NEWS & VIEWS DIABETES
β cells at last Josué K. Mfopou and Luc Bouwens
The race to generate β cells from stem cells has taken another big turn. We can already generate definitive endoderm from human embryonic stem cells and functional insulin-producing cells from transplanted pancreatic progenitors. Now, differentiating glucose-responsive insulin-producing cells in vitro that function like adult human β cells has been achieved. Mfopou, J. K. & Bouwens, L. Nat. Rev. Endocrinol. advance online publication 11 November 2014; doi:10.1038/nrendo.2014.200
Intensive insulin therapy can considerably reduce the incidence and severity of complications associated with type 1 diabetes mellitus; however, only the replacement of lost β cells (islet transplantation) can restore physiological control of blood levels of glucose and could potentially cure patients with diabetes mellitus. 1 Unfortunately, the scarcity of islet donors has restricted the number of patients who have been able to receive this therapy. In clinical practice, two or three donors are usually required for a transplantation procedure in a single patient. The discovery of human pluripotent stem cells (hPSC) was, therefore, readily adopted as a potential means of providing cell therapy to millions of patients with type 1 diabetes mellitus or advanced type 2 diabetes mellitus, as it was assumed that an unlimited number of β cells could be generated upon their differentiation. This hope, as shown by early studies in the field, did not sufficiently consider the complexity of incorporating features of pancreatic development into a model of pluripotent stem cell differentiation in vitro. Despite the difficulties, the development of a therapy for diabetes mellitus based on stem cells (Figure 1) has now reached another milestone with the publication of a study by Pagliuca and colleagues.2 In this study, the researchers demonstrated that functional β cells can be obtained in vitro from hPSC that were sequentially treated with a total of 11 extracellular growth and differentiation factors for 4–5 weeks. These findings are valuable for developing cell-based therapies for diabetes mellitus, and might also provide a platform for disease modelling and for screening thousands of small
molecules (with potential effects on human β‑cell function, proliferation or survival) for the development of antidiabetic drugs. As in the majority of previous studies in the field,3–8 the study by Pagliuca and coworkers built on the knowledge gathered from examining pancreatic development in model organisms. Pagliuca et al. also followed the fairly safe strategy of modulating the extracellular microenvironment to induce cell differentiation rather than introducing DNA encoding transcription factors inside the hPSC. They tested 150 different combinations of factors that had to be added sequentially to their cultures to induce proper cell differentiation.
Previous studies have indicated that insulin-producing cells derived in vitro from hPSC are polyhormonal, are similar to fetal β cells and do not respond to glucose challenge.3–6 However, the stem-cell-derived β (SCβ) cells described in this new study display the major and specific characteristics of a genuine adult β cell (including calcium flux, glucose-responsiveness and expression of a single hormone—insulin). The SCβ cells also expressed several β‑cell differentiation markers (PDX1, NKX6.1 and ZnT8) and had ultrastructural morphological features similar to those of adult human β cells. In addition, in a mouse model of diabetes mellitus, transplantation of SCβ cells quickly restored and maintained normal blood levels of glucose. The thorough characterization of the SCβ cells in the paper by Pagliuca and associates is more comprehensive than that reported for insulin-producing cells derived from hPSC that were glucose responsive upon culture in a 3D matrix during the matura tion stage of in vitro differentiation.7 Thus, the work by Pagliuca and colleagues is an important technical and biological breakthrough in the field of pancreatic β‑cell differentiation from hPSC in vitro. The research has unravelled the minimal
Blastocysts from embryo donors
Somatic cells such as fibroblasts
Genetic modification (KLF4, OCT2, SOX2, MYC)
Pluripotent stem cells In vitro culture and clonal selection Definitive endoderm
Pancreatic progenitor cells Last step achieved by Pagliuca and colleagues
Stepwise differentiation by sequential supplementation of extracellular modulators of cell signalling
Figure 1 | β-cell differentiation from pluripotent stem cells. The third most important milestone (differentiating glucose-responsive insulin-producing cells) has just been set in this process. Pagliuca and colleagues achieved in vitro differentiation of functional β cells using a scalable 3D culture system.
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NEWS & VIEWS conditions required to efficiently turn hPSC-derived pancreatic progenitor cells into endocrine progenitor cells that are amenable to acquiring a phenotype very close to that of genuine islet β cells. These conditions could only be poorly defined in previous in vivo experiments, wherein hPSC-derived pancreatic progenitors required 3–4 months to mature into β cells, which contrasts with the 4–5 weeks required for SCβ to mature in vitro.8 The study has also consolidated the central position that a good understanding of tissue and organ developmental biology occupies in the race for generating different cell types of interest from hPSC. However, despite the mention that the protocol works on a total of four cell lines in the authors’ laboratory, the importance of this breakthrough will depend on the reproducibility of its findings by other investigators using different hPSC lines.
