Epithelial Stem Cells and Differentiation PDF

ANAT 416 Epithelial Stem Cells and Differentiation Prof. Luke McCaffrey, PhD Goodman Cancer Institute Department of Oncology Department of Biochemistry Department of Medicine, Division of Experimental Medicine Epithelial cells: Essential building blocks of tissues and organs Line body and organ surf...

ANAT 416 Epithelial Stem Cells and Differentiation Prof. Luke McCaffrey, PhD Goodman Cancer Institute Department of Oncology Department of Biochemistry Department of Medicine, Division of Experimental Medicine Epithelial cells: Essential building blocks of tissues and organs Line body and organ surfaces Over 80% of cancers arise from epithelial tissues there are a lot of different patterns that epithelial cells can grow in: squamous, cuboidal, columnal, stratified vs simple Epithelial cells have a permeability layer thats mainly govenerned by tight junctions General organization of epithelial cells Apical membrane Absorption/secretion Tight junction Cell-cell adhesion Separates apical and lateral membranes Roles of polarized epithelia: Barrier between compartments Secretion Absorption Directional signal transduction Tissue organization Growth control 1. permeability barrier so that molecules and pathogens cannot pass through epithelial layer 2. acts to physically separates the basolateral and apical domains Lateral membrane Cell-cell adhesion Basal membrane Cell-matrix adhesion Organization of epithelial cells (Organelles) they have organized organelles —> nucleus tends to be at the bottom Golgi sits on top of that Nat Rev Mol Cell Biol 15, 225–242 (2014). A major phenotype of epithelial cells is Cell-Cell adheision —> tight, adherens, desmosomes, integrin Epithelial cell adhesion ZO1 ZO1 ZO2 There is a fluidity of these tisxues —> this comes from the cytoskeleton All adhesion molecules connect to the cytoskeleton ZO3 ZO3 Nectin Afadin Nectin β-catenin α-catenin Tight junction ZO2 Afadin Adherens junction E-cadherin E-cadherin β-catenin α-catenin Desmoplakin Plakoglobin Plakophilin Desmoglein Desmocollin Plakophilin Plakoglobin Desmoplakin Desmosome desmoglein + desmocollin Integrin adhesions Epithelial cell adhesion ZO1 ZO1 ZO2 Tight junction ZO2 ZO3 ZO3 Adhesions are dynamic!! Actin microfilaments Nectin Afadi n Nectin Cytoskeleton really connects all these cell-cell adhesions β-catenin α-catenin Afadi n E-cadherin E-cadherin they are not static cells that are glued together —> they can slide past eachother Adherens junction β-catenin α-catenin Desmoplakin Plakoglobin Plakophilin Keratin intermediate filaments Desmoglein Desmocollin Plakophilin Desmosome Plakoglobin Desmoplakin Extra-cellular matrix Integrin adhesions Apical-basal polarity is maintained by mutual exclusion of protein complexes -this property is lost early in tumour cells Crumbs complex Crb3 – transmembrane glycoprotein Pals1 – multidomain scaffold Patj – multidomain scaffold mutual exclusion: proteins will inhibit other complexes from forming in their domain. Many are scaffoling proteins that simply bind to other proteins —> except aPKC which phosphorylates other complexes, pushing them off the plasma membrane. The Par complex does this, but the Scrib complex recruites proteins and pushes the par complex off Par complex Par3 – multidomain scaffold Par6 – multidomain scaffold aPKC – kinase -emerges at sites of tight junctions, allows the membrane to split into apical and basal membranes Scrib complex Scrib – multidomain scaffold Dlg – multidomain scaffold Llgl – multidomain scaffold this is a basolateral determinate, so wherever this complex is, it will make it a basolateral membrane Epithelia have an INCREDIBLE capacity for self organisation Nat Rev Mol Cell Biol 15, 225–242 (2014). How do epithelial tubes form? 1. Tissue Folding Example, neural tube Neuroepithelium constitutes stem cells for central nervous system Neuroepithelial cells fold to form a tube Folding is driven by cell shape changes at the “hinge” apical constriction – reduces the size of the apical domain to generate wedge-shaped cells -driven