By MingMing Yang
The 2012 Nobel Prize in Physiology or Medicine was jointly awarded to John B. Gurdon and Shinya Yamanaka, for the discovery that mature cells can be reprogrammed to become pluripotent. This was when the phrase induced pluripotent stem (iPS) cells came into researchers’ attention. Gurdon’s investigations, using frog models back in 1962, discovered that the nucleus of a mature, specialized cell, can return to an immature, pluripotent state. Over 40 years later in 2006, Yamanaka and his co-workers developed iPS cells by only introducing four genes. These discoveries have provided new tools for scientists around the world and led to remarkable progress in many areas of medicine(The Nobel Prize in Physiology or Medicine 2012. ).
Human embryonic stem (ES) cells are human blastocyst-derived pluripotent cells capable of unlimited, undifferentiated proliferation in vitro, and were first acquired in 1998. Direct differentiation of human ES cells into specific cell types could be used as a potential lifelong treatment for diseases such as Parkinson’s disease and insulin-dependent diabetes mellitus, in which ES cells can be used to replace the dysfunctional cell types(Thomson, 1998). However, there are several problems surrounding the research and use of human ES cells, including ethical concerns, as it involves the use of human embryos, and certain practical and safety issues, like immunorejection or uncontrolled proliferation of cells after transplantation(Kelly, 2017). These factors slowed down the research of human ES cells.
Having recognised the immense potential of human ES cells, researchers tried to find alternatives for human ES cells with the same ability but reduced concerns for use, and this led to the development of iPS cells. In 2006, Yamanaka et. al generated iPS cells from mouse embryonic fibroblasts and adult mouse tail-tip fibroblasts by the retrovirus-mediated transfection of four transcription factors, namely Oct3/4, Sox2, c-Myc, and Klf4. The mouse iPS cells generated were said to be indistinguishable from ES cells in morphology, proliferation, gene expression, and teratoma formation(Takahashi & Yamanaka, 2006). To allow this technique to be used on patients in the hope to create patient-disease specific stem cells for treatment, the group of researchers successfully used the same four factors to generate iPS cells from adult human somatic cells, by optimizing retroviral transduction in human fibroblasts and subsequent culture conditions. The iPS cells derived were said to be comparable to human ES cells in their differentiation potential in vitro and in teratomas, which paved a way for further research into regenerative medicine(Takahashi et al., 2007).
However, there are still a few challenges regarding the potential clinical use of iPS cells. One of the problems is the low efficiency of the conversion to iPS cells, which need to be overcome for wide-spread clinical use to be possible. It has been reported that, typically, less than 1% of transfected fibroblasts become iPS cells. Another major safety concern is the risk of insertional mutagenesis into the genome of the target cells, as iPS cells were initially generated from retroviruses. A number of ways of generating integration-free iPS cells have then been reported, including the use of adenovirus(Stadtfeld et al., 2008), Sendai virus(Fusaki et al., 2009), and plasmids(Okita et al., 2008), but these safer alternatives all come with a trade-off of even lower efficiency(Moradi et al., 2019). Furthermore, prevention of tumorigenesis is also a challenge, as the expression of Oct4 , Sox2 , Klf4, and c–Myc genes is associated with the development of multiple tumours known in oncogenetics. In particular, the improper expression of c–Myc, a known oncogene, is observed in 70% of human cancers(Medvedev, Shevchenko & Zakian, 2010).
Despite the challenges and concerns that scientists are trying hard to eliminate, the potential applications of iPS cells can be revolutionary. For instance, iPS cells can be employed in drug screening and toxicity testing in vitro for the development of new drugs and is a good alternative to testing in vivo in animal models. As iPS cells have the potential to differentiate into any cell types from the somatic cells of any individual, they may enable pharmaceutical in vitro testing for both toxicity and efficacy over any individual genotype, by obtaining cells from different patients. Comparing with previous in vivo animal testing, using in vitro models that resemble conditions in human patients hugely increases the efficiency and accuracy of drug screening. The ability to use patient-specific cells for screening also makes the concept of bringing personalized medicine into clinics possible(Ebert, Antje D., Liang & Wu, 2012).
iPS cell is also an ideal potential candidate for human disease modelling. Upon acquiring differentiated target cells from reprogrammed patient specific human iPS cells, they can be used as in vitro disease models as they retain disease-related phenotypes of specific patients. This paved a way for the understanding of complex or rare genetic diseases, and further characterization of some common diseases, as the models can be used for investigating pathophysiology and testing therapeutic strategies like gene editing(Ebert, Antje D., Liang & Wu, 2012). The first study to show that human iPS cells can be used to model the specific pathology seen in a genetically inherited disease was conducted by Ebert et al., who generated iPS cells from skin fibroblast taken from a patient with spinal muscular atrophy, an autosomal recessive inherited forms of neurological disease leading to infant mortality. Those cells expanded robustly in culture and maintained the disease genotype(Ebert, Allison D. et al., 2009). To understand the cause of the dominantly inherited neurodegenerative disease, Huntington’s Disease (HD), and develop new therapeutics, Zhang et al. utilized a recently established HD-specific iPS cell line to generate a human HD cell model derived from a HD patient, which have endogenous CAG repeat expansion that is suitable for mechanistic studies and drug screenings for HD(Zhang et al., 2010).
