The Effects of Aging on Human Bone Marrow Stromal Cells Alec Richardson 3/17/09

What is Aging? Changes in an organism over time Can be physiological or behavioral or both Key component is time – Aging seen as the result of numerous changes over time Viewed as a decline in function – perhaps better to think of it as a change in function Affects people differently and the same (m/f) Different vulnerabilities to disease Aging results in a higher probability of death No proven biological basis

Aging Theories Inside: mechanisms that protect against disease become intrinsically disrupted Changes in transcription/translation, increased cell death, disruption of organ function Outside: environment affects genomic integrity or other physiologic parameters Results in similar changes that lead to vulnerability and malfunction Probably a mixture of both inside and outside factors Statistics: Billions of bodily events occur every second These events can be harmless or harmful based on the genetic makeup of the organism and ‘luck’ e.g. tumor suppression mechanisms As the number of events accumulates, the probability of mistakes and problems increases

Aging in Bone Focus on one particular organ Intimate relationship between bone and other organs How does bone change over time in humans? Disease, disruption in synergy between other organs, changes in cell #, vulnerability to fracture, load bearing capacity Production of cells, support, storage, detox Mineralization, stem cells, hormones, immune system, ions, homeostasis

Aging Models Models of accelerated aging E.g. w/t mice Differences between young and old organisms Artificially aging of organisms

Human Bone Marrow Stromal Cells Precursor cells in bone are necessary to replenish supplies of vital osteogenic cell types such as osteoblasts Hypothesis: The aging process results in a decrease in the number of HBMSCs and/or a decrease in the ability of such cells to differentiate and produce mineralization. If true, this theory could explain why aging results in vulnerability to fracture and diseases such as osteoporosis in older populations

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 Stem Cell Mesenchymal Stem Cell Osteoprogenitor Pre-osteoblast Osteoblast BMPs PTH Runx2 Osx Adipocyte Chondrocytes Myocytes Fibroblasts Osteoblast senescence Bone- Lining cell Osteocyte MSC senescence Lineage switching Transdifferentiation

Experimental Model CFU-F Assay Previous papers show CFU-Fs to contain osteoprogenitor cells Human donors Young=Age 34-49, Old=Age 70-85 Stains Crystal Violet, Alkaline Phosphatase, Alizarin Red 14 days, 4 re-feedings Differentiation Factors added to all plates Β-Glycerophosphate, Ascorbic Acid, Dexamethasone Seeding density for all plates: 40 cells/cm^2 Image J software – used for all stains except Aliz. Red

Crystal Violet (+ D.F.) Age 77 Age 43

Alizarin Red Age 77 Age 43

Alkaline Phosphatase Age 77 Age 43

Future Directions Compare passaged and un-passaged cells Ficoll gradient to isolate mononuclear cells Passage to P1 or P2