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MSC Differentiation Working Group Meeting August 30, 2007

MSC Differentiation Working Group Meeting August 30, 2007. Robert J. Pignolo Alec M. Richardson. Stem Cell. Mesenchymal Stem Cell. Osteoprogenitor. Pre-osteoblast. Osteoblast. MSC Differentiation. BMPs Collagen Osteocalcin Osteopontin Collagenase Other NCPs

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MSC Differentiation Working Group Meeting August 30, 2007

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  1. MSC DifferentiationWorking Group MeetingAugust 30, 2007 Robert J. Pignolo Alec M. Richardson

  2. Stem Cell Mesenchymal Stem Cell Osteoprogenitor Pre-osteoblast Osteoblast MSC Differentiation BMPs Collagen Osteocalcin Osteopontin Collagenase Other NCPs Mineralization Histone Collagen TGFβ1 Osteopontin Alk Phos BSP Collagen IGF-I, PGE2 Vitamin D Steroids BMPs TGFβ BMPs PTH Runx2 Osx Adipocyte Chondrocytes Myocytes Fibroblasts Bone- Lining cell Osteocyte R. Pignolo and F. Kaplan, Chapter 40: Bone Biology in Inverventional Spine, 2007.

  3. Stem Cell Mesenchymal Stem Cell Osteoprogenitor Pre-osteoblast Osteoblast Possible mechanisms of age-related bone loss BMPs Collagen Osteocalcin Osteopontin Collagenase Other NCPs Mineralization Histone Collagen TGFβ1 Osteopontin Alk Phos BSP Collagen IGF-I, PGE2 Vitamin D Steroids BMPs TGFβ BMPs PTH Runx2 Osx Adipocyte Chondrocytes Myocytes Fibroblasts Osteoblast senescence Bone- Lining cell Osteocyte MSC senescence Lineage switching Transdifferentiation

  4. Osteoblast differentiation is impaired in BMSCs derived from mouse models of accelerated aging

  5. Stem Cell Mesenchymal Stem Cell Osteoprogenitor Pre-osteoblast Osteoblast Possible mechanisms of preferential osteoblast differentiation in POH BMPs Collagen Osteocalcin Osteopontin Collagenase Other NCPs Mineralization Histone Collagen TGFβ1 Osteopontin Alk Phos BSP Collagen IGF-I, PGE2 Vitamin D Steroids BMPs TGFβ BMPs PTH Runx2 Osx Adipocyte Chondrocytes Myocytes Fibroblasts Bone- Lining cell Osteocyte Differential signaling Lineage switching Transdifferentiation

  6. Osteogenic Potential of Bone Marrow Stromal Cells Gnas Vit C/ßGP DEX BMP2 +/+ + - - +/- + - - +/+ + + - +/- + + - + - + +/+ +/- + - +

  7. Osteogenic Potential of Soft Tissue Stromal Cells Gnas Vit C/ßGP DEX BMP2 +/+ + - - +/- + - - +/+ + + - +/- + + - + - + +/+ +/- + - +

  8. Marrow stromal cells • Give rise to progeny that support developing hematopoietic cells • Capable of becoming reticular cells, adipocytes, vascular endothelial cells, smooth muscle cells, macrophages, chondrogenic cells, and osteogenic cells • Marrow stromal progenitors (bone marrow stromal cells, BMSCs) or mesenchymal stem cells (used interchangeably )

  9. Clonogenic marrow stromal progenitors in vitro • Cells with the potential to form fibroblast colony-forming units (CFU-F), morphologically resembling fibroblasts with variably low incidence (mouse, 0.1-5 x 10-5; human, 1-20 x 10-5) • Isolated on the basis of rapid adherence, absence of phagocytic activity, and extended proliferation in vitro • Further identified on the basis of positive (e.g., Sca-1 in mice; STRO-1 in humans) and negative selective markers

  10. Mouse BMSC colony forming units

  11. Phenotypic characteristics of marrow stromal precursor cells • Stromal progenitors can be isolated from the adult mouse based on the selection of Sca-1+Lin-CD31-CD45- cells • Similarly, in humans STRO-1+VCAM-1/CD106+ restriction identifies an enriched population of cells with the capacity for differentiation into multiple mesenchymal lineages, including osteoblasts • Negative selection can also enrich for marrow stromal precursors by removing contaminating hematopoietic cells

  12. Technical considerations Optimal seeding density to obtain discrete CFU-F Optimal refeeding schedule and time course to obtain differentiated colonies Choice of differentiation factors Experimental questions Effect of age or medical condition (e.g., POH) on osteoblast differentiation Effect of soluble factors, differential gene expression, or gene mutations on osteoblast differentiation Quantitative analysis of CFU-F to assess osteoblast differentiation

  13. Factors for osteoblast differentiation

  14. Technical considerations Optimal seeding density to obtain discrete CFU-F Optimal refeeding schedule and time course to obtain differentiated colonies Choice of differentiation factors Experimental questions Effect of age or medical condition (e.g., POH) on osteoblast differentiation Effect of soluble factors, differential gene expression, or gene mutations on osteoblast differentiation Quantitative analysis of CFU-F to assess osteoblast differentiation

  15. Experimental Design Day 0 3 6 9 12 14 Seed cells Stain w/ toluidine blue Rfd Rfd Rfd Rfd

  16. Seeding density (cells/cm2) 500 100 50 25* Effect of seeding density on human bone marrow CFU-F

  17. Seeding density (cells/cm2) 20 10 5 2.5 Effect of seeding density on human bone marrow CFU-F

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