Document Detail

Fluorescence activated cell sorting reveals heterogeneous and cell non-autonomous osteoprogenitor differentiation in fetal rat calvaria cell populations.
MedLine Citation:
PMID:  12938161     Owner:  NLM     Status:  MEDLINE    
Identification of osteoblast progenitors, with defined developmental capacity, would facilitate studies on a variety of parameters of bone development. We used expression of alkaline phosphatase (ALP) and the parathyroid hormone/parathyroid hormone-related protein receptor (PTH1R) as osteoblast markers in dual-color fluorescence activated cell sorting (FACS) to fractionate rat calvaria (RC) cells into ALP(-)PTH1R(-), ALP(+)PTH1R(-), ALP(-)PTH1R(+), and ALP(+)PTH1R(+) populations. These fractionated populations were seeded clonally (n = 96) or over a range of cell densities ( approximately 150-8,500 cell/cm(2); n = 3). Our results indicate that colony forming unit-osteoblast (CFU-O)/bone nodule-forming cells are found in all fractions, but the frequency of CFU-O and total mineralized area is different across fractions. Analysis of these differences suggests that ALP(-)PTH1R(-), ALP(-)PTH1R(+), ALP(+)PTH1R(-), and ALP(+)PTH1R(+) cell populations are separated in order of increasing bone formation capacity. Dexamethasone (dex) differentially increased the CFU-O number in the four fractions, with the largest stimulation in the ALP(-) cell populations. However, there was no significant difference in the number or size distribution of CFU-F (fibroblast) colonies that formed in vehicle versus dex. Finally, both cell autonomous and cell non-autonomous (i.e., inhibitory/stimulatory effects of cell neighbors) differentiation of osteoprogenitors was seen. Only the ALP(-)PTH1R(-) population was capable of forming nodules at the clonal level, at approximately 3- or 12-times the predicted frequency of unfractionated populations in dex or vehicle, respectively. These data suggest that osteoprogenitors can be significantly enriched by fractionation of RC populations, that assay conditions modify the osteoprogenitor frequencies observed and that fractionation of osteogenic populations is useful for interrogation of their developmental status and osteogenic capacity.
Kelly A Purpura; Peter W Zandstra; Jane E Aubin
Publication Detail:
Type:  Journal Article; Research Support, Non-U.S. Gov't    
Journal Detail:
Title:  Journal of cellular biochemistry     Volume:  90     ISSN:  0730-2312     ISO Abbreviation:  J. Cell. Biochem.     Publication Date:  2003 Sep 
Date Detail:
Created Date:  2003-08-25     Completed Date:  2004-02-09     Revised Date:  2006-11-15    
Medline Journal Info:
Nlm Unique ID:  8205768     Medline TA:  J Cell Biochem     Country:  United States    
Other Details:
Languages:  eng     Pagination:  109-20     Citation Subset:  IM    
Copyright Information:
Copyright 2003 Wiley-Liss, Inc.
Department of Chemical Engineering and Applied Chemistry, University of Toronto, Toronto, Ontario, Canada.
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MeSH Terms
Alkaline Phosphatase / metabolism
Biological Markers
Bone Development / physiology
Cell Differentiation / physiology*
Cell Separation
Cells, Cultured
Fetus / anatomy & histology*,  physiology
Flow Cytometry*
Osteoblasts / cytology,  physiology*
Receptors, Parathyroid Hormone / metabolism
Regression Analysis
Skull / cytology*,  embryology,  metabolism
Stem Cells / cytology,  physiology*
Reg. No./Substance:
0/Biological Markers; 0/Receptors, Parathyroid Hormone; EC Phosphatase

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