Bromodomain and Extra Terminal Proteins Inhibitors Promote Pancreatic Endocrine Cell Fate
ABSTRACT
Bromodomain and Extra-terminal (BET) proteins are epigenetic readers that interact with acetylated lysines of histone tails. Recent studies have demonstrated their role in cancer progression, as they recruit key components of the transcriptional machinery to modulate gene expression. However, their role during embryonic development of the pancreas has never been studied. Using mouse embryonic pancreatic explants and hiPSCs, we show that BET proteins inhibition with I-BET151 or JQ1 enhances the number of neurogenin3 endocrine progenitors. In mouse explants, BET proteins inhibition further led to increased expression of β cell markers, but in the meantime strongly downregulated Ins1 expression. Similarly, while acinar markers, such as Cpa1 and CelA were upregulated, Amy expression was repressed. In hiPSCs, BETi strongly repressed C-PEP and GCG during endocrine differentiation. Explants and hiPSCs were then pulsed with BETi to increase NEUROG3 expression, and further chased without inhibitors. Endocrine development was enhanced in explants, with higher expression of insulin and maturation markers, such as UCN3 and MAFA. In hiPSC, the outcome was different, as C- PEPTIDE expression remained lower than in control, but Ghrelin expression was increased. Altogether, by using two independent models of pancreatic development, we show that BET proteins regulate multiple aspects of pancreas development.
INTRODUCTION
During pancreatic development, multipotent endodermal pancreatic progenitors proliferate in response to signals derived from surrounding mesodermal cells and next differentiate into cells with exocrine and endocrine properties, including β cells (1,2). Chronic failure to reduce high blood glucose levels results in diabetes, which in most cases is due to impaired functional β cell mass. Recent effort therefore concentrated on developing in vitro protocols to produce functional β cells from induced pluripotent stem cells (iPSCs) or embryonic stem cells, to replenish the decreasing β cell mass (3). These protocols are based on our knowledge of the molecular mechanisms underlying in vivo β cell development (4,5). Despite recent considerable progress, generating a homogeneous population of fully mature, glucose-responsive β cells remains a challenge (6).The mouse embryonic pancreas starts developing at ~E9.0 with the dorsal and ventral budding of the foregut endoderm under the influence of surrounding mesodermal structures. At this stage, multipotent proliferating epithelial pancreatic progenitors express a specific set of transcription factors such as PDX1 and NKX6.1 (7). They can undergo exocrine and endocrine cell fate until ~E11.5, and then progressively segregate into acinar progenitors or bipotent endocrine/duct progenitors (8,9). Endocrine progenitors then transiently express the basic helix-loop-helix transcription factor neurogenin-3 (NEUROG3) to give rise to all pancreatic endocrine cells (10).Major progress was made during the past years on growth factors, such as FGF7 and FGF10, that signal through FGFR2b (11–15) and activate the proliferation of pancreatic progenitors. It is also the case for small molecules acting through yet to be discovered pathways (16), or upon co-culture on a layer of 3T3-J2 feeder cells (17).
On the other hand, information regarding signals that modulate the differentiation of pancreatic progenitors into functional β cells remains scarce, and the objective of the present work was to increase our knowledge on this topic.The BET family of proteins comprises four members, BRD2, BRD3, BRD4 and BRDT. The latter is specifically expressed in the testis while the three others are more ubiquitously expressed (18– 20). The BET proteins recently emerged as a major class of epigenetic readers and modulators of gene expression by their ability to recognize and bind, through their two bromodomains, N-acetylated-lysine residues of histone tails (21). They subsequently induce an opened-chromatin structure and can tether various transcription factors at target promoters and enhancer regions to promote transcription (22). The BET family of proteins has largely been associated with cancer progression and recent efforts have concentrated on developing potent and specific inhibitors of BET proteins (BETi), such as (+)-JQ1 and I-BET151 (21,23,24). BET proteins play a crucial role throughout development, as Brd2 and 4 null mice are embryonic lethal (25,26). Recent work have shown that BRD4 participates in adipogenesis and myogenesis by modulating gene transcription at specific enhancer regions (27), yet BET proteins role in organogenesis and cell fate remains poorly characterized.Here we evaluated the effect of BET protein inhibition during pancreas development. We used two different in vitro models: i) a culture of rodent fetal pancreas under conditions that replicate the major steps of in vivo pancreatic development (13) ii) in vitro differentiation of human induced pluripotent stem cells (hiPSCs) into insulin-producing cells (28,29). We report that BET inhibition using either I-BET151 or JQ1 increases the pool of NEUROG3+ endocrine progenitors in mouse embryonic pancreatic explants and in pancreatic progenitors derived from hiPSCs.
