Abstract

Induced pluripotent stem cells (iPSCs) were first created in 2006 when it was shown that four gene factors could be used to reprogramme somatic cells to a stem cell-like state. Using this protocol, scientists could have a large, ethical supply of stem cells for research. This article considers some of the uses of iPSCs in developing degenerative disease therapies and some of the hurdles yet to be overcome before iPSCs can be used clinically.

Stem cells are undifferentiated pluripotent cells that can give rise to any of the body’s cells. There are many different types of stem cells in the body, but they all share major characteristics including clonality and the ability to self-renew (Evans and Kaufman, 1981). There are numerous benefits of using stem cells in research including scientists’ ability to manipulate them into the desired differentiated cell type. Embryonic stem cells (ESCs), especially, have enabled research into degenerative human diseases and offer potential cures for many disease types. However, there are numerous ethical issues associated with ESCs due to their provenance. Differentiated adult tissue cells (somatic cells) have recently been shown to be reprogrammable, creating induced pluripotent stem cells (iPSCs) (Takahashi and Yamanaka, 2006). This process avoids many of the ethical issues associated with ESCs. This article will discuss the recent progresses made with using iPSCs and the challenges yet to be overcome.

The importance of stem cells in regenerative disease models

Degenerative diseases are characterised by the progressive loss of particular cell types. Some well known examples include Alzheimer’s disease, Parkinson’s disease and multiple sclerosis. However, despite the frequency of degenerative diseases, research into degeneration has been hindered due to the lack of representative in vitro models. So far, research has relied on the pluripotent characteristics of ESCs and has shown that lab-grown ESCs have the potential to replace lost tissues, for example by differentiating into brain, nerve and bone tissues amongst others (Lin, 2011; Handschel et al., 2011).

In spite of the advantages of ESCs, there are limitations to their use. ESCs cannot be cultured in sufficient quantities for regenerative medicine, partly due to their provenance: obtaining cells from embryos raises major ethical issues.

As a consequence, recent research has focused on finding alternative methods of generating representative disease models. Many barriers have arisen such as mature neurones not being able to divide, immortalised cell lines not being truly pluripotent and adult stem cells already being committed to a particular cell type. In this case, the cells rarely survived the neuronal differentiation process (Peng and Zeng, 2011).

The discovery of iPSCs

In 2006, it was discovered that gene factors could be used to induce somatic cell reprogramming. It was shown that any adult mouse tissue cell can be reprogrammed to an iPSC using a set of four gene factors (Takahashi and Yamanaka, 2006). Just a year later, it was shown that the same four gene factors could also be used to genetically reprogramme human somatic cells (Takahashi et al., 2007). The four factors used by Takahashi and Yamanaka were Oct4, Sox2, Klf4 and c-Myc (OSKM), though later work successfully substituted Klf4 and c-Myc with Lin28 and Nanog respectively (giving OSLN).

This technique enabled scientists to culture iPSCs from any somatic cell, providing an unlimited supply of stem cells. Additionally, ESCs and iPSCs have been shown to share many characteristics including morphology, proliferation, gene expression and surface antigens (Takahashi et al., 2007; Kolios and Moodley, 2013). The reprogramming process bypasses the ethical issues and the quantitative limitations of ESCs. Disease-specific models can now be cultured, overcoming many limitations of previously available systems (Peng and Zeng, 2011).

Brief overview of the steps for reprogramming

Reprogramming is initiated by introducing the four factors, OSKM or OSLN, into mature adult somatic cells. These factors bind in a specific order to their targets and induce the cellular stress response to viruses and oncogenes. This in turn recruits p53, which is crucial in ensuring that only cells with genomic integrity survive to the pluripotent stage. It has been shown that c-Myc is fundamental in both the early stages of translation and in decreasing expression of mouse embryonic fibroblasts (MEF)-enriched miRNAs, which are barriers to reprogramming (Yang and Rana, 2013).

The next step in reprogramming is mesenchymal-to-epithelial (MET) transition, which is essential for some cells to start their de-differentiation process. During MET transition, the reprogrammed cells start to display pluripotency markers. Of these markers, SSEA-1 is the first to be expressed and indicates potential iPSCs. The expression of additional factors mark a successful and complete reprogramming (Yang and Rana, 2013).

The potential for iPSCs

The unlimited supply and differentiation capacities of iPSCs means models of many diseases can now be created for research. These models enable scientists to gain a better understanding of the mechanisms of diseases, potentially leading to cell-based therapy.

Another major clinical opportunity for iPSCs is tolerance to treatment. Somatic cells can be taken and reprogrammed from the person requiring treatment, meaning a personalised diagnosis and the conservation of their specific cell markers. This should prevent immune rejection (Park et al., 2008). Disease models are expected to be more accurate with iPSCs; as the cells are taken directly from the diseased patients, the genetic makeup of the disease can be conserved (Dimos et al., 2008).

Drug development is another area made easier with iPSCs. Reprogramming means large quantities of pluripotent stem cells. iPSCs can be created as long as researchers have access to adult somatic cells. Drug development requires numerous assays and an increase in the quantity of pluripotent stem cells is invaluable for progress. Furthermore the reprogramming protocol is fairly straightforward (Oh et al., 2012). However, it should be noted that, at present, it is not yet known how iPSCs would behave in a clinical environment compared to ESCs (Kolios and Moodley, 2013).

Limitations to iPSC use: safety concerns

The main iPSC safety concern is genetic stability. The use of retroviral vectors and oncogenes such as c-Myc and Klf4 are a major cause of concern for clinical studies. The transcription factors are typically introduced into the somatic cells using vectors, generating a possibility of cancer formation (Kolios and Moodley, 2013; Okita et al., 2007).

