Mesenchymal Stem Cells Flashcards
What are MSCs?
- → Non-haematopoetic stem-like cells that can be isolated from most mesenchymal tissues such as bone marrow, fat, blood vessels and umbilical cord
- In culture, MSCs defined as plastic-adherant, fibroblasts-like cells which are able to self-renew and differentiate into bone, adipose and cartilage
Nomenclature:
• Marrow stromal cells, mesenchymal stem cells etc… nomenclature still controversial
How were MSCs first described
• Freidenstein 1974
• Identified cells in the stroma of the bone marrow that could form bone, fat and cartilage cells
• Cells demonstrated:
− tight adherence to tissue culture plastic
− multipotent differentiation in culture and in vivo
− spindle-like morphology
− single cell-derived colonies
• Described them initially as CFU-fibroblastic
What was the early understanding of MSCs?
Owen and Freidenstein, 1988: Stromal cells , marrow derived osteogenic precursors
• Evidence for stromal stem cells present in the soft connective tissue associated with marrow and bone surfaces
• Able to give rise to a number of different cell lines
• Fibroblastic colonies formed when marrow cells cultured in vivo
• CFU-F assays demonstrated that some CFU-F have high ability for self-renewal and multipotency whereas some have more limited potential
• Different stromal cell lines can be promoted under different culture conditions → more specific markers for the different lines required
Where do we find MSCs?
• Stem cell niche → stem cell niche controls MSC function
• Niches vary from tissue to tissue but generally:
− MSCs surrounded by stromal cells
− Cell-cell adhesion, receptor ligand binding and gap junctions will mediate interaction
− Niche also has ECM that provides structural support
− Soluble factors provide trophic support
Why are MSCs important?
- Regulation of HSCs
− CAR cells (CXCL12 abundant reticular cells) thought to be progeny of MSCs
− Endosteal niche – CAR cells secrete factors responsible for mainternance of HSCs:
− Differentiation of osteoblasts (which give support for HSCs)
− Differentiation of adipocytes (which negatively regulate HSCs)
− Quiescence, retention and maintenance of HSCs
− Perivascular niche – CAR cells secrete factors responsible for activation of HSCs:
− Activation, division, differentiation and mobilisation of HSCs - Tissue repair:
• In vivo, the classic paradigm always been that activation and mobilization of MSCs, fibroblasts and cells of the immune system repair an injured tissue by MSC integration and differentiation.
• We are now increasingly finding cell empowerment → MSCs are trophic factories, produce molecules that stimulate other cells.
− In response to cells of the immune system, tissue damage, high cytokine levels, hypoxia etc… get activation of MSCs to produce immunosuppressive factors, GFs to stimulate fibroblasts to produce new tissue, GFs that stimulate endothelial cells for angiogenesis, and factors that stimulate progenitor cells of that particular tissue to differentiate and repair – rather than MSCs being the ones to differentiate.
What is the differentiation potential of MSCs?
• Multipotent differentiation potential towards mesenchymal lineages
− Bone, cartilage, muscle, marrow, ligaments, adipose, connective tissue
• In vivo, we particularly see osteocytes and adipocytes
• Don’t really see much chondrocytes (cartilate) – isn’t a direct blood supply to articular cartilage. But can see this in the lab.
• We also have potential transdifferentiation to different germ lineages, but this is controversial, especially in vivo.
What processes can be affected by MSC secreted factors?
− Immunomodulation → HL, TGF-B, PGE-2, LIF…
− Anti-apoptosis → VEGF, HGF, IGF, TGF-b, GM-CSF…
− Angiogenesis → VEGF, PIGF, IL-6, bFGF
− Chemoattraction → CCLs and CXCLs
− Anti-scarring → HGF, bFGF, ADM
− Support of growth and differentiation of stem and progenitor cells →SCF, LIF, M-CSF, SDF-1
What are isolation procedures for MSCs from bone marrow and adipose
From bone marrow:
• Bone marrow taken from patients undergoing hip replacement therapy
• Put bone marrow in density gradient centrifugation → seperates marrow into plasma, mononuclear cells, granulocytes and RBCs.