This increased number of endocrine progenitors resulted in enhanced endocrine differentiation from pancreatic explants, while GHRL expression was increased in hiPSCs. Our data demonstrate the importance of the BET protein family during pancreatic endocrine lineage differentiation.Pregnant C57Bl6/J mice were purchased from the Janvier Breeding Center and killed by CO2 asphyxiation according to the French Animal Care Committee’s guidelines. Dorsal pancreatic buds were dissected as previously described (13).Pancreatic buds culture, treatment with BETi and BrdU incorporation.E11.5 pancreatic buds were cultured as previously described (13). The medium was changed daily and supplemented with either 0.1% DMSO, 500nM I-BET151 (Sigma Aldrich) or 100nM JQ1 (Abcam). For cell proliferation assay, 10μM BrdU (Sigma Aldrich) was added during the last hour of culture.Culturing and differentiation of induced pluripotent stem cells.Wild type SBAD03-01 and SBAD03-04 hiPSCs were obtained through the IMI/EU sponsored StemBANCC consortium via the Human Biomaterials Resource Centre, Birmingham University (http://www.birmingham.ac.uk/facilities/hbrc). Cells were cultured and differentiated as previously described (30), BET inhibitors or DMSO control were added daily to culture medium during the stage 4 (pancreatic endoderm) or 5 (endocrine progenitors) of the differentiation. Data presented in the manuscript are from SBAD03-01 line, similar data were obtained with SBAD03-04 hiPSCs (see Fig. S1).Total RNA was extracted as previously described (30,31) Real-time PCR was performed with a QuantStudio 3 or OneStepPlus Real-time PCR system (Applied Biosystems) or a Mx3005P qPCR system (Stratagene).
Each reaction consisted of either a mix of Power Sybr Green PCR Mastermix (Applied Biosystems) with a specific pair of designed primers or a mix of Taqman Universal PCR master mix with a specific labeled probe (Applied Biosystems). Data are presented relative to Cyclophylin A (for rodent samples) or to HPRT1 and ACTB (for hiPSCs samples).and quantification.Mouse fetal pancreases were processed for immunohistochemistry, as previously described (31). All primary antibodies and dilutions are described in the Online Supplemental Materials. The fluorescent secondary antibodies were purchased from Jackson ImmunoResearch (1/400). The biotin-labeled secondary antibodies were purchased from Vector Laboratories (1/200). NEUROG3 and MAFA were detected using the Vectastain elite ABC kit (Vector Laboratories). The nuclei were stained using the Hoechst 33342 fluorescent stain (0.3 mg/ml; Invitrogen). Surface area quantifications were performed on one out of three consecutive sections (that is, sections separated by 12μm) to avoid counting the same cell twice. The signal was quantified using ImageJ software (National Institute of Health, NIH), and summed to obtain the surface area per explant, expressed in mm2. NEUROG3 and MAFA positive nuclei were manually counted on one out of three consecutive sections with ImageJ, then summed to obtain the number of positive nuclei per explant.hiPSC were fixed with 4% PFA at room temperature for 30 minutes, and then processed for immunocytochemistry as previously described (32).Flow cytometry and cell sorting.Cells were sorted as previously described (33,34). hiPSC cells were harvested to a single-cell solution using TrypLE Select and subsequently fixed and stained as previously described (29). Additional information on antibodies is described in the online supplementary data.
