1. The Effect of Aging on Human Bone Marrow Stromal Cell Differentiation Alec Richardson

2. The Question How does donor age affect stem cell differentiation?

3. Marrow Stromal Cell Differentiation 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.

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

5. HBMSC Inventory Patient femoral head specimens 45 total specimens Age range = 34-79 Approximately ten specimens per decade Three T-25 flasks seeded per specimen Samples frozen at P2 At least 13 flasks frozen per specimen (up to 24) Specimens stored in formalin

6. The Assay 100 cm^2 plates Low seeding density Small population (1-20 X 10^-5) form fibroblast colony-forming units (CFU-F) CFU-F provides measurement of # stem cells and ability to differentiate (Hypothesis: Age affects both) Measure # CFU-F’s, size of CFU-F’s, # Alizarin Red/Alkaline Phos./Osteocalcin/Type 1 Collagen positive colonies Crystal violet stain

7. Seeding Density Experiment #1 Donor: Age 64 Male (Flushed) Densities: 100,100,150,150,200,400,700,1000 Cell Count = 1.0 x 10^5 cells/mL 14 days, 4 feedings

8. Experiment #1

9. 1000

10. Experiment #2 Donor: Age 34 Male (Flushed) Densities: 50,50,50,75,75,75 Cell count = 1.6 x 10^5 cells/mL

11. Experiment #2

12. 5075

13. Experiment #3 Donor: Age 43 Female (Scooped) Densities: 50,100,100,150,150,200 Cell count = 1.8 x 10^5 cells/mL

14. Experiment #3

15. 50

16. Experiment #3 100

17. Experiment #3 200

18. Experiment #3 50 100 200

19. Future Directions Differentiation Factors: Ascorbic Acid, Beta- glycerophosphate, BMP2, Vitamin D3, DEX, BMP4, TGF beta1 Alizarin Red, Alkaline Phosphatase, Osteocalcin, Type 1 Collagen Donor sex Donor medical condition Quantitation (Image J)