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MSC Differentiation Working Group Meeting August 30, 2007 - PowerPoint PPT Presentation


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MSC Differentiation Working Group Meeting August 30, 2007. Robert J. Pignolo Alec M. Richardson. Stem Cell. Mesenchymal Stem Cell. Osteoprogenitor. Pre-osteoblast. Osteoblast. MSC Differentiation. BMPs Collagen Osteocalcin Osteopontin Collagenase Other NCPs

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Presentation Transcript
msc differentiation

Stem

Cell

Mesenchymal

Stem Cell

Osteoprogenitor

Pre-osteoblast

Osteoblast

MSC Differentiation

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

Bone-

Lining

cell

Osteocyte

R. Pignolo and F. Kaplan, Chapter 40: Bone Biology in Inverventional Spine, 2007.

possible mechanisms of age related bone loss

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

possible mechanisms of preferential osteoblast differentiation in poh

Stem

Cell

Mesenchymal

Stem Cell

Osteoprogenitor

Pre-osteoblast

Osteoblast

Possible mechanisms of preferential osteoblast differentiation in POH

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

Bone-

Lining

cell

Osteocyte

Differential signaling

Lineage switching

Transdifferentiation

osteogenic potential of bone marrow stromal cells
Osteogenic Potential of Bone Marrow Stromal Cells

Gnas

Vit C/ßGP

DEX

BMP2

+/+

+ - -

+/-

+ - -

+/+

+ + -

+/-

+ + -

+ - +

+/+

+/-

+ - +

osteogenic potential of soft tissue stromal cells
Osteogenic Potential of Soft Tissue Stromal Cells

Gnas

Vit C/ßGP

DEX

BMP2

+/+

+ - -

+/-

+ - -

+/+

+ + -

+/-

+ + -

+ - +

+/+

+/-

+ - +

marrow stromal cells
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
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
phenotypic characteristics of marrow stromal precursor cells
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

Quantitative analysis of CFU-F to assess osteoblast differentiation
quantitative analysis of cfu f to assess osteoblast differentiation1
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

Quantitative analysis of CFU-F to assess osteoblast differentiation
experimental design
Experimental Design

Day

0

3

6

9

12

14

Seed

cells

Stain w/

toluidine

blue

Rfd

Rfd

Rfd

Rfd