by two things: cell shape changes, and cell intercolation (cells coming together and weaving) “hinge” hinging is driven bt apical constriction cell intercalation apical constriction Requires the polarity protein Scrib “Circletail” mouse has a mutation that truncates the protein Disrupts apical constriction and intercalation of cells to close the neural tube. Dev Biol 478, 59–75 (2021). How do epithelial tubes form? 2. A Cord hollowing Example, embryonic mammary gland A placode is established that grows out as a solid bud or cord of epithelial cells Micro lumen form in the solid cord and expand/merge to form an expanded lumen they push a part out of the center and create a tube Solid cord Microlumen formation Microlumen fusion Dev Biol 341(1), 34–55 (2010). Open lumen Curr Topics Dev Biol 154, 245–283 (2023). Formation of microlumen in non-polarized cells in many ways, it’s like cell division Non-polarized cell midbody AMIS = Apical Membrane Initiation Site Midbody formation is a symmetry-breaking event Apical pioneers (Par complex) accumulate at AMIS Apical determinants are recruited to the AMIS to form a pre-luminal apical patch (PAP) Expansion of the lumen to form an open lumen Repulsion by apical glycoproteins Pumping of fluid into the lumen PAP = Pre-luminal Apical Patch Traffic 17(12), 1244–1261 (2016). How do epithelial tubes form? 2. B Cavitation Similar to cord hollowing, but includes apoptosis to clear internal cells Micro lumen form in the solid cord and expand/merge to form an expanded lumen detatch the cells in the middle so they can die Inner cells detach and undergo apoptosis Solid cord Microlumen formation Microlumen Fusion, exclusion of internal cells Apoptosis Open lumen Dev Biol 341(1), 34–55 (2010). Epithelial cells display phenotypic flexibility Epithelial-Mesenchymal Transition (EMT) Epithelial cell Mesenchymal cell EVERYTHING is different between epithelial to mesenchymal cells Core EMT changes Cytoskeletal remodelling Loss of apical-basal polarity Cell-cell adhesion weakening Cell-matrix adhesion Cell individualization Establishment of frontback cell polarity Change in cell motility (collective vs mesenchymal) All of these changes are necessary to be called EMT if you have one marker, thats not enough - everything has to change for EMT. But, there are intermediate states between the two. Nat Rev Mol Cell Biol 21, 341–352 (2020). Stem cell renewal and differentiation most epithelial cells have some sort of stem cell renewal process a stem cell that renews less frequently, there are less errors in replication and the errors will be passed on in a less harmful way Stem cell Restricted progenitor Transit amplifying cell Differentiated cell Stem cell potency Multipotent stem cells – Multiple cell potential – Can differentiate into many closely related cell types – Example: Hematopoietic stem Bipotent stem cells – Produce two types of related cells – Example: Prostate basal cells give rise to basal and luminal stem cells Unipotent stem cells (progenitors) – Produce a single cell type – Example: Adult mammary gland Stem cell differentiation Stem cells – have the potential to derive multiple different cell lineages – have unlimited self-renewal capacity Progenitor cells – Often multipotent – have limited renewal capacity Transit amplifying cells – Rapidly dividing progeny of stem or progenitor cells – Allows expansion of cells from small number of stem cells Differentiated cells – Specialized functions (absorption, secretion, electrical conductance, barrier, structure/support, etc.) Lineage tracing YFP YFP YFP mRNA Lineage tracing Nat Rev Cancer. 