow  skin  skeletal muscle  heart muscle  intestine  liver  brain Location of stem cells  HSC niche Hematopoietic stem cells (HSCs) occur in bone marrow, but also in the skin and heart Stem cells and precursor cells are formed from hematopoietic stem cell through asymmetric divisions As a result of successive divisions, differentiation and maturation, the precursor cells produce differentiated blood cells of different lineages  Hematopoietic stem cells Hematopoietic precursor cells give rise to erythrocytes (red blood cells (RBCs)) and leukocytes (white blood cells (WBCs)):  erythrocytes: without cell nuclei, capable of transporting oxygen and carbon dioxide in the body  thrombocytes (platelets): involved in process of clot formation and wound healing  granulocytes: WBCs capable of phagocytosis (i.e., engulfing and eliminating foreign cells) include basophils, neutrophils, eosinophils  macrophages: scavenger cells, responsible for defending the body against bacteria, fungi and parasites - differentiated monocytes  lymphocytes: B-cells and T-cells responsible for producing antibodies and defending the body against pathogens, respectively Hematopoietic stem cells  HSC niche HSC niches consist of:  the cellular part (i.e., osteoblasts, macrophages, mesenchymal stem cells and endothelial cells)  the extracellular matrix HSCs functions are regulated by:  adhesion proteins, ligands and cytokines  oxygen and calcium ion concentration  HSC niche There are two types of HSC niches:  Endosteal: located near the periosteum of the internal medullary cavity of bone  Vascular: located near the thin-walled sinusoidal vessels  HSC niche Periosteum Bone Marrow  MSC organ niche Mesenchymal stem cells (MSC) are multipotent stem cells present in the following tissues and locations:  bone marrow  adipose  periosteum  skeletal muscle  skin  bones  lungs  periodontal ligaments and dental pulp Occurrence of MSC  Skin SSC organ niches Somatic skin stem cells include:  epidermal stem cells  hair follicle stem cells  melanocyte stem cells Epidermal stem cells are located in basal layer of the epidermis and are responsible for regeneration of various layers of the epidermis Hair follicle stem cells are responsible for regeneration of hair and sebum glands among others Melanocyte stem cells are responsible for the reproduction of skin pigment cells SSC organ niche of the epidermis Location of epidermal stem cells SSC niche of the hair follicle  Intestinal SSC niche Intestinal somatic stem cells are found in the epithelium that forms a lining on the bottom of crypts (particular glands) of the small intestine Intestinal stem cells form dividing precursor cells that:  move continuously upward  differentiate into secretory and absorptive cells  are shed from the upper layer of villi Intestinal SSC organ niche  Skeletal muscle SSC niche Skeletal muscle somatic stem cells, also known as satellite cells, are:  involved in the growth and regeneration of muscle tissue  located in the niche between the muscle fiber membrane (sarcolemma) and the basement membrane surrounding each muscle fiber Satellite cells  Very small embryonic stem cells Very small embryonic-like stem cells (VSELs) are remnants of embryonic tissues scattered throughout the body VSELs in adult tissues:  occur in small numbers  are smaller than erythrocytes (< 7-8 µm)  are mobilized in peripheral blood in response to tissue and organ damage  Very small embryonic stem cells VSELs have:  large nuclei  high nuclear to cytoplasmic ratio  undifferentiated chromatin  epigenetic marks (histone methylation and acetylation) Sources of stem cells  Obtaining ESCs Animal embryonic stem cells were obtained from:  mouse embryos (1981)  rhesus embryos (1995)  rat embryos (2008) Human embryonic stem cells were isolated in 1998 ESCs from different animal species often have different requirements for culture conditions Before they are defined as ESCs, pluripotent properties must be positively verified  Obtaining ESCs Embryonic stem cells (ESC) are mainly obtained in vitro The first step is to place blastocyst stage embryos obtained from the female reproductive tract into a petri dish During cell culture, blastocysts stick to a layer of suitably prepared nutrient cells (fibroblasts) at bottom of dish Embryonic cells spread out on the bottom of dish creating so- called colonies  Obtaining ESCs The next step is to isolate the cells from the colonies and transfer them to a new dish. This process is called passaging cells. Obtaining a homogeneous group of ESC cells requires multiple passages. In order to maintain the pluripotent properties of the cells, they must be cultured in strictly defined conditions:  on a layer of fibroblasts  in a culture medium containing leukaemia inhibitory factor (LIF), which protects cells from losing pluripotency Scheme for obtaining ESC ICM isolation of growing a colony transferring cells obtaining ESCs ESCs of a from blastocyst from an blastocyst embryonic node to new dish T - trophectoderm cells ICM - Inner Cell Mass  Therapeutic use ESC Currently, there are no approved uses for human embryonic (ESC) and fetal (FSC) cells in any type of medical therapy  Therapeutic use ESC Reasons:  strategy raises number of ethical controversies in our cultural and religious area  ESC culturing is very difficult and multi-stage (costly)  difficulties in directing ESC cells, (i.e. transforming them into a desired tissue)  lack of tissue compatibility with the HLA (Human Leukocyte Antigen) proteins of a recipient  stem cells isolated from embryos often form teratomas in the recipient's body  Umbillical cord blood A source of fetal