Methods of obtaining pancreatic endocrine cells

Abstract

The present invention relates to methods of producing pancreatic endocrine cells and uses of the cells obtained using the methods. The method utilises inhibitors or combinations of factors to provide increased quantities of endocrine material, for example for transplantation purposes.

Claims

1. A method for ex-vivo reprogramming comprising: a) providing pancreatic cells to be reprogrammed, b) reprogramming the pancreatic cells, comprising: (i) treating the pancreatic cells with PAX4, PDX1, MAFA and NGN3 transcription factors and (ii) inhibiting ARX expression and/or function c) thereby obtaining beta-like cells that express insulin mRNA.

2. The method according to claim 1, wherein the beta-like cells obtained in step (c) are capable of producing insulin protein in response to glucose stimulation.

3. The method according to claim 1, wherein the beta-like cells obtained in step (c) produce insulin protein at a level of at least 5% of that of adult human islets.

4. The method according to claim 1, wherein ARX expression is inhibited.

5. The method according to claim 4, wherein ARX expression is inhibited using RNA interference (RNAi).

6. The method according to claim 4, wherein ARX expression is inhibited by siRNA.

7. The method according to claim 1, wherein the cells are cultured on laminin throughout the method, or wherein during the reprogramming the cells are cultured in glucose, wherein the glucose concentration is between 0-5 mM, or wherein the cells are cultured in adherent culture prior to reprogramming, optionally for about 2 days, or a combination thereof.

8. The method according to claim 1, wherein the pancreatic cells provided in step (a) are human pancreatic cells, or wherein the pancreatic cells provided in step (a) comprise exocrine cells, optionally from an exocrine enriched fraction of a pancreas, or a combination thereof.

9. The method according to claim 1, wherein the reprogramming further comprises the step of pre-treating the cells, before treatment with the one or more transcription factors, with an inhibitor of epithelial to mesenchymal transition, or reprogramming further comprises the step of pre-treating the cells, before treatment with the one or more transcription factors, with a chromatin modifying agent, or a combination thereof.

10. The method according to claim 9, wherein the inhibitor of epithelial to mesenchymal transition is a TGFbetal signaling pathway inhibitor, a Rho-associated protein kinase (Rock) signaling pathway inhibitor, or a combination thereof.

11. The method according to claim 9, wherein the chromatin-modifying agent is a DNA-methyltransferase inhibitor, a histone deacetylase (HDAC) inhibitor, or a combination thereof.

12. The method according to claim 1, further comprising culturing the cells in media comprising betacellulin, exendin-4, nicotinamide, or a combination thereof.

13. The method according to claim 1, wherein the reprogramming comprises treatment with zinc.

Description

FIGURES

(1) FIG. 1. Combinations of transcription factors (TFs) induce reprogramming of human exocrine enriched fraction (EEF). (A) EEF cells were cultured as a monolayer for two days and then treated with SB, Y2, Aza and NaBu for 3 days. The cells were then transduced with various combinations of adenoviruses containing TFs (AD-TFs) as indicated and further cultured in presence of BEN for 6 days. (B) Insulin mRNA levels were measured by RT/QPCR. (C) Layout of the different transcription factor combinations used during the transdifferentiation process. Combinations of TFs that generated highest levels of insulin mRNA were indicated in yellow. N/A refers to untreated EEFs and SF to soluble factors in absence of Ad-TFs.

(2) FIG. 2. Four combinations of TFs induce expression of insulin (FIG. 1) and endogenous pancreatic TFs PDX1, NeuroD1, NGN3, MAFA, PAX4, PAX6 and NKX6.1 suggesting that the exogenous TFs are inducing reprogramming or transdifferentiation of the EEFs towards -cells. Condition 1 is untreated cells, conditions 30 is cells treated with soluble factors in absence of Ad-TFs. Condition 20, PDX1/NGN3/NKX6.1/NeuroD1; condition 24, PDX1/MAFA/NKX6.1/NeuroD1; condition 25, PDX1/MAFA/NGN3/NeuroD1; and condition 29, PDX1/MAFA/NGN3/PAX4. SFs are BEN, Y2, SB, 5-Aza-2deoxycytidine (Az) and sodium butyrate (NaBu).

