Establishment Of Safe,Immune Tolerated Human Embryonic Stem Cells And Their Application Potential

Human embryonic stem cells(hESCs)are capable of unlimited self-renewal and retain the pluripotency to differentiate into all cell types,which hold great promise in the field of regenerative medicine.One of the key bottlenecks is the immune rejection of hESC-derived allografts by the recipient.However,all of the current immunosuppressive strategies will inevitably increase the cancer risk of hESCs and their derivatives.To resolve this issue,we established an immune-tolerated hESCs cell line with a safety checkpoint by insertion of CTLA4-Ig,PD-L1,and TK into HPRT1 locus(hESC-CPTK)using bacteria artificial chromosome(BAC)homologous recombination.The co-expression of CTLA4-Ig and PD-L1 can induce immune tolerance in vivo.TK-expressing cells can be effectively eliminated by FDA-approved gene therapy drug ganciclovir(GCV)both in vitro or in vivo.A humanized mouse reconstituted with human immune system was used to evaluate the function of hESCs-CPTK.The results of flow cytometry and immunofluorescence staining demonstrated that the T cell infiltration rate of hESCs-CPTK derived teratoma was significantly lower than wild-type hESCs derived teratoma in humanized mouse.The hESCs-CPTK derived teratoma can be eliminated by intraperitoneal injection of GCV in vivo.The pluripotency of hESCs-CPTK was characterized by flow cytometry and real time PCR.The hESCs-CPTK were differentiated into cardiomyocytes.The hESCs-CPTK derived cardiomyocytes were immune tolerated and effectively eliminated by GCV in humanized mouse.In addition,we established a rat model reconstituted with human immune system,and successfully implanted hESCs derived cardiomyocytes into the heart of immunodeficient myocardial infarcted(MI)rat,which improved the feasibility to study hESCs-CPTK cells for their therapeutic potential on myocardial infarction.In conclusion,we employed gene editing to induce immune tolerance and reduce cancer risk of hESCs simultaneously,and validated these functions in a humanized mouse model reconstituted with human immune system.hESCs-CPTK are capable of differentiating into cardiomyocytes that can potentially be used in myocardial infarction treatment.Furthermore,the rat models will shed light on the treatment of myocardial infarction using hESCs-CPTK.