Confluent Min6 cells were treated with either 0.1% DMSO or 1μM JQ1 for 24h at 37°C in a humidified 95% air/5% CO2 gas mixture. Chromatin Immunoprecipitation was performed as described by Cotney et al. (35) using 5μg of BRD2, 3 and 4 rabbit polyclonal antibodies (A302-583A; A302-368A; A301- 985150; Bethyl Laboratories). Immunoprecipitated chromatin was analyzed by Real-Time PCR. Primer sequences are detailed in the Online Supplemental Materials.Pools of at least 5 pancreatic buds were incubated overnight in culture medium containing 2.8mM glucose (Sigma-Aldrich) and 2% FCS (Eurobio), then incubated in Krebs-Ringer buffer (KRB) [125mMol/L NaCl, 4,7mMol/L KCl, 2,5mMol/L CaCl2, 1,2mMol/L MgSO4, 1,2mMol/L KH2PO4, 25mMol/L NaHCO3, pH 7.4], supplemented with 0.2% bovine serum albumin (BSA) (Sigma-Aldrich) and 2.8mM glucose for 60 min. Insulin secretion was assessed by sequential static incubations of 60 min in KRB containing 45μM IBMX (Sigma-Aldrich) and increasing concentrations of glucose (2.8mM to 22.4mM) and finally 30mM KCl without IBMX.Pancreatic explants were homogenized in RIPA buffer and protein concentration was assayed using a Bicinchoninic Acid (BCA) Protein Assay Kit (Pierce). Secreted Insulin and content were measured in duplicate by ELISA according to manufacturer’s instructions (Crystal Chem Mouse Ultrasensitive Insulin ELISA).All quantitative data are presented as the mean ± SD. Statistical significance was set at 5% and was determined using ordinary one-way ANOVA with Dunett post hoc test except for ChIP data that were determined using repeated-measure one-way ANOVA.
RESULTS
At E11.5, we separated the EpCAM+ fraction that contains epithelial progenitors from EpCAM- mesenchymal fraction by FACS and performed real-time PCR. Brd2, 3 and 4, but not Brdt, mRNAs were detected in both the epithelial and mesenchymal fractions (Fig. 1A). At E16, EpCAM+ cells were separated from EpCAM- cells by FACS and further divided into three fractions that contain exocrine cells, endocrine progenitors and hormone-producing cells (34). Brd2, 3 and 4 were detected in the mesenchymal as well as in all three EpCAM+ fractions, with the highest expression in the hormone producing cells (Fig. 1B). We next cultured E11.5 pancreatic dorsal buds for 1, 3, 5 or 7 days under culture conditions that successfully replicate in vivo development of exocrine and endocrine lineages, d3 being comparable to E15.5 and d7 to E17.5-18.5 (36). We observed a sustained expression of Brd2, Brd3 and Brd4 during the 7 days culture period (Fig. 1B). These results hence show that Brd2, 3 and 4 are expressed as early as E11.5 and that their expression is maintained during pancreas development.BET bromodomain inhibition induces Neurog3 expression in both mouse and human.We treated mouse pancreatic explants with I-BET151, and analyzed by real-time PCR the expression of Neurog3, a specific marker of pancreatic endocrine progenitors (10). In control explants, Neurog3 mRNA level peaked after 3 days of culture, when endocrine progenitors develop, and decreased thereafter as they further differentiate into hormone-expressing cells (10,36). I-BET151 treatment enhanced Neurog3 expression after 3 days of culture with a 10-fold induction over control conditions at day 5 and day 7 (Fig. 2A). Similar induction of Neurog3 levels was obtained with another BET inhibitor, JQ1 (Fig. 2A). Immunohistochemical analysis at day 5 showed an increase of NEUROG3+ cells upon treatments with either I-BET151 or JQ1 (Fig. 2B and 2C for quantification). Finally, both inhibitors enhanced the expression of NeuroD and Fev, two downstream targets of NEUROG3 (37,38) (Fig. 2D).
