TGFb signaling hyperactivation-induced germ cell-like tumorigenicity during ESC-derived neural progenitors

Despite the therapeutic promise for pluripotent stem cells (PSCs)-based transplantation, a crucial challenge for PSC implementation is the frequent development of teratomas or tumors in animal models or clinical patients. Therefore, clarifying the mechanism of cell therapy-derived carcinogenesis is of great importance to reduce the risk of tumorigenicity in stem cell therapy. Here we differentiate Oct4-GFP mouse embryonic stem cells (mESCs) into neural progenitor cells (NPCs) or neurons and find that a minority of Oct4+ cells are sustained at an undifferentiated state. These cells are enriched and proliferated in standard ESC medium. Interestingly, the differentiation potential of these enriched cells is tightly restricted with much higher tumorigenic activity, so defined as differentiation-resistant ESCs (DR-ESCs). Transcriptome and epigenomic analysis shows that DR-ESCs are characterized by primordial germ cell-like gene signatures (Dazl, Rec8, Stra8, Blimp1, etc) and specific histone modification patterns distinct from mESCs. Moreover, the DR-ESCs possess the germ cell potential to generate Sycp3+ haploid cells and are able to reside in sperm-free spermaduct induced by busulfan. Finally, we find that TGFβ signaling is over-activated in DR-ESCs, and inhibition of TGFβ signaling induces the fully differentiation of DR-ESCs and eliminates the tumorigenicity of mESC-derived NPCs. These data demonstrate that these TGFβ hyperactivated germ cell-like DR-ESCs are the main contributor for the tumorigenicity of ESCs-derived target cell therapy and that inhibition of TGFβ signaling in ESC-derived NPC transplantation can help to drastically reduce the risk of tumor development. To fully analyze the molecular mechanisms of carcinogenesis of pluripotent stem cells, we enriched the differentation-resistant cells from mouse neural progenitor cells which are derived from Oct4-GFP mouse embryonic stem cell neural differentiation system by sorting GFP positive cells. Transcriptional analyses by RNA sequencing and epigenetic analyses by chromatin immunoprecipitation followed by sequencing for H3K4me3 (Abcam 8580), H3K4me1 (Abcam 8895), H3K27me3 (Millipore 07-449) and H3K27ac (Active Motif 39133) were performed.