Although hiPSCs hold great promise as platforms to study and treat human disorders, it is critical to carefully assess their safety for clinical and biomedical application. In particular, hiPSCs generated using retroviruses and lentiviruses have chromosomal integrations that may lead to unpredictable genetic dysfunction. Our present results demonstrated that there was residual exogenous gene expression even when lentivirus-based hiPSCs were terminally differentiated into NPCs or neurons. In contrast, expression of the reprogramming genes was not readily detected in retrovirus-based hiPSCs after neural differentiation, indicative of almost complete silencing of exogenous genes. At present, it is not known why exogenous reprogramming genes exhibit differential silencing in lentivirus- and retrovirus-based hiPSC lines. Interestingly, during hematopoietic differentiation of mouse ESCs, gene expression from the retrovirus long terminal repeat (LTR), but not from the lentivirus, was severely reduced (32
), which appears to be caused by methylation of the retroviral LTR promoter (33
). Thus, it is possible that differential promoter methylation underlies the distinct silencing between lentivirus- and retrovirus-based hiPSCs.
Although NPCs derived from retrovirus- and lentivirus-based hiPSCs displayed limited expandability and early senescence, neuronal differentiation appeared to occur normally in all hiPSCs, as evidenced by efficient generation of NPCs and DA neurons under our optimized procedure. These observations suggest that retrovirus- and lentivirus-based hiPSCs may be useful for a variety of biological and neurorepair studies, even if they are an unsuitable source of cells for future personalized medicine. A recent study demonstrated that hiPSCs generated by the lentiviral method exhibit neural differentiation capacity with significantly reduced efficiency and increased variability compared with hESCs (13
). We also recently showed that derivative cells (e.g., hemangioblasts, endothelial cells, and hematopoietic cells) could be generated from lentivirus- and retrovirus-derived hiPSC lines with phenotypes similar to those derived from hESCs, but with a dramatic decrease in efficiency (34
). In distinct contrast to the hESC derivatives, hemangioblasts derived from virus-based hiPSCs showed significantly increased apoptosis, severely limited expansion capability, and substantially decreased hematopoietic colony-forming capability. Taken together, these results suggest that limited expandability and early senescence of derivative precursor cells is a common phenomenon for lentivirus- and retrovirus-based hiPSCs with genomic disruptions. Interestingly, we found that P53 expression was induced in apoptotic NPCs derived from lentivirus- and retrovirus-based hiPSCs before they underwent massive apoptosis. Why P53 is induced in these cells, but not in NPCs derived from hESCs or protein-based hiPSCs, warrants further investigation.
To overcome the potential caveats associated with residual exogenous reprogramming genes and chromosomal integrations, several groups have recently reported new approaches of hiPSC generation, including use of minimal numbers of reprogramming genes (35
), use of small molecules (36
), excision of the remaining transgenes (38
), and use of nonintegrating vectors (41
). It will be important to determine whether these hiPSCs behave similarly to hESCs and protein-based hiPSCs in terms of differentiation and function of their derivative progenitor cells.
To our knowledge, this is the first study to directly compare the differentiation and cellular properties of hiPSCs generated by chromosome integrating and nonintegrating methods. A potential limitation of this work is that we used disparate hiPSC lines derived from different cell sources, at different passages, and handled by different laboratories, any of which can substantially affect the properties of individual hiPSC lines. Thus, a more straightforward comparison could be made using individual hiPSC lines generated from the same cell source via different reprogramming methods and used at similar passage and culture conditions. Despite these possible limitations, our data demonstrate fundamental and consistent differences in cellular and differentiation properties within each group of hiPSC lines without any exception, which strongly supports that these results are not due to experimental artifacts. Thus, our results suggest that protein-based hiPSCs may serve as a promising source of cells for clinical translation. These cells behaved similar to hESCs without abnormal senescence/apoptosis, they did not show any exogenous reprogramming gene expression, and DA neurons derived from protein-based hiPSCs significantly improved behavioral defects in a rodent model of PD. A high number of TH+
neurons (>25,000 cells/graft) survived over months after transplantation with prominent behavioral effect. This is in clear contrast to most hESC/hiPSC transplantation studies, which show only modest behavioral improvements in rodent PD models with much fewer surviving TH+
neurons (e.g., refs. 43
). Although it is not clear whether these differences are related to the hiPSC lines used in the present studies and how they were derived, it is likely that our optimized differentiation protocol and/or the stage of cell transplantation contributed to the different functional outcomes. In line with this, no functional recovery was observed when we transplanted the differentiated cells. Although tumorigenic masses were observed when a large number of cells was transplanted, this was most likely caused by the remaining undifferentiated cells. This underscores the importance of establishing clinically safe methods for complete removal of undifferentiated cells, as evidenced by a recent comprehensive study of multiple mouse iPSC lines showing the correlation of teratoma-forming propensities and remaining undifferentiated cells (12
). Our results suggest that protein-based reprogramming may be a viable approach for generating a patient-specific source of cells for treatment of PD and other degenerative diseases.