Post on 29-Dec-2015
transcript
The Effect of Aging on Human Bone Marrow Stromal Cell
Differentiation
Alec Richardson
The Question
• How does donor age affect stem cell differentiation?
Marrow Stromal Cell Differentiation
StemCell
MesenchymalStem Cell
Osteoprogenitor Pre-osteoblast Osteoblast
ChondrocytesMyocytesFibroblasts
Bone-Liningcell
Osteocyte
Adipocyte
BMPsTGFβ BMPs
Runx2 Osx
PTH
IGF-I, PGE2 Vitamin D Steroids
Histone Collagen TGFβ1Osteopontin
Alk Phos BSP Collagen
BMPs Collagen Osteocalcin Osteopontin Collagenase Other NCPsMineralization
R. Pignolo and F. Kaplan, Chapter 40: Bone Biology in Inverventional Spine, 2007.
Osteoblast differentiation is impaired in BMSCs derived from mouse models of accelerated
aging
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
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
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
Experiment #1
0
20
40
60
80
100
120
140
160
# colonies
1000 700 400 200 150 150 100 100
Seeding Density (cells/cm^2)
10/12/07 CFU Assay
Experiment #1
1000
Experiment #2
• Donor: Age 34 Male (Flushed)
• Densities: 50,50,50,75,75,75
• Cell count = 1.6 x 10^5 cells/mL
Experiment #2
0
10
20
30
40
50
60
70
80
90
# colonies
50 50 50 75 75 75
Seeding Density (cells/cm^2)
10/31/07 CFU Assay
Experiment #2
50 75
Experiment #3
• Donor: Age 43 Female (Scooped)
• Densities: 50,100,100,150,150,200
• Cell count = 1.8 x 10^5 cells/mL
Experiment #3
0
20
40
60
80
100
120
140
160
# colonies
50 100 100 150 150 200
Seeding Density (cells/cm^2)
11/14/07 CFU Assay
Experiment #3
50
Experiment #3
100
Experiment #3
200
Experiment #3
50
100
200
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)