…the importance of this breakthrough will depend on the reproducibility of its findings...
The SCβ cells are phenotypically very close, but not transcriptionally identical, to primary β cells. In addition to the strategies highlighted by the authors to tackle this issue, it will be of interest to examine whether this discrepancy fades with time spent in culture or upon SCβ cell transplantation in vivo, given that the reported protocol is still a big reduction of the 13–15 weeks required for islet development in the human embryo. Nevertheless, the SCβ cells are obtained at a sufficiently high efficiency (33%) in the current system so that any future moves towards clinical implementation would probably not face challenges with mass production. As the SCβ cells could respond to several glucose challenges and their transplantation in mice resulted in detectable levels of human C‑peptide and normalization of blood
glucose levels within 2 weeks, they might represent an optimal candidate for testing in clinical trials in patients with diabetes melli tus. However, important questions need to be solved before reaching this step. Many laboratories around the world are working toward the goal of using genuine or ‘surrogate’ insulin-producing cells as a basis for treating patients with type 1 diabetes mellitus and advanced type 2 diabetes melli tus. The current paper is very important in this context; however, it will undoubtedly be some time before these results are translated into treatment for patients with diabetes mellitus. For example, the FDA has just approved the first clinical trial using a product derived from embryonic stem cells that consists of pancreatic progenitor cells delivered in a macro-encapsulation device; however, this cell product was developed on the basis of research that was published 8 years ago.3 An advantage of the product developed by Pagliuca and co-workers is that it consists of fairly pure β cells. However, some encapsulation technology to prevent graft rejection will also be needed. Cellular grafts encapsulated in biomaterials often trigger a process that begins with inflammation and ends with the implant being surrounded by macrophages and fibrotic tissue. This reaction to a foreign body is absent in transplantation experiments using immune-deficient animals,2 but might severely compromise the survival and function of encapsulated cells in patients.9 For this reason (and others, such as the lack of direct vascularization of the encapsulated cells in a graft of sufficient size for use in humans), islet transplantation in combination with encapsulation has not yet been successful in clinical trials. Encapsulation is also needed to prevent the risk of outgrowth of teratoma cells or cells containing potentially oncogenic mutations.10 The presence of such cells actually needs to be ruled out by more sensitive techniques than those used by Pagliuca et al., even though
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mice examined 18 weeks after transplantation were declared to be free of tumours. Furthermore, the SCβ cells might function differently in the human microenvironment. Therefore, caution is needed when we talk of a therapeutic breakthrough for diabetes mellitus. Cell Differentiation Unit, Diabetes Research Center, Vrije Universiteit Brussel (VUB), Laarbeeklaan 103, 1090 Brussels, Belgium (J.K.M., L.B.). Correspondence to: L.B. [email protected]
Competing interests The authors declare no competing interests. 1.
Bouwens, L., Houbracken, I. & Mfopou, J. K. The use of stem cells for pancreatic regeneration in diabetes mellitus. Nat. Rev. Endocrinol. 9, 598–606 (2013). 2. Pagliuca, F. W. et al. Generation of functional human pancreatic β cells in vitro. Cell 159, 428–439 (2014). 3. D’Amour, K. A. et al. Production of pancreatic hormone-expressing endocrine cells from human embryonic stem cells. Nat. Biotechnol. 24, 1392–1401 (2006). 4. Nostro, M. C. et al. Stage-specific signaling through TGFβ family members and WNT regulates patterning and pancreatic specification of human pluripotent stem cells. Development 138, 861–871 (2011). 5. Rezania, A. et al. Maturation of human embryonic stem cell-derived pancreatic progenitors into functional islets capable of treating pre-existing diabetes in mice. Diabetes 61, 2016–2029 (2012). 6. Bruin, J. E. et al. Characterization of polyhormonal insulin-producing cells derived in vitro from human embryonic stem cells. Stem Cell Res. 12, 194–208 (2014). 7. Takeuchi, H., Nakatsuji, N. & Suemori, H. Endodermal differentiation of human pluripotent stem cells to insulin-producing cells in 3D culture. Sci. Rep. 4, 4488 (2014). 8. Kroon, E. et al. Pancreatic endoderm derived from human embryonic stem cells generates glucose-responsive insulin-secreting cells in vivo. Nat. Biotechnol. 26, 443–452 (2008). 9. de Vos, P., Faas, M. M., Strand, B. & Calafiore, R. Alginate-based microcapsules for immunoisolation of pancreatic islets. Biomaterials 27, 5603–5617 (2006). 10. Hong, S. G., Dunbar, C. E. & Winkler, T. Assessing the risks of genotoxicity in the therapeutic development of induced pluripotent stem cells. Mol. Ther. 21, 272–281 (2013).
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