Stem cells

The magic brew

Researchers have engineered embryonic stem-like cells from normal mouse skin cells. If this method can be translated to humans, patient-specific stem cells could be made without the use of donated eggs or embryos.

Two reports in this issue1,2 and one elsewhere3 describe a seemingly simple method for changing differentiated adult cells into pluripotent stem cells. The 'gold-standard' test for pluripotency is the ability of a cell to contribute extensively to all adult cell types, including the germ line. The cells generated by these authors pass this test. The researchers introduced four gene-transcription factors into fibroblast cells originating from mouse skin, and specifically selected those cells that, in response to these factors, expressed genes indicative of a pluripotent state. Not only did all three teams manage to isolate cell lines that resembled mouse embryonic stem (ES) cells, but when they injected these cells into early embryos, the cells differentiated into all normal adult cell types.

A previous study4 had shown that differentiated adult cells could be transformed into pluripotent cells when fused with ES cells. This hinted that factors found in ES cells might be essential to conferring pluripotency on other cells. However, the transcriptional profiles, modifications to chromatin (complexes of DNA and histone proteins) and DNA methylation status of ES cells are very different from those of adult cells, indicating that pluripotency is probably under complex layers of control. It was, therefore, a surprise when Takahashi and Yamanaka5 reported last year that they could produce cell lines with some of the properties of ES cells by introducing just four transcription factors associated with pluripotency — Oct3/4, Sox2, c-Myc and Klf4 — into mouse skin fibroblasts, and then selecting cells that expressed a marker of pluripotency, Fbx15, in response to these factors. These cells were called induced pluripotent stem (iPS) cells.

However, the generated iPS cells differed from ES cells in their gene-expression and DNA-methylation patterns. And when these cells were injected into normal mouse blastocysts (70–100-cell embryos), no live chimaeras — animals carrying cells throughout their bodies from both the original blastocyst and from iPS cells — were born.

Yamanaka and colleagues1, as well as Wernig et al.2 and Maherali et al.3, surmised that if selection of iPS cells were based on the expression of genes that are more essential for pluripotency than Fbx15, this might improve the generation of truly pluripotent reprogrammed cells. They derived embryonic or adult fibroblasts that were engineered to express drug-selectable markers under the control of one or other of the two best-studied genes crucial for pluripotency — Nanog and Pou5f1 (Fig. 1). After introducing the four factors (Oct3/4, Sox2, c-Myc and Klf4) by the technique of retroviral transfection, cells were subjected to drug selection. All three groups could derive stable cell lines. In terms of transcriptional, imprinting (expression of alleles predetermined by the parent from which they originated) and chromatin-modification profiles, these were essentially identical to ES cells. Maherali et al.3 also report appropriate reactivation of the inactivated X chromosome in a female iPS cell line, and all authors present images of chimaeric mice, as well as evidence of germline transmission of the genetic content of iPS cells.

Figure 1: Generating induced pluripotent stem cells.
figure1

a, To turn adult skin fibroblasts into embryonic stem cells, researchers generated mice that carried a drug-selectable marker conferring resistance to either neomycin2 or puromycin1,3. Resistance to these antibiotics was linked to expression of either Pou5f1 (ref. 2) or Nanog (refs 1,3), which are markers of pluripotency. b, The authors then isolated fibroblasts from these genetically modified mice and, c, introduced genes for four transcription factors — Oct3/4, Sox2, Klf4 and c-Myc — into these cells by retroviral transfection. d, The transfected cells were subjected to appropriate drug selection. Cells that did not express the gene for drug resistance — that is, Pou5f1 or Nanog — died. e, Thus, rare colonies that resembled embryonic stem cells were isolated, and expanded into stable induced pluripotent stem (iPS)-cell lines. f, When injected into blastocysts of normal mice, iPS cells could contribute to all cell types of the body, including the germ line. g, When the chimaeric animals resulting from these blastocysts were crossed with normal mice, this led either to the birth of live offspring carrying the genetic content of an iPS cell1 or to development to the embryo stage2,3.

