Ageing of bone is associated with decreased bone mineral density (BMD) and is caused by increased bone resorption by osteoclasts that is not sufficiently compensated by an increase in bone formation by osteoblast. In addition, increased adipose tissue volume is observed in the bone marrow cavity caused by enhanced adipogenesis in aging and osteoporotic people. Since osteoblasts and adipocytes share a common precursor in the bone marrow (Mesenchymal Stromal Cells, MSC), we wanted to assess the early regulatory events that occur upon differentiation of human MSCs into both lineages. To address this question, we have generated gene expression profiles of early osteogenic and adipogenic differentiating human MSCs (11 time points within 4 days) by Illumina microarray analyses. The data were dissected by bioinformatic means (a.o. gene ontology (GO) and upstream regulator analyses) and functional overexpression studies. Unsupervised cluster analyses of our gene expression profiles illustrated that the adipogenic and osteogenic differentiated cells can be separated within 3 hours after the initiation of differentiation and reach a stable phenotype after 2-3 days. GO analyses demonstrated that transcription factor (TF) activity (p<10−9) was significantly enriched among the differentially expressed genes within the first 3 hours. This was followed by more lineage specific categories such as extracellular matrix proteins (12 hours, p<10-8) in osteogenic and oxidation-reduction (24 hours, p<10−2) in adipogenic differentiating hMSC. Analyses of TF activity identified 10 TFs that were (in)activated (z-score <-2 or >2) within the first 3 hours in both lineages. The activity of 4 and 20 TFs changed specifically upon osteoblast or adipocyte differentiation, respectively. These included known TFs involved in hMSC differentiation as well as TFs that have not been described before to be involved in the differentiation of osteoblasts. Overexpression of two TFs, HOXA9 and IRF2, illustrated enhanced mineralization confirmed their role in osteogenic differentiation of human MSCs.
Taken together, we describe at the genetic level the osteogenic and adipogenic MSC differentiation. TFs are among the first proteins that are differentially expressed and (in)activated upon differentiation. We have identified interesting TFs that hold the potential as early regulators of lineage decision.
Disclosure: The authors declared no competing interests. This work was supported by the Netherlands Institute of Regenerative Medicine.