8. Remodelling

Learning Outcomes: L16-17

Explain the organisation of the extracellular matrix (ECM) and the different ECM components involved Explain the mechanisms governing ECM assembly Describe how cell function can be regulated by the ECM Explain the transcriptional control of embryonic stem cells and the defining characteristics of stem cells Describe how stem cells can be induced to generate specific differentiated cell types and how differentiated function can be experimentally tested Explain how pluripotency can be induced in somatic cells (SCNT and iPS cells) Give examples of the therapeutic potential of stem cells

The ECM

Tissues composed of cells and often ECM  ECM composed of proteins and proteoglycans Connective tissues form framework of vertebrate body but amounts vary for different organisms Physical properties of different tissues can vary from hard structures (bone) to transparent (cornea) Cells have physical interaction with ECM: integrins on cell membrane Interaction important for cell survival

ECM: Support Structure

Inert scaffold to stabilise physical structure of tissues  Helps define cellular phenotype of cells residing within it: what cell is and what it does  Acts as storage compartment for cell signalling factors

Composition of ECM: Collagens

~25% of total mamamalian proteins- most abundant protein  Homo- or hetero- trimers-> form triple helical structure (alpha chains) Give tissues tensile strength  Gly-X-Y repeats for aa seq (X and Y often proline, hydroxyproline, hydroxylysine and glycine essential every 3rd residue) Vitamin C essential cofactor- hence hydroxylation important for collagen function Effects of impaired collagen synthesis through vitamin C deficiency Lack of collagen/inappropriately synthesised= lose structural support, prone to easy wounding and don’t repair well

Structure of Collagen Fibres

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Collagens: Why is Glycine Needed?

Small aa that enables triple helical twisting to form a collagen molecule Collagen molecules associate with each other through covalent bonding to form fibril Fibrils associate with eachother to form fibers

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Different Types of Collagen

Collagens are diverse but there are some similarites in their structure Different organisations of homo or hetero trimers but always 3 alpha chains  Different functions depending on structure and location in body

Collagen Synthesis + Secretion from Cell

Synthesis through ribosomes into pro-alpha chain  Hydroxylation of predominantly lysine and proline- uses vitamin C  Glycosylation of alpha chain  Spontaneous self assembly of alpha chain into triple helix (pro-peptide at end that dont form helix) Procollagen secreted into extraceullular space Cleavage of pro-peptides forms collagen which forms fibrils then fibres outside cell  

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Collagen Synthesis + Secretion from Cell

Collagen Gene Mutations

Osteogenesis Imperfecta (OI, brittle bone disease) Caused by mutations in α1(I) or α2(I) genes ​​​​​​​ Dermatosparaxis- inherited disorder Causes fragile and loose skin with substantial bruising and bleeding  Caused by mutations in N-terminal propeptidase that removes pro-peptides in type I and III collagen ​​​​​​​Proteolytic processing of procollagen required for collagen assembly into fibrils N-terminal propetidase= Metalloproteinase ADAMTS-2  

ECM Components: Proteoglycans + GAGs

Glycosaminoglycans (GAGs) attached to core protein to form proteoglycan (except hyaluronan) Eg. Aggrecan= core protein decorin with lots of GAGs attached Large space filling molecule, hydrated water trapping structure Space filling function and compressive strength important in cartillage Aggrecan lost in arthiritis 

Aggrecan Structure

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Four Classes of GAGs

GAGs formed by polymerisation of specific dissacharides and modifications: 1. Hyaluronan (HA)- binds other proteoglycans to build bigger structures 2. Chondroitin Sulphate (CS) 3. Heparan Sulphate (HS) 4. Keratan Sulphate (KS)

ECM Components: Proteoglycans + GAGs

Variety of different proteoglycans enabled by different combinations of core proteins with GAGs and numbers of GAGs affect their function Some basic structures->

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Assembly of Proteoglycans

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Linking sugars enable attachment of GAGs to core protein n= multiples of GAGs away from core protein

