Project description:Fibroblasts can be directly reprogrammed to induced renal tubular epithelial cells (iRECs) using four transcription factors. These engineered cells may be used for disease modeling, cell replacement therapy or drug and toxicity testing. Direct reprogramming induces drastic changes in the transcriptional landscape, protein expression, morphological and functional properties of cells. However, how the metabolome is changed by reprogramming and to what degree it resembles the target cell type remains unknown. Using untargeted gas chromatography-mass spectrometry (GC-MS) and targeted liquid chromatography-MS, we characterized the metabolome of mouse embryonic fibroblasts (MEFs), iRECs, mIMCD-3 cells, and whole kidneys. Metabolic fingerprinting can distinguish each cell type reliably, revealing iRECs are most similar to mIMCD-3 cells and clearly separate from MEFs used for reprogramming. Treatment with the cytotoxic drug cisplatin induced typical changes in the metabolic profile of iRECs commonly occurring in acute renal injury. Interestingly, metabolites in the medium of iRECs, but not of mIMCD-3 cells or fibroblast could distinguish treated and non-treated cells by cluster analysis. In conclusion, direct reprogramming of fibroblasts into renal tubular epithelial cells strongly influences the metabolome of engineered cells, suggesting that metabolic profiling may aid in establishing iRECs as in vitro models for nephrotoxicity testing in the future.
Project description:Sertoli cells are essential nurse cells in the testis that regulate the process of spermatogenesis and establish the immune-privileged environment of the blood-testis-barrier (BTB). The induction of human Sertoli cells from fibroblasts could provide cellular sources for fertility and transplantation treatments. Here, we report the in vitro reprogramming of human fibroblasts to Sertoli cells and characterize these human induced Sertoli-like cells (hiSCs). Initially, five transcriptional factors (NR5A1, GATA4, WT1, SOX9 and DMRT1) and a gene reporter carrying the AMH promoter were utilized to obtain the hiSCs. We further reduce the number of reprogramming factors to two, i.e., NR5A1 and GATA4, and show that these hiSCs have transcriptome profiles that are similar to those of primary human Sertoli cells. Consistent with the known cellular properties of Sertoli cells, hiSCs attract endothelial cells and exhibit high number of lipid droplets in the cytoplasm. More importantly, hiSCs can sustain the viability of spermatogonia cells harvested from mouse seminiferous tubules. In addition, hiSCs suppress the production of IL-2 and proliferation of human T lymphocytes. When hiSCs were cotransplanted with human embryonic kidney cells, these xenotransplanted human cells survived longer in mice with normal immune systems. hiSCs also allow us to determine a gene associated with Sertoli-only syndrome (SCO), CX43, is indeed important in regulating the maturation of Sertoli cells.
Project description:Reprogramming of somatic cells to pluripotency, thereby creating induced pluripotent stem (iPS) cells, promises to boost cellular therapy. Most instances of direct reprogramming have been achieved by forced expression of defined exogenous factors using multiple viral vectors. The most used four transcription factors, OCT4, SOX2, KLF4 and C-MYC, can induce pluripotency in mouse and human fibroblasts. Here we report that a forced expression of a new combination of transcription factors (TCL-1A, C-MYC and SOX2) is sufficient to promote the reprogramming of human fibroblast into pluripotent cells. These three-factor pluripotent cells are similar to human embryonic stem cells (hESC) in morphology, in the ability to differentiate into cells of the three embryonic layers, and at the level of global gene expression. Induced pluripotent human cells generated by combination of other factors will be of great help for the understanding of reprogramming pathways. This in turn will allow us to better control cell-fate and apply this knowledge to cell therapy.
Project description:Direct lineage reprogramming represents a remarkable conversion of cellular and transcriptome states. However, the intermediates through which individual cells progress are largely undefined. Here we used single cell RNA-seq at multiple time points to dissect direct reprogramming from mouse embryonic fibroblasts (MEFs) to induced neuronal (iN) cells. By deconstructing heterogeneity at each time point and ordering cells by transcriptome similarity rather than time we reconstructed a continuous reprogramming path. We find that overexpression of a single 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. However, overexpression of Ascl1 alone leads to abundant alternative fates that are suppressed by the combination of additional factors (Myt1l, Pou3f2). We find transgene silencing and emergence of alternative fates are the major efficiency limits of direct reprogramming. These data provide a high-resolution approach for understanding transcriptome states during lineage differentiation.