There are new techniques emerging that prevent genetic instability. Reprogramming can be achieved using just two of the four gene factors mentioned. Oct4 and Soc2 can induce reprogramming without the other oncogenic factors in the presence of a histone deacetylase inhibitor (Huangfu et al., 2008).

Alternatively, microRNAs, along with Oct4, Sox2 and Klf4, can induce reprogramming and actually increase the rate of efficiency with respect to the OSKM factors alone. New viral vectors and recombinant proteins have also been considered as alternatives to the OSKM factors (Ebben et al., 2011).

Limitations to iPSC use: supply concerns

As research progresses, the main provenance of iPSCs will likely be from diseased patients’ somatic cells. This will make iPSCs much more easily available than ESCs, but will not necessarily solve supply problems completely. Reprogramming is not an efficient process, and many somatic cells do not complete it (Polo et al., 2012).  Stem cells are also known for their delicacy and specific culture requirements. A lot of laboratory equipment is too abrasive for stem cells and is susceptible to regularly blocking. This said, recent progress in automated liquid handlers design means that robots capable of handling stem cells do now exist (e.g. Redd&Whyte’s Preddator).

Conclusions

Since the first creation of iPSCs in 2006, research has come a long way. We are now able to create patient-specific and disease-specific degenerative disease models. However, before clinical trials with iPSCs can occur, some important barriers remain to be overcome. The full potential of iPSCs to improve our understanding of diseases is not yet clear, but progress in this field is clearly happening quickly.

About The Author: Clare Stewart is a biochemistry student at the University of Manchester, she has written this post on behalf of Redd & Whyte

References: 

Dimos, J. T., Rodolfa, K. T., Niakan, K. K., Weisenthal, L. M., Mitsumoto, H., Chung, W., Croft, G. F., Saphier, G., Leibel, R., Goland, R., Wichterle, H., Henderson, C. E. & Eggan, K. (2008) Induced pluripotent stem cells generated from patients with ALS can be differentiated into motor neurons. Science, 321(5893), 1218-1221.

Ebben, J. D., Zorniak, M., Clark, P. A. & Kuo, J. S. (2011) Introduction to Induced Pluripotent Stem Cells: Advancing the Potential for Personalized Medicine. World Neurosurgery, 76(3-4), 270-275.

Evans, M. J. & Kaufman, M. H. (1981) Establishment In Culture Of Pluripotential Cells From Mouse Embryos. Nature, 292(5819), 154-156.

Handschel, J., Naujoks, C., Depprich, R., Lammers, L., Kubler, N., Meyer, U. & Wiesmann, H. P. (2011) Embryonic stem cells in scaffold-free three-dimensional cell culture: osteogenic differentiation and bone generation. Head & Face Medicine, 7.

Huangfu, D. W., Osafune, K., Maehr, R., Guo, W., Eijkelenboom, A., Chen, S., Muhlestein, W. & Melton, D. A. (2008) Induction of pluripotent stem cells from primary human fibroblasts with only Oct4 and Sox2. Nature Biotechnology, 26(11), 1269-1275.

Kolios, G. & Moodley, Y. (2013) Introduction to Stem Cells and Regenerative Medicine. Respiration, 85(1), 3-10.

Lin, S. L. (2011) Concise Review: Deciphering the Mechanism Behind Induced Pluripotent Stem Cell Generation. Stem Cells, 29(11), 1645-1649.

Oh, Y. Z., Wei, H. M., Ma, D. R., Sun, X. M. & Liew, R. (2012) Clinical applications of patient-specific induced pluripotent stem cells in cardiovascular medicine. Heart, 98(6), 443-449.

Okita, K., Ichisaka, T. & Yamanaka, S. (2007) Generation of germline-competent induced pluripotent stem cells. Nature, 448(7151), 313-U1.

Park, I. H., Lerou, P. H., Zhao, R., Huo, H. G. & Daley, G. Q. (2008) Generation of human-induced pluripotent stem cells. Nature Protocols, 3(7), 1180-1186.

Peng, J. & Zeng, X. M. (2011) The role of induced pluripotent stem cells in regenerative medicine: neurodegenerative diseases. Stem Cell Research & Therapy, 2.

Polo, J. M., Anderssen, E., Walsh, R. M., Schwarz, B. A., Nefzger, C. M., Lim, S. M., Borkent, M., Apostolou, E., Alaei, S., Cloutier, J., Bar-Nur, O., Cheloufi, S., Stadtfeld, M., Figueroa, M. E., Robinton, D., Natesan, S., Melnick, A., Zhu, J. F., Ramaswamy, S. & Hochedlinger, K. (2012) A Molecular Roadmap of Reprogramming Somatic Cells into iPS Cells. Cell, 151(7), 1617-1632.

Takahashi, K., Tanabe, K., Ohnuki, M., Narita, M., Ichisaka, T., Tomoda, K. & Yamanaka, S. (2007) Induction of pluripotent stem cells from adult human fibroblasts by defined factors. Cell, 131(5), 861-872.

Takahashi, K. & Yamanaka, S. (2006) Induction of pluripotent stem cells from mouse embryonic and adult fibroblast cultures by defined factors. Cell, 126(4), 663-676.

Yang, C. S. & Rana, T. M. (2013) Learning the molecular mechanisms of the reprogramming factors: let's start from microRNAs. Molecular Biosystems, 9(1), 10-17.