Amplifying Beta Cell Differentiation with Small Molecules BET (Bromodomain And Extraterminal Family Of Bromodomain-Containing Proteins) Inhibitors

Abstract

The present invention provides an in vitromethod for obtaining cells of the pancreatic endocrine lineage, comprising a step of culturing pancreatic progenitor cells, wherein said pancreatic progenitor cells are in a cell culture medium comprising at least one BET inhibitor.

Claims

1. In vitro method for obtaining cells of the pancreatic endocrine lineage, comprising a step of culturing pancreatic progenitor cells, wherein said pancreatic progenitor cells are in a cell culture medium comprising at least one BET inhibitor, and wherein said pancreatic progenitor cells are obtained by differentiation of stem cells obtained by techniques that do not involve the destruction of a human embryo.

2. The in vitro method according to claim 1, wherein the at least BET inhibitor is comprised in a concentration from 10 nM to 10 M.

3. The in vitro method according to claim 1 or 2, wherein the at least BET inhibitor is targeting BD1 and/or BD2.

4. The in vitro method according to claim 3, wherein the at least BET inhibitor targeting BD1 and/or BD2 is selected in the group comprising BET 151, JQ1, BET762, OXT-015, TEN-010, CPI-203, CPI 0610, LY29002 and RVX8, preferentially BET 151 and JQ1.

5. The in vitro method according to claim 1, wherein said pancreatic progenitor cells are obtained from embryonic stem cells, perinatal stem cell, somatic stem cells, and bioengineered stem cells, preferably said stem cells are hESC or iPSC, in particular hi PSC.

6. The in vitro method according to any of the previous claims wherein said pancreatic progenitor cells are cultured in said cell culture medium for at least 8 hours, preferably for at least 24 hours, more preferably for 48 hours, even more preferably 72 hours.

7. A cell of the pancreatic endocrine lineage obtainable by a method according to any of the previous claims.

8. A cell of the pancreatic endocrine lineage according to claim 6, for use as a medicament

9. A cell of the pancreatic endocrine lineage according to claim 6 or 7, for its use as a medicament for treating or preventing a pancreatic disorder, preferably chosen in the list consisting of pancreatitis, such as acute pancreatitis and chronic pancreatitis, diabetes mellitus, exocrine pancreatic insufficiency (EPI), cystic fibrosis (also known as mucoviscidosis), congenital malformations, such as pancreas divisum and annular pancreas, neoplasms (such as serous cystadenoma of the pancreas, solid pseudopapillary neoplasm or Zollinger-Ellison syndrome), and Hemosuccus pancreaticus.

10. A cell of the pancreatic endocrine lineage for use according to claim 8, wherein said pancreatic disorder is diabetes mellitus, preferably type I or type II diabetes.

11. Use of a cell of the pancreatic endocrine lineage obtainable by a method of any one of claims 1 to 5 for the in vitro production of insulin.

12. Use of a cell of the pancreatic endocrine lineage obtainable by a method of any one of claims 1 to 5 for the in vitro identification of compounds capable of modulating insulin production.

13. At least one BET inhibitor for use for treating or preventing a pancreatic disorder, preferably chosen in the list consisting of pancreatitis, such as acute pancreatitis and chronic pancreatitis, diabetes mellitus, exocrine pancreatic insufficiency (EPI), cystic fibrosis (also known as mucoviscidosis), congenital malformations, such as pancreas divisum and annular pancreas, neoplasms (such as serous cystadenoma of the pancreas, solid pseudopapillary neoplasm or Zollinger-Ellison syndrome), and Hemosuccus pancreaticus.

14. At least one BET inhibitor for use according to claim 13, wherein said pancreatic disorder is diabetes mellitus, preferably type I or type II diabetes.

15. Pharmaceutical composition comprising at least one BET inhibitor according to claim 13 or 14 and a pharmaceutically acceptable carrier.