Apart from drug screening and disease modelling, the iPS cell technology also provides an opportunity for the use of cell transplantation therapy in treating many genetic or degenerative diseases. This can be achieved by reprogramming somatic cells harvested from a patient in vitro into iPS cells, differentiate them into the affected cell type after replacing the diseased gene with a healthy one if needed, and implanting the healthy cells back into the patient to replace the diseased ones(Al Abbar et al., 2020). For example, Swistowsk et al. used a completely defined (xeno-free) system to successfully generate functional dopaminergic neurons from human iPS cells. This could potentially be used for treating or modeling Parkinson’s disease (PD)(Swistowski et al., 2010). Based on this technique, recently in 2018, a group in Japan started a clinical trial to treat PD patients by using iPSC-derived dopaminergic progenitors after extensive safety evaluation(Doi et al., 2020).
This technique could also be a promising approach in treating insulin-dependent diabetes mellitus, in which the regeneration of functional pancreatic ß cells from patient-specific somatic cells can be used to replace the malfunctional insulin-secreting cells. In fact, a group of researchers had successfully derived β-like cells from iPS cells, similar to the endogenous insulin-secreting cells in mice. These β-like cells secreted insulin in response to glucose and corrected a hyperglycaemic phenotype in two mouse models of type 1 and 2 diabetes via cell transplant(Alipio et al., 2010). Tateishi et al. also demonstrated that insulin-producing islet-like clusters (ILCs) can be generated from human skin fibroblast-derived iPS cells under feeder-free conditions. The iPS-derived ILCs not only contain C-peptide-positive and glucagon-positive cells but also release C-peptide upon glucose stimulation(Tateishi et al., 2008). These findings raised the possibility that patient-specific iPS cells could potentially provide a treatment for diabetes in the future.
Above all, human iPS cells reprogrammed from somatic cells represent a promising unlimited cell source for generating patient-specific cells for biomedical research and personalized medicine. Although there are still concerns and areas of uncertainty regarding its use, iPS cells currently appear to be the best alternative to the use of human ES cells, without ethical and immunological concerns. With continuing research and deeper understanding, it is hopeful that iPS cells can enter the medical field and benefit a large population in the near future.
The Nobel Prize in Physiology or Medicine 2012. Available from: https://www.nobelprize.org/prizes/medicine/2012/press-release/ [Accessed Nov 27, 2020].
Al Abbar, A., Ngai, S. C., Nograles, N., Alhaji, S. Y. & Abdullah, S. (2020) Induced Pluripotent Stem Cells: Reprogramming Platforms and Applications in Cell Replacement Therapy. BioResearch Open Access. 9 (1), 121-136. Available from: https://www.liebertpub.com/doi/10.1089/biores.2019.0046. Available from: doi: 10.1089/biores.2019.0046. [Accessed Dec 8, 2020].
Alipio, Z., Liao, W., Roemer, E. J., Waner, M., Fink, L. M., Ward, D. C. & Ma, Y. (2010) Reversal of hyperglycemia in diabetic mouse models using induced-pluripotent stem (iPS)-derived pancreatic β-like cells. Proceedings of the National Academy of Sciences of the United States of America. 107 (30), 13426-13431. Available from: https://www.ncbi.nlm.nih.gov/pmc/articles/PMC2922145/. Available from: doi: 10.1073/pnas.1007884107. [Accessed Dec 8, 2020].
Doi, D., Magotani, H., Kikuchi, T., Ikeda, M., Hiramatsu, S., Yoshida, K., Amano, N., Nomura, M., Umekage, M., Morizane, A. & Takahashi, J. (2020) Pre-clinical study of induced pluripotent stem cell-derived dopaminergic progenitor cells for Parkinson’s disease. Nature Communications. 11 (1), 1-14. Available from: https://www.nature.com/articles/s41467-020-17165-w. Available from: doi: 10.1038/s41467-020-17165-w. [Accessed Dec 8, 2020].