− From MSCs we are interested in the mononuclear layer
• Put the mononuclear layer in culture → the cells that elongate and adhere are MSCs
From adipose (liposuction)
• Centrifuge the solution
• Adipose end up at the bottom, plate these out
→ Quite crude methods, to be more specific use FACS
What are the minimal criteria for MSCs?
• Established in 2006 by the International Society for Cellular Therapy
• Isolated on the basis of:
− Mononuclear cells
− Adherance – plastic adherent and grow as a monolayer
− Positive/negative expression of cell surface markers
➢ CD271 and Stro-1 quite specific and only expressed by small subset
➢ 100% of MSC prep may fit the minimal criteria. Within that, could look for expression of specific CD markers
➢ May find CD271 expressed on 0.01% of those cells, those are a subset
➢ Can do the same with Stro-1
➢ These subsets may have a different differentiation capacity → Stro-1 more oestogenic, CD271 more chondrogenic.
− Differentiation capacity towards adipocytes, chondrocytes, and osteoblast (as the minimum)
• MSCs still relatively poorly defined
What are differences between BM and AD MSCs?
Bone marrow MSCs Positive markers: • CD29 • CD44 • CD105
Negative markers:
• CD14
• CD45
Adipose derived MSCs • Easy to isolate • More abundant that bone marrow MSCs • Higher proliferation rate • Differences in CD profile: − AD-MSCs → CD34, high CD30, CD36 − BM-MSCs → no CD34, lower CD30, , no CD36 • Differences in differentiation: − Osteogenesis very similar − Chondrogenesis very different → adipose derived stem cells don’t make chondrocytes well
What GFs influence MSCs?
• AD-MSCs proliferate faster, BM-MSCs undergo better chondrogenesis
• AD-MSCs may lack TGF receptor, thus BMP6 might improve differentiation
− Indeed, paper published → Reduced Chondrogenic Potential of Adipose Tissue Derived Stromal Cells Correlated with an Altered TGF-B Receptor and BMP Profile is Overcome by BMP-6.
GFs and Effects
• TGF-B & BMP-2 → increases proliferation and cartilaginous ECM production, downregulates collagen type I expression
• BMP-4 → accelerates the progression of cartilage differentiation
• GDF-5 → increases cartilaginous ECM production
• FGF-2 → increases proliferation, increases proteoglycan production
What TFs control MSC differentiation?
Adipocyte differentiation:
• PPARy → from committed progenitor to mature preadipocyte
• C/EBPb → commited progenitor and early preadipocyte
• FABPs → mature preadipocyte and adipocyte
• Lipids → mature preadipocyte and adipocyte
Osteocyte differentiation:
• Runx2 → committed progenitor to preosteoblast. Master regulator of osteogensis
• Osterix → Preosteoblast
• Alkaline phosphatase → preosteoblast to early osteoblast
• Osteocalcin → mature osteoblast
• Osteopontin → mature osteoblast
What is the model for chondrogenesis in vivo
Key TFs in osteogenesis and chondrogenesis are Runx2 vs SOX-9
- From the multipotent mesenchymal progenitors (Notch, Runx2) some will become osteochondro progenitor cells (Col1, CD44, N-cadherin)
- From these we get mesenchymal condensation and expression of Sox-9
- SOX-9 is the master regulator of chondrogenesis → directly linked to expression of type 2 collagen
- Proliferating chondrocytes will either give cells that will upregulate type 2 collagen and aggrecan, or hypertrophic chondrocytes that upregulate Runx2
- Hypertrophied chondrocytes either die, or through maintained Runx2 expression, can transfer to osteogenic lineage
What is the model for chondrogenesis in vitro?
• Recreate the 3D microenvironment → use alginate beads. Keeps them rounded and held within a matrix
• Use of a differentiating media → dexamethasone and TFG-B
• Promotes chondrogenesis:
− Rounded morphology
− Upregulation of Sox-9, type II collagen and aggrecan
− First demonstrated by Johnstone et al, 1998
• 2D culture → Sox-9 overexpression required to induce MSC chondrogenesis in a monolayer, but we don’t get aggregan expression and also get type I collagen.
• 3D culture → complete differentiation – sustained upregulation of SOX-9, type 2 collagen and aggrecan
How does donor age of MSCs affect differentiation?
- Mean telomere length decreases with age
- Total population doublings decrease with age
- Differentiation is negatively effected by age
