A possible therapeutic approach to curing PD is to generate immune compatible dopaminergic neurons by reprogramming somatic cells (e.g., skin fibroblasts or blood cells) from PD patients and then differentiating them into dopaminergic neurons. To do this in a clinically compliant manner, it is important to show that scalable methods of GMP-compatible cell culture exist, and that iPSC lines are overall similar to hESCs so that the body of existing literature on hESCs can be utilized. It is equally important to show that iPSC lines can be obtained from disease models of PD, and that these lines can be propagated and differentiated appropriately, and used for comparative analysis. Finally, it is also important to show that a stage(s) where the effect of a mutation can be assessed and that the mutation can be corrected or a mechanism be identified.
In this article, we showed that iPSC lines derived from different somatic cell populations can be grown in a hESC-based protocol using a xenogenic-free defined medium and differentiation protocol optimized for hESCs. Using this 4-step scalable process (propagation of hESCs → generation of NSCs → induction of dopaminergic precursors → maturation of dopaminergic neurons), we tested two iPSC lines derived from different somatic cells (adult fibroblast and mesenchymal stem cell) and generated NSCs and subsequently dopaminergic neurons from them. We showed that each of the manufacturing steps could be performed using xeno-free defined conditions in iPSCs similar to hESCs. Neurons generated by this process appeared to be authentic A9 dopaminergic neurons as assessed by in vitro (marker expression) and in vivo (transplantation in PD animal model) assays. This observation was not entirely unexpected as it has been observed that the reprogramming factors are not needed forever. Indeed, once the cells are reprogrammed, they express endogenous pluripotency genes and silence the exogenous ones. Thus, like ESCs or other pluripotent cells, iPSCs should readily differentiate into appropriate lineages and respond in a manner indistinguishable from ESCs. Indeed, this observation has been utilized cleverly by several groups to develop zero-footprint technology [20
] that allows one to reprogram somatic cells using factors or genes that can then be permanently eliminated leaving cells that theoretically, at least, should be indistinguishable from ESCs derived in a conventional fashion, and thus media and reagents developed for ESCs should work with iPSCs.
Our results of directly comparing hESCs with iPSCs suggest that it is true that iPSCs are similar to hESCs. Both functional studies and transcriptome analyses suggest that overall iPSCs derived from somatic cells are largely similar to hESCs. We focused our transcriptome analysis in particular on selected pathways that we and others have suggested may be different between ESCs and iPSCs given the different histories of the ancestor cell that generates a pluripotent population. These include maternal and paternal allele-specific gene expression (imprinting), cell cycle, and senescence, and subtle biases in differentiation due to the source of cells and incomplete reprogramming. Although we did not detect any significant differences, we emphasize that subtle biases are probably best tested in competitive functional assays. Our transplant results suggest that this may be a useful model in which mixed labeled populations can be transplanted in a competitive assay akin to that descried earlier by Jaenisch and coworkers where they showed mouse iPSCs differentiated into functional neurons and glia after transplantation into the developing brain [10
Given the overall similarity and the ability to bypass the immune response, interest in using iPSCs for therapy has been high. Recently, iPSC lines from sporadic PD patients have been generated [8
] and several groups have now shown that gene targeting can be performed with roughly the same efficiency as in hESCs, and that iPSCs from people carrying known mutations can be harvested and propagated [22
]. Although PD is usually sporadic, genetic studies have, however, identified mutations in several genes, including LRRK2, parkin, α-synuclein, uchL1, PINK1, DJ-1
, and ATP13A2
, in familial PD. We, therefore, initiated an effort to generate iPSC lines from familial PD patients with defined mutations. As a proof-of-principle experiment, we derived an iPSC line from skin biopsies obtained from a PD patient with a defined mutation in LRRK2
as mutations in LRRK2
account for approximately 7% of familial PD cases and a significant portion of sporadic PD cases [24
]. Using the identical differentiation protocol, we derived Sox1+
NSCs from the LRRK2
iPSC line. Further analysis of potential phenotype during dopaminergic differentiation associated with the mutation in familial PD patient lines is the scope of our future work.
Full utilization of iPSCs for personalized medicine and disease modeling requires the development of methods of efficient genetic modification including gene targeting and efficient expression or reporters. Recently, it has been shown that gene targeting in iPSCs can be achieved by homologous recombination mediated by zinc finger nucleases [22
]. Efficient gene transfer in iPSC-derived differentiated cells such as neurons would be valuable for both therapy and gene discover, but has been a challenge because of the difficulty of transfecting postmitotic cells. Nonviral physical- or chemical-based methods such as electroporation, nucleofection, and lipofection are inefficient in transfecting postmitotic cells [26
]. Viral vectors such as lentiviral and retroviral systems can transduce neurons with high efficiency, but are primarily used for stable genomic modification and transduction is associated with random integration of transgene. Here, we used an episomal baculoviral-based vector to achieve efficient expression of transgene in iPSC-derived NSCs and particularly in postmitotic neurons. This nonintegration method of high efficiency transfection in human neurons may enhance the utilization of iPSCs for disease modeling as it provides a powerful tool to study neuronal cell biology.
In conclusion, we believe that we have provided strong evidence of the utility of the iPSCs approach to both gene discovery and therapy.