Understanding the mysteries of iPS cells

The development of induced pluripotent stem (iPS) cells is one of the newest and most exciting areas of stem cell research.

Takahashi and Yamanaka, in a landmark paper in 2006 [1], were able to demonstrate that cultured and fully differentiated mouse fibroblasts could be transformed into pluripotent embryonic-like stem cells able to produce all three germ layers in addition to chimeric animals. But what made this finding even more astonishing was that only four transcription factors needed to be transduced for this transformation. These newly derived iPS cells have tremendous promise because they lack the typical ethical and immunological problems of other approaches. Ethically, no human embryos would have to be used in their creation, and immunologically, a patient’s own genetically identical cells could be transplanted. The heretofore locked door to truly regenerative medicine finally seems to have found its key in iPS cells.

At StemCONN 2009, a Connecticut-wide scientific symposium that took place at the Omni Hotel in New Haven in March, Konrad Hochedlinger of Massachusetts General Hospital and Harvard Medical School addressed the question of how broadly the iPS conversion strategy can be applied, i.e., whether this method of cellular reprogramming is specific only to cultured mouse fibroblasts. These starting cells are far removed genetically and environmentally from the typical cells that reside in the body.

Hochedlinger presented data showing that even terminally differentiated pancreatic cells were able to be reprogrammed using the same four factors: c-myc, Klf4, Oct4, and Sox2 [2]. Other cell types also have been shown to be iPS compatible, including lymphocytes [3], liver cells [4], intestinal cells [5], and neural progenitors [6], suggesting that in vitro reprogramming may be a universal process. However, slight alterations in the combinations of factors required for reprogramming can be seen depending on the endogenous expression patterns of certain cell types. Specifically, c-myc, Klf4, and Sox2 are expressed in multiple adult tissues, and their pattern of expression can affect iPS creation. For example, neural progenitor cells lack a Sox2 transduction requirement because of the already naturally high level of this transcription factor in this cell type. However, Oct4 appears to be the only irreplaceable, and debatably the most important, determinant of direct reprogramming.

These landmark findings led Hochedlinger to propose that the stoichiometry of each factor is critically significant in the reprogramming process, since too much of any one transgene appears to be toxic to the cells, while insufficient amounts will not lead to faithful iPS creation.

Hochedlinger explained that based on the current understanding, iPS cells and embryonic stem (ES) cells are similar, both molecularly and functionally [7]. For example, iPS cells show reactivation of pluripotency genes, telomerase activity, and a silent X chromosome in female cells, as well as genome-wide transcriptional and epigenetic patterns that are characteristic of ES cells. Moreover, iPS cell chimeras give rise to offspring, indicating that these cells can contribute to the germline.

But the comparison of iPS cells and ES cells is not simple, because other studies have shown defective differentiation and commitment into mature cardiac myocytes when using an iPS starting population vs. ES cells. Furthermore, about one-third of iPS chimeras succumb to tumors that are likely related to the reactivation of retroviral transgenes and insertional mutagenesis, exposing a critical technical problem associated with the original methods of producing iPS cells: Retroviruses or lentiviruses must be deployed to stably integrate exogenous DNA into the host cells’ genome. Unfortunately, this integration frequently falls into oncogene or tumor suppressor loci, leading to malignant transformation. Newer techniques use non-integrating viruses, transiently transfected adenoviruses, or even small molecule activators. These methods, which diminish the negative consequences of prior techniques, are essential before any promising human treatment method could be considered. Yet the robustness and efficiency of these methods remains to be proven.

Currently, most protocols demonstrate efficiencies of only between 0.01 percent to 0.1 percent. Hochedlinger suggested that much of this inefficiency arises from the small probability that all four viral vectors (each containing one of the necessary transgenes) infect one cell while still maintaining a stable copy number and randomly inserting into a “safe” genomic region. Without a “safe” integration site, the viruses used to create iPS cells could damage an endogenous gene that is essential for the reacquisition of the pluripotent, self-renewing state. In addition, it has been proposed that the low efficiency is also dependent on the presence of rare tissue-resident stem cells within the starting population [7]. It is possible that this stem cell population alone is responsible for iPS creation, because a relatively immature cell fate may mean that these cells are better primed genetically for pluripotent reprogramming. Hochedlinger, however, was unconvinced that adult stem cells are the selective target population and instead focused on the observation that endodermal derivatives are more amenable to reprogramming than cells of mesodermal origin. Nonetheless, the question remains an open one with ongoing work on both sides of the debate yielding conflicting results as to the need for a specific iPS cell of origin.

As work continues to understand the mechanism and cellular requirements for reprogramming, it is certain that further experimentation to optimize the induction of pluripotency will lead to an efficient mechanism. iPS cell yields of much greater than 0.1 percent are necessary in order to functionally repair the typical organ system composed of millions of cells. Only with improvements in these yields can true therapeutic application of this cellular therapy be realized.

Abbreviations

iPS

induced pluripotent stem

ES

embryonic stem