Induced pluripotent stem cells

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Induced pluripotent stem cells, also known as iPSCs, are pluripotent stem cells derived from fully-differentiated ("adult") somatic cells[1][2]. While they are often mistakenly referred to as "adult stem cells", this is not the proper term for them because biologists more commonly use the phrase "adult stem cells" to refer to the partially-differentiated stem cells residing in somatic tissues.

iPSCs are generated by transfecting fully differentiated cells with a combination of transcription factors that cause the cells to de-differentiate into a pluripotent state, mimicking the pluripotency of embryonic stem cells (ES cells). As pluripotent cells, iPSCs can generate cells of any type; they can even be used to generate "organoids" (structures mimicking the organization of complex tissues) in-vitro (in culture)[3][4].

Current methods of generating iPSCs are not without limitations. While the process of inducing de-differentiation does yield pluripotent cells, these cells retain some epigenitic imprinting of the cell type from which they were derived. As such, iPSCs do not perfectly recapitulate the behavior of ES cells. Additionally, cell transplantation studies have demonstrated that iPSCs are significantly (~3-4 times, depending on the source tissue and method of induction) more tumorigenic in-vivo than embryonic stem cells, limiting their current therapeutic potential[5][6].

Contents

History

The first iPSCs were produced in 2006 in the lab of Shinya Yamanaka at Kyoto University in Japan. Yamanaka and colleagues transfected mouse fibroblasts (in this case, skin cells) with a combination of four transcription factors (Oct3/4, Sox2, c-Myc, and Klf4) to generate cells with similar morphology, gene expression, and "growth properties" to ES cells[7]. A year later, a team led by James Thomson at the University of Wisonsin generated the first human iPSCs by applying the same technique to human fibroblasts[8].

Since then, researchers have been focused on addressing the shortcomings of the original technique, notably its low efficiency[9] and the tumorigenicity [10] of the resulting iPSCs, and many novel variations of the original method have been published. No current protocol for generating iPSCs is capable of producing cells that perfectly (and reliably) mimic the behavior of ES cells [11][12][13]; accordingly, techniques for generating iPSCs will remain an area of intense research for the forseeable future.

In 2012, Shinya Yamanaka was awarded the Nobel Prize in Medicine for his 2006 paper describing the production of iPSCs[14].

Generation of iPSCs

The original method for producing iPSCs used a retroviral vector to express Oct3/4, Sox2, c-Myc, and Klf4 in cultured fibroblasts. Successfully induced cells were identified by their expression of Nanog (an ES cell marker) and isolated by selection for Fbx15+ (another ES cell marker) cells. iPSC lines established using this technique remain widely used in research.

Newer approaches for generating iPSCs generally vary from the original method in the vector and/or genes used, they may also use a different selection protocol for isolating the resulting iPSCs. For instance, one of the first variations on the original protocol, also published by Yamanaka, selected for Nanog+ cells instead of Fbx15+ cells to isolate iPSCs. This had the effect of increasing "ES cell like" behavior and decreasing tumorigenicity in the resulting cells, at the expense of signficantly reducing the efficiency of induction[15].

Other variations have used lentiviral and plasmid vectors to transfect the cells. Depending on the specific protocol used, these approaches can increase induction efficiency and ES cell characteristics in the resulting iPSCs, while at the same time somewhat reducing tumorigenicity.

Varying the transfected genes has also shown promise in improving iPSC generation. For example, culturing the cells with valproic acid allows c-Myc to be excluded from the transfection vector, greatly reducing tumorigenecity at the expense of efficiency. Alternatively, substituting LIN28 for c-Myc and Klf4 has been shown to increase transduction efficiency and reduce tumorigenicity (c-Myc and Klf4 being oncogenes)[16][17].

Some recent methods have dispensed with transfection altogether, using recombinant protein growth factors instead[18]. iPSCs generated with protein growth factors are sometimes referred to as protein induced pluripotent stem cells (piPSCs) to differentiate them from iPSCs generated by transfection. These methods are safer for therapeutic use, as the resulting cells are much less tumorigenic than transfected iPSCs; however they are usually far less efficient than methods relying on transfection.

Regardless of the method used, the mechanism of de-differentiation is the same. The cultured cells are induced to epigenetically reprogram themselves from a non-dividing (or slowly dividing) terminally differentiated state into an actively cycling pluripotent state[19].

Applications

Currently, iPSCs are primarily used in research. They often function as a substitute for human ES cells (the use of which is tightly regulated) in studies on cell differentiation and tissue development. More commonly, iPSCs are used in experiments where the use of ES cells would be inappropriate (e.g. lab animal models requiring perfectly histocompatible cell grafts).

