Dissecting direct reprogramming from fibroblast to neuron using single-cell RNA-seq.

Direct lineage reprogramming represents a remarkable conversion of cellular and transcriptome states. However, the intermediate stages through which individual cells progress during reprogramming are largely undefined. Here we use single-cell RNA sequencing at multiple time points to dissect direct reprogramming from mouse embryonic fibroblasts to induced neuronal cells. By deconstructing heterogeneity at each time point and ordering cells by transcriptome similarity, we find that the molecular reprogramming path is remarkably continuous. Overexpression of the proneural pioneer factor Ascl1 results in a well-defined initialization, causing cells to exit the cell cycle and re-focus gene expression through distinct neural transcription factors. The initial transcriptional response is relatively homogeneous among fibroblasts, suggesting that the early steps are not limiting for productive reprogramming. Instead, the later emergence of a competing myogenic program and variable transgene dynamics over time appear to be the major efficiency limits of direct reprogramming. Moreover, a transcriptional state, distinct from donor and target cell programs, is transiently induced in cells undergoing productive reprogramming. Our data provide a high-resolution approach for understanding transcriptome states during lineage differentiation.

Dormant phase and multinuclear cells: two key phenomena in early culture of murine bone marrow mesenchymal stem cells.

Special features of mesenchymal stem cells (MSCs) have made them a popular tool in cell therapy and tissue engineering. Although mouse animal models and murine MSCs are common tools in this field, our understanding of the effect of in vitro expansion on the behavior of these cells is poor and controversial. In addition, in comparison to human, isolation of MSCs from mouse has been reported to be more difficult and some unexplained features such as heterogeneity and slow growth rate in the culture of these cells have been observed. Here we followed mouse bone marrow MSCs for >1 year after isolation and examined the effect of expansion on changes in morphology, growth kinetics, plasticity, and chromosomal structure during in vitro culture. Shortly after isolation, the growth rate of the cells decreased until they stopped dividing and entered a dormant state. In this state the size of the cells increased and they became multinuclear. These large multinuclear cells then gave origin to small mononuclear cells, which after a while resumed proliferation and could be expanded immortally. The immortal cells had diminished plasticity and were aneuploid but could not form tumors in nude mice. These results suggest that mouse bone marrow MSCs bear several modifications when expanded in vitro, and therefore, the interpretation of the data obtained with these cells should be done more cautiously.