Science in Society Archive

The Promise of Induced Pluripotent Stem Cells

Ordinary cells can be induced to become pluripotent stem cells, making it unnecessary to use human embryos, but is it safe? Dr Eva Sirinathsinghji

Pluripotent stem cells – cells that can multiply indefinitely to differentiate into all cells of the body - have long generated great excitement in the scientific and public communities due to their potential use for tissue regeneration. The excitement has grown even more so recently with the development of a new technique for artificially generating pluripotent cells without the use of embryos. These ‘induced pluripotent stem cells’ (iPS cells) sent a shockwave through the scientific community as they demonstrated that an adult cell can revert back to a pluripotent state. Yamanaka and colleagues, based in Kyoto, showed that by expressing just four genes in adult skin cells, they became similar to embryonic stem cells, with the unique capacity to self-renew and turn into almost every cell type in the human body.

Previously, there have been a number of methods for sourcing pluripotent stem cells, from ordinary embryos, or embryos created by somatic cell nuclear transfer (used to clone Dolly the sheep) and cell fusion. Since the first generation of mouse iPS cells in 2006 [1] and human iPS cells in 2007 [2], this technique has ballooned into a huge and rapidly evolving research field.  

Of all of the techniques, that of iPS cells is the only one to date that circumvents the use of embryos, and also has the ability to generate patient-specific cell lines, avoiding immune rejection following transplantation. In theory, a patient requiring stem cell transplant only needs to donate skin cells, which can be re-programmed and multiplied to sufficient number of cells, induced to differentiate into a cell-type required, and then transplanted back into the patient. It seemed surprisingly simple and reproducible, and aroused huge interest for its potential use in regenerative medicine and in scientific research. The safety of such clinical procedures has been questioned in the journal Nature, however, where iPS cells were shown to trigger an immune response following transplantation in to mice with the same genetic makeup as the transplanted cells [3].

What are iPS cells?

iPS cells were first generated by the forced expression of a specific set of genes in adult specialised cell types such as skin cells. These genes were previously known to play an important role in the maintenance of pluripotency, and so could reprogram differentiated cells back to an undifferentiated, pluripotent state. By looking at 24 genes, the researchers were able to narrow down a combination of only 4 that have the ability to reprogram cells: c-Myc, Klf-4, Oct3/4 and Sox2. They were introduced into skin cells, and over a period of 3-4 weeks a proportion of the skin cells changed their morphology and growth and molecular characteristics to resemble embryonic stem cells.  These iPS cells were shown to be able to turn into brain, heart as well as bone cells.

A detailed look at the technique of iPS cell production

This technique was first performed in human cells by using viral gene vectors (carriers) to insert the genes of interest into the host genome. These viral vectors (with disease-causing genes of the viruses removed) are used in other areas of medical research such as gene therapy.

The drawbacks of the classic viral vector approach include the permanent insertion of the DNA into the host genome, which can lead to disruption of the normal functioning of genes, a phenomenon referred to as ‘insertional mutagenesis’ (see [4] Naked and Free Nucleic Acids - Unregulated Hazards, ISIS Report for hazards of viral vectors and gene therapy) . This is a major limitation to the clinical value of these iPS cells for transplant. The first publications found at least 20 insertions of the combination of 4 genes in the iPS cell lines generated. With such high numbers of viral DNA present in these cells, the risks of insertional mutagenesis is even greater.  In addition, although the expression of 4 genes appears to be temporary, their reactivation could have serious implications for the risks of tumour formation. These genes are not only important in maintaining pluripotency, but are also known to play a role in various types of cancers. Cancer also involves reprogramming of differentiated cells to a proliferative state, which shares many characteristics of embryonic stem cells. Indeed, reactivation of c-Myc resulted in tumour formation in ~20 percent of mice generated solely from iPS cells [5].

Successful cellular reprogramming has now been done with fewer genes, importantly, without c-Myc [6]. Chemical compounds such as valproic acid have also been used to increase the efficiency of reprogramming [7]. These compounds alter DNA structure, making them more accessible to gene regulation processes, a conformational feature that is closely associated with undifferentiated cells. More recently, iPS cells have been generated without viral vectors, but instead with the direct delivery of the protein products of the necessary genes [8]. By modifying the proteins to harbour a peptide anchor, the proteins were able to be channelled into the cell to induce the reprogramming. This eliminates the previously mentioned concerns of permanently altering the genome of iPS cells, making them more suitable for therapeutic use.

The ultimate test to show that iPS cells are truly pluripotent is the generation of viable adults derived solely from these cells. This was first done in 2009 by tetraploid complementation assays [9] (see [10] Unacceptable Death Rates End Cloning Trials in New Zealand, SiS 50, for a succinct explanation of the tetraploid complementation assay). Viable mice were generated, although many died at embryonic stages or at birth, a phenomenon common to this technique.

Potential uses

iPS cell reprogramming addresses interesting and important questions about what is required to maintain a cell in a pluripotent state, and how dynamic the cells in our body really are. By identifying key underlying genes, our understanding of pluripotency is advancing.  These de-differentiation studies can be applied to cancer research. They also provide human cells that could be valuable for modelling of disease, providing a compliment, or even an alternative to animal research. Some disease-specific cell lines were quickly established, with one publication showing the production of 10 cell lines from patients with diseases including Down’s syndrome, Huntington’s disease, juvenile diabetes as well as Parkinson’s disease [11].