(3) FIG. 3. Condition 29 is the only combination of TFs to provide glucose stimulated insulin (C-peptide) secretion. These data suggest that the presence of exogenous PAX4 is essential for efficient reprogramming of EEFs towards functional glucose sensitive -cells.

(4) FIG. 4. Culture on laminin promotes the reprogramming of EEFs towards insulin-expressing -cells by combinations of PDX1/NGN3/MAFA and PAX4.

(5) FIG. 5. Culture in low glucose promotes efficient reprogramming of EEFs towards insulin-expressing -like cells.

(6) FIG. 6. Knock-down of the endogenous TF ARX by siRNA on day 6 promotes efficient reprogramming of EEFs (FIG. 6B). Experimental protocol is illustrated in FIG. 6A. N/A refers to untreated EEFs and SF to soluble factors in absence of Ad-TFs. SCRB refers to a non-specific, non targeting siRNA. (B) insulin expression (C) glucagon expression; (D) somatostatin expression.

(7) FIG. 7. Knock-down of the endogenous TF ARX increases glucose-sensitive insulin (C-peptide) secretion in reprogrammed exocrine tissue. This effect is dependent on exogenous PAX4.

(8) FIG. 8. Reprogrammed islets express insulin at levels around 15% of that in adult human islets. To reprogram EEF cells were cultured as a monolayer for two days and then treated with SB, Y2, Aza and NaBu for 3 days. The cells were transduced with PDX1/MAFA/NGN3/PAX4 and cultured in presence of BEN for 6 days. Silencing of endogenous transcription factor (TF) ARX by siRNA was carried out at day 6 post addition of SB, Y2, Aza and NaBu.

(9) FIG. 9. Role of endocrine transcription factors on transdifferentiation. RT-qPCR analysis of the endocrine hormones insulin, glucagon and somatostatin after treatment with each transcription factor combination. Layout of the different transcription factor combinations used during the transdifferentiation process is shown in FIG. 1C. Data are representative of triplicate experiments and are relative to glyceraldehyde 3-phosphate dehydrogenase.

(10) FIG. 10. ARX inhibition enhances beta cell maturation and decreases alpha cell differentiation. (A) C-peptide release by untreated (N/A), transdifferentiated cells in the absence of siARX (REP), siARX transdifferentiated cells (siARX) and in the absence of PAX4 (siARX-PAX4). C-peptide was detected from the culture medium by a specific human C-peptide ELISA. Data are representative of triplicate experiments. (B) Insulin, C-peptide and Proinsulin content of untreated, transdifferentiated cells in the absence of siARX (REP), siARX transdifferentiated cells (siARX) and human islets. The content of each peptide was detected by specific human ELISAs after cell lysis and normalised to the total protein content. Data are representative of triplicate experiments. (C) Glucagon content of untreated, transdifferentiated cells in the absence of siARX (REP), siARX transdifferentiated cells (siARX) and human islets. Glucagon was detected by a specific human ELISA after cell lysis and normalised to the total protein content. Data are representative of triplicate experiments. (D) Immunocytochemistry for glucagon and C-peptide in transdifferentiated cells in the absence of siARX (REP) and siARX transdifferentiated cells (siARX). Data are representative of triplicate experiments. Scale bar=20 m.