But questions remain about the exact sequence of molecular events that leads to this dramatic reprogramming, and whether additional changes, beyond the expression of the four transcription factors, are involved. The process of reprogramming is slow — colonies take up to 20 days to develop into real ES-like cells, and their frequency is quite low. Is this because only a few cells happen to express the right combination or levels of the four factors because of the random integrations of the retroviruses? Or are there additional events, perhaps associated with retroviral insertion, that are required for full transformation?

Continued expression of the four external factors may not be needed for the maintenance of iPS cells; in fact, these cells seem to show low-level expression of these factors. Maherali et al.3 directly tested the requirement for continued expression of Oct3/4 and found that it was dispensable for iPS survival. Such tests need to be done for the other factors.

Side effects of generating stem cells in this way are also problematic. Yamanaka et al.1found that 20% of the iPS-derived offspring developed tumours, presumably related to the activation of one of the transfected genes, c-myc. Some iPS-cell chimaeras have also developed tumours (R. Jaenisch, personal communication).

In addition, it is not known whether the same set of factors can reprogramme other, more specialized cell types. Clearly, the current methodology is more of a proof-of-principle than a fully rationalized series of molecular events that leads to reproducible reprogramming of adult cells. But it is an exciting proof-of-principle nonetheless. Multipotent progenitor cell lines — cells that can give rise to several, but not all, other cell types — have been developed from an increasing number of different fetal and adult sources; but none of these consistently shows the full pluripotency possessed by ES cells or these new iPS cells.

From proof-of-principle in mice to application in humans is still a leap. But since the publication of these studies online, media commentaries have focused on this possibility. The pluripotent nature of human ES cells holds enormous potential for future cell-based therapies for degenerative diseases and traumatic injuries. Use of human embryos to derive such cells remains controversial in many jurisdictions. The possibility of making cell lines with all of the properties of ES cells directly from non-controversial adult sources such as skin holds obvious appeal. It could also be a powerful way of making patient-specific stem cells to provide tissue-matched cells for therapy, and a source of cells for research into the pathogenesis of complex diseases.

Until now, the proposed method to make patient-specific ES-cell lines has been somatic-cell nuclear transfer (SCNT), or cloning. This involves reprogramming DNA from an adult cell by transplanting it into the cytoplasmic environment of an unfertilized egg6 — or, more recently, of a newly fertilized egg7. ES cells derived through SCNT have been generated in mice, albeit at a fairly low frequency. In humans, the first reported success8 from Woo Suk Hwang's group in South Korea is probably a parthenogenetic ES cell derived from a blastocyst, where the egg is activated without the sperm9; later claims by this group were found to be fraudulent. Descriptions of the generation of SCNT-derived primate ES cells at a recent stem-cell meeting in Cairns, Australia, indicate that it might be possible to derive human ES-cell lines using a similar method. However, it is still not clear whether human ES cells can be made at any reasonable frequency after SCNT. An efficient way of directly reprogramming adult cells, which also avoids the controversial use of donated eggs or embryos, would clearly be technically advantageous.

Will the same magic brew of molecular factors work to generate iPS cells in humans? Many groups will certainly be rushing to test this. But translation from proof-of-principle to any therapy has many challenges. First, human cells would have to be given a built-in drug-selectable pluripotent marker by some efficient means. Second, potentially cancer-causing factors such as c-Myc must be avoided. Third, factors would need to be introduced by a method other than retroviral transfection; retroviruses can cause activation of cancer-causing genes and are therefore risky. Transient gene expression by direct introduction of membrane-permeable transcription factors into cells might be one way to achieve this, and screens for small molecules that can replace the gene products would also be useful. Despite these challenges, direct reprogramming of adult cells is clearly the way of the future, and promises to open up new frontiers in human biology and future therapy.

References

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    Maherali, N. et al. Cell Stem Cell 1, 55–70 (2007).

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    Kitai, K. et al. Cell Stem Cell (in the press).

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