WK 8 Stem cells- cell determination and differentiation_B.Malik2024.pdf

Stem cell, cell differentiation and determination 5BY514 / 5BY547 Dr. Bilal Malik [email protected]  Part 1 Understand types of stem cells in multicellular orga...

Stem cell, cell differentiation and determination 5BY514 / 5BY547 Dr. Bilal Malik [email protected]  Part 1 Understand types of stem cells in multicellular organisms Understand of how cells become specialized Aims of the Understand cell differentiation and determination session Understand the applications / potential use of stem cells Part 2 The development of tissues and organs from cells The role of extracellular matrix in tissue formation ©Dr. Bilal Malik2024, [email protected]  What are stem cells? Types of stem cells Embryogenesis Cell plasticity and differentiation Key Gene expression in differentiated content cells Cell determination Formation of tissue from cells The extracellular matrix Importance of stem cell in Medicine ©Dr. Bilal Malik2024, [email protected]  What are your thoughts on Use of Stem cells to treat Alzheimer’s disease?  What are stem cells? Cells that have the ability to? o Self-renew o Develop into specialised cell types Embryonic and somatic/adult. Characterised based on their potency (capacity for differentiation). Other cell types with specialized functions could be generated from stem cells ©Dr. Bilal Malik2024, [email protected]  oocyte Types of Stem cells sperm Totipotent Cells zygote blastocyst reprogramming of oocyte sperm somatic cells ESC iPSC Pluripotent Cells Multipotent Cells endoderm mesoderm ectoderm Somatic Cells ©Dr. Bilal Malik2024, [email protected] Adapted from https://www.technologynetworks.com/cell-science/articles/cell-potency-totipotent-vs-pluripotent-vs-multipotent-stem-cells-303218 and https://sciencekeys.com/tissue-culture-biology-science-  Types of stem cell – Embryonic stem cell Pre implantation - Totipotent - Pluripotent ©Dr. Bilal Malik2024, [email protected]  Embryonic stem cells – Totipotent Cell can generate any cell type of the body somatic cells germ cells Extra embryonic components required for development i.e. placenta. Can form a whole organism – identical twins First few days of development – zygote and morula. ©Dr. Bilal Malik2024, [email protected]  Embryonic stem cells – Pluripotent Cells can give rise to many cell types i.e. somatic germ cells. Can’t develop into placenta Embryonic stem cells are pluripotent stem cells derived from the inner mass of a blastocyst. Blastocyst – an early stage pre-implantation embryo, consisting of 50 -150 cells which occurs 4 - 5 days post fertilisation ©Dr. Bilal Malik2024, [email protected]  Embryogenesis -Blastocyst A blastocyst consists of: Inner cell mass forms the embryo and some extra embryonic structures Trophoblast forms the placenta ©Dr. Bilal Malik2024, [email protected]  Embryogenesis -Gastrulation Three germ layers are formed: Ectoderm Endoderm Mesoderm Can differentiate into all adult cells in body http://www.hhmi.org/biointeractive/differentiation-and-fate-cells ©Dr. Bilal Malik2024, [email protected]  Key stages of embryogenesis Fertilisation Cleavage Blastulation ©Dr. Bilal Malik2024, [email protected]  Types of stem cell – Somatic stem cells Specialised tissues - Multipotent - Unipotent Undifferentiated cells found in tissues and organs of the juvenile and adult body Maintain homeostasis and can regenerate local tissue Exist in distinct cell niches within the human body alongside differentiated cells Found in almost every organ of the body but reduce in number during aging process ©Dr. Bilal Malik2024, [email protected]  Somatic stem cell - Unipotent Differentiated cells which give rise to a single mature cell type. E.g. epidermal stem cell, which may harbour the potential to regenerate skin. ©Dr. Bilal Malik2024, [email protected]  Somatic stem cell - Unipotent Progenitor cells, which are less plastic and more differentiated. Give rise to multiple but limited number of lineages, usually a restricted/related group of cells. ©Dr. Bilal Malik2024, [email protected]  Stem cell- HESC and iPSC Human embryonic stem cells (hESCs) are similar to induced pluripotent stem cells (iPSC) in phenotype, karyotype, phenotype, telomerase activity and capacity for differentiation. iPSCs are considered ethically and morally superior to hESCs as they are not generateds from the destruction of embryo Differentiated somatic cells are reprogrammed to pluripotent state by manipulating “Yamanaka factors”: sex determining region Y box- containing gene 2 [SOX2], OCT3/4, tumor suppressor Krüppel-like factor 4 [KLF4], and proto-oncogene c-MYC ©Dr. Bilal Malik2024, [email