MSC Differentiation

Working Group MeetingAugust 30, 2007

Robert J. Pignolo

Alec M. Richardson

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.

MSC 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

Possible mechanisms of age-related bone loss

Osteoblast senescenceMSC senescenceLineage switchingTransdifferentiation

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

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

Possible mechanisms of preferential osteoblast differentiation in POH

Differential signalingLineage switchingTransdifferentiation

Osteogenic Potential of Bone Marrow Stromal Cells Vit C/ßGP DEX BMP2Gnas

+/+

+/+

+/+

+/-

+/-

+/-

+ - -

+ - -

+ + -

+ + -

+ - +

+ - +

Osteogenic Potential of Soft Tissue Stromal Cells

Vit C/ßGP DEX BMP2Gnas

+/+

+/+

+/+

+/-

+/-

+/-

+ - -

+ - -

+ + -

+ + -

+ - +

+ - +

Marrow stromal cells

Give rise to progeny that support developing hematopoietic cells

Capable of becoming reticular cells, adipocytes, vascular endothelial cells, smooth muscle cells, macrophages, chondrogenic cells, and osteogenic cells

Marrow stromal progenitors (bone marrow stromal cells, BMSCs) or mesenchymal stem cells (used interchangeably )

Clonogenic marrow stromal progenitors in vitro Cells with the potential to form fibroblast colony-

forming units (CFU-F), morphologically resembling fibroblasts with variably low incidence (mouse, 0.1-5 x 10-5; human, 1-20 x 10-5)

Isolated on the basis of rapid adherence, absence of phagocytic activity, and extended proliferation in vitro

Further identified on the basis of positive (e.g., Sca-1 in mice; STRO-1 in humans) and negative selective markers

Mouse BMSC colony forming units

Phenotypic characteristics of marrow stromal precursor cells Stromal progenitors can be isolated from the adult

mouse based on the selection of Sca-1+Lin-CD31-CD45- cells

Similarly, in humans STRO-1+VCAM-1/CD106+ restriction identifies an enriched population of cells with the capacity for differentiation into multiple mesenchymal lineages, including osteoblasts

Negative selection can also enrich for marrow stromal precursors by removing contaminating hematopoietic cells

Quantitative analysis of CFU-F to assess osteoblast differentiation Technical

considerations Optimal seeding density

to obtain discrete CFU-F Optimal refeeding

schedule and time course to obtain differentiated colonies

Choice of differentiation factors

Experimental questions Effect of age or medical

condition (e.g., POH) on osteoblast differentiation

Effect of soluble factors, differential gene expression, or gene mutations on osteoblast differentiation

Factors for osteoblast differentiationFactor Other Name Stock Sol’n

(conc./solv.)Final Conc. Final Conc.

Range

Ascorbic Acid Sodium L-ascorbate 10mg/ml(200X) in H20

50ug/ml N/A

Beta-glycerophosphate

Glycerol 2-phosphate disodium salt hydrate

2M (200X)in H20

10mM 7.5-10mM

BMP2 Bone morphogenetic protein 2

10ug/ml(33.3X) in media+FBS

300ng/ml

100-300 ng/ml

Vitamin D3 10E-4M (2000X) in DMSO or EtOH

5E-8M E-8M- E-7M

DEX Dexamethasone 8mM (8000X) or 1mM (1000X) in EtOH

E-6M E-6M- E-8M

BMP4 Bone morphogenetic protein 4

10ug/ml(33.3X) in media+FBS

300ng/ml

100-300 ng/ml

TGF beta1 Transforming growth factor beta1

1ug/ml (100X) in media+FBS

10ng/ml 1-10 ng/ml

Quantitative analysis of CFU-F to assess osteoblast differentiation Technical

considerations Optimal seeding density

to obtain discrete CFU-F Optimal refeeding

schedule and time course to obtain differentiated colonies

Choice of differentiation factors

Experimental questions Effect of age or medical

condition (e.g., POH) on osteoblast differentiation

Effect of soluble factors, differential gene expression, or gene mutations on osteoblast differentiation

Experimental Design

0 3 6 9 12 14Day

Seedcells Rfd Rfd Rfd Rfd Stain w/

toluidineblue

Effect of seeding density on human bone marrow CFU-F

Seeding density(cells/cm2)

500

100

50

25*

Effect of seeding density on human bone marrow CFU-F

Seeding density(cells/cm2)

20

10

5

2.5