Scientists from Washington University (Saint Louis, MO) and Harvard University (Cambridge, MA) have been able to take skin cells from people with Type 1 Diabetes (T1D) and reprogram them to become cells closely resembling embryonic stem cells. They then converted the reprogrammed cells into β-like cells capable of releasing insulin in response to glucose challenges both in vitro (in dishes) and in vivo (in diabetic mice), (Reference 1, Millman et al. 2016). This discovery increases our hopes that in the near future, we will be able to take skin cells from a T1D patient, convert them into β-like cells in a dish and then return them to the patient’s body to cure the disease.
We have already described the basic technology regarding obtainment of β-cells from skin cells: Type 1 Diabetes Research Advances To Develop A Cure, See Part 4 for more information.
In the fall of 2014 a team of scientists at Harvard University (Reference 2, Pagliuca et al. 2014) and the Kieffer/Beta Logic team in Canada (Reference 3, Rezania et al. 2014) reported the conversion of human embryonic stem cell (hESC) lines into insulin-producing β-like cells capable of reversing diabetes in mice that were rendered diabetics (key abbreviations are in Footnote 1). Moreover, the Harvard University team had also succeeded in programming skin cells from healthy people to become cells closely resembling hESCs which are referred to as induced pluripotent stem cell (iPSC), (Reference 2, Pagliuca et al. 2014). Next, they mixed the iPSCs with growth media and by sequential addition of soluble factors, such as growth factors, cytokines and small molecules, coaxed them to differentiate into insulin-producing β-like cells. When these β-like cells were implanted in a mouse model of T1D, they responded to glucose variations like normal β cells would do and cured the disease. These scientific/technological accomplishments are pretty remarkable because assembling a cocktail of factors and developing a procedure to differentiate ESCs into insulin-producing β-cells, is a very complex undertaking (Footnote 2); but the greatest challenge was to start with skin cells and reprogram them into iPSCs and differentiate them into insulin-producing β-cells. It is not surprising that these accomplishments are generating excitement because they have established the technology to produce an unlimited number of β-like cells for transplantation in T1D patients (though a direct transplantation into humans has not yet reached the clinical stage). The procedures to differentiate hESCs to β-cells developed by the Harvard (Reference 2, Pagliuca et al., 2014) and Kieffer/Beta Logic groups (Reference 3, Rezania et al. 2014) are remarkable indeed, but rather complex and still led to β-like cells preparations containing ~15% cells expressing undesirable hormones (polyhormonal cells). Moreover, no one had reported the conversion of skin cells obtained from T1D patients into β-cells capable of producing insulin. To address these drawbacks, and to also better understand the interplay among the factors that regulate the process of stem differentiation into β-like cells, many groups have developed new protocols; a few are described:
Scientists at the University of California at San Francisco both removed and added differentiation molecules at specific steps of the differentiation protocol to ensure proper temporal gene activation (Reference 5; Russ et al., 2015). By doing so they obtained robust β-like cell preparations that could be executed in less than 3 weeks (shortening the earlier procedures) and contained only ~ 3% polyhormonal cells as contaminant. When transplanted in diabetic mice, these β-like cells were glucose-responsive and corrected the disease.
In a more recent report, also from the University of California at San Francisco, researchers shortened the process of converting human skin cells into pancreatic β-cells (Reference 6, Zhu et al., 2016, Footnote 4). They used a mixture of factors and molecules typically employed to generate induced pluripotent stem cells (iPSCs) from human skin cells, but they did not bring the reprogramming process all the way back to the iPSCs state. By using a strategy called Cell-Activation and Signaling-Directed (CASD), they transformed skin cells into early developmental cells called endoderm progenitor cells; these are like stem cells, but have the ability to differentiate into only limited cell types such as pancreatic, and liver cells. Moreover, their studies also identified novel sets of signaling factors capable of differentiating these endodermal progenitor cells into functional β-like cells. When implanted into diabetic mice, the β-like cells developed by these investigators were able to regulate glucose concentrations in these animals.
A team of scientists at the ETH Zurich's Department of Biosystems Science and Engineering in Basel (CH) led by Martin Fussenegger, took a novel approach: They converted stem cells extracted from fatty tissue into mature functional β-like cells (Reference 4, Saxena et al., 2016). In earlier work this team extracted stem cells from fatty tissues of a 50-year-old female and converted them into induced pluripotent stem cells (iPSCs) by a technique that uses synthetic modified mRNA encoding transcription factors, rather than relying upon the use of recombinant DNA and viral vectors (Reference 7, Heng et al., 2013). In their more recent work they took their iPSCs and converted them into functional β cells (Reference 4, Saxena et al., 2016). To accomplish this, they focused on three pancreatic transcription factors referred to as, Ngn2, Pdx1, and MafA. These factors had already been proven to be critical to the stem cells differentiation process into β cells in the pancreas (in vivo); but their behavior is complex as their concentrations rise and fall in intricate ways during cell differentiation. By synthesizing a set of genes, they were able to closely mimic the major steps of the differentiation pathway. Their procedure yielded β-like cells that produced insulin in response to elevation in glucose, at least in dishes (in vitro).
