Transcriptional plasticity of stromal cells amplifies their differentiation efficiency in vitro

Human bone marrow-derived stromal cells (also termed mesenchymal stem cells or MSCs) are progenitors capable of differentiating into bone-forming osteoblasts and fat-storing adipocytes. Due to the loss of bone mass being associated with increased marrow fat, the trans-differentiation of osteoblasts into adipocytes has been hypothesized as a contributor to osteoporotic bone loss and fragility. Reprogramming of transcriptional networks is a prerequisite for cellular differentiation, however, to which extent cell-type specific transcriptional networks modulate cellular plasticity within stromal cells remains unknown. In this study, we performed global gene expression analysis at the single cell level in immortalized human stromal cells from bone marrow (hBMSC-TERT4) that were repeatedly exposed to osteogenic and adipogenic inducers in vitro. Surprisingly, cell-type specific gene networks are not suppressive but rather promote the development of an opposing phenotype; for example, osteoblast differentiation of adipogenic pre-stimulated stromal cells is enhanced compared to undifferentiated ones. This is supported by the presence of cells simultaneously showing an osteogenic and adipogenic phenotype highlighting a strong molecular plasticity of transcriptional networks in stromal cells. These observations provide a strong molecular support for the notion of not only progenitor speciation but also the plasticity of differentiated cells contributing to the balance of bone mass and marrow fat content. Overall design: Single cell and bulk RNA-expression profiles were determined in immortalized human bone marrow-derived stromal cells that were exposed to osteogenic or adipogenic inducers as well as to sequential treatments of both differnetiation cocktails to obtain cells differentiating towards the osteogenic or adipogenic lineage, either directly from undifferentiated cells or from intermediate states that have been previsouly committed to the opposing lineage.