Project description:Definitive hematopoiesis emerges via an endothelial-to-hematopoietic transition in the aorta-gonad-mesonephros (AGM) region and placenta. We have recently demonstrated the induction of hematopoietic stem/progenitors (HSPCs) from mouse fibroblasts with a combination of transcription factors progressing through endothelial-like precursors. Here, guided by our in vitro programming experiments we analyzed mouse placentas for the presence of the precursor phenotype. We identified a small population of CD34+ Sca1+Prom1+ (34PS) cells in mid-gestation placentas that do not express the pan-hematopoietic marker CD45. After isolation and culture 34PS cells acquire CD45 and generate large hematopoietic as well as cobblestone colonies. Prom1+ cells localize to the placental vascular labyrinth where HSPCs emerge. 34PS cells express markers associated with the hemogenic endothelium (CD31, Tie2, VE-Cadherin, Sox17, Runx1, Scl) and also markers identified by direct induction (Itga6/CD49f). This population is heterogeneous for the early hematopoietic marker CD41 and expresses the programming transcription factors. Remarkably, global gene expression profiles of placental 34PS cells correlate with AGM-derived hemogenic endothelium and fibroblast-derived precursors. Finally, when co-cultured with stroma placental 34PS cells give rise to B/T lymphoid cells as well as mixed colonies containing erythroid, myeloid and megakaryocytic cell lineages. In summary, we show that direct in vitro conversion provided a cell surface phenotype for the isolation of hemogenic precursors in vivo. Our findings provide insights into the specification of definitive hemogenesis in the placenta, in depth characterization of hemogenic precursor populations and the first evidence that direct in vitro conversion approaches can be used as a valuable tool to address basic developmental questions in vivo.
Project description:We report the generation of induced oligodendrocyte precursor cells (iOPCs) by direct lineage conversion. Forced expression of the three transcription factors Sox10, Olig2 and Zfp536 was sufficient to convert mouse and rat fibroblasts into iOPCs with morphologies and gene expression signatures that resemble OPCs.
Project description:Direct lineage conversion holds great promise in the regenerative medicine field for restoring damaged tissues using functionally engineered counterparts. However, current methods of direct lineage conversion, even those employing virus-mediated transgenic expression of tumorigenic factors, are extremely inefficient (~25%). Thus, advanced methodologies capable of revolutionizing efficiency and addressing safety issues are key to clinical translation of these technologies. Here, we propose an exosome-guided, non-viral, direct-lineage conversion strategy to enhance transdifferentiation of fibroblasts to induced cardiomyocyte-like cells (iCMs). Exosomes produced during the cardiac differentiation process of embryonic stem cells (ESCs) are able to achieve extremely high reprogramming efficiency (>60%) by generating functional iCMs from mouse embryonic fibroblasts via a cardiac precursor-like stage rather than a pluripotent state. The resulting iCMs possess typical cardiac Ca2+ transients and electrophysiological features, and exhibit global gene expression profiles similar to those of cardiomyocytes. The optimized reprogramming conditions produce beating iCM clusters ~3-fold more efficiently than conventional methods. This is the first demonstration of the use of exosomes derived from ESCs undergoing cardiac differentiation as biomimetic tools to induce direct cardiac reprogramming with greatly improved efficiency, establishing a general, more readily accessible platform for broadly generating a variety of specialized somatic cells through direct lineage conversion.
Project description:Direct lineage conversion holds great promise in the regenerative medicine field for restoring damaged tissues using functionally engineered counterparts. However, current methods of direct lineage conversion, even those employing virus-mediated transgenic expression of tumorigenic factors, are extremely inefficient (~25%). Thus, advanced methodologies capable of revolutionizing efficiency and addressing safety issues are key to clinical translation of these technologies. Here, we propose an exosome-guided, non-viral, direct-lineage conversion strategy to enhance transdifferentiation of fibroblasts to induced cardiomyocyte-like cells (iCMs). Exosomes produced during the cardiac differentiation process of embryonic stem cells (ESCs) are able to achieve extremely high reprogramming efficiency (>60%) by generating functional iCMs from mouse embryonic fibroblasts via a cardiac precursor-like stage rather than a pluripotent state. The resulting iCMs possess typical cardiac Ca2+ transients and electrophysiological features, and exhibit global gene expression profiles similar to those of cardiomyocytes. The optimized reprogramming conditions produce beating iCM clusters ~3-fold more efficiently than conventional methods. This is the first demonstration of the use of exosomes derived from ESCs undergoing cardiac differentiation as biomimetic tools to induce direct cardiac reprogramming with greatly improved efficiency, establishing a general, more readily accessible platform for broadly generating a variety of specialized somatic cells through direct lineage conversion.
Project description:Direct conversion of pericytes (PCs) or mouse embryonic fibroblasts (MEFs) into induced oligodendrocytes (iOPCs) by ectopic expression of Olig2, Sox10 and Nkx6.2 was assessed by transcriptome profiling (RNA-seq). Samples were collected before and after direct conversion (for PCs at 3 different time points/passages - p5, p15 and p25)