protected] Volarevic et al. 2018  Cell plasticity and differentiation Plasticity Ability of tissue-specific stem cells to switch or develop to new identities depending on inductions. The term plasticity also means stem cell phenotypic potential. Differentiation The process through which a young and immature cell changes in to a specialized cell. Differentiated cells lose their precursor characteristics and to acquire new. Terminally differentiated cells are cells which have differentiated to form specific cell types required by the organism, for example a muscle cell ©Dr. Bilal Malik2024, [email protected]  Cell plasticity and differentiation Plasticity Totipotent Differentiation Pluripotent ESC Multipotent endoderm Unipotent Fully differentiated (260 types) ©Dr. Bilal Malik2024, [email protected]  Cell differentiation and determination Recap: Cellular differentiation The process through which a stem cell changes into a specialised cell, losing its precursor properties and acquiring new characteristics. Involves a change in gene expression Cell type is characterised by the cell’s protein expression profile. Cellular determination Initially identical cells become committed to different pathways of development2. An irreversible process ©Dr. Bilal Malik2024, [email protected] 2: https://www.britannica.com/science/cell-biology/Intercellular-communication Q Stem cell fate  Stem cell fate ©Dr. Bilal Malik2024, [email protected] Volarevic et al. 2018  What controls stem cell fate? Asymmetric cell division Cell polarity requires specific determinants to be asymmetrically localised within a cell. Determinants are segregated during cell division. Daughter cells will have different fates. ©Dr. Bilal Malik2024, [email protected]  What controls stem cell fate? Inductive signals Extrinsic (external) - Caused by cell niche, developmental or environmental signals (diffusion, direct contact, gap junctions) Intrinsic (internal) - Caused by changes in DNA methylation or chromatin modification Once a cell’s fate has been chosen, the cell is said to be determined; this is a stable change. ©Dr. Bilal Malik2024, [email protected] Royall, L. N. and S. Jessberger (2021)  What controls stem cell fate? ©Dr. Bilal Malik2024, [email protected] Donelly et al. (2018)  Cell differentiation and determination Due to activation or repression of genes Transcription factors can activate or repress transcription due to binding with specific sites Constitutive gene expression may require the continual presence of a transcription factor. Examples o DNA methylation can modify gene activity. o The state of chromatin packaging is also important for gene activity/inactivity and histone modification can regulate gene activity ©Dr. Bilal Malik2024, [email protected]  Cell differentiation and determination Dependent on epigenetics – does not involve a change in genome Relies on chemical and physical signals to express different subsets of genes Results in cells containing different proteins and therefore they develop into morphologically distinct cell types. ©Dr. Bilal Malik2024, [email protected]  Cell differentiation and determination Expression of specific transcription factors commits the cell to becoming a bone cell. The cell is now considered to be determined. ©Dr. Bilal Malik2024, [email protected] Amarasekara et al. 2021  Transcription factors involved in myogenesis ©Dr. Bilal Malik2024, [email protected] Applications of Stem cell  Application of stem cell – Regenerative Medicine ©Dr. Bilal Malik2024, [email protected]  Application of stem cell – Regenerative Medicine Disease modelling, drug discovery and cell therapy Potential treatment of degenerative, autoimmune and genetic disorders. Regenerative medicine and tissue engineering o Helping body to heal itself o Replacing donor transplants o Blood donation o Tissue replacement after injury or disease ©Dr. Bilal Malik2024, [email protected]  Application of stem cell – Regenerative Medicine Repopulation and regeneration of depleted neuronal circuitry by exogenous stem cells Hematopoietic stem cell transplantation therapy for malignant and non-malignant diseases Stem cell transplantation for the regeneration of periodontal tissues Stem cell inner ear transplantation ©Dr. Bilal Malik2024, [email protected]  Application of stem cell – Regenerative Medicine ©Dr. Bilal Malik2024, [email protected] Volarevic et al. 2018  Application of stem cell – Scientific research First human Cell Line - HeLa Cell Line (Henrietta Lacks 1951) Epithelial cell from biopsy of cervical cancer tumor (immortal) Unlimited proliferation To study disease states in vitro, e.g. cancer research, immunology