Hyaluronan: GAG that Links Proteoglycans

Hyaluronan made of repeating disaccharide units ~5000 Hyaluronan binds proteoglycan (aggrecan) through link proteins Multiple proteoglycans can bind to hyaluronan

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Proteoglycans and Cell Signalling

Major role in signalling between cells- they bind various secreted molecules, enhance or inhibit signalling activity of growth factors  FGF signalling (fibroblast): Heparan sulphate proteoglycan (HSPGs) help control FGF signalling strength- FGF binding FGF-R regulated by its presentation to receptor by proteoglycan Free FGF doesnt have bioactivity Enhance signal= FGF binds HS proteoglycan (syndecan) in cell membrane- depending on proteoglycan expression levels and proximity to FGF-R can present FGF to receptor and increase the signal Inhibit signal= HSPGs free in extracellular space may not be near receptors and can sequester FGF

Proteoglycans and FGF Signalling

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ECM Components: Elastin

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Gives elasticity to tissues, help regulate tissue function Consists of covalently linked monomers Elasticity to connective tissues: Elastin is the dominant ECM component in arteries ~50% Also in lungs and elastic ligaments Maintains structure and shape

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Elastin Associated Disorders

Cutis Laxa Rare inherited disorder of connective tissues Skin inelastic and hangs loosely Caused by mutation in genes that affect elastin formation and function May cause hypermobility of joints

ECM Components: Fibronectin

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Large glycoprotein, helps matrix organisation Multi-adhesive protein (binds many things) Homodimer has many binding motifs for proteoglycans, cells, collagen RGD domain= recognised by integrins- enables interactions between cells

Regulation of Cell Behaviour by ECM

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Cells need to attach to ECM to grow and proliferate (and survive)- blood cells dont  =Anchorage Dependence  Mediated by integrins and signals they generate Intracellular actin cytoskelton overlaps with extracellular fibronectin via integrin interactions

Regulation of Cell Behaviour by ECM

How ECM geometry and organisation can regulate cell function Experiment: Cells grown on different fibronectin shapes Make fibronectin cell sized and shape into tear drop, apply one cell (only thing that can bind) Cell takes up teardrop shape- orders actin cytoskeleton into that shape and forms lamellipodium Pulled into shape of migrating cell- so wants to migrate, ECM controls function

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The Basal Lamina: Basement Membrane

Basement membrane is a specialised ECM structure made up of different ECM components Separates epidermis (epidermal cells) from dermis (skin) Dermis made from collagen network underlying the connective tissue

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The Basal Lamina: Basement Membrane

Basement membrane composed of: Entactin- multiadhesive protein  Perlecan- HSPG Lamin Type IV collagen- forms sheet like structure Acts as selective barrier for cell movement

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Basement Membrane Components

Type IV collagen: Defines back bone of flat sheet like structure due to its organisation Triple helical structure of alpha chains  Monomer has globular head (C-), kinked structure Head allows end-on-end associations and lateral associations= forms planar network of collagen Laminin: Crucifix shaped protein  Binds to many other things: HSPG, collagen, fibronectin, entactin

Basement Membrane Components

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Stem Cell Key Features

Self renewal- Divide asymmetrically to give rise to another stem cell (maintain stem cell pool) and transit amplifying cell (differentiate) Differentiation- goes through precursor stage and becomes specialised cell ESC have ability to live longer- increased telomerase activity, more rounds of cell division

Stem Cell Key Features

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Potency of ESC

Potency= repertoire of different cell types SCs can differentiate into  Totipotent= Ability to develop into entire organism Including supportive cells (extraembryonic tissues), enable embryo to implant and survive(umbilical cord and placenta) Stages(early embryogenesis): fertilised egg, daughter cells upto day 4 following fertilisation, before blastocyst Pluripotent=Ability to develop into virtually every cell type (3 germ layers) Don't form supportive tissue needed for foetal development, unable to generate new organism on their own Stages: ES cells of inner cell mass in blastocyst