2013 Oct;13(10):727-38. Epithelial tissues are derived from embryonic stem cells and maintained by adult stem cells there was a lot of interest in the stem cell in maintaining the mammary gland Functional Assays to track stem cells in mamm. cells: Case study 1: Mammary Gland 1. you inject what you think is a stem cell into a native tissue, you can inject cells into where the mammary Embryo Adult gland would be and see. K5 K8 Experimental design K5 K8 Embryo Adult K14 – all cells K14 – basal cells K8 – some cells K8 – luminal cells Mammary gland made up of two cell types: luminal cells that surround a central lumen , most cancer cells arise from this Myoepithelial layer - contractile epithelial layer during pregnrancy helps milk squeeze down the milk ducts ONLY when you inject stem cells into the embryo mammary gland do you get basal cells forming. In the embryo, they labelled K14 cells with a tracer - Cre, activated only in Basal cells. —> next page Nature 479, 189-193 (2011). Epithelial tissues are derived from embryonic stem cells and maintained by adult stem cells Case study 1: Mammary Gland Conclusion: Embryonic K14+ cells give rise to both cell types in the adult gland. Basal cells Luminal cells Luminal Basal 1. Label K14+ cells in the embryonic mammary gland at E17 2. YFP expression measured in basal and luminal cell types in the adult (5 week) mammary gland they find that ALL of the basal and luminal cells are green. They find that K14 stem cells are bipolar Nature 479, 189-193 (2011). Epithelial tissues are derived from embryonic stem cells and maintained by adult stem cells Case study 1: Mammary Gland Luminal cells Basal Conclusion: Adult K14+ cells only give rise to K14+ basal cells. unipotent - only can give rise to basal cells Basal cells Luminal 1. Label K14+ cells in the adult mammary gland 2. YFP expression measured in basal and luminal cell types 10 weeks later Nature 479, 189-193 (2011). Epithelial tissues are derived from embryonic stem cells and maintained by adult stem cells Case study 1: Mammary Gland Basal cells Luminal cells -only under situations where there are NO luminal or myoepithelial cells can it become a bipotent stem cell Conclusion: Adult K8+ cells only give rise to K8+ luminal cells. Embryonic mammary stem cells are bipotent, and adult mammary stem cells are unipotent. Luminal Basal 1. Label K8+ cells in the adult mammary gland 2. YFP expression measured in basal and luminal cell types 10 weeks later Nature 479, 189-193 (2011). Epithelial tissues are derived from embryonic stem cells and maintained by adult stem cells Case study 2: Multipotent Intestinal stem cells Colon-based columnar (CBC) cells were proposed to contain intestinal stem cells CBCs exclusively express high levels of Lgr5 Lgr5 binds Rspo to sustain Wnt signaling EdU labels proliferating cells Lgr5 promoter drives GFP expression CBC = colon based columnar cells LRC = label retaining cells Science. 2010 Jan 29;327(5965):542-5; PNAS 109(2), 466-471 (2011) Multicolour lineage tracing shows that Lgr5+ stem cells maintain the intestinal epithelium Lgr5 Cre Each villus is a single colour -> therefore each is maintained by a different stem cell pool Multicolour lineage tracing shows that Lgr5+ stem cells maintain the intestinal epithelium: Used this to prove how stem cells divide cells are randomly labelled as one of these four colours each of these crypt has the stem cells required to maintain the villi above it Lgr5+ stem cells at the base of intestinal crypts Lgr5+ intestinal stem cells Lgr5+ stem cells reside at the base of the intestinal cleft Random labeling creates heterogeneous stem cell colours in each crypt Villi tend towards a single dominant colour How do multi-coloured stem cells lead to a single-coloured villus? – A sub population of Lgr5+ cells are stem cells? – Competition between stem cells? did all stem cells have to asymetrically divide? Exp Cell Res. 2011 Nov 15;317(19):2719-24 How do cells maintain self-renewal and contribute to differentiation? Symmetric stem cell divisions expand the stem cell population – One stem cell may differentiate Differentiation Asymmetric divisions regenerate a stem cell and generate a cell that will differentiate Neutral drift: an alternative to asymmetric cell division they argued that asymmetric cell divisions CANNOT occur in mammary tissue Lgr5+ intestinal stem cells Lgr5+ stem cells reside at the base of the intestinal cleft Random labeling creates heterogeneous stem cell colours in each crypt How does this lead to a single coloured