TGFβ signaling hyperactivation-induced tumorigenicity during the derivation of neural progenitors from mouse ESCs

Clinical therapies of pluripotent stem cells （PSCs）-based transplantation have been hindered by frequent development of terato- mas or tumors in animal models and clinical patients. Therefore, clarifying the mechanism of carcinogenesis in stem cell therapy is of great importance for reducing the risk of tumorigenicity. Here we differentiate Oct4-GFP mouse embryonic stem cells （mESCs） into neural progenitor cells （NPCs） and find that a minority of Oct4＋ cells are continuously sustained at Oct4＋ state. These cells can be enriched and proliferated in a standard ESC medium. Interestingly, the differentiation potential of these enriched cells is tightly restricted with much higher tumorigenic activity, which are thus defined as differentiation-resistant ESCs （DR-ESCs）. Transcriptomic and epigenomic analyses show that DR-ESCs are characterized by primordial germ cell-like gene sig- natures （Dazl, Rec8, Stro8, BUmp1, etc.） and specific epigenetic patterns distinct from mESCs. Moreover, the DR-ESCs possess 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 overactivated in DR-ESCs, and inhibition of TGFβ signaling eliminates the tumorigenicity of mESC-derived NPCs by inducing the full differentiation of DR-ESCs. These data demonstrate that these TGFβ-hyperactivated germ ceU-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 could drastically reduce the risk of tumor development. Keywords： embryonic stem cells, differentiation-resistant ESCs, tumorigenicity, germ cell, TGFβ signaling

Allele-specific genome editing of imprinting genes by preferentially targeting non-methylated loci using Staphylococcus aureus Cas9(SaCas9)

Allele-specific DNA methylation is the most important imprinting marker localized to differentially methylated regions(DMRs),and aberrant genomic imprinted DNA methylation is associated with some human diseases,including Prader-Willi syndrome and cancer.Thus,the development of an effective strategy for the precise editing of allele-specific methylated genes is essential for the functional clarification of imprinting elements and the correction of imprinting disorders in human diseases.To discover a feasible allele-specific genome editing tool based on the CRISPR/Cas system,which is an efficient genetargeting technique in various organisms,we examined the targeting efficiency of Staphylococcus aureus Cas9(SaCas9)and Streptococcus pyogenes Cas9(SpCas9)in response to DNA methylation interference.We found that the targeting efficiency of SaCas9,but not SpCas9,was enhanced by targeted DNA demethylation using the d Cas9-Tet1 catalytic domain(CD)but suppressed by targeted DNA methylation using Dnmt3l-Dnmt3a-d Cas9.An in vitro cleavage assay further demonstrated that SaCas9 nuclease activity was inhibited by 5-methylcytosine(5mC)in a synthesized Cp G-containing context.Further analysis with Ch IP-Q-PCR demonstrated that the non-methylated sequence targeting of Sa Cas9 depends on the binding preference of SaCas9 to non-methylated sequences.Taking advantage of this feature of SaCas9,we have successfully obtained non-methylated allele-biased targeted embryos/mice for two imprinting genes,H19 and Snrpn,with relatively high efficiencies of 28.6%and 47.4%,respectively.These results indicate that the targeting efficiency of SaCas9 was strongly reduced by DNA methylation.By using SaCas9,we successfully achieved allele-specific genome editing of imprinting genes by preferentially targeting non-methylated loci.