IV. Cell-based therapy against diabetes
Cell-based therapy against diabetes is currently limited by a lack of donated organs and difficulties with increasing insulin-secreting cells in vivo. The in vitro generation of pancreatic β cells and their in vivo transplantation in diabetic patients offer the prospect of bypassing these restrictions (Ludovic Vallier lecture).
A. Reversal of β-cell dedifferentiation
The redifferentiation of dedifferentiated adult human β cells (eg, in aging or pathophysiological conditions) represents an interesting therapeutic approach. In particular, there is a reservoir of dedifferentiated β cells (named β-cell–derived [BCD] cells) for endogenous renewal (Shimon Efrat lecture), and redifferentiation of these BCD cells can be achieved using a mix of soluble factors (“redifferentiation cocktail” [RC]) that induce the transcription factors PAX4 and ARX (also including OX9, FOXA2, PDX1, and Ngn3). However, RC treatment leads to redifferentiation of only a fraction of BCD cells. Shimon Efrat et al demonstrated that blocking ARX expression by short hairpin RNA (shRNA) in RC-treated BCD drastically improved insulin expression and proposed that the c combination of RC and ARX-shRNA treatment may help to generate abundant insulin-producing cells ex vivo.16 In addition, the inhibition of TGF-β, Wnt, and Notch pathways has been shown to redifferentiate adult human β cells expanded in vitro,17 while overexpression of the miRNA miR-375 promotes their redifferentiation and thus represents an interesting target.18 This approach is promising for ex vivo expansion of adult human β cells for transplantation, as this process
results mainly in BCD cells.
The redifferentiation of β cells could also occur with diabetes treatment; relieving the pressure on β cells (ie, lowering glucose by exogenous insulin treatment) could restore the sulfonylurea response and set up a virtuous circle (Maria S. Remedi lecture). Remedi et al showed that a mouse model of KATP gain-of-function in β cell developed diabetes and mimicked the features of human neonatal diabetes.19 As said previously, in the diabetic state, β cells lose their mature identity and dedifferentiate to Ngn3+ and insulin- cells. Wang et al demonstrated that dedifferentiated cells can subsequently redifferentiate to mature Ngn-, insulin+, β cells after lowering of blood glucose by insulin therapy. These results may help explain the recovery of β-cell function and drug response in T2D patients following insulin therapy. In accordance with the Domenico Accili lecture, they showed that β-cell dedifferentiation, rather than apoptosis, is the main mechanism of loss of insulin+ cells, and redifferentiation accounts for restoration of insulin content and antidiabetic drug response in these KATP
B. In vitro generation of β cells for transplantation
Successful regeneration of functional β-cell mass in diabetic patients via cell-based therapy would restore normal insulin secretion and cure the disease. However, developing methods to differentiate human embryonic stem cells (hESCs) and human induced pluripotent stem cells (hIPSCs) into pancreatic β cells remains a major challenge (Ludovic Vallier lecture),20 and the in vivo transplantation is delicate.
The transdifferentiation of pancreatic cells of other lineages into β cells is also a promising approach.
The first step is to obtain stem cells able to differentiate into β cells: hESCs and hIPSCs offer unique opportunities. Indeed both hESCs and hIPSCs share the important properties of self-renewal and pluripotency: they are theoretically capable of generating unlimited amounts of any differentiated cell in the human body.21 Figure 4 shows the protocols used to obtain these pluripotent cells, in particular the reprogramming techniques for the dedifferentiation of somatic cells using the transcription factors Oct4, Sox2, Klf4, and c-Myc. The Hongkui Deng lecture investigated the alternative of reprogramming somatic cells into pluripotent cells with chemicals instead of transcriptions factors. Chimeric mice generated from regular induced pluripotent stem cells (IPSCs) induced with transcription factors developed cancer and died after 30-60 days, whereas chimeric mice generated from chemically induced pluripotent stem cells survived.
