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The Effects of Aging on Human Bone Marrow Stromal Cell Differentiation

The Effects of Aging on Human Bone Marrow Stromal Cell Differentiation. Alec Richardson. Stem Cell. Mesenchymal Stem Cell. Osteoprogenitor. Pre-osteoblast. Osteoblast. Possible mechanisms of age-related bone loss. BMPs Collagen Osteocalcin Osteopontin Collagenase

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The Effects of Aging on Human Bone Marrow Stromal Cell Differentiation

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

  2. 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

  3. Latest Experiment • 6 plates (100 cm^2) • 3 from old donor (age 77) • 3 from young donor (age 43) • Crystal Violet, Alizarin Red, Alkaline Phosphatase • Seeding density for all plates: 40 cells/cm^2 • 14 days, 4 re-feedings • Differentiation Factors added to all plates • Β-Glycerophosphate, Ascorbic Acid, Dexamethasone

  4. Crystal Violet Age 43 Age 77

  5. Alizarin Red Age 43 Age 77

  6. Alkaline Phosphatase Age 43 Age 77

  7. The Secretion of Soluble Growth Factors by Early Passage Osteoblasts

  8. The Secretion of Soluble Growth Factors by Early Passage Osteoblasts • Conditioned media will function as differentiation factor • Hypothesis: Early passage osteoblasts secrete greater levels/more potent differentiation/growth factors and will be better able to produce mineralization than late passage osteoblasts

  9. Method • 1. Seed 24 well plate with HBMSC from young donor • 2. After 24 hrs, add conditioned media from early and late passage osetoblasts • Must normalize for higher confluent density in early vs. late passage osteoblasts (2.5X) • 3. Add new media to osteoblast flasks after each refeeding • add conditioned media to 24 well plate 4 times during 14 day period • 4. Stain with Alk. Phos., Aliz. Red

  10. Early Passage Conditioned Media Alk Phos. Aliz. Red Alk Phos. Aliz. Red Late Passage Conditioned Media

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