Description

DESCRIPTION OF THE FIGURE

[0083] FIG. 1: BET inhibitors induce a major increase of neurogenin 3 expression. A, Mouse pancreatic buds were cultured in presence of either DMSO, 0.5M I-BET 151 or 0.1M (+)-JQ1. After 1, 3, 5 or 7 days, total RNA was extracted. Relative expression of Ngn3 was measured by RT-qPCR normalized with Cyclophylin A expression. Values represent the average of three independent experiments with standard deviation error bars. B and C, NGN3 expression was analyzed by immunohistochemistry on paraffin embedded mouse pancreatic buds that were cultured for five days in presence of DMSO, 0.5M I-BET 151 or 0.1M (+)-JQ1. B, Representative image of NGN3 staining. Scale bar measures 100m. C, Total NGN3 positive nuclei per rudiment were visually counted. D. Mouse pancreatic buds were culture in presence of either DMSO, 0.5 M I-BET 151 or 0.1M (+)-JQ1 during 5 days and then cultured for 9 additional days with complete medium only. Ins1, Ins 2 and MafA expression was measured by RT-qPCR normalized with Cyclophylin A expression. Values represent the average of three independent experiments with standard deviation error bars. *P0.05 **P0.01 ***P0.001.

[0084] FIG. 2: Relative Ngn3 mRNA expression in iPSC-derived endocrine progenitors treated with BET inhibitors. A Overview of the protocol for directed differentiation of pluripotent stem cells towards endocrine progenitors. Schematic overview outlining the five stages of the protocol for differentiating pluripotent stem cells towards pancreatic endocrine progenitors.

[0085] The timing of the individual stages (in days) and proteins and chemicals used for each step of the protocol are listed under each stage. iPSC-derived pancreatic endoderm was differentiated towards endocrine progenitors for three days in the presence of varying (+)-JQ1 B or IBET-151 C concentrations. Following the differentiation, mRNA was harvested from the cells and Ngn3 mRNA expression was analysed by qPCR. Graphs shows meanSD of one experiment with two technical replicates.

[0086] FIG. 3: Viability and cell number following BET inhibitor treatment. iPSC-derived pancreatic endoderm was differentiated towards endocrine progenitors for three days in the presence of varying (+)-JQ1 A or IBET-151 B. Following the differentiation, cells were harvested from wells and cell number and viability was assessed by a Nucleocounter image cytometer. (.circle-solid.) shows the percentage of live cells (left Y-axis of graphs). (.square-solid.) shows cell number adjusted to growth area (right y-axis of graphs). Graphs shows meanSD of one experiment with two technical replicates.

[0087] FIG. 4: Neurogenin 3 protein expression in iPSC-derived endocrine progenitors treated with BET inhibitors. iPSC-derived pancreatic endoderm was differentiated towards endocrine progenitors for three days in the presence of 500 nM (+)-JQ1, 2000 nM IBET-151 or vehicle control (DMSO). Following the differentiation, cells were fixed and processed for immunohistochemistry. Cells were stained for Neurogenin 3 (NGN3) using a polyclonal

[0088] Neurogenin 3 antibody and the nuclei of all cells were visualized using DAPI. Scalebar in images are 500uM in A and 200 uM in B. Representative images of three independent experiments are shown. The same exposure time was used for both control and BET inhibitor treated wells.

[0089] FIG. 5: Quantification of Neurogenin 3 protein expressing cells following BET inhibitor treatment. iPSC-derived pancreatic endoderm was differentiated towards endocrine progenitors for three days in the presence of 400 nM (+)-JQ1, 2000 nM IBET-151 or vehicle control (DMSO). Cells were subsequently harvested and percentage of cells expressing Neurogenin 3 was analyzed by flow cytometry. A Representative dot plots of cells treated with DMSO, (+)-JQ1 or IBET-151 for three days. X-axis shows Neurogenin 3 signal (Ngn3), Y-axis shows side scatter signal (SSC). Gates were set according to isotype controls. Numbers in dot plots shows percentage of cells within the gate. B Percentage of Neurogenin 3 (Ngn3) positive cells analyzed across three independent differentiation experiments, using a different iPSC line for each experiment. Boxes in graph show min to max with line at the mean. Individual biological replicates are shown as dots on the graph. C A human ESC-line genetically modified to express green fluorescence protein (GFP) under the control of Neurogenin 3 was differentiated to pancreatic endoderm and subsequently differentiated to the endocrine progenitor stage for three days in the presence of 400 nM (+)-JQ1, 2000 nM IBET-151 or vehicle control (DMSO). X-axis shows the GFP signal, Y-axis shows side scatter signal (SSC).

EXAMPLES

Example 1

Dissection and Culture

[0090] Mouse pancreatic buds were dissected from E11.5 C57Bl6/J embryos and placed at the air/medium interface on 0.45m filters in culture dishes containing RPMI medium supplemented with 10% fetal calf serum, 1% penicillin-streptomycin, 1% non-essential amino acids and 10mM HEPES. Pancreatic buds were cultured at 37 C. with 5% CO.sub.2.