The surge of NEUROG3+ cells was not the result of an increased proliferation of multipotent pancreatic progenitors during early stages of explants development as: i) There was no major variations of multipotent progenitor markers Pdx1, Sox9 and Ptf1a after 24h of exposure to BETi (Fig S2); ii) immunohistochemical analyses of BrdU incorporation indicated that the proliferation of PDX1+ pancreatic progenitors did not increase. In fact, in BrdU incorporation by PDX1+ cells upon BETi treatment was decreased by 10% (Fig S3A and B). The NEUROG3 surge did not result from an increased proliferation of endocrine progenitors. Indeed, BrdU incorporation remained nearly undetectable in NEUROG3+ cells from pancreatic buds cultured with I-BET151 or JQ1 (Fig. S3C). TUNEL staining revealed no variations in apoptotic events (Fig. S3D and E). We next examined the effect of BETi on human endocrine progenitors derived from hiPSC. They were first differentiated toward multipotent pancreatic progenitors, and further cultured in presence of I- BET151 and JQ1. Both inhibitors increased NEUROG3 expression (Fig. 3B). Immunohistochemistry and flow cytometry analysis further confirmed enhanced NEUROG3 expression (Fig. 3C and D), with an almost doubling of the NEUROG3+ population (Fig. 3E). The increase of NEUROG3 expression followed a dose-response curve with no decline in cell viability (Fig. S4).These results indicate that BET bromodomain inhibition increases the pool of endocrine progenitors both in mouse embryonic pancreatic explants and in a model of multipotent pancreatic progenitors derived from hiPSC.We next assessed whether the increased pool of NEUROG3+ cells that developed upon BETi treatment gives birth to more hormone producing cells in mouse pancreatic buds. Somatostatin (Sst) and Glucagon (Gcg), respectively markers of δ and α cells, were significantly increased by I-BET151 and JQ1 treatments (Fig. 4A). Ghrelin expression (Ghrl), a marker of ε lineage expressed at low levels in control conditions was markedly increased, peaking at d5 of culture with a 25-fold and a 47-fold increase with I- BET151 and JQ1 respectively (Fig. 4A).
Immunohistological analysis indicated a sharp increase in the number of GHRL+ cells at d7 with both inhibitors (Fig. 4B and C for quantification). Most GHRL+ cells stained negative for GCG and all GHRL+ cells stained negative for PDX1, INS and SST (Fig. 4B and S5). Altogether, these results show that BET bromodomain inhibition induces an increase of α, δ, and ε cell markers. The expression of acinar markers was also evaluated, and we observed a strong decrease in Amy expression, whereas Cpa1 and CelA were upregulated (Fig. 4 D). This suggests that BET inhibition enhances acinar development but that in the meantime, BET activity is required for amylase expression.Chronic treatment with BETi activates the β cell lineage but inhibits insulin gene expression.We next determined whether I-BET151 and JQ1 treatments influence β cell development in mouse pancreatic buds. As expected, Pcsk1/3, MafA and Iapp expressions increased during the culture period in control conditions, while Nkx6.1, which encodes a transcription factor first expressed in all early multipotent pancreatic progenitors and next restricted to β cells, decreased (Fig. 5A, white columns). BETi sharply increased the expression of all four β cell markers (Fig. 5A). The increase in MafA mRNA was further confirmed by immunohistochemistry at d7 of culture. I-BET151 and JQ1 treatments increased the number of MAFA+ cells by 2.5±0.6 and 2.8±0.6 folds respectively (Fig. 5B and C for quantification). These results demonstrate that β cell development is induced upon BETi treatment. However, to our surprise, we observed a major reduction of Ins1 expression upon BETi treatment. Ins1 mRNA levels were reduced by 47- and 20-fold after 7 days of culture with I-BET151 and JQ1 respectively (Fig. 5D). This was further confirmed by immunohistochemistry (Fig. 5E and F for quantification) and by ELISA (Fig. 5G). On the other hand, Ins2 mRNA levels were only mildly reduced following I-BET151 and JQ1 treatments (Fig. 5H). This difference between Ins1 and Ins2 expression following BETi treatment was next analyzed by immunohistochemistry. We used antibodies against either C-peptide 1 or C-peptide 2 as surrogate markers of INS1 and INS2 expression. We observed an almost complete loss of C-peptide 1 signal with both inhibitors, while many cells remained positive for C-peptide 2 (Fig. S6). Such differential effect of BETi on Ins1 and Ins2 expression was also observed in the mouse insulinoma cell line MIN6 (Fig S7A), and in primary mouse islets (Fig S7B).
