Mesenchymal stem cells

• What are mesenchymal stem cells?

  Mesenchymal stem cells (MSCs) are multipotent stromal cells that have the ability to self-renew and also exhibit multilineage differentiation

  • Also known as bone marrow stromal cells or mesenchymal stromal cells
• How can mesenchymal stem cells be isolated from the bone marrow?

  • Iliac crest aspiration via. wide-bore needle - withdraw cellular content from bone marrow
  • Density gradient centrifugation - mononuclear cells (including MSCs) sit at the interface between ficoll and serum (plasma) whereas other cells will be pelleted

  
• What are the adherent properties of MSCs?

  Most cells within the BM are non-adherent (e.g. blood cells) but MSCs are adherent and form fibroblast-like cells when grown in culture

  • Spindle-shaped
  • Form fibroblastic colonies - colony forming unit fibroblastic number (CFU-F) quantifies the number of MSCs progenitors in bone marrow samples

  
• What did Pittenger et al (1999) describe?

  The multipotentiality of human MSCs

  • Treated MSCs in 3 different ways - MSCs differentiated into fat, cartilage, or bone tissue depending on the combination of regulatory signals used
  • Some signals are necessary, but other growth factors simply aid the transition
  • Fat, cartilage, and bone = main 3 lineages - other lineages possible but multipotent and not pluripotent
• What are the common positive stains for fat, cartilage, and bone?

  • Fat = Oil Red O (stains neutral triglycerise and lipids)
  • Cartilage = Collage II (stains collagen type II, a major component of cartilage) and Toluidine blue (stains glycose aminoglycans deposited within cartilage tissue)
  • Bone = Alkaline phosphatase (stain for alkaline phosphatase expression, key marker of pluripotent stem cells) and Alizarin Red (stains the calcium containing osteocytes undergoing osteogenesis)
• What are the names of the processes for development of fat, cartilage, and bone?

  • Fat = adipogenesis
  • Cartilage = chondrogenesis
  • Bone = osteogenesis
• What are the regulatory signals that induce MSC differentiation?

  Fat

  • 1-methyl-3-isobutylxanthine (IBMX)
  • Dexamethasone
  • Insulin
  • Indomethacin

  Cartilage

  • High density
  • TGF-B3
  • Serum-free
  • (Additional aid) IGF-1, BMPs 2, 4, 6, 12 and 13

  Bone

  • Dexamethasone
  • B-glycerol phosphate
  • Ascorbate
  • 10% w/v FBS
  • (Additional aid) BMP-2, bFGF
• When are mesenchymal lineage markers expressed?

  • Early culture express multiple mesenchymal lineage markers - the cells are non-committed and have differences in their ability to commit to different lineages
• What is the relationship between osteogenic and adipogenic lineage commitment and differentiation?

  Inverse relationship - differentiation towards an osteoblastic phenotype occurs at the expense of an adipocytic phenotype

  
  

  This balance is regulated by numerous, intersecting signalling pathways that converge on the regulation of TF factors: PPARγ and Runx2 which are considered master regulators of adipogenesis and osteogenesis
• Describe the signalling pathtways for MSC differentiation into fat

  • Major regulator is PPARy
  • PPARy is blocked by Wnt - Wnt signalling promotes osteogenesis but inhibits adipogenesis
  • Addition of insulin/indomethacin/IBMX in culture promotes PPARy expression - drives adipogenic differentiation of MSC

  
• Describe the signalling pathtways for MSC differentiation into bone

  • Major regulator is TF Runx2
  • Addition of Dex and B-glycerol phosphate (in culture) promotes Runx2 expression which drives osteoblastic differentiation
  • Ascorbic acid aids in production of collagen I (main ECM component of bone tissue)
  • Bone morphogenetic proteins (BMPs) aid this by stimulating Runx2 expression and hence osteogenesis
  • Also promoted by Wnt signalling (also responsible for self-renewal of MSCs)

  
• Describe the signalling pathtways for MSC differentiation into cartilage

  • Both Wnt and Runx2 inhibit chondrogenesis - leads to osteogenesis instead
  • Main regulator is Sox9 - promoted by Shh and BMPs
  • Generate pre-hypertrophic chondrocytes - addition of TGFb promotes SMAD3 which inhibits Runx2, but TGFb also promotes Sox9
  • Generate fully differentiated hypertrophic chondrocytes - Indian hedgehog (Ihh) + Wnt + Runx2 is a positional feedback loop, Ihh promotes terminal chondrogenesis
  • Dependent on where cells are in within cartilage tissue - experience either terminal differentiation or don't
• What are hypertrophic chondrocytes?

  Enlarged cartilage cells - swell with water (larger, rounder). They lie between the cartilage and bone components, secreting several factors which promote the cartilage-to-bone transition, including MMPs, Ihh, BMPs, VEGF, RANKL/OPG

  
  

  Hypertrophic chondrocytes may also directly become bone cells, contributing to bone formation
• Are MSCs pluripotent or multipotent?