Treatments

[0091] IBET-151 was obtained from Sigma-Aldrich and (+)/-JQ1 from Abcam. Stock solutions were prepared in DMSO. Inhibitors or DMSO (0.1% final concentration) were added to the culture medium which was changed daily.

Real Time PCR Analyses

[0092] Total RNA from three or more pancreatic buds was extracted using Qiagen RNA extraction microkit and 250ng were reverse transcribed using maxima first strand cDNA synthesis kit from Thermo Fisher. Real-time PCR analysis of Ngn3, MafA and Cyclophylin A were performed in 1 Sybr Green Powermix in QuantStudio 3 Applied Biosystem system. Real-time PCR analysis of Ins1 and Ins2 were performed in 1 TaqMan Gene Expression Mastermix in a QuantStudio 3 Applied Biosystem system. Relative expression of Ngn3, MafA and Cyclophylin A were calculated using the comparative method of relative quantification (2.sup.CT) normalized to cyclophilin A expression. Values represent the average of three experiments with standard deviation error bars. Statistical analysis was performed using unpaired Student t test.

Immunohistochemistry and Quantification

[0093] Pancreatic buds were fixed in 3.7% formaldehyde, pre-embedded in agarose gel (4% of type VII low gelling temperature agarose (Sigma-Aldrich)) and embedded in paraffin. Sections (4 m thick) were collected and processed for 3,3' diaminobenzidine immunohistological staining of NGN3, as previously described (Attali et al., 2007).

[0094] Photographs representative of a whole pancreas were taken using a transmitted light microscope (Leitz DMRB, Leica) and digitized using a Hammamatsu cooled 3CCCD camera. Total number of NGN3 positive nuclei per rudiment were then manually counted. Values represent the average of three experiments with standard deviation error bars. Statistical analysis was performed using unpaired Student t test.

Results

[0095] We studied the effects of IBET-151 and JQ1 on mouse embryonic pancreatic buds, which were cultured for 1, 3, 5 or 7 days in presence of each inhibitor (0.5 M of IBET-151 or 0.1 M of (+)-JQ1). Here we show that Ngn3 relative mRNA levels are strongly increased after 3 days of treatment, and remain increased after 7 days (FIG. 1A). These results were further confirmed by quantitative immunohistochemistry. They indicate that the number of NGN3 positive nuclei is increased by BET inhibitors treatment (FIGS. 1B and 1C). To evaluate the potential ability of the increased NGN3 positive population to undergo endocrine differentiation into mature B cells, E11.5 pancreatic buds were cultured for 5 days with IBET-151 or (+)-JQ1. Buds were then washed with fresh culture medium devoid of inhibitors and kept in culture for 9 additional days. Interestingly, Ins1 and Ins2 expressions were increased by 3 fold when pancreatic buds had previously been exposed to IBET-151 or (+)-JQ1 (FIG. 1D). Moreover, the expression of MafA, another marker of mature B cells, was increased by 10 folds (FIG. 1D). Altogether, these results indicate that IBET-151 and (+)-JQ1 stimulate multipotent progenitors toward endocrine differentiation, and that the increased NGN3 positive population can ultimately lead to more insulin and MafA expression.

[0096] These results hence suggest that these two inhibitors stimulate multipotent progenitors toward endocrine differentiation.

Example 2

Directed Differentiation of Pluripotent Stem Cells to Pancreatic Endocrine Progenitors.

[0097] Human induced pluripotent stem cells (iPSC) and human embryonic stem cells (ESC) were cultured on standard tissue culture plastic ware coated with hESC-qualified matrigel in mTeSR1.sup.medium. Cells were passaged every three to four days as single cells using TrypLe Select. Rock inhibitor (5 M) was included at the first day of passaging. Three different iPSC lines derived from a total of two individual donors and one ESC line were applied.