stem cells derived from extra-embryonic tissues (FSC) is umbillical cord blood Biological advantages of umbillical cord blood:  high proliferative capacity  high ex vivo colony-forming capacity (outside the living organism)  autocrine capacity to produce growth factors  long telomeres  Umbillical cord blood Clinical advantages of umbillical cord blood:  rapid availability  short waiting time  low risk of  infectious disease transmission  transplant rejection  acute and chronic graft-versus-host disease (GVHD)  possibility of use in presence of HLA incompatibility between the recipient and the donor  Umbillical cord blood Clinical drawbacks of umbillical cord blood:  lower number of hematopoietic stem cells than of bone marrow and peripheral blood  delayed hematological renewal in the recipient's marrow  risk of transmission of overlooked genetic diseases Therapeutic application of HSCs Hematopoietic stem cells are used in the treatment of:  Non-solid tumors (acute lymphoblastic leukaemia, acute and chronic myeloid leukaemia, various types of lymphomas)  non-cancer diseases (aplastic anaemia, congenital anaemias, severe immune disorders, metabolic diseases) Hematopoietic stem cells can be obtained from:  peripheral blood (80% of procedures)  bone marrow (20% of procedures)  Therapeutic application of HCS Preparation of HSCs from peripheral blood is performed using apheresis Apheresis stages:  mobilization of bone marrow from the donor  separation of HSCs using a cell separator  administration of prepared HSCs to recipient via an IV drip infusion  Therapeutic application of HCS Mobilization involves administering the following medications to the donor five days before blood collection:  corticosteroids,  stimulating factor G-CSF Granulocyte Colony-Stimulating Factor: (G-CSF) stimulates hematopoiesis and release of cells from bone marrow into peripheral blood  Therapeutic application of HCS HSC separation involves:  inserting needles into two donor veins  taking blood from one donor vein  separating the stem cells  returning the blood (plasma, etc.) to other donor vein The entire procedure takes about four hours and does not require anaesthesia or hospitalization  HSC separation Stage I Suspension of blood cells is incubated with solution of specific antibodies for surface proteins characteristic of HSCs Antibodies are combined with fluorophore (i.e., fluorescent tags/labels (dyes) that emit light at specific wavelength) Antibodies attach to the CD34 protein present on the surface of HSCs  HSC separation Stage II After incubation with antibody, the blood cell suspension is passed through capillaries Light emitted by laser excites the fluorophores attached to antibody so that they emit light of a specific wavelength The fluoresced cells with the tagged surface markers (CD34 protein) are separated from the other cells/components, which allows obtaining the target cell population HSCs constitute from 5 to 20% of all cells in suspension. HSC separation diagram Stage I Stage II recorder fluorochrome capillary antibody marker HSC cells without marker cells with marker  Therapeutic application of HSCs The second method of obtaining HSCs is to collect bone marrow from the hip bone plate This procedure involves extracting bone marrow via a biopsy needle inserted into the bone About one liter of bone marrow mixed with blood is collected The procedure is performed under general anaesthesia and lasts up to 60 minutes The donor remains in hospital for the duration of the procedure (two days) Therapeutic application of HCS  Therapeutic application of SSC Somatic stem cells are used to treat some types of corneal injuries Stem cells, located in the area of the eye called the corneal limbus, are responsible for producing new corneal cells Limbal stem cells are also known as corneal epithelial stem cells Transplanting limbal stem cells from the cornea of healthy eye can help repair damaged cornea Therapeutic application of SSC  Obtaining MSCs Sources of mesenchymal stem cells:  bone marrow (BM-MSC - Bone Marrow-derived Mesenchymal Stromal Cells)  adipose tissue (ADSC - Adipose-Derived Stem Cells) In bone marrow stem cells population is heterogeneous and predominate HSCs In adipose tissue MSCs constitute more homogeneous population than in bone marrow Therapeutic application of MCS MSC are used in treatment of skin burns, trophic ulcers and diabetic foot Stages of obtaining MSC:  collection of adipose tissue by liposuction (suction)  isolation of the MSC preparation (removal of adipose tissue);  enzymatic digestion  multiple centrifugation  multiple washing Isolated MSC are transferred to culture media, where they are multiplied Therapeutic application of MCS Therapeutic application of MCS  Induced stem cells Induced pluripotent stem cells (iPCS) Stem cells that have been artificially derived from non- pluripotent somatic cells Reprogramming of cell nuclear changes gene expression profile of mature somatic cell and transforms it into a stem cell  Induced stem cells Features of iPSCs:  large nucleus clearly demarcated from the cytoplasm  small amount of cytoplasm  ability to rapidly divide and self-renew  expression of specific proteins (c-kit, Thyl)  Uses of iPSC Research in areas of:  disease etiology  embryogenesis  abnormal development (of cells, tissues and organs)  gene action (e.g., expression)  drug testing (toxicity, mutagenicity, carcinogenicity, teratogenicity) Uses of iPSC  Potential applications Potential clinical applications of stem cells include the treatment of:  myocardial infarction  stroke  parkinsonism  spinal cord injury  diabetes  myopathy  liver injury