Mapping SOX9 transcriptional dynamics during multi-lineage differentiation of human mesenchymal stem cells

Abstract

The master transcription factor SOX9 is a key player during chondrocyte differentiation, cartilage development, homeostasis and disease. Modulation of SOX9 and its target gene expression is essential during chondrogenic, osteogenic and adipogenic differentiation of human mesenchymal stem cells (hMSCs). However, lack of sufficient knowledge about the signaling interplay during differentiation remains one of the main reasons preventing successful application of hMSCs in regenerative medicine. We previously showed that Transcription Factor – Fluorescence Recovery After Photobleaching (TF-FRAP) can be used to study SOX9 dynamics at the single cell level. We showed that changes in SOX9 dynamics are linked to its transcriptional activity. Here, we investigated SOX9 dynamics during differentiation of hMSCs into the chondrogenic, osteogenic and adipogenic lineages. We show that there are clusters of cells in hMSCs with distinct SOX9 dynamics, indicating that there are a number of subpopulations present in the heterogeneous hMSCs. SOX9 dynamics data at the single cell resolution revealed novel insights about its activity in these subpopulations (cell types). In addition, the response of SOX9 to differentiation stimuli varied in these subpopulations. Moreover, we identified donor specific differences in the number of cells per cluster in undifferentiated hMSCs, and this correlated to their differentiation potential.

Mapping RUNX2 transcriptional dynamics during multi-lineage differentiation of human mesenchymal stem cells

“…This indicates that different subpopulation of cells do not respond to differentiation stimuli of particular lineage and only selected subpopulation of cells can differentiate into a particular lineage. These data are also in line with our previous findings with SOX9 dynamics in hMSCs (16).…”

“…We have shown that number of cells present per subpopulation might determine differentiation potential of a donor towards a particular lineage. Our RUNX2 dynamics study confirms our findings in our previous similar study on SOX9 dynamics in hMSCs (16).…”

“…Morphology or CD marker based estimations suggest existence of three subpopulations in hMSCs (33). We have previously reported that at least four distinct dynamic rates of SOX9 present in the undifferentiated hMSCs and subsequent FACS analysis showed every subpopulation has distinct SOX9 dynamic rates (16). In line with this report, we have also observed four distinct RUNX2 dynamic rates in undifferentiated hMSCs.…”

“…We have previously shown how SOX9 protein dynamics change among the subpopulation of hMSCs during chondro-, osteo-and adipogenic differentiation (16). In this report, using TF-FRAP, we have studied how RUNX2 protein dynamics changes during chondro-, osteo-and adipogenic differentiation of hMSCs.…”

“…We performed a similar study, in which we determined the mobility of SOX9 in the same donors (16). The cluster IDs of RUNX2 and SOX9 do not correspond.…”