Abstract
BackgroundThe differentiation of human bone marrow derived skeletal stem cells (known as human bone marrow stromal or mesenchymal stem cells, hMSCs) into osteoblasts involves the activation of a small number of well-described transcription factors. To identify additional osteoblastic transcription factors, we studied gene expression of hMSCs during ex vivo osteoblast differentiation.ResultsClustering of gene expression, and literature investigation, revealed three transcription factors of interest – ZNF25, ZNF608 and ZBTB38. siRNA knockdown of ZNF25 resulted in significant suppression of alkaline phosphatase (ALP) activity. This effect was not present for ZNF608 and ZBTB38. To identify possible target genes of ZNF25, we analyzed gene expression following ZNF25 siRNA knockdown. This revealed a 23-fold upregulation of matrix metallopeptidase 1 and an 18-fold upregulation of leucine-rich repeat containing G protein-coupled receptor 5 and RAN-binding protein 3-like. We also observed enrichment in extracellular matrix organization, skeletal system development and regulation of ossification in the entire upregulated set of genes. Consistent with its function as a transcription factor during osteoblast differentiation of hMSC, we showed that the ZNF25 protein exhibits nuclear localization and is expressed in osteoblastic and osteocytic cells in vivo. ZNF25 is conserved in tetrapod vertebrates and contains a KRAB (Krueppel-associated box) transcriptional repressor domain.ConclusionsThis study shows that the uncharacterized transcription factor, ZNF25, is associated with differentiation of hMSC to osteoblasts.
Highlights
The differentiation of human bone marrow derived skeletal stem cells into osteoblasts involves the activation of a small number of well-described transcription factors
We report that zinc finger protein 25 (ZNF25) has a Krueppel-associated box (KRAB) domain, a transcriptional repressor, which is conserved in tetrapod vertebrates
Results Human mesenchymal stem cell (hMSC)-TERT differentiate into osteoblastic cells in vitro and form heterotopic bone in vivo hMSC-TERT differentiate readily into osteoblastic cells, evidenced by enhanced expression of osteoblast marker genes (ALPL, BGLAP, Collagen 1 (COL1A1)) (Fig. 1a), increased alkaline phosphatase activity and formation of in vitro mineralized matrix stained positive for Alizarin red during differentiation (Fig. 1b). hMSC-TERT cells are able to form heterotopic bone when implanted subcutaneously, with hydroxyapatite tricalcium phosphate (HA/TCP), in immune deficient mice (Fig. 1c)
Summary
The differentiation of human bone marrow derived skeletal stem cells (known as human bone marrow stromal or mesenchymal stem cells, hMSCs) into osteoblasts involves the activation of a small number of well-described transcription factors. Adult human skeletal stem cells ( known as bone marrow stromal or mesenchymal stem cells, hMSCs) are present in the bone marrow stroma. They are defined by their ability to both self-renew and differentiate into mesoderm-specific lineage cells including osteoblasts, adipocytes and chondrocytes [1, 2].
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