Project description:Macrophages derived from somatic cells by reprogramming technologies have the potential to enable the development of cell-based therapies for numerous malignant diseases. Here we report on the establishment of a novel methodology allowing for the conversion of fibroblasts into functional macrophages with pro-inflammatory phenotype just by c-Myc overexpression. Ectopic expression of c-Myc in fibroblasts in induced pluripotent stem cell (iPSC) culture medium induced the rapid appearance of CD45+ hematopoietic cell complex (HCC) intermediates with engraftment capacity as well as the repopulation of distant hematopoietic compartments. HCC intermediates were stably maintained in suspension culture and continuously generated functional and highly pure-induced macrophage (iMac) just by M-CSF cytokine stimulation. Single-cell transcriptomic analysis of HCC intermediates revealed that c-Myc up-regulates the expression of MafB, a major regulator of macrophage differentiation, to promote macrophage differentiation. Characterization of the macrophage activity showed NF-kB signaling activation and pro-inflammatory phenotype. iMac transplantation significantly reduced leukemia and breast cancer progression in animal xenograft models. Our finding indicates that reprogramming strategies of fibroblasts could help circumvent long-standing obstacles to gaining “off-the-shelf” macrophages for anti-cancer immunotherapy.
Project description:Macrophages derived from somatic cells by reprogramming technologies have the potential to enable the development of cell-based therapies for numerous malignant diseases. Here we report on the establishment of a novel methodology allowing for the conversion of fibroblasts into functional macrophages with pro-inflammatory phenotype just by c-Myc overexpression. Ectopic expression of c-Myc in fibroblasts in induced pluripotent stem cell (iPSC) culture medium induced the rapid appearance of CD45+ hematopoietic cell complex (HCC) intermediates with engraftment capacity as well as the repopulation of distant hematopoietic compartments. HCC intermediates were stably maintained in suspension culture and continuously generated functional and highly pure-induced macrophage (iMac) just by M-CSF cytokine stimulation. Single-cell transcriptomic analysis of HCC intermediates revealed that c-Myc up-regulates the expression of MafB, a major regulator of macrophage differentiation, to promote macrophage differentiation. Characterization of the macrophage activity showed NF-kB signaling activation and pro-inflammatory phenotype. iMac transplantation significantly reduced leukemia and breast cancer progression in animal xenograft models. Our finding indicates that reprogramming strategies of fibroblasts could help circumvent long-standing obstacles to gaining “off-the-shelf” macrophages for anti-cancer immunotherapy.
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.