Ebert, A. D., Yu, J., Rose, F. F., Mattis, V. B., Lorson, C. L., Thomson, J. A. & Svendsen, C. N. (2009) Induced pluripotent stem cells from a spinal muscular atrophy patient. Nature. 457 (7227), 277-280. Available from: https://www.ncbi.nlm.nih.gov/pmc/articles/PMC2659408/. Available from: doi: 10.1038/nature07677. [Accessed Dec 8, 2020].
Ebert, A. D., Liang, P. & Wu, J. C. (2012) Induced Pluripotent Stem Cells as a Disease Modeling and Drug Screening Platform. Journal of Cardiovascular Pharmacology. 60 (4), 408-416. Available from: https://www.ncbi.nlm.nih.gov/pmc/articles/PMC3343213/. Available from: doi: 10.1097/FJC.0b013e318247f642. [Accessed Dec 7, 2020].
Fusaki, N., Ban, H., Nishiyama, A., Saeki, K. & Hasegawa, M. (2009) Efficient induction of transgene-free human pluripotent stem cells using a vector based on Sendai virus, an RNA virus that does not integrate into the host genome. Proceedings of the Japan Academy. Series B, Physical and Biological Sciences. 85 (8), 348-362. Available from: doi: 10.2183/pjab.85.348. [Accessed Dec 7, 2020].
Kelly, J. (2017) Practical and Ethical Issues Limiting the Clinical Use of Human Embryonic Stem Cells. Arch Stem Cell Res. 4 (1), 1018.
Medvedev, S. P., Shevchenko, A. I. & Zakian, S. M. (2010) Induced Pluripotent Stem Cells: Problems and Advantages when Applying them in Regenerative Medicine. Acta Naturae. 2 (2), 18-28. Available from: https://www.ncbi.nlm.nih.gov/pmc/articles/PMC3347549/. [Accessed Dec 7, 2020].
Moradi, S., Mahdizadeh, H., Šarić, T., Kim, J., Harati, J., Shahsavarani, H., Greber, B. & Moore, J. B. (2019) Research and therapy with induced pluripotent stem cells (iPSCs): social, legal, and ethical considerations. Stem Cell Research & Therapy. 10 (1), 341. Available from: https://search.datacite.org/works/10.1186/s13287-019-1455-y. Available from: doi: 10.1186/s13287-019-1455-y.
Okita, K., Nakagawa, M., Hyenjong, H., Ichisaka, T. & Yamanaka, S. (2008) Generation of mouse induced pluripotent stem cells without viral vectors. Science (New York, N.Y.). 322 (5903), 949-953. Available from: doi: 10.1126/science.1164270. [Accessed Dec 7, 2020].
Stadtfeld, M., Nagaya, M., Utikal, J., Weir, G. & Hochedlinger, K. (2008) Induced pluripotent stem cells generated without viral integration. Science (New York, N.Y.). 322 (5903), 945-949. Available from: doi: 10.1126/science.1162494. [Accessed Dec 7, 2020].
Swistowski, A., Peng, J., Liu, Q., Mali, P., Rao, M. S., Cheng, L. & Zeng, X. (2010) Efficient Generation of Functional Dopaminergic Neurons from Human Induced Pluripotent Stem Cells Under Defined Conditions. Stem Cells (Dayton, Ohio). 28 (10), 1893-1904. Available from: https://www.ncbi.nlm.nih.gov/pmc/articles/PMC2996088/. Available from: doi: 10.1002/stem.499. [Accessed Dec 8, 2020].
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. Available from: http://hdl.handle.net/2433/49782.
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. Available from: http://www.sciencedirect.com/science/article/pii/S0092867406009767. Available from: doi: 10.1016/j.cell.2006.07.024. [Accessed Nov 27, 2020].
Tateishi, K., He, J., Taranova, O., Liang, G., D’Alessio, A. C. & Zhang, Y. (2008) Generation of Insulin-secreting Islet-like Clusters from Human Skin Fibroblasts. Journal of Biological Chemistry. 283 (46), 31601-31607. Available from: http://www.jbc.org/content/283/46/31601. Available from: doi: 10.1074/jbc.M806597200. [Accessed Dec 8, 2020].
Thomson, J. A. (1998) Embryonic Stem Cell Lines Derived from Human Blastocysts. Science. 282 (5391), 1145-1147. Available from: https://search.datacite.org/works/10.1126/science.282.5391.1145. Available from: doi: 10.1126/science.282.5391.1145.
Zhang, N., An, M. C., Montoro, D. & Ellerby, L. M. (2010) Characterization of Human Huntington’s Disease Cell Model from Induced Pluripotent Stem Cells. PLoS Currents. 2 Available from: https://www.ncbi.nlm.nih.gov/pmc/articles/PMC2966296/. Available from: doi: 10.1371/currents.RRN1193. [Accessed Dec 8, 2020].