Originally taken in US without consent, provided freely within scientific community and commercialised Laws in place to protect donor Multiphoton fluorescence image of HeLa cells stained with the actin binding toxin phalloidin (red), microtubules (cyan) and cell nuclei (blue). Nikon RTS2000MP custom laser scanning microscope. National Institutes of Health (NIH) - National Institutes of Health (NIH) ©Dr. Bilal Malik2024, [email protected]  Application of stem cell – Cloning Cloning Making exact genetic copies of living things Therapeutic cloning Reprogramming nucleus of an adult cell by transfer to the cytoplasm of an enucleated egg and isolating ES cells after formation of blastocyst in vitro Reproductive cloning Reprogramming nucleus of an adult cell by transfer to the cytoplasm of an enucleated egg and re-implanting the embryo to enable the formation of a viable fetus ©Dr. Bilal Malik2024, [email protected]  Application of stem cell – Cloning ©Dr. Bilal Malik2024, [email protected] https://www.britannica.com/science/cloning  Application of stem cell –Time line 2006: Professor Shinya 2009: ChondroCelect®, an Yamanaka shows that adult, autologous cell therapy 1997: Dermagraft, a tissue fully specialised mouse cells where a patient’s cartilage 2015 The Europe Commission engineered human donor can be reprogrammed to cells are grown in the approves the sale of Holoclar to skin replacement therapy, is become cells that behave laboratory and used to treat treat people with severely damaged 1978: Haematopoietic stem marketed in the UK for the like embryonic stem cells cartilage defects in knees, is corneas. It is the first stem-cell cells are discovered in treatment of diabetic foot (induced pluripotent stem approved for commercial use therapy to reach the market. human cord blood. ulcers. – Earmouse! cells). by the EMA 2001: The Human Fertilisation and 2008: A Colombian 2012: Biopolymer 2017 ACI 1963: Dr Georges 1996: Dolly the Embryology Act is amended to permit woman receives the first hydrogels are developed approved Mathé pioneers sheep is created by research on hESC for strictly regulated human windpipe by RCUK-funded for use by NHS the use of bone Research Council purposes reconstructed using stem scientists for use in the marrow scientists using cells. The transplant team treatment of corneal transplants in the somatic cell was jointly led by RCUK blindness caused by treatment of nuclear transfer. researcher Professor limbal stem cell leukemia Dolly is the first Martin Birchall deficiency mammal to be cloned from an adult somatic cell ©Dr. Bilal Malik2024, [email protected] https://stfc.ukri.org/files/regenerative-medicine-timeline/  Stem cell- ethical and safety issues ©Dr. Bilal Malik2024, [email protected]  Stem cell- ethical and safety issues What do you think are the key ethical issues around the use of stem cells. Think about where they come from, who uses them, what can we do with them ©Dr. Bilal Malik2024, [email protected]  Stem cell- ethical and safety issues -Where they come from 1) Can we ever intentionally destroy a human embryo? 2) Can we benefit from others' destruction of embryos? 3) Can we create an embryo to destroy it? ©Dr. Bilal Malik2024, [email protected]  Stem cell- ethical and safety issues Ethical issues- Destruction of a human embryo is a major factor that may have limited the development of hESC-based clinical therapies; use of somatic cells and derivation of induced pluripotent stem cells (iPSCs) has helped overcome this Ethical issue - Unlimited differentiation potential of iPSCs which can be used in human reproductive cloning, as a risk for generation of genetically engineered human embryos and human-animal chimeras, is major ethical issue Safety issues- Undesired differentiation and malignant transformation are major, immune issues (possibility of rejection), complex tissue formation (vascularisation and innervation) ©Dr. Bilal Malik2024, [email protected] Volarevic et al. 2018  Stem cell- ethical and safety issues ©Dr. Bilal Malik2024, [email protected] Volarevic et al. 2018  Stem cell therapy – long term safety concerns Mortality rate of patients treated with stem cell therapy reported to be higher than their general population peers Contribution of late complications to significant long-term morbidity and mortality. Late effects in HCT (Hematopoietic cell transplantation) survivors include organ specific complications, late infections, secondary cancers, quality of life impairments, psychosocial issues, sexual and fertility concerns There are also issues around return to school / work and financial implications. Lifelong follow-up of HCT survivors is