villus? they argued that you would always have multicoloured villi but you DON’T so you have neutral drift rather that asymmetric division. There is not something driving it, it is a good tumour suppresor mechanism. Exp Cell Res. 2011 Nov 15;317(19):2719-24 Adult epithelial stem cells can self-organize “organoids” with tissue-specific structures and function Organoids Three-dimensional (3D) in vitro systems, which model organs in terms of differentiated cell types and their spatial arrangement, morphology, and functionality Independent of auxiliary systems, such as blood vessels, a nervous system, and stromal cells. Utilizes defined culture conditions including an extracellular matrix scaffold plus specific growth factors Self-organization -> local interactions between cells that are initially disordered lead to the emergence of tissue-like patterns J. Mol Med 99, 449-462 (2021). responses to drugs = more accurate in organoid models than rissue models Tissue-derived and pluripotent stem-cell derived organoids Benefits Can be derived from human tissues Can be genetically manipulated Can be used for therapeutic evaluation or screening Limitations Only multi-potent stem cells and their progeny Co-cultures with additional components in possible in some cases, but culture conditions are a challenge Apical membrane is on inside Frontiers Cell Dev Biol 10, doi.org/10.3389/fcell2022.854740 (2022). Difficult to target can’t absorptive side, which is manipulatewhere drugs would lumen like normally in enter real tissue Functional engraftment of intestinal organoids to treat disease Case study 3: Colitis Ulcerative colitis: an inflammatory bowel disease (IBD) that causes inflammation and ulcers in the digestive tract. Treatment: anti-inflammatory or immune suppression medication; surgery to remove the colon Nat Prot 17, 649-671 (2022); Nat Med 18(4):618-623, (2012). Functional engraftment of intestinal organoids to treat disease Case study 3: Colitis you can use organoids in a variety of different tissues for regerenation is very useful, they can be implanted for higher/ faster rates of recovery. Organoids implant into regions with the most epithelial damage following DSS treatment. Mice with transplanted organoids recovered faster following DSS treatment. Nat Med 18, 618-623 (2012). Organoids can be used to study rare diseases Case study 4: Cerebral organoids model human brain development and microencephaly. Microcephaly is a condition in which a baby’s head is much smaller than expected for their age. This is due to the brain not developing properly during pregnancy or soon after birth. Common symptoms: small head and small brain Seizures Delayed speech Movement and balance difficulty Problems feeding, eating and swallowing; Vision and hearing problems. Causes: Genetic abnormalities Brain injury before birth Complications during labour and delivery Stroke or hemorrhage Malnutrition after birth Nature 501, 373-379 (2013). Case study 4: Cerebral organoids model human brain development and microencephaly. what they found: these will spontaneously organize into brainlike tissues went in with different markers and different regions of the brain, would all be formed in these brain organioids, and that regions of brains that were close together would also organize close together in the organoids - indicating some signalling that occured in the organoids Nature 501, 373-379 (2013). Case study 4: Cerebral organoids model human brain development and microencephaly. Nature 501, 373-379 (2013). Case study 4: Cerebral organoids model human brain development and microencephaly. Patient with severe microencephaly has mutation in CDK5RAP2 – microtubule associated protein linked to brain size variations.. Generate brain organoids from iPS cells derived from patient fibroblasts. mutation causes early differentiation of organoid cells into neurons and this could maybe explain neuroencephaly Premature neuronal differentiation in patient organoids. This could explain the disease phenotype. GFP+ neurons; GFP+ progenitors Nature 501, 373-379 (2013).