Ye et al demonstrated that neural stem cells (NSCs) from the ectoderm and small intestinal epithelial cells (IECs) from the endoderm can be reprogrammed by small molecule compounds into pluripotent stem cells (they also reported a chemical approach from mouse fibroblasts).22
Both hESCs and hIPSCs are capable of proliferating indefinitely in vitro while maintaining the capacity to differentiate into a broad number of cell types, including pancreatic progenitors; the second step is to apply a protocol of differentiation. As Ludovic Vallier presented, robust protocols allowing for the production of homogenous populations of β cells in defined culture conditions have not yet been established. Current methods involve undefined animal products, such as feeders, FBS, and Matrigel, and only allow for the generation of heterogeneous populations of cells, thus increasing the risk of teratoma formation after transplantation. As shown in Figure 5, the pancreas arises on approximately embryonic day 8.5–9.5 from adjacent regions of the developing primitive foregut under the influence of inductive signals secreted by the nearby mesoderm, among them signals which probably command the production of transcription factors necessary for pancreatic specification.20 Cho et al, furthermore, showed that activin/TGF-β signalling blocks pancreatic specification induced by retinoic acid. Using this knowledge, they managed to differentiate hIPSCs in culture into a near homogenous population of pancreatic progenitors displaying functional characteristics specific to β cells.20 These cells could be used, in the future, for clinical applications.
The generation of insulin-producing cells in vitro starting from ESCs/IPSCs is challenging per se, but in addition cells produced exclusively in vitro do not properly respond to glucose; having 3D culture systems may improve their functionality (Anne Grapin-Botton lecture). Indeed, it is known that 3D cues are needed for appropriate endocrine cell differentiation, such as apico-basal and planar polarity complex.3 Groups of primary cells, ESCs and IPSC, can be grown in vitro, differentiate into a 3D structure, and display similar organization and functionality as an organ, thanks to Matrigel, which acts as a substitute
for mesoderm. The conditions, ie, chemicals and and growth factors, required for the expansion into different structures, eg, mini-pancreas, hollow spheres, and organoids, are recapitulated in Figure 6.
C. Genetically engineered human β-cell lines
Although rodent models are effective systems to study β-cell development, function, and dysfunction, there are inherent limitations in extrapolating observations in these models to what is occurring in humans. In this context, the mass production of functional β cells is a valuable tool to study the modulation of functional β-cell mass in humans (Raphaël Scharfmann lecture). The human pancreatic β-cell lines EndoC-βH1 and EndoC-βH2 have been generated by targeted oncogenesis in fetal pancreas (via the use of a lentivirus encoding for SV40 T and human telomerase) and amplified through passages as xenotransplants in SCID mice. This method carries the risk of infection by xenotropic viruses, and both the EndoC lines have been infected by xenotropic retrovirus Bxv1; however, the involuntary propagation of Bxv1 from these cells could be easily avoided with good laboratory practices.23 EndoC-βH1 was the first cell line developed. The cells are able to secrete insulin when stimulated with glucose or secretagogues, and they have been successfully used in a large number of laboratories for β-cell–related studies (review by Scharfmann et al).24 However, in this cell line, proliferation is under the control of the oncogenes used for transformation; thus a second generation of human β-cell line, EndoC-βH2, was developed. The transgenes can be conditionally excised upon Cre expression, and this procedure enhances β-cell–specific features (insulin expression and content). A main achievement using this cell line is the finding that the transcription factor RFX6 is present in adult human β cells and regulates insulin secretion by modulating Ca2+ homeostasis,25 its mutation being linked to neonatal diabetes (Raphaël Scharfmann lecture).
D. In vivo transdifferentiation in mouse models
Patrick Collombat et al showed that the selective inhibition of the Arx gene in α cells is sufficient to promote the conversion of adult α cells into β-like cells at any age (Figure 7).26 Recently, they submitted evidence that Pax4, a direct target of Ngn3, is a key player in pancreas development and plasticity in adult mice (Patrick Collombat lecture).27 Briefly, the ectopic expression of Pax4 in α cells is sufficient to induce their neogenesis and conversion into β-like cells, not only during development in rodents, but also in adult rodents. Therefore, differentiated endocrine α cells can be considered a putative source for insulin-producing β-like cells.
However the transgenic approach is difficult to develop in humans. A lot of screening was done to find molecules or chemical compounds that mimic the effects of Pax4. Some potential compounds, under development, are able to significantly stimulate islet hyperplasia (caused by insulin+ cell hyperplasia, generated form glucagon+ cells, as for the Pax4 effect) (Patrick Collombat lecture).