Rapid Development of Pluripotent Stem Cells as a Potential Therapeutic Modality

Embryonic stem (ES) and induced pluripotent stem (iPS) cells have attracted attention as powerful tools for both basic biological studies and novel future therapies. ES and iPS cells possess enormous proliferative and broad developmental potential. Both of these characteristics, if understood and regulatable, could lead to a significant capacity for the generation either in vitro or in vivo of differentiated cells that make up functional tissues. Such tissues could be molecularly characterized, even at the clonal level, and transplanted for repair of damaged tissues, augmentation of tissue function, or correction of genetic abnormalities. An important goal—possible with the use of iPS cells—is to derive transplantable tissues in a patient-specific fashion, thus overcoming the significant issues of graft rejection and graft-vs.-host disease that are problematic in current tissue transplantation therapies. Progress in the early development of this field has been impressively rapid, in many cases providing solid proof of principle for the use of these cells in disease models. However, there remain substantial obstacles to translation of these basic studies, and the field has also been prone to the type of overoptimistic predictions of clinical utility in the lay press reminiscent of the gene therapy field in the late 1980s.

In this issue of Molecular Therapy, Müller et al.¹ review the concepts and progress in the development of iPS cells and the potential use of these cells as tools for therapeutic applications. iPS cells share many characteristics with ES cells but are derived from somatic tissues. The reprogramming of murine and human somatic cells to iPS cells requires expression of a small and defined number of factors for a limited period of time. As noted in the review, although the seminal studies in the field empirically defined these factors as OCT4, SOX2, c-MYC, KLF4, and NANOG, subsequent studies have shown that fewer factors are required and have established that certain somatic cells can be reprogrammed with higher efficiency and/or with a few factors, namely, OCT4, SOX2, and NANOG, or in some cases, just two factors.

Early studies, now increasingly refined, used virus vectors for the transfer and expression of complementary DNAs encoding these factors. In addition to the morphological similarities of iPS cells to ES cells, the cells demonstrate similar in vitro differentiation capacities, and molecular studies have demonstrated that iPS cells and ES cells have highly similar expression profiles. In vivo pluripotency has been demonstrated by tissue chimerism in murine experiments but depends on development of teratomas consisting of all three germ layers, with human iPS cells. Interestingly, expression of reprogramming genes is not consistently sustained after development of iPS cells, and this lack of expression is associated with methylation of vector promoter sequences. At the same time, promoter sequences of the endogenous OCT4 and NANOG promoters are demethylated.

Early in the development of iPS technology, it was clear that integrating vectors expressing the reprogramming factors were an obstacle to therapeutic translation. As is well known to the readers of Molecular Therapy, integrating viral vectors can and have produced insertional mutagenic lesions that can be tumorigenic in both mouse nonclinical studies and human gene therapy trials. In addition, low-level residual dysregulated expression of reprogramming factors, even if infrequent, may have deleterious effects on the proliferative or differentiation capacity of the reprogrammed cells. Impressively, as the review in this issue of Molecular Therapy was in preparation and in review, successful approaches to addressing these issues were published by several groups. These approaches include the use of LoxP sites and Cre-induced excision and piggyBac transposon excision of integrated reprogramming vector sequences.^2,3 Most recently, Yu et al. reported the successful development of iPS cells from human fibroblasts free of vector and transgene sequences using a single transfection with an oriP/EBNA1-based episomal vector.⁴ In their study, in some of the derived clones that were stable with extended in vitro passage and analyzed in detail, no integrated or episomal vector DNA remained and no expression of the plasmid DNA could be detected, even by sensitive polymerase chain reaction assays.

As noted in the review by Müller et al., iPS cells offer exciting potential for future cellular therapies. Although a great many obstacles still exist, the rapid progress made even as the review was being written apparently removes one such obstacle, in that it seems that iPS cells can now be generated without genomic integration or continuing expression of reprogramming vectors.