By Kevin E. Noonan --
More important (and perhaps even more sought after) than the alchemist's Philosopher's Stone have been the efforts over the past several years to find a substitute for embryonic stem (ES) cells. In view of ethical considerations and the Bush Administration's religion-based (and politically-motivated) restrictions on federal funding for human ES cell research, researchers have attempted, with varying degrees of success (see "Limitations on the Usefulness of Adult Stem Cells"), to use stem cells isolated from adult tissues, as well as placentally-derived hematopoietic stem cells instead of human ES derived from embryos (most of which are slated for destruction as an unused consequence of in vitro fertilization efforts). Although the full scope of the utility and potential of human ES cells has yet to be established, it is generally recognized that until now these cells possess developmental properties and potentialities different from (more robust and versatile) adult stem cells. Additional concerns involve isolating a sufficient number of stem cells for regenerative medicine even if a change of administration in Washington in 20 months leads to a change in the current policies.
Reports this week suggest a new alternative: genetically-engineered fibroblasts having increased developmental potential. One report, from a group including Dr. Rudolf Jaenisch (at left) at the Massachusetts Institute of Technology and its related research organizations, the Whitehead Institute and the Broad Institute (a joint effort between MIT and Harvard), was published on June 6th on the Nature website (Wernig et al., 2007, "In vitro reprogramming of fibroblasts into a pluripotent ES-cell-like state"). In this report, Jaenisch and co-workers showed that adult mouse fibroblasts (the precursor cells to connective tissue) could be "reprogrammed" to have increased developmental potency. This reprogramming involved introducing into these cells and ectopically expressing four transcription factors: OCT4 (also known as OCT3/4 or POU5F1), SOX2, C-MYC, and KLF4. Cells produced by nuclear somatic introduction of these genes into fibroblast cells were found to have methylation patterns, chromatin configurations, and gene expression patterns characteristic of human ES cells. Functionally, the cells could be used to form viable chimeras (embryos created classically by fusion of different zygotes; see papers by Clement Markert from the 1980's), can generate late-term embryos and can contribute to the germ line in mice produced using them. The authors characterize the attributes as being "indistinguishable" from ES cells.
This work fulfilled earlier studies by a Japanese group (Takahashi et al., 2006, "Induction of pluripotent stem cells from mouse embryonic and adult fibroblast cultures by defined factors") that achieved expression of these four transcription factors in fibroblasts but could not be shown to be functionally-equivalent to ES cells. The Jaenisch group used a different selection criteria to produce its reprogrammed fibroblasts, by selecting cells having induced expression of the endogenous Oct4 gene through homologous recombination and selection with a drug marker gene, followed by infection with retroviral vectors encoding each of the four transcription factors. The reprogrammed fibroblasts having reactivated endogenous Oct4 or Nanog genes (linked by homologous recombination to the bacterial neo gene) were epigenetically identical to ES cells by several important criteria, and generated viable chimeras and late-gestation embryos after injection into tetraploid blastocysts.
Such reprogrammed pluripotent stem cells have an advantage over ES cells in their capacity to be "customized" to an individual patient, since they could in theory be developed from the individual's own fibroblasts. These reprogrammed cells also were able to grow independently of feeder cells, and expressed normal OCT4, NANOG, and SOX2 RNA and protein levels. Importantly, although the reprogramming appeared to be triggered by expression of the exogenous transcription factors under control of the retroviral long terminal repeats (LTRs), maintenance of the pluripotent state was the result of expression of the endogenous transcription factor genes, particularly Oct4. In fact, methylation patterns of the retroviral promoter sequences indicated that these sequences were transcriptionally silenced in the reprogrammed cells. This is important for translation of these results from mouse to human cells, since it is known that inappropriate, retroviral LTR-mediated expression can cause (or at least increase the risk of) malignancy. Indeed, the authors identify their use of retroviral-mediated vectors as a major impediment to applying these methods to human cells.
In another report from the Yamanaka group (Okita et al., 2007, "Generation of germline-competent induced pluripotent stem cells") - responsible for the first demonstration for reprogramming fibroblasts using exogenously-introduced transcription factors - similar results were obtained. However, this group targeted endogenous Nanog gene expression, rather than Oct4 expression in the reprogrammed fibroblasts, but showed increased ES cell-like gene expression and methylation patterns. These cells also differed from their previously-reported reprogrammed cells by being competent to make chimeras. Unlike the Jaenisch group's results, however, the chimeras obtained in these studies showed a high (20%) incidence of tumors that the authors attributed to activation of the c-myc transgene.
Yet another demonstration of this reprogramming was reported in the inaugural issue of Cell Stem Cell by Dr. Konrad Hochedlinger from the Massachusetts General Hospital Center for Regenerative Medicine and the Harvard Stem Cell Institute, Dr. Kathrin Plath from the Institute for Stem Cell Biology and Medicine at UCLA, and their colleagues (Maherall et al., 2007, "Directly reprogrammed fibroblasts show global epigenetic remodeling and widespread tissue contribution"). This report contained as additional evidence reactivation of the X chromosome in female-induced stem cells (that was silenced in differentiated cells), as well as random X inactivation upon redifferentiation.
Once more, the inexorable advance of technology may outpace attempts by policy-makers and politicians to direct the developmental course of stem cell science. It is gratifying that this work was done in the U.S., in the face of the several obstacles imposed by federal funding limitations, but it must be acknowledged that these advances were achieved using mouse cells rather than human cells. It is certainly likely that the experimental flexibility of the mouse ES model would have directed the course of this research through the mouse intermediate in any event. But it is certainly the case that efforts by several states, including California and (relevant to the Jaenisch group) Massachusetts will be necessary to provide the financial support (and incentives) for further development of a technology that could avoid many of the troublesome ethical challenges facing use of human embryos to produce ES cells.
For additional information on this and related topics, please see:
- "Massachusetts to Invest $1 Billion on Medical and Science Research," May 10, 2007
- "It's Time to Stop the Hypocrisy over Stem Cell Patents - Part II," April 26, 2007
- "It's Time to Stop the Hypocrisy over Stem Cell Patents - Part I," April 17, 2007
- "WARF Stem Cell Patent Claims Rejected in Re-examination," April 3, 2007
- "NIH Chief Dissents on Federal Stem Cell Funding Ban," March 20, 2007
- "Stem Cells a Go! in California," February 28, 2007
- "Limitations on the Usefulness of Adult Stem Cells," February, 28, 2007
Comments