recommended, ©Dr. Bilal Malik2024, [email protected]  Summary Stem cells have the ability to self renew and differentiate into specialised cell types Cells start out within an organism as totipotent cells, capable of producing all the cells that are required for the mature organism The ability of a cell to form multiple cell types is defined as the cells plasticity. A cells fate is determined via epigenetic mechanisms (diverse network of physical and chemical signalling pathways) Formation of terminally differentiated cells allows for the development of tissues and organs. ©Dr. Bilal Malik2024, [email protected]  Conclusions iPSC opened a new era of personalized medicine. Patient-specific iPSCs have potential use in and may be helpful in drug screening, generating in vitro models of human diseases, and novel reproductive and therapeutic techniques ©Dr. Bilal Malik2024, [email protected] Building tissues and organs After the break Coffee break  Building tissues and organs The development of tissues and organs from cells The role of extracellular matrix in tissue formation  How cells assemble into tissues and organs Cells are the building blocks of multicellular organisms, there are more than 250 different cell types in the human body. Cells are organised into cooperative assemblies called tissues (tissues = cells + extracellular matrix) While differentiation results in specific cell types, morphogenesis is the process whereby the shape (morph) of the cell, tissue or organ is generated (genesis). Tissue structures and shapes can be formed by the organisation of groups of cells into polarised arrangements and by coordinating their polarity in space and time. ©Dr. Bilal Malik2024, [email protected]  Tissue types Tissue = Cells + extracellular matrix. Perform specialised function. Four major types: Connective Nervous Muscle Epithelial Organs are built from tissues. ©Dr. Bilal Malik2024, [email protected]  Tissue types ©Dr. Bilal Malik2024, [email protected]  Tissue types Lets consider the human hand ©Dr. Bilal Malik2024, [email protected]  Relative density of tissue and extracellular matrix Connective tissue Epithelial cells ©Dr. Bilal Malik2024, [email protected]  Connective tissues ©Dr. Bilal Malik2024, [email protected]  Connective tissues Loose connective tissue Lymph gland Dense irregular Muscles Dense regular ©Dr. Bilal Malik2024, [email protected] Ligaments and tendons  Tissue type 1 Composition of bone tissue Bone is composed of 3 major constituents Living cells (osteoblasts; osteocytes; osteoclasts) Non-living organic proteins (collagen, muco-polysaccharides) Non-living inorganic crystals (hydroxy-carbonate apatite (HCA)) Matrix is produced and mineralised by osteoblasts and osteocytes Resorption occurs by osteoclasts ©Dr. Bilal Malik2024, [email protected]  Hierarchical Organisation of bone Organisation provides form and function Collagen & inorganic crystals provide strength ©Dr. Bilal Malik2024, [email protected]  Tissue type 2: Muscle tissue ©Dr. Bilal Malik2024, [email protected]  Tissue type 3: Epithelium ©Dr. Bilal Malik2024, [email protected]  Tissue type 3: Nervous tissue Yellow = Nerve fibres Red = Extracellular matrix Blue = Ganglion cells ©Dr. Bilal Malik2024, [email protected]  The extracellular matrix Most mammalian tissues are embedded in an extracellular matrix The ECM is made and secreted by cells It fills the gaps between cells and binds cells and tissues together Most abundant in connective tissues Several types of matrices Contains three types of molecule: Structural proteins (collagens and elastins) Protein-polysaccharide complexes to embed the structural proteins (proteoglycans) Adhesive glycoproteins to attach cells to matrix (fibronectins and laminins) ©Dr. Bilal Malik2024, [email protected]  The ECM – Structural and adhesion proteins ©Dr. Bilal Malik2024, [email protected]  Structural proteins - Collagen Major structural component of the ECM Fibrous glycoproteins Most abundant protein in animal tissue. Large family of proteins (≥31 members). Responsible for tensile Fibril forming collagens Network-forming, anchoring fibrils and transmembrane collagens. ©Dr. Bilal Malik2024, [email protected]  Structural proteins - Collagen ©Dr. Bilal Malik2024, [email protected]  Structural proteins – Elastin Elastic fibres Abundant in organs that regularly stretch e.g. the lungs, Flexible. Composed principally of a protein elastin, cross-linked by covalent bonds formed between the side chains of leucine residues. Crosslinks allow elastin fibres to recoil to their original shape after extension. ©Dr. Bilal Malik2024, [email protected]  ECM Polysaccharide The fibrous collagen