Blastocyst Structure

Fluid filled ball of cells Outer layer= trophectoderm from Exe tissue (trophoblasts) Inner cell mass= where pluripotent ESCs reside  

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Determining Pluripotency-Teratoma Assay

In vivo method of determining pluripotency , teratoma=benign cancer with tissues from all 3 germ layers  Experiment:  Take population of suggested pluripotent SCs,inject into immunocompromised mouse If pluripotent-> develops teratoma, all 3 germ layers Analysis: ​​​​​​​Histological analysis- look at sections of teratoma, do we see structures/cells from each germ layer eg gut-like epithelium, cartilage, neuroepithelial rosettes Stem cell assay- ​​​​​​​Histological analysis alone not enough, need to create a defect and see if suggested pluripotent cells restore functionality in defect

Histological Analysis

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Transcriptional Control of ESCs

Pluripotent TFs: Oct4, Nanog, Sox2 (act collectively to regulate pluripotency) Expressed early in pluripotent ESCs in inner cell mass How they regulate: Activate pluripotency associated pathways, active promoters of self-renewal genes Suppress pathways that progress cell differentiation, silent promoters of developmental genes Upon differentiation ESCs lose expression of TFs

Transcriptional Control of ESCs

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Therapeutic Potential of ESCs

Generate specific cell types/tissue structures-replace worn/diseased body parts: Neurodegenerative diseases Diabetes Corneal defects Cardiovascular diseases Musculoskeletal disorders Other applications include: Toxicity testing- replace animal testing? Model systems- 3D co-culture system engineered from stem cells that can differentiate and mimic developmental processes

Potential difficulties: Need differentiation of cells into specific cell types to form functional tissues Need ALL cells to COMPLETELY differentiate- no pluripotent SC which could give rise to a teratoma

Therapeutic Potential: Parkinson's

Characteristic of Parkinson's= Dopamine producing neurones in CNS die off Loss of dopamine binding causes neurones to fire out of control TF Nurr1 involved in differentiation of neuronal precursors into dopamine-producing neurones Growth factors FGF8 and sonic hedgehog (Shh) are required for dopamine producing neurones in normal midbrain TFs and GFs= signals that can be applied to stem cells and induce differentiation

Therapeutic Potential: Parkinson's

Experiment: Indication of formation of dopamine producing neurones from ESCs GAPDH= loading control  WT ESC lines exposed to GFs- increased expression of markers for DA-producing neurones ESC lines that overexpress Nurr1 (TF) exposed to GFs- significant further increase of expression of markers    

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Indicated ESCs may be forming DA-producing neurones, need functional test to be sure Experiment(functional test): Use animal model where DA-producing neurones killed off to mimic Parkinson's Inject stem cells (from previous) and look for restored functionality- removal of Parkinson's characteristic 

Therapeutic Potential: Parkinson's

Therapeutic Potential

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Example of transplantation of neuronal cells differentiated from human ESCs Paralysis in rat model- equivalent to spinal cord injury  Following transplantation, injection of neurones into spinal cord lesions and restore some function

Therapeutic Cloning and ESCs

 Somatic cell nuclear transfer(SCNT): Fusion of a somatic (adult) cell nucleus with emptied egg(enucleated) Reverts adult cell back to its pluripotent state Technique used to create dolly the sheep, used to create an organism = reproductive cloning When used to generate a blastocyst from which ICM  pluripotent cells are isolated and expanded in vitro= therapeutic cloning Differentiate them for therapeutic processes, genetically identical to donor nucleus so wont be rejected  

Natural Reproduction vs SCNT

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Adult Stem Cells

Effectively in every tissue and maintain tissue ability to self-renew Ability deteriorates with age Tissue repair and remodelling  Multipotent

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Adult Stem Cell: Multipotent

Stem cells that can differentiate into more than one cell type But more restricted differentiation potential than pluripotent stem cells Generally have ability to differentiate into the cells of tissues in which they reside

