1. MSC Differentiation Working Group Meeting August 30, 2007 Robert J. Pignolo Alec M. Richardson

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

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

6. Osteogenic Potential of Bone Marrow Stromal Cells Vit C/ßGPDEXBMP2Gnas +/+ +/- + - - + + - + - +

7. Osteogenic Potential of Soft Tissue Stromal Cells Vit C/ßGPDEXBMP2Gnas +/+ +/- + - - + + - + - +

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. Quantitative analysis of CFU-F to assess osteoblast differentiation 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

13. Factors for osteoblast differentiation FactorOther NameStock Sol’n (conc./solv.) Final Conc.Final Conc. Range Ascorbic AcidSodium L-ascorbate10mg/ml (200X) in H 2 0 50ug/mlN/A Beta-glycero phosphate Glycerol 2-phosphate disodium salt hydrate 2M (200X) in H 2 0 10mM7.5-10mM BMP2Bone morphogenetic protein 2 10ug/ml (33.3X) in media+FBS 300 ng/ml 100-300 ng/ml Vitamin D310E-4M (2000X) in DMSO or EtOH 5E-8ME-8M- E-7M DEXDexamethasone8mM (8000X) or 1mM (1000X) in EtOH E-6ME-6M- E-8M BMP4Bone morphogenetic protein 4 10ug/ml (33.3X) in media+FBS 300 ng/ml 100-300 ng/ml TGF beta1Transforming growth factor beta1 1ug/ml (100X) in media+FBS 10ng/ml1-10 ng/ml

14. Quantitative analysis of CFU-F to assess osteoblast differentiation 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

15. Experimental Design 03691214Day Seed cells Rfd Stain w/ toluidine blue

16. Effect of seeding density on human bone marrow CFU-F Seeding density (cells/cm 2 ) 500 100 50 25*

17. Effect of seeding density on human bone marrow CFU-F Seeding density (cells/cm 2 ) 20 10 5 2.5