Stuart P. Atkinson

Induced Pluripotent stem cells (iPSCs) have the potential to revolutionise stem cell biology and regenerative medicine.

Stuart P. Atkinson

Induced Pluripotent stem cells (iPSCs) have the potential to revolutionise stem cell biology and regenerative medicine. The reprogramming of human somatic cells to a pluripotent phenotype by the forced expression of transcription factors was reported in 2007 (Papers 1 and 2) and has lead to an acceleration in research on prospective translational aspects of stem cell biology. Thus far, reprogramming efforts have involved the expression of pluripotency associated transcription factors such as Oct4, Nanog, Sox2, Lin28, Myc, and Klf4 in cell lines utilising viral transduction methods. Although this technology holds great promise, there are potential problems, such as the potential oncogenic nature of over-expressing transcription factors such as Myc, and integrative virus’. Recent research topics in iPSC technology have been the identification of cells which can be reprogrammed using a minimum of exogenous factors and also the replacement of reprogramming factors with small molecule drugs (Papers 3 and 4). These studies have begun to address some of the concern over the potential oncogenicity of both the factors used and also the vectors utilised for their transfer.

However, the use of exogenous genes and vectors will always leave some risk of possible complications if technology based on these procedures is ever to venture into the human clinical arena. Therefore, any techniques which could eliminate these risks would be of great importance and use.

The ability to induce and manipulate endogenous expression of stem cell genes in human somatic cells without genetic manipulation has been reported previously (Paper 5) by manipulations in culture conditions, namely oxygen concentration and FGF2 levels. This technique, however, did not allow the reversion of the cells utilised back to a pluripotent-like state.

It has now been shown that through the manipulation of the culture conditions of a specific adult cell type, pluripotency can indeed be induced without the requirement for genetic manipulation or the use of viral vectors. This exciting paper in this months issue of Stem Cells from the laboratory of Iqbal Ahmad at the University of Nebraska Medical Center (Paper 6), describes a novel two stage reprogramming process in which the induction of pluripotency from rat limbal epithelial progenitors (LEPs) is observed following alterations in the growth micro-environment.

In the first stage, dissociated LEPs isolated from rat eyes were grown under neural induction conditions in the presence of FGF2 and Noggin for 7 days. Stage 2 involved the addition of mouse embryonic stem cell (mESC) conditioned medium for a further 10-14 days which allowed for colony formation under feeder-free conditions. Cells expressed endogenous Klf4, Sox2 and c-Myc during stage one, whilst the second stage promoted induction of Oct4 and Nanog in the LEPs and reprogrammed the rat LEPs to generate iPSCs.

In addition to being morphologically similar to mESC, further analysis of LEP-derived iPSCs revealed that they expressed alkaline phosphatase activity and SSEA1, similar to mESCs. Temporal analysis of the reprogramming process showed an initial surge of Oct4 expression, then Nanog, alongside a concomitant down-regulation in the limbal specific markers, p63 and a-enolase. DNA demethylation was observed at specific regions of the Nanog and Oct4 promoters, indicative of epigenetic reprogramming. In addition, an evaluation of pluripotency-associated gene expression demonstrated that the LEP-derived iPSCs displayed elevated levels of these genes, although relative levels were overall lower than that observed in mESC.

An estimated reprogramming efficiency was reported at 0.0025%, lower than that reported for 3 or 4 factor reprogramming of mouse embryonic fibroblasts (MEFs), and single factor reprogramming of adults neural stem cells (NSCs). Interestingly, the efficiency of reprogramming was increased 4-fold by the addition of the histone deacetylase inhibitor (HDACi) valproic acid, perhaps suggesting that additional epigenetic plasticity needs to be afforded to these cells in order for full reprogramming to occur. Indeed, the DNA methylation studies suggest that epigenetic reprogramming may be incomplete or insufficient.

Subsequent analysis, however, demonstrated the pluripotent ability of the iPSCs, as they were able to 1) form embryoid bodies which exhibited markers of embryonic ectoderm, endoderm and mesoderm, 2) develop into well differentiated teratomae, identical to mESC-derived teratomae in all but size and 3) differentiate into functional neurons, cardiomyocytes and hepatocytes. Such tests have been discussed as being the “gold standard” for proving pluripotentiality, at least in human iPSCs (Paper 7 and Author Replies).

This research leaves us with many interesting questions. Can this type of reprogramming be applied in other adult stem cells or other terminally differentiated somatic cell types? Further, can this type of reprogramming be achieved with human cells? Limbal progenitors can be obtained and amplified quite readily and may represent an exciting target for reprogramming efforts (Paper 8) and other adult stem cells may prove to be approachable targets. Although generally lower efficiency in reprogramming from human cells may hinder this approach, this method of reprogramming has the potential to lead to safer cell replacement therapy, such as pancreatic beta cell replacement in Type I diabetes , the treatment of macular dystrophies (Paper 9 for a recent review in Stem Cells), such as photoreceptor replacement in Age-related Macular Degeneration, or to treat various neurodegenerative disorders . This technology may also lend itself to a more faithful recapitulation of models of disease and early development, by dispensing with any potentially interfering exogenous factors.

1. Cell - Induction of pluripotent stem cells from adult human fibroblasts by defined factors (2007)
2. Science - Induced pluripotent stem cell lines derived from human somatic cells (2007)
3. Stem Cells - Reprogramming of neural progenitor cells into induced pluripotent stem cells in the absence of exogenous Sox2 expression (2008)
4. Stem Cells - Generation of Human Induced Pluripotent Stem Cells in the Absence of Exogenous Sox2 (2009)
5. Cloning Stem Cells - Induction of stem cell gene expression in adult human fibroblasts without transgenes (2009)
6. Stem Cells - Non Cell-Autonomous Reprogramming of Adult Ocular Progenitors: Generation of Pluripotent Stem Cells Without Exogenous Transcription Factors (2009)
7. Cell Stem Cell - Guidelines and techniques for the generation of induced pluripotent stem cells (2008)
8. Regen Med - Loss of corneal epithelial stem cell properties in outgrowths from human limbal explants cultured on intact amniotic membrane (2008)
9. Stem Cells - Genetic basis of inherited macular dystrophies and implications for stem cell therapy (2009)

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