Many researchers hope to make iPS cells for regenerative medicine, with the added potential of making patient-specific cells, avoiding the risks of immune rejection. These cells could also be used for tailor-made treatment programs [12]. Certain disease treatments can work on only a percentage of patients, so pre-testing them on patient’s cells may increase the predictability of drug efficacy and toxicity in each patient.

A more sinister use of iPS cells is the generation of transgenic animals for food consumption. The use of iPS cells could circumvent cloning experiments that have shown to cause high levels of death and suffering in livestock, (see [13]).  

Drawbacks

As with ES cells there is a significant risk of tumour formation if iPS cells are to be used for transplantation. Tumours can form when a few remaining undifferentiated cells continue to proliferate following transplantation. This is a well established concern that is widely discussed, and for good reason. Added limitations of some iPS cells are the possibilities of insertional mutagenesis. Incomplete re-programming may also lead to inability to differentiate fully into adult specialised cells. However, recent studies of both ES and iPS cells show a similar differentiation capacity, depending on the cell line used [12]. A further concern is the accumulation of mutations and genomic rearrangements during the iPS cell generation process. A paper recently published in Nature, showed that 124 mutations were discovered in 22 different iPS cell lines [14]. Moreover, a number of these mutations were discovered in genes related to cancers, with mutations found in the same gene across numerous lines. Developing the same mutation across cell lines suggests that there may a selective advantage to their acquirement. This limitation may never be overcome if cells are grown for long periods of time, something difficult to avoid when high cell numbers are needed for transplantation. 

With these associated risks of ES and iPS cell therapy,  alternative research into methods of stimulating one’s own stem cells for regenerative therapy may prove more effective, (see [15] Stem Cells Repair Without transplant, SiS 50). Other research also focuses on the prospects of transdifferentiation of cells from one cell type to another, without reverting to a pluripotent state in the process [16].  

To conclude

iPS cells may prove important for drug discovery and research into disease mechanisms, providing a unique source of human cells that can be derived from the skin of diseased or healthy individuals. Their use for tissue regeneration, however, poses considerable health risks, with research into their clinical value still in the earliest stages.

Article first published 24/05/11



References

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  2. 2. Takahashi K, Tanabe K, Ohnuki M, Narita M, Ichisaka T, Tomoda K, Yamanaka S. Induction of pluripotent stem cells from adult human fibroblasts by defined factors. Cell, 2007, 131, 861-72
  3. 3. Zhao T, Zhang Z-L, Rong Z, Xu Y. Immunogenicity of induced pluripotent stem cells. Nature 2011, doi:10.1038/nature10135
  4. 4. Ho MW. Ryan A, Cummins J and Traavik T. Naked and Free Nucleic Acids – Unregulated Hazards, TWN Report, 2000
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  6. 6. Nakagawa M, Koyanagi M, Tanabe K, Takahashi K, Ichisaka T, Aoi T, Okita K, Mochiduki Y, Takizawa N, Yamanaka S. Generation of induced pluripotent stem cells without Myc from mouse and human fibroblasts. Nat Biotech 2008, 26, 101-6
  7. 7. Huangfu D, Maehr R, Guo W, Eijkelenboom A, Snitow M, Chen AE, Melton DA. Induction of pluripotent stem cells by defined factors is greatly improved by small-molecule compounds Nat Biotechnol. 2008, 26, 795-7.
  8. 8. Kim D, Kim CH, Moon JI, Chung YG, Chang MY, Han BS, Ko S, Yang E, Cha KY, Lanza R, Kim KS. Generation of human induced pluripotent stem cells by direct delivery of reprogramming proteins Cell Stem Cell. 2009, 4, 472-6.
  9. 9. Boland MJ, Hazen JL, Nazor KL, Rodriguez AR, Gifford W, Martin G, Kupriyanov S, Baldwin KK. Adult mice generated from induced pluripotent stem cells Nature. 2009 461, 91-4.
  10. 10. Ho MW Unacceptable Death Rates End Cloning Trials in New Zealand Science and Society 50, in press.
  11. 11. Park IH, Arora N, Huo H, Maherali N, Ahfeldt T, Shimamura A, Lensch MW, Cowan C, Hochedlinger K, Daley GQ. Disease-specific induced pluripotent stem cells Cell. 2008, 134, 877-86
  12. 12. Itzhaki et al., Modelling the long QT syndrome with induced pluripotent stem cells Nature. 2011, 471, 225-9
  13. 13. Bock C, et al., Reference Maps of human ES and iPS cell variation enable high-throughput characterization of pluripotent cell lines Cell. 2011, 144, 439-52
  14. 14. Gore A, et al., Somatic coding mutations in human induced pluripotent stem cells Nature. 2011, 471, 63-7
  15. 15. Sirinathsinghji E Stem Cells Repair Without Transplant Science and Society 50  (to appear).  
  16. 16. Vierbuchen T, Ostermeier A, Pang ZP, Kokubu Y, Südhof TC, Wernig M. Direct conversion of fibroblasts to functional neurons by defined factors. Nature 2010, 463, 1035-41.

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