Because patient-derived iPSCs would allow for perfectly histocompatible autografts, they are preferred over ES cells by researchers working to develop stem cell therapies. Several iPSC therapies are in clinical trials around the world (most in the United States), however none of them are currently widely available or routinely used. The major barriers to developing iPSC therapies are the tumorigenicity of iPSCs generated using current techniques, as well as the low efficiency, labor intensiveness, and relatively high cost (compared to other modern therapies) of these techniques. However, as researchers continue to develop better methods for producing iPSCs, it is thought that iPSC therapies for various diseases (ranging from type 1 diabetes to cardiomyopathies) will become more clinically viable[20][21].

The simplest approach to addressing the tumorigenicity problem is to screen generated iPSCs for tumorigenic mutations; however this process by itself is currently prohibitively labor-intensive and expensive. Alternatively, some researchers have tried genetically modifying the iPSCs to make them inherently non-tumorigenic or easily killable should they become tumorigenic; but this approach also adds a technically difficult (not to mention, somewhat unreliable) and costly step to potential iPSC therapies[22].

Tumorigenicity of iPSCs is a real concern. Many studies transplanting iPSC derived cells into animal models report subsequent cancer rates approaching 100% (as opposed to ~10 to 25% for grafts derived from ES cells). Grafts from iPSCs generated using newer techniques are demonstrated to be safer, but are still generally 2 to 4 times as tumorigenic as similar grafts derived from ES cells. Until this problem is overcome, the clinical use of iPSCs will remain reserved for treatments of last resort[23][24].

References

  1. This video is a good introduction to the topic: http://www.jove.com/video/3804/reprogramming-human-somatic-cells-into-induced-pluripotent-stem-cells
  2. http://genesdev.cshlp.org/content/22/15/1987.full.pdf+html
  3. http://www.ncbi.nlm.nih.gov/pmc/articles/PMC3226288/pdf/scrt58.pdf
  4. http://www.ncbi.nlm.nih.gov/pmc/articles/PMC3236565/pdf/nihms-339822.pdf
  5. http://www.ncbi.nlm.nih.gov/pmc/articles/PMC3419439/pdf/SCI2012-521343.pdf
  6. http://www.ncbi.nlm.nih.gov/pmc/articles/PMC3457607/pdf/CFG2012-538639.pdf
  7. http://www.cell.com/retrieve/pii/S0092867406009767
  8. http://www.sciencemag.org/content/318/5858/1917.long
  9. e.g. http://www.ncbi.nlm.nih.gov/pmc/articles/PMC3448677/pdf/pone.0045633.pdf
  10. e.g. http://www.ncbi.nlm.nih.gov/pmc/articles/PMC3204002/pdf/281_2011_Article_266.pdf
  11. http://www.ncbi.nlm.nih.gov/pmc/articles/PMC3457607/pdf/CFG2012-538639.pdf
  12. http://www.ncbi.nlm.nih.gov/pmc/articles/PMC3114956/pdf/10815_2011_Article_9552.pdf
  13. http://www.ncbi.nlm.nih.gov/pmc/articles/PMC3159104/pdf/ars.2010.3814.pdf
  14. http://www.washingtonpost.com/world/europe/2012-nobel-prize-announcements-being-with-medicine-award/2012/10/08/3f0284fe-110f-11e2-9a39-1f5a7f6fe945_story.html
  15. http://www.ncbi.nlm.nih.gov/pubmed/17554338
  16. http://www.ncbi.nlm.nih.gov/pmc/articles/PMC3159104/pdf/ars.2010.3814.pdf
  17. http://www.ncbi.nlm.nih.gov/pmc/articles/PMC2924949/pdf/nihms-219126.pdf
  18. http://masspec.scripps.edu/publications/news_art/2009_04_CellSteml.pdf
  19. http://www.bu.edu/abl/files/annual_review_gen.pdf
  20. http://www.ncbi.nlm.nih.gov/pubmed/20335067
  21. http://onlinelibrary.wiley.com/doi/10.1002/stem.727/pdf
  22. http://www.ncbi.nlm.nih.gov/pmc/articles/PMC2733374/pdf/stem0027-1050.pdf
  23. http://www.ncbi.nlm.nih.gov/pmc/articles/PMC3419439/pdf/SCI2012-521343.pdf
  24. http://www.ncbi.nlm.nih.gov/pmc/articles/PMC3070641/pdf/1479-5876-9-29.pdf
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