[0098] For differentiation, cells were seeded as single cells in mTeSR1 with 5 M Rock inhibitor into tissue culture plates (Corning CellBind) at densities ranging between 300-400.000 cells/cm.sup.2. Cells were cultured for 24 h at 37 C., 5% CO.sub.2. Following incubation, medium was aspirated and the cells were washed once in PBS before adding the differentiation medium. Differentiation was carried out essentially as described in Rezania et al. (Rezania et al., 2014b). The differentiation protocol is outlined in FIG. 2A. Medium was replenished daily according the list below: [0099] Stage 1Definitive endoderm (3 days): [0100] Day 1:MCDB131-1 medium* with 100 ng/ml Activin and 3 M CHIR [0101] Day 2:MCDB131-1 medium* with 100 ng/ml Activin and 0.3 M CHIR [0102] Day 3:MCDB131-1 medium* with 100 ng/ml Activin [0103] Stage 2primitive gut tube (2 days): [0104] Day 4-5:MCDB131-1 medium* with 0.25 mM Ascorbic acid and 50 ng/ml KGF [0105] Stage 3Posterior foregut (2 days): [0106] Day 6-7:MCDB131-2 medium** with 0.25 mM Ascorbic acid, 50 ng/ml KGF, 1 M Retinoic acid, 0.25 M Sant-1, 100 nM LDN and 200 nM TPB. [0107] Stage 4Pancreatic endoderm (3 days): [0108] Day 8-10 MCDB131-2 medium** with 0.25 mM Ascorbic acid, 2ng/ml KGF, 0.1 M Retinoic acid, 0.25 M Sant-1, 200 nM LDN and 100 nM TPB. [0109] Stage 5Endocrine progenitors (3 days): [0110] Day 8-10:MCDB131-3 medium*** with 0.05 M Retinoic acid, 0.25 M Sant-1, 100 nM LDN, 10 M Alk5ill, 10g/ml heparin and 1 M T3. At this stage, varying concentrations of BET inhibitors (IBET-151 or (+)-JQ1) or concentration matched vehicle control (DMSO) were included in the differentiation medium.

Medium Details:

[0111]

TABLE-US-00001 *MCDB131-1 **MCDB131-2 ***MCDB131 medium MCDB131 medium MCDB131 medium MCDB131 medium 0.1% Pen/Strep 0.1% Pen/Strep 0.1% Pen/Strep 1.5 g/L NaHCO3 2.5 g/L NaHCO3 1.5 g/L NaHCO3 1 x Glutamax 1 x Glutamax 1 x Glutamax 10 mM Glucose 10 mM Glucose 20 mM Glucose final 0.5% BSA 2% BSA 2% BSA 0.25 mM Ascorbic Acid 1:200 ITS-X 22 mg/ml AA solution 1:200 ITS-X 10 M Zinc sulfate

[0112] This protocol consistently yields >90% Sox17-positive cells with <5% Oct4 cells at the end of stage 1 (definitive endoderm) and between 40-70% PDX1/NKX6-1 co-positive cells at the end of stage 4 (pancreatic endoderm) depending on cell lines used (data not shown).

[0113] Viability of the cells and total number of cells was analyzed using a Nucleocounter NC3000 Cell analyzer (Chemometec).

Flow Cytometry Analysis

[0114] Differentiation efficiency was analyzed by flow cytometry essentially as described in van de Bunt et al., 2016. Briefly, cells were harvested from wells by TrypLe select and subsequently quenched for 20 min in 4% formalin on ice. Fixed cells were washed once in PBS and then permeabilized for 30 min on ice in PBS containing 5% donkey serum and 0.2% Triton-X100. Following permeabilization, cells were stained with primary antibodies diluted in PBS+5% donkey serum+0.1% Triton-X100 for 30 min at room temperature (directly conjugated antibodies) or overnight at 4C. (unconjugated antibodies). Cells were washed once in PBS with 1% bovine serum albumin. Unconjugated antibodies were detected with fluorophore conjugated secondary antibodies. The following antibodies were used:

TABLE-US-00002 Catalog Final Antigen Conjugate Vendor no. dilution Sox17 Alexa488 BD Pharmingen 562205 1:40 PDX1 Alexa488 BD Pharmingen 562274 1:40 NKX6.1 Alexa647 BD Pharmingen 563338 1:40 Oct4 Alexa647 BD Pharmingen 560329 1:10 Neurogenin 3 None R&D systems AF3444 1:200
RNA Isolation, cDNA Synthesis and Quantitative PCR (qPCR).

[0115] RNA was isolated from cells using NucleoSpin RNA/protein isolation kit (Macherey-Nagel). RNA was quantified using a nanodrop and 500-1000 g RNA pr. sample was converted to cDNA using iScript reverse transcription kit (Bio-Rad). Gene expression was evaluated using Tagman gene expression assay for Neurogenin 3(Hs01875204, Applied Biosystems).