Therapeutic potential: Allows use of autologous cells Fewer safety concern than ESCs- no risk of teratoma Fewer ethical concerns than ESC- no creating embryos in clinic Examples of adult stem cell for therapeutic processes: Haematopoietic stem cells (HSC) Mesenchymal  Epidermal- skin grafts/burns Neural- Spinal cord injury Limbal (corneal)- treat corneas

Haematopoietic Stem Cells: HSCs

HSC transplants are effectively blood and marrow transplants  Massive increase in autologous (own cells) and allogenic (different donor to recipient) blood marrow transplants through the years More autologous in recent years

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Mesenchymal Stem Cells (MSC)

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Found in BM and give rise to structural tissues like bone and cartilage Good for diseases that affect skeleton-arthiritis Osteogenesis Imperfecta (OI, brittle bone disease): Genetic, mutation in genes coding type I collagen Potential treatment using adult SC therapy  Allogenic BM transplant using MSC Restore strength of skeleton to some extent and enables normal collagen production

Cornea and Limbal Stem Cells

Cornea made up of collagen (stroma) surrounded by epithelium tissue (outer layer of cornea) Limbus region stem cells give rise to epithelial corneal cells that maintain health and support corneal structure

   

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Limbal Stem Cell Therapy

Corneas subject to damage/injury- loss of corneal function/damage may benefit from limbal stem cell therapy to restore lost function How it works: Cornea and surrounding limbus- biopsy to get viable limbal stem cells  Expand cells by growing in petri dish Replacing 3D structure: Use amniotic membrane- has same physical properties as cornea (translucent, type I collagen) Populate membrane with limbal stem cells + induce differentiation Can transplant whole cornea structure  

Limbal Stem Cell Therapy

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Limbal Stem Cell Therapy

2 different approaches: ​​​​​​​Generate enough limbal stem cells to use in cell based therapy- injection of stem cells into damaged cornea Generate stem cells to create 3D structure that replaces cornea Histological analysis= look for markers of epithelial corneal cells  Applications of limbal stem cell therapy: ​​​​​​​Limbal stem cell deficiency- cell based therapy  Loss of corneal structure- injury/burns- 3D transplant  Corneal clouding due to aging- transplant 

Induced Pluripotent Stem Cells (iPS)

Oct-4, Sox2 and Nanog TFs and others involved in regulating ESC pluripotency, understanding regulatory control of pluripotency underpinned iPS cell development: Introducing genes associated with ESC plutipotency (TFs) into somatic cells eg fibroblasts could reprogram somatic cell to pluripotent stem cell Introduction of 4 or fewer factors was sufficient  Some genes include: Oct-4, Sox-2, Nanog, Lin28, Klf4, c-Myc​​​​​​​

Induced Pluripotent Stem Cells (iPS)

Experiment: Using viral delivery system, introduced factors into somatic fibroblasts Look for formation of stem-cell like colonies (compact clusters) Do induced pluripotent stem cells express telomerase, ESC surface markers, and differentiate into 3 germ layers (Teratoma)?

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Induced Pluripotent Stem Cells (iPS)

Issues: Reprogramming typically inefficient (<1%)- depends on number of cells that take up virus/vector carrying reprogramming factors and number of ones that do that actually undergo reprogramming Use viral delivery systems- safety concern Use oncogenes (cMyc)- cancer risk  Epigeneitc memory of parent cell (unlike SCNT)- can return back to ESC but retain epigenetics of original fibroblast Teratoma risk

Example of iPS Use

Treat sickle cell anaemia in mouse model: Caused by mutation in haemoglobin gene  How it works: Isolate cells from mouse with anaemia and expand them  Infect cells with reprogramming factors and reverse back to pluripotent state Correct mutation using genetic engineering (CRISPR/Cas9) and differentiate into haemoglobin carrying cells  Put back in mouse model and look for restored functionality   

Example of iPS Use

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Potential of iPS Cells

Take own cells, revert back to ESC and differentiate (with or without genetic mutation) Grow differentiated cell type and replace back into individual  No risk of immunological rejection- same genetics Used for transplant or in vitro screening 

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