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)