[0116] Neurogenin 3 transcripts were normalized to the average expression of two housekeeping genes (ACTB, Hs01060665_g1 and HPRT1, Hs99999909_m1, both from Applied Biosystems). Relative expression was calculated using the Ct method (FIGS. 2B and 2C).

Immunohistochemistry Analysis

[0117] Immunohistochemistry analysis was performed as described in van de Bunt et al., 2016. Briefly, cells were fixed directly in tissue culture plates and subsequently permeabilized in PBS+05% Triton-X100 for 10 minutes and blocked in a tris-buffer containing 0.5% Tyramide Signal Amplification (TSA) immunohistochemistry kit blocking reagent for 30 min at room temperature. Cells were incubated with an anti-Neurogenin 3 antibody diluted in PBS+0.1% Triton-X100 (R&D systems, AF3444) overnight at 4 C. Cells were washed thrice in PBS and specific binding of the Neurogenin 3 antibody was revealed using a fluorescence coupled secondary antibody. Nuclei of all cells was revealed using 4,6-diamidino-2-phenylindole.

Results and Discussion

[0118] The effect of the BET inhibitors on Neurogenin 3 induction was tested in the context of human pluripotent stem cell differentiation. hiPSC were differentiated towards the pancreatic lineage using directed differentiation as described in the materials and methods (FIG. 2A). The hiPSC-derived pancreatic progenitors (also termed pancreatic endoderm) were differentiated towards the pancreatic endocrine lineage for three days in the presence of six different concentrations of either of the two BET inhibitors. Following the differentiation, induction of Neurogenin 3 expression was assessed by qPCR. A clear dose-dependent increase of Neurogenin 3 mRNA expression was observed for both of the BET inhibitors, with the maximum expression achieved at 300-400 nM JQ1 or 2000 nM IBET151. Potential toxic effect of the BET inhibitors on the differentiated cells was also evaluated. In the same experiment as described above, cells were harvested following the three day treatment and viability and cell number was determined using a Nucleocounter. No obvious difference in both cell number and viability was observed across all tested concentrations of the BET inhibitors compared to the controls (concentration matched DMSO) (FIG. 3A, B). These results suggest that the BET inhibitors can induce expression of Neurogenin 3 mRNA in hiPSC differentiated towards pancreatic progenitors.

[0119] To determine whether the induction of Neurogenin 3 mRNA by the BET inhibitors also resulted in increased expression of Neurogenin 3 protein in the differentiated hiPSC, cells were fixed following BET inhibitor treatment and Neurogenin 3 protein expression was evaluated by immunofluorescence microscopy and flow cytometry. FIGS. 4A and B shows representative images of hiPSC-derived endocrine progenitor cells treated for three days with JQ1, IBET 151 or DMSO as control. A clear increase in the number of cells positive for Neurogenin 3 protein is detected. The staining intensity of Neurogenin 3 in individual cells appears stronger in the cells treated with the BET inhibitors compared to the DMSO control, suggesting that there is more Neurogenin 3 protein present in individual cells (FIG. 4A, B). The percentage of cells expressing Neurogenin 3 protein following treatment with the BET inhibitors was determined using flow cytometry. Across three biological experiments, the number of cells expressing Neurogenin 3 protein was approximately 1.6 fold higher when treated with one of the BET inhibitors compared to the control treated cells (FIGS. 5A and B). In order to test the effect of the BET inhibitors on a hESC we applied a genetically modified hESC line that express green fluorescence protein (GFP) under the control of the Neurogenin 3 promoter. When this hESC line was differentiated to pancreatic progenitors and further towards the endocrine lineage for three days, more cells expressing GFP was observed when the cells were differentiated in the presence of either of the two BET inhibitors compared to the control. Together, these results demonstrate the ability of JQ1 and IBET 151 to induce the expression of Neurogenin 3 mRNA and protein during the differentiation of human pluripotent stem cells (both hiPSC and hESC) towards the pancreatic endocrine lineage.

Conclusion/Summary

[0120] JQ1 and IBET 151 dose-dependently induces Neurogenin 3 mRNA expression in hiPSC-derived pancreatic progenitors differentiated towards the endocrine lineage. [0121] JQ1 and IBET 151 induce Neurogenin 3 protein expression in pancreatic endocrine progenitors. [0122] No obvious toxicity or influence on cell number was detected on the differentiated human pluripotent stem cells by either of the BET inhibitors. [0123] The induction of Neurogenin 3 expression during the pancreatic endocrine differentiation is applicable to both hESC and hiPSC.

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