In order to determine if such effect was due to differential binding of BET proteins to the Ins1 and Ins2 promoters, chromatin immunoprecipitation (ChIP) of BRD2, 3 and 4 was performed in MIN6 cells. We observed strong bindings of BRD2 and BRD4 to both promoters, which strongly decreased upon JQ1 treatment. Interestingly, BRD2 and 4 were bound at the proximal region of Ins1 promoter, whereas they bound at a more distal position of Ins2 promoter (Fig 6).This indicates that rather than reducing the ability of amplified NEUROG3+ endocrine progenitors to pursue β cell differentiation, BETi promote β cell development but in the meantime downregulate Ins1 expression with little effect on Ins2.Long-term effect of transient BETi on endocrine differentiationOur results demonstrate that exposure to either I-BET151 or JQ1 enhances pancreatic endocrine cell development. However, Ins1 levels are strongly downregulated upon such chronic treatment. In order to determine whether BETi exposure during explant development could ultimately lead to an increase of insulin-producing β cells, we performed pulse-chase experiments. We cultured (pulse period) pancreatic explants with BETi during 5 days. At this stage, Neurog3 expression is at its highest. Pancreatic explants were then cultured for 9 additional days (chase period) without BETi and analyzed (Fig. 7A). Under this setting, at d14, Ins1 and Ins2 mRNA levels increased 3 to 4 folds (Fig. 7B) and Insulin content was also strongly increased (Fig. 7C). MafA, but not MafB, mRNA level was also strongly upregulated, with 13- and 16-fold increase following I-BET151 and JQ1 pulse/chases respectively (Fig. 7D). It was also the case for Ucn3 mRNA levels (22- and 23-fold increase) (Fig. 7D). Gcg, Sst and Ghrl expressions were also all increased (Fig. 7D), as well as other β cell markers, such as Nkx6.1, Pcsk1/3, Pdx1 and Iapp (Fig. S10A). Immunohistochemical labeling of MAFA (Fig. 7E, and 7F for quantification) and UCN3 (Fig. 7G) following BETi pulse/chase confirmed this massive increase observed by real time PCR.
There was no co-expression of Insulin with either SST or GCG, showing that the resulting β cells are not poly-hormonal (Fig. S10B). ELISA assay of insulin secretion did not show any response upon glucose stimulation (Fig. 7H) suggesting that the resulting β cells yet remain not fully mature. Similar to what was observed in pancreatic buds, transient BETi treatment of hIPSCs during stage 5 reduced C-PEP+ cells and INS expression (Fig. 8A and B). GCG+ and SST+ cells were also reduced (Fig. 8A), while GHRL expression was upregulated (Fig. 8B). Cells were then further cultured without BETi during the maturing endocrine cells stage (chase during stage 6). C-peptide expression increased following removal of BETi (Fig. 8, compare panels A and B to C and D), but remained lower than in control conditions (Fig. 8C, D). GCG and SST expressions were similar to control condition (Fig. 8C). On the other end, GHRL expression increased (Fig. 8C and 8D). Importantly, co-immunocytochemistry of C-PEP with either SST and GHRL or GCG and GHRL showed that most endocrine cells at the end of stage 6 were mono-hormonal (Fig. S11).Taken together, our data indicate that treatment with BETi induces an increased pool of Neurog3+ endocrine progenitors that will further differentiate into β, α, δ or ε cells in mouse pancreatic buds or ε cells in hiPSCs.