(11) FIG. 11. Reprogrammed insulin producing cells prevent STZ-induced diabetes in vivo. (A) Body weight and blood glucose levels were measured in NOD/SCID mice grafted with transdifferentiated cells (Transdif Cells), exocrine pancreatic cells (Exoc Cells) or in non grafted mice (Ctrl) over a 38 day period after surgery. A single dose (150 mg/kg) streptozotocin was administered one day prior to surgery. n=5 Transdiff cells; n=3 Exoc cells; n=2 Ctrl. (B) Serum C-peptide levels were measured in NOD/SCID mice grafted with transdifferentiated cells (Transdif Cells) or exocrine pancreatic cells (Exoc Cells) and in non grafted mice (Ctrl) after a 4 h starvation period (fast) or under ad libitum feeding (fed) conditions. n=5 Transdiff cells; n=3 Exoc cells; n=2 Ctrl. (C) Immunostaining for insulin and glucagon of grafted kidneys following kidney removal. Yellow dash lines indicate the border between the kidney (k) and the graft. The red circle in panel a indicates the difference in glucagon staining observed within the cluster. A 5 higher magnification of the cells inside this circle is shown in panel c. 10 higher magnifications of the cells inside (e) and outside (f) the circle is shown. Panel d shows a 5 higher magnification of insulin staining within the area marked by the red square in panel b. A 10 higher magnification of insulin positive cells present in the centre of the cluster is shown in panel g. Scale bar for a and b=100 m. Scale bar for c-g=20 m. (D) Immunofluorescent staining for PDX1 in kidneys grafted with transdifferentiated cells. Scale bar=50 m. A 5 higher magnification inlet is shown.

(12) FIG. 12. Transdifferentiated pancreatic mesenchymal stem cells (MSCs) release glucagon in a regulated manner in vivo. (A) Blood glucose levels were measured in NOD/SCID mice grafted (A+Bu+4TFs+BEN) and in non grafted mice (Ctrl) over an 18 week period. n=5 animals in each group. (B) Glucagon was measured from the serum of grafted animals after a 4 h starvation period (fast) or under ad libitum feeding (fed) conditions. n=5 animals in each group. (C) NOD/SCID mice were rendered diabetic with one dose (150 mg/kg) of streptozotocin, one day prior to surgery. Blood glucose levels were measured in grafted (A+Bu+4TFs+BEN) and in non grafted mice (Ctrl) over an 18 week period. n=5 animals in each group. (D) NOD/SCID mice were rendered diabetic with one dose (150 mg/kg) of streptozotocin, one day prior to surgery (D) Glucagon was measured from the serum of grafted animals after a 4 h starvation period (fast) or under ad libitum feeding (fed) conditions. n=5 animals in each group. A, 5-Aza-2deoxycytidine (aza); Bu sodium butyrate (NaBu).

(13) FIG. 13. Representative electron microscopic images of cells reprogrammed with siARX. Unlike non reprogrammed cells, reprogrammed cells are rich in dense secretory granules (A). Scale bar=2 m. High magnification images (B and C) of dense core vesicles with different morphologies in reprogrammed cells. Scale bar=0.5 m (B) and 0.1 m (C).

(14) FIG. 14. RT-qPCR analysis of the three main endocrine hormones insulin (INS), glucagon (GCG) and somatostatin (SST) and the transcription factors PDX1, PAX4, MAFA, NEUROD, NGN3 and NKX6.1 in untreated (N/A) or cells reprogrammed (siARX) in the absence or presence of ZnCl.sub.2 (10 M). Expression was normalised to glyceraldehyde 3-phosphate dehydrogenase. Data are representative of triplicate experiments and represented as mean+/standard error of the mean.

(15) FIG. 15. C-peptide ELISA measurements of cell extracts from untreated cells (N/A) or cells reprogrammed (siARX) in the absence or presence of ZnCl.sub.2 (10 M). C-peptide levels were expressed level to protein content and represent 3SD (n=3). ***p<0.001 relative to NA and **p<0.01 relative to siARX.

EXAMPLES

Example 1

Materials and Methods

(16) Culture of Human Exocrine Pancreatic Fractions.

(17) All human tissue was procured with appropriate ethical consent. Human pancreata (n=42) were isolated from brain-dead adult donors in the Scottish Islet Isolation Laboratory (SNBTS, Edinburgh, UK). The mean donor age was 39.4 years (range 23-61 years) and BMI 27.2 kg/m2 (range 22-36.5 kg/m2).

(18) Culture where EMT is Inhibited.

