Expression Profile of Osteoblast Lineage at Defined Stages of Differentiation

Ivo Kalajzic,Ada Staal,Wen-Pin Yang,Yuli Wu,Susan E Johnson,Jean H.M Feyen,Winfried Krueger,Peter Maye,Fang Yu,Yifang Zhao,Lynn Kuo,Rishi R Gupta,Luke E.K Achenie,Hsin-Wei Wang,Dong-Guk Shin,David W Rowe

doi:10.1074/jbc.m413834200

Abstract

The inherent heterogeneity of bone cells complicates the interpretation of microarray studies designed to identify genes highly associated with osteoblast differentiation. To overcome this problem, we have utilized Col1a1 promoter-green fluorescent protein transgenic mouse lines to isolate bone cells at distinct stages of osteoprogenitor maturation. Comparison of gene expression patterns from unsorted or isolated sorted bone cell populations at days 7 and 17 of calvarial cultures revealed an increased specificity regarding which genes are selectively expressed in a subset of bone cell types during differentiation. Furthermore, distinctly different patterns of gene expression associated with major signaling pathways (Igf1, Bmp, and Wnt) were observed at different levels of maturation. Some of our data differ from current models of osteoprogenitor cell differentiation and emphasize components of the pathways that were not revealed in studies based on a total cell population. Thus, applying methods to generate more homogeneous populations of cells at a defined level of cellular differentiation from a primary osteogenic culture is feasible and leads to a novel interpretation of the gene expression associated with increasing levels of osteoprogenitor maturation.

Highlights

The inherent heterogeneity of bone cells complicates the interpretation of microarray studies designed to identify genes highly associated with osteoblast differentiation
Cultures were established from GFP transgenic mice, and the onset of GFP expression during the osteoblast differentiation was obtained by fluorescent imaging
The biological properties of the cells identified by the marker genes reflected an incremental increase in osteoblast differentiation that is supported by the present molecular studies of the isolated cell populations

Summary

Introduction

The inherent heterogeneity of bone cells complicates the interpretation of microarray studies designed to identify genes highly associated with osteoblast differentiation. In a number of immortalized cell lines, and the lineage progression to osteogenic nodule formation has been studied in primary murine, rat, and human cultures when grown in the presence of ascorbic acid, ␤-glycerophosphate, and dexamethasone From these studies, a generalized pattern of diminished cell proliferation followed by an increase in the expression of genes associated with bone matrix production has been observed. A 2.3-kb Col1a1 promoter fragment driving GFP (pOBCol2.3GFP, referred to as 2.3GFP) remains inactive until the nodules begin to mineralize [8] Based on these expression patterns we have associated the early expression of the 3.6GFP with cells reaching the preosteoblast level of differentiation, which continues during early osteoblast development, whereas the 2.3GFP expression represents a cell that has acquired the ability to form a mineralized matrix. Because the 2.3GFPpositive cells represent less than 15% of the total cell population of a well mineralized osteoblast culture, it is likely that a microarray study based on a heterogeneous cell mixture would not accurately represent the expression of a fully differentiated osteoblast [8]

Methods

Results

Conclusion