and elastin structural proteins are embedded in gels formed from polysaccharides - glycosaminoglycans (GAGs). Highly negatively charged GAGs attract lots of cations, which in turn attract water forming a porous, hydrated gel – gives mechanical support to the ECM. Most GAGs are bound to proteins to form proteoglycans. ©Dr. Bilal Malik2024, [email protected]  Matrix adhesion proteins - Fibronectin Matrix adhesion proteins link matrix components to one another and to the surfaces of cells. They interact with collagen and proteoglycans to specify matrix organisation and are major binding sites for cell surface receptors, i.e. integrins Fibronectin are the principal adhesion protein of connective tissues. Dimeric glycoprotein - cross-linked into fibrils in the ECM. Has binding sites for collagen and GAGs, cross-linking them. Has a distinct site for recognition by cell surface receptors – Integrins. ©Dr. Bilal Malik2024, [email protected]  Matrix adhesion proteins – Laminins & Nidogen Laminins Principal component of and major organisers of basal laminae. Can self-assemble into mesh-like networks. Cross or T-shaped heterotrimers of alpha, beta and gamma subunits. Different subunits have binding sites for cell surface receptors and other components of the ECM (agrin and other proteoglycans). Nidogen Tightly associated with laminins. Binds type IV collagen. ©Dr. Bilal Malik2024, [email protected]  Cell –matrix adhesion proteins - Integrin Intracellular - cytoskeleton Integrins mediate linkage between fibronectin in the extracellular matrix and the cytoskeleton allowing the external cellular environment to influence intracellular functions Extracellular - ECM ©Dr. Bilal Malik2024, [email protected]  Structural arrangement of ECM Laminin, nidogen, collagen and proteoglycans form cross-linked networks within the ©Dr. Bilal Malik2024, [email protected] basal laminae  Cell- cell and cell-matrix interaction -many ways ©Dr. Bilal Malik2024, [email protected]  Cell- cell and cell-matrix interaction Four main functions, three main types Anchoring junctions – provide strength, shape Occluding (tight) junctions – control flow of solutes Channel (gap) junctions – allow communication and transport Signal junctions – facilitate communication (could also be involved in anchoring/ providing barrier and allowing transport ©Dr. Bilal Malik2024, [email protected]  Cell junctions ©Dr. Bilal Malik2024, [email protected]  Cell junctions organisation ©Dr. Bilal Malik2024, [email protected]  Cell-cell adhesion Cell-cell adhesion is selective – cells only adhere to specific types. Mediated by transmembrane adhesion proteins known as cell adhesion molecules (CAMs). These proteins span the cell membrane, one end linking to the cytoskeleton and other end linking to structures outside it Four groups: Cadherins (anchoring junctions) Selectins Integrins (anchoring junctions) Immunoglobulin (Ig superfamily) Selectins, integrins and most cadherins require Ca2+, Mg2+ or Mn2+. ©Dr. Bilal Malik2024, [email protected]  Cell-cell adhesion – Anchoring junctions ©Dr. Bilal Malik2024, [email protected]  Cell-cell adhesion – cadherins Classic cadherins β-catenin and P120 bind to the cytosolic tail – maintain stability β-catenin binds α-catenin – associates adherens junction with actin cytoskeleton – mechanism unknown. Play an important role in cell polarity. Depend on Ca 2+ therefore “Calcium adhering” Many different types, form a superfamily (180 members) Highly selective, therefore can form many different tissues Required for formation of morula and mediate cells adhesion Disrupted in cell culture by the protease, trypsin Bind directly or indirectly to adapter proteins that bind to actin filaments ©Dr. Bilal Malik2024, [email protected]  Cell-cell adhesion – cadherins ©Dr. Bilal Malik2024, [email protected]  Cell-cell adhesion – Tight junctions ©Dr. Bilal Malik2024, [email protected]  Cell-cell adhesion – Tight junctions ©Dr. Bilal Malik2024, [email protected]  Cell-cell adhesion – Tight junctions Specialised contacts between epithelial cells. Important for epithelial cell sheet function as barriers between fluid compartments e.g. blood brain barrier. Minimal adhesive strength, therefore associate with adherens junctions and desmosomes. ©Dr. Bilal Malik2024, [email protected]  Cell-cell adhesion – Gap junctions ©Dr. Bilal Malik2024, [email protected]  Cell-cell adhesion – Gap junctions ©Dr. Bilal Malik2024, [email protected]  Cell-cell adhesion – Gap junctions Within an individual tissue cells are linked by gap junctions. Provide direct connections between cytoplasm of adjacent cells. Regulated channels allowing ions and small molecules (