(19) Following islet isolation the low purity exocrine fractions were transported to Aberdeen where the cells were immediately cryopreserved in liquid nitrogen at a density of 300,000 exocrine clusters per vial. The cells were cryopreserved in 90% fetal bovine serum (FBS, Gibco, Life Technologies, Paisley, UK) and 10% DMSO (Sigma Aldrich, Dorset, UK). Human exocrine fractions were thawed and plated on tissue culture 9 cm.sup.2 dishes (Greiner, Stonehouse, UK) and cultured for two days in RPMI 1640 (Gibco, Life Technologies) supplemented with 10% foetal bovine serum (FBS), 10 mM HEPES, 1 mM sodium pyruvate (all from Gibco) and 75 M -mercaptoethanol (Sigma Aldrich).

(20) After 48 h the cells were incubated for another 72 h in serum free medium (SFM) prepared with RPMI 1640, insulin-transferrin-selenium (Gibco) and 1% bovine serum albumin (Sigma), supplemented with 10 M SB431542, 2 M Y27632, 1 M 5-Aza-2deoxycytidine and 10 mM sodium butyrate (all from Sigma). On the next day the cells were incubated for 4 h with the adenoviruses encoding pancreatic transcription factors. On the following day the medium was changed for SFM supplemented with 1 nM betacellulin (R&D systems, Abingdon, UK), 10 nM exendin-4 and 10 mM nicotinamide (both from Sigma). The medium was changed every two days for another 6 days before harvesting.

(21) Culture to Obtain MSCs for Reprogramming where EMT is not Inhibited.

(22) Following islet isolation for clinical application the low purity exocrine fractions were transported to Aberdeen where the cells were immediately plated at a density of 300,000 exocrine clusters on 75 cm.sup.2 tissue culture flask (Greiner, Stonehouse, UK) and cultured in serum complete medium (SCM) prepared using RPMI 1640 (Gibco, Life Technologies, Paisley, UK) supplemented with 10% foetal bovine serum (FBS), 10 mM HEPES, 1 mM sodium pyruvate (all from Gibco) and 75 M -mercaptoethanol (Sigma Aldrich, Dorset, UK).

(23) Human exocrine pancreatic cells were passaged every 7 days with a solution of Trypsin (0.05%)-EDTA (0.02%, Gibco). Serum free medium (SFM) was prepared using RPMI 1640 supplemented with 1% bovine serum albumin (BSA, Sigma), 10 g/ml insulin and 5.5 g/ml transferrin (both from Roche Diagnostics, West Sussex, UK).

(24) Preparation of Adenoviruses.

(25) Recombinant adenoviruses encoding the mouse sequences of PDX1, MAFA, NGN3 and PAX4 (Swales et al., 2012) were prepared using the Ad-Easy system (Agilent Technologies, Edinburgh, UK). The adenoviruses containing PDX1 and NGN3 also expressed GFP through a downstream CMV promoter. Viral transduction was performed in SFM for 4 h at a multiplicity of infection (MOI) of 100 for each virus.

(26) Quantitative Reverse Transcription Polymerase Chain Reaction (qRT-PCR).

(27) QRT/PCR was performed as previously described (Lima et al., 2012. Data were analysed using the 2.sup.CT method. Statistical analysis was performed using PRISM software and the student's t-test or one-way ANOVA followed by the Dunnet's post-hoc test, as appropriate. The list of TaqMan probes are listed in Table 1:

(28) TABLE-US-00002 TABLE 1 List of Taqman gene expression primers: Gene Assay ID GAPDH Hs99999905_m1 INS Hs00355773_m1 GCG Hs00174967_m1 SST Hs001174949_m1 PDX1 Hs00236830_m1 NGN3 Hs01875204_s1 MAFA Hs01651425_s1 NKX6.1 Hs00232355_m1 NEUROD1 Hs00159598_m1 PAX6 Hs00240871_m1

(29) Immunocytochemistry and Immunohistochemistry.

(30) Immunocytochemistry and immunohistochemistry were performed as previously described (Cho, C-H, Hannan, N, Docherty, F M., Docherty, H M. Docherty, K. Vallier L.; Lima et al., 2012, using the antibodies listed in Table 2:

(31) TABLE-US-00003 TABLE 2 antibodies used in immunohistochemistry and immunocytochemistry Antigen Antibody host Source Dilution used C-