DISCUSSION
The molecular mechanisms underlying the transitions from pancreatic progenitors toward mature endocrine cells remain not fully elucidated. Histone modifications are key epigenetic events that play major roles in cell proliferation and differentiation (39), and we previously showed that inhibiting histone deacetylases (HDAC) modulates cell fate during rat pancreas development (31). Here, we extended our comprehension of epigenetic modulation of embryonic pancreas development by studying histone code readers BET proteins. We provide evidence that BET inhibition promotes the pool of NEUROG3+ endocrine progenitors, which subsequently gives rise to an increased pool of endocrine cells.To explore the role of BET proteins in embryonic pancreas development, we used an in vitro model of dorsal embryonic pancreatic buds that recapitulates the major steps of endocrine and exocrine development that occur in vivo (13,36). We first observed that Brd2-4 expression at E11.5 was similar in the epithelial population that is enriched in pancreatic progenitors and in mesenchymal cells. This result was expected as Brd2-4 have been described as ubiquitously expressed in the majority of tissues (22), with some exceptions, such as the highly enriched expression of BRD2-4 in crypts of the small intestine, when compared to villi (40). Interestingly, Brd2-4 expression at E16.5 is higher in a fraction enriched in hormone expressing cells compared to fractions enriched in acinar or pancreatic progenitor cells, suggesting an important role of BET proteins in establishing or maintaining endocrine cell fate.
In mouse pancreatic explants cultured with I-BET151 or JQ1, Neurog3 expression was amplified both at the mRNA and protein level. NEUROG3 activity was also increased as demonstrated by the increased expression of two of its targets, NeuroD1 (38) and Fev (37). This increase of NEUROG3+ cells was not the consequence of an upstream effect on proliferation of PDX1+ pancreatic progenitors or NEUROG3+ endocrine progenitors themselves. In mouse pancreatic buds, the expression pattern of Neurog3 was shifted, as it peaked at day 5 of culture and remained 10 times more expressed than in controls at day 7. A possibility is that BETi increase the half-life of NEUROG3. Indeed, NEUROG3 has been described as an unstable protein rapidly degraded by ubiquitin-mediated proteolysis (41) and it can also be suspected that Neurog3 mRNA is unstable. Finally, it is interesting to note that the positive effect of BETi on NEUROG3 expression is context- and tissue-dependent as BETi (CPI203 or I- BET151) treatment induces a loss of progenitor cell markers such as Neurog3 in enteroendocrine cell differentiation in the mouse adult small intestine (40).
In mouse explants, BETi treatment increased the expressions of endocrine markers, Ghrl being the most strongly upregulated. Ghrelin is expressed by a rare population of pancreatic endocrine cells named ε-cells (42), and GHRELIN+ cells that developed with BETi resemble ε-cells. They do not express PDX1, insulin or somatostatin, and only a few coexpress glucagon (Fig. S5). GHRELIN+ cell number was shown to be increased in the pancreas of mice deficient for either nkx2.2, pax6 or pax4 (42,43). Our data however suggest that ghrelin induction upon BETi treatment does not occur through Nkx2.2 or Pax6, whose expressions did not decrease following explants treatment (Fig. S8A). The moderate reduction of pax4 expression (Fig. S8B) seems unlikely to explain the observed effect on ghrelin, as supernumerary GHRELIN+ cells of pax4-/- mice co-express glucagon and low PDX1 levels (44), which was not the case in our model. The mechanism involved in the effects of BETi on the development of ε cells hence remains to be clarified. We also observed a strong decrease in amylase expression in pancreatic buds treated with BETi, yet other markers such as Cela1 or Cpa1 were upregulated by BETi (Fig.4D and S9), suggesting an incomplete development of the acinar compartment.Nkx6.1 and MafA are key transcription factors essential for β cell differentiation and maturation, respectively. Their up-regulation should then reflect an increase of β cell differentiation. We were therefore surprised to observe that Ins1 expression was dramatically reduced following treatment of pancreatic buds with BETi whereas Ins2 did not vary with JQ1 and only by 2 folds with I-BET151. Our data indicate that it was also the case in two models of mature β cells, adult mouse islets and MIN6 cells, where Ins1, but not Ins2 was downregulated by I-BET151 and JQ1. Brd4 has been the most studied BET member, as it was shown to modulate RNA polymerase II activity, either by interacting with members of the transcription initiation complex, such as pTEFb and Mediator, (45–47) or by directly releasing proximally paused RNA polymerase II (48).
