Safety Of Directed Differentiation Of Genetically Modified Human Induced Pluripotent Stem Cells Into Motor Neurons

Human induced pluripotent stem cells(hiPSCs)are reprogrammed stem cells induced by transgenes or small molecules,which can be differentiated into almost any cell type of the human body.As an important renewable stem cell resource,hiPSCs have been widely used in preclinical and clinical studies for the treatment of degenerative diseases.However,the potential safety risks of hiPSCs need to be addressed before transplantation.Due to complexity and variability of hiPSCs differentiation,it is difficult to control their directed differentiation completely and accurately,which may lead to the formation of teratoma or the generation of undesired cells.An effective approach is multi-gene modification of hiPSCs with transcription factors for cell fate,which can program hiPSCs into specific type of cells.After that,we can transplant these modified hiPSCs into animal models to evaluate the safety and efficacy of stem cell therapy.Privously we have established a hiPSCs cell line(NILB-hiPSCs)which conditionally express motor neuron specific transcription factors NGN2,ISL1,and LHX3(NIL)via Doxycycline(Dox)induction,and constitutely expressed anti-apoptotic gene BCL-XL.In this study,we transplanted NILB-hiPSCs into the subcutaneous tissues of immunodeficient mice,and then evaluated the tumorigenicity,survival and differentiation of transplanted cells,which may explore the safety and clinical-value of hiPSCs with conditional gene modification in cell therapy.First,NILB-hiPSCs were transplanted into the subcutaneous tissues of immunodeficient mice.NILB-hiPSCs proliferated rapidly in the transplanted sites,and formed a teratoma-like structure with increasing size at 4 week post transplantation without Dox induction.While,after Dox induction,both the number of NILB-hiPSCs and the size of the engraftments decreased,and no teratoma formed.These data indicated that NILB-hiPSCs does not lead to tumor formation after subcutaneous injection with Dox treatment.Secondly,to determine the differentiation of NILB-hiPSCs in subcutaneous tissues,immunohistochemistry for motor neuron marker were performed.We found many transplanted cells expressed these markers,including ?-tubulin,MAP2,HB9 and Ch AT,suggesting that the transplanted NILB-hiPSCs had directionally converted to mature motor neurons in vivo after Dox induction.Unexpectedly,we observed massive ventricle-like cavities in the engraftments,which expressed Neural precursor cells(NPCs)markers SOX2 and PAX6,implying that NILBhiPSCs differentiate into NPCs partially after Dox induction.The grafts were isolated and cultured further in vitro,these cells were differentiated into motor neurons rapidly and effectively.Further to investigate the causes of NPCs differentiation,we tested three related factors,including Dox concentrations,medium conditions,and culture systems.The results showed that the differentiation process was direct,and did not pass through the NPCs state in vitro.It was indicated that unknown and unique microenvironment exiting in vivo might contribute to the generation of NPCs when differentiating in vivo.Taken together,NILB-hiPSCs differentiated into motor neurons,and no tumor formation was noted in the subcutaneous tissue after Dox induction.In addition,the engrafted cells partially converted into NPCs,and further differentiated into motor neurons in vitro,which may serve as potential candidates to neural circuit reconstruction.The results of the current study provided important reference of the safety issue in stem cell therapy.