  Multipotent - replicate only for a finite time, self-renew to form new MSCs and differentiate to other phenotypes

  
  

  This means MSCs will not form teratomas
• What are the main souces of mesenchymal stem cells?

  Found in almost every tissue within the body (* = main sources) - this factor is one reason why we see MSC heterogeneity

  • Bone marrow *
  • Adipose tissue *
  • Skeletal muscle
  • Umbilical cord *
  • Synovium
  • Circulatory system
  • Spleen
  • Kidney
  • Lung
  • Dental pulp
  • Conjunctiva
  • Thymus
  • Amniotic fluid
  • Foetal tissues
• Are MSCs functionally equivalent?

  No

  • Different sources may yield cells with similar phenotypic characteristics but there are still differences in surface markers, proliferation, and differentiation
  • Sometimes have different names depending on where the MSCs are derived from

  
• What must be considered when selecting a source of HSCs for therapeutic use?

  Heterogeneity of source cells

  • BM - osteo-, chondro-, and adipogenic differentiation potential
  • Adipose - osteo-, chondro-, and adipogenic differentiation potential
  • Umbilical cord blood - osteo- and chondrogenic but NOT adipogenic differentiation potential
• What is the effect of ageing on MSCs?

  Ability to differentiate MSCs decreases with age so get less MSCs as we're older - therapeutically better to derive MSCs from younger people, particularly cord blood is attractive

  
• What is the MSC niche?

  MSCs reside in many different tissues throughout the body - conditions and signalling environments within the different tissues are different

  Bone marrow

  • MSCs secrete soluble factors which control HSC maintenance

  Other factors - blood vessels, ECM, gradient of oxygen, MSCs, various differentiated cells
• What is the role of MSCs?

  • Providing daughter cells that differentiate and participate in repair
  • Mobilisation and homing to distant sites of injury
  • Secretion of paracrine factors that support wound repair by recruiting other cell types and modulating the immune response
• What are the therapeutic applications of MSCs?

  • Off-the-shelf therapy (autologous or allogeneic) - MSCs elicit some immune response
  • Prevention of GvHD - MSCs have immunomodulatory functions functions
  • Therapy for strokes, heart attacks, or other injury - MSCs home to these sites or attracted by chemokines in blood
  • Delivery of therapeutic proteins
  • Cell-free exosome therapeutics (harvested exosomes from MSCs)
  • Tissue engineering
• What is the effect of MSC priming?

  Increases therapeutic efficacy by applying external stimuli including GFs, small molecules, hypoxia, and 3D cultures

  • Interferon gamma and TNFa - improve immunomodulatory function
  • Lipopolysaccharide - improve immunosuppressive function
  • Hypoxia - secretes more factors involved in angiogenesis
  • 3D culture - improve innate MSC abilities
• What is the therapeutic use of MSCs for a heart attack?

  • MSCs home to the site of damage
  • Release paracrine factors to aid in repair
  • Recruit cardiac progenitor cells (CPCs) and transdifferentiate into endothelial cells to partake in vasculogenesis
  • Recruit cardiac stem cells (CSCs) and transdifferentiate into cardiomyocytes to aid in cardiomyogenesis

  Currently more evidence for paracrine factors than vasculogenesis/cardiomyogenesis

  
• What is the therapeutic use of MSCs for cancer?

  • MSCs are involved in homeostasis and repair - homing to sites of injury
  • As tumours are like a wound that doesn't heal, MSCs can home to tumours and negatively affect tumour growth
  • Different cytokines/chemokines are released and shown to attract MSCs
  • Hypoxia, ECM composition, extracellular acidity and inflammatory component of the stroma are crucial mediators of the outcome of MSC action in tumours

  
• How does MSC priming work?

  Pre-treat MSCs and effectively make them into anti-tumour cells through activation using toll-like receptors (TLRs) - activates immune cells via. binding of pathogen-derived molecules

  TLR4 priming - with lipopolysaccharide produces proinflammatory MSCs (MSC1)

  • Increases in proinflammatory cytokines and cells
  • Increases inflammation in different animal models
  • Decreases growth of tumours

  TLR3 priming - with poly(I:C) produces anti-inflammatory MSCs (MSC2)

  • Decreases T cell activation
  • Decreases inflammation in different animal models
  • Increases tumours and metastasis

  
• What is the therapeutic use of MSCs for cancer treatment?

  • Delivery of proteins/drugs to kill cells within tumours - MSCs can home to tumour sites
  • Combination cell-gene therapy where MSCs transfected with a TRAIL genes which kills tumour cells, and takes advantage of homing ability of MSCs - potential as off-the-shelf therapy - not immunogenic so large numbers grown up, modified, and delivered
  • Anti-angiogenic strategies - engineer MSCs with different proteins that are anti-cancer then induce apoptosis/reduce metastasis or miRNAs (e.g. paclitaxel, doxycycline) that downregulate tumour growth