Interestingly, Brd4 was shown to specifically associate with active promoters and enhancers of a given cell type and tether various transcription factors through its ET domain (49,50). The insulin promoter is hyperacetylated in β cells (51), and Ins1 is among the most expressed genes in β cells. ChIP of BRD2, 3 and 4 in BETi treated MIN6 cells, indicated that BRD4, and to a lesser extent, BRD2, were strongly bound at the proximal promoter of Ins1 (-85/+16). We also found them to bind Ins2 promoter, but at a more distal position (-683/-771). This could explain the differences observed in BETi responses, as proximally bound BRD4 could be more likely to influence RNA polymerase II activity. Such approach and high throughput ChIPseq analysis on purified β cells or NEUROG3+ progenitors would be of great use for deciphering the underlying mechanisms of BET on pancreas development and Insulin regulation. However, to date, there are no available specific markers to efficiently purify such cells from mouse pancreatic buds.Our data indicate that BETi can increase the proportion of NEUROG3+ endocrine progenitors both in a model of rodent pancreatic development and in a model of beta cell development from hiPSCs. However, the failure of the generated β cells to properly express insulin represents a limiting step. Our pulse-chase approach in rodent however indicates that NEUROG3+ endocrine progenitors amplified with BETi can develop into INS+ β cells following BETi removal. Moreover, newly-formed β cells expressed higher levels of MafA and Ucn3, both considered as markers of β cell maturity (52,53). Their maturity, however, is not yet complete, as their sensitivity to glucose in terms of insulin secretion was not activated, suggesting that MafA or Ucn3 expression can not be used as sole markers of cell maturity. It is established that in rodent, beta cells appear quite late during development, when compared for example to α cells (1).
It has also been shown that early NEUROG3+ cells differentiate into alpha cells and those appearing later on, into beta cells (54). It could thus be postulated that the timing of differentiation correlates with maturity and β cells generated upon BETi treatment are more mature as NEUROG3 expression peak was maintained for a longer period. Our results in hiPSCs show that BET proteins are essential for endocrine progenitor differentiation, as BET inhibition during Endocrine Progenitor stage enhanced NEUROG3+ cells and resulted in a total lack of C-PEP+ and GCG+ cells. Such effect was not observed when hiPSCs were cultured with BETi during earlier Pancreatic Endoderm stage (Fig. S11). Further removing BETi from amplified NEUROG3+ progenitors replenished the C- PEP+ population that however did not quite reach control levels. Further testing additional conditions may permit to reproduce the increase in β cell population observed in the mouse pancreatic buds. Indeed, the discrepancies between the two models may come from different susceptibility time windows to BET inhibition, which should be tested. They also might be due to the absence, in hiPSC, of mesenchymal tissue, which participates in the control of β cell differentiation (55) and could mediate some BETi effects in explants. Finally, we observed a rise of ε cells in hiPSCs that were pulsed with BETi during the Endocrine Progenitor stage (Fig. 8), which is consistent with what was observed in mouse pancreatic buds. This should therefore be of particular interest to study ε cells differentiation, which I-BET151 remains poorly understood.Altogether, our results obtained across two very different models of pancreas development revealed novel roles of BET on pancreatic endocrine progenitors and their differentiation into endocrine cells.