However, until a few days ago, nobody had yet reported the conversion of skin cells obtained from T1D patients into β-cells capable of producing insulin. Now that this feat has been accomplished; in a collaborative research scientists from Washington University (Saint Louis, MO) and Harvard University (Cambridge, MA) (Footnote 3) have been able to take skin cells from three people aged 26, 29, and 25 that were diagnosed with T1D at 25, 29, 23, respectively and reprogram these cells to become pluripotent stem cells (T1D iPSCs) that closely resembled embryonic stem cells (ESCs). As a control, they took cells from non-diabetic (ND) people (aged 36, 34, and 23) and also reprogrammed them to become pluripotent stem cells (ND iPSCs). They were able to instruct both ND iPSCs (as they did in their original studies, Reference 2) and the newly developed T1D iPSCs to become functional pancreatic β-cells, which they called ND SC-β cells and T1D SC-β cells, respectively, to indicate that the former come from Non-Diabetics Stem Cells, and the latter from Type 1 Diabetic Stem Cells. The authors found that ND SC-β cells and T1D SC-β cells were functionally indistinguishable in their capability to release insulin in response to increases of glucose concentration in vitro (in dishes) and, more importantly, were able to control blood glucose in diabetic mice. The ND SC-β cells and T1D SC-β cells were very similar to both the ESC- and ND SC-β cells obtained in their previous work and had protein markers characteristic of mature human β cells; though they were similar to the latter cells, they were not identical to them. The ND SC-β cells and T1D SC-β cells similarly increased insulin release in response to anti-diabetic drugs and responded to cytokine stress.
Before this paper (Reference 1, Millman et al. 2016), it was not taken for granted that functional T1D SC-β cells could be obtained, nor that they would be indistinguishable from both ND SC-β cells and ESC-β cells. This suspicion was legitimate as T1D SC-β cells are generated from skin cells of T1D people and could contain some defects; based on the results of this paper this seems not to be the case (Reference 1, Millman et al. 2016). Yet, as the authors point out, increasing the number of T1D skin cell donors and monitoring the performance of transplanted T1D SC-β cells over the span of years would give a more firm answer as to their functional equivalence to ND SC-β cells and ESC-β cells.
The results of this research are another step forward in the quest for a cure for Type 1 Diabetes, but there is still a long way to go until this technology will become useful to patients because of a few unanswered questions such as: (1) How similar are the externally made β-cells (ex-vivo) to the native ones (in vivo)? It looks like the work described in References 1-7 is closing the gap between the two cells species; (2) Can these ex-vivo made β-cells be implanted safely in humans without triggering the induction of cancer or other undesired effects (once the transplant is complete it cannot be reversed)? This question has not been answered yet, but again the work described in References 1-7 gives us hope that we are getting closer and closer to make ESC-β, ND SC-β, and T1D SC-β cells equivalent to the real pancreatic β-cells and should be able to minimize cancer and other side effects when transplanted in humans; (3) Transplantation of β-cells derived from embryonic stem cells is an allogeneic (cells come from another person) transplant and the cells will be attacked by the host immune system as foreign entities (the so-called graft-versus-host disease or GVHD). In principle, the allogenic β-cells’ problem could be by-passed by producing autogenic β-cells (from patient own cells), and indeed this is what these investigators have accomplished (Reference 1), but until the transplantation is performed in patients we will not know the correct answer. Yet, (4) the newly implanted autogenic β-cells will also be attacked by the same mechanism that has caused the disease in the first place, and this would require the patients to still receive chronic anti-suppressive drugs for the rest of their lives unless re-education of the faulty immune system is accomplished (this is the ultimate goal!); indeed, efforts are underway to re-educate the Type 1 Diabetics broken immune system to recognize self and spare it (See this blog Type 1 Diabetes Research Advances To Develop A Cure, Attempts to re-educate the immune system, Part 7). Moreover, other means of β-cell protection are used e.g., β-cell encapsulation. Authors of the paper in Reference 1 (Millman et al. 2016) in collaboration with other groups have already incorporated their ND SC-β cells into semi-permeable devices they developed (References 8 and 9, Vegas et al 2016a and 2016b, respectively). When tested in mice ND SC-β cells secreted insulin in response to raising glucose and were protected by the semi-permeable devices from being killed off by the immune system. Two biotechnology companies, Viacyte in San Diego (USA), and Beta O2 in Israel have underway Phase I/II clinical trials testing their pancreatic islets encapsulation technology. These are islets containing authentic β cells and should soon give a feeling for the technology soundness.
In conclusion, though significant hurdles still need to be overcome before a direct transplantation in people will be carried out, this discovery (Reference 1) may allow to study mechanisms of T1D onset and progression and adds to our hopes that in the near future we will be able to return the transformed human β-cells to the T1D patient’s body to cure this disease.
- human embryonic stem cells (hESCs);
- β-cells derived from human embrionic stem cell (hESC-β cells);
-human induced pluripotent stem cells (hiPSCs); same as stem cells generated from non diabetic donors (ND hiPSC);
-stem cells generated from T1D donors (T1D hiPSC);
- β-cells derived from stem cells generated from non diabetic donors (ND hiPSC-β cells);
-stem cells generated from T1D donors (T1D hiPSC);
- Saxema et al., 2016, Reference 4; describe the differentiation mixture: “efforts to generate beta-cells from human embryonic stem cells (hESCs) have led to reliable protocols involving the sequential administration of growth factors (activin A, bone morphogenetic protein 4 (BMP-4), basic fibroblast growth factor (bFGF), FGF-10, Noggin, vascular endothelial growth factor (VEGF) and Wnt3A) and small-molecule compounds (cyclopamine, forskolin, indolactam V, IDE1, IDE2, nicotinamide, retinoic acid, SB-431542 and γ-secretase inhibitor) that modulate differentiation-specific signaling pathways”.
- Notice that most of the authors of the present paper (Reference 1) were also part of the team that developed the technology described in (Reference 2).
- Some authors of paper 5 are also authors of paper 6
- Millman JR, Xie C, Van Dervort A, Gürtler M, Pagliuca FW, and Melton DA. Generation of stem cell-derived β-cells from patients with type 1 diabetes. Nature Communications, 2016; 7: 11463 DOI: 10.1038/ncomms11463
- Pagliuca, F.W., Millman, J.R., Gu¨ rtler, M., Segel, M., Van Dervort, A., Ryu, J.H.,Peterson, Q.P., Greiner, D., and Melton, D.A. (2014). Generation of functionalhuman pancreatic b cells in vitro. Cell 159, 428–439.
- Rezania, A., Bruin, J.E., Arora, P., Rubin, A., Batushansky, I., Asadi, A.,O’Dwyer, S., Quiskamp, N., Mojibian, M., Albrecht, T., et al. (2014). Reversal of diabetes with insulin-producing cells derived in vitro from human pluripotent stem cells. Nat. Biotechnol. 32, 1121–1133.
- Saxena P, Heng BC, Bai P, Folcher M, Zulewski H, Fussenegger, M. A programmable synthetic lineage-control network that differentiates human IPSCs into glucose-sensitive insulin-secreting beta-like cells. Nature Communications, published online April 11th 2016. DOI: 10.1038/NCOMMS11247.
- Russ HA, Parent AV, Ringler JJ, Hennings TG, Nair GG, Shveygert M, Guo T, Puri S, Haataja L, Cirulli V, Blelloch R, Szot GL, Arvan P, Hebrok M (2015) Controlled induction of human pancreatic progenitors produces functional beta-like cell in vitro. EMBO J. 2;23(13):1759-7
- Zhu S, Russ HA, Wang X, Zhang M, Ma T, Xu T, Tang S, Hebrok M, Ding S (2016) Human pancreatic beta-like cells converted from fibroblasts. Nat Commun. 7:10080. doi: 10.1038/ncomms10080.
- Heng, B. C. et al. mRNA Transfection-Based, Feeder-Free, Induced Pluripotent Stem Cells From Adipose Tissue of a 50 Year-Old Patient. Metab. Eng. 18, 9–24 (2013).
- Vegas, A.J., Veiseh, O., Doloff, J.C., Ma, M., Tam, H.H., Bratlie, K., Li, J., Bader, A.R., Langan, E., Olejnik, K., et al. (2016a). Combinatorial hydrogel library enables identification of materials that mitigate the foreign body response in primates. Nat. Biotechnol. 34, 345–352.
- Vegas, A.J., Veiseh, O., Gu¨ rtler, M., Millman, J.R., Pagliuca, F.W., Bader, A.R., Doloff, J.C., Li, J., Chen, M., Olejnik, K., et al. (2016b). Long-term glycemic control using polymer-encapsulated human stem cell-derived beta cells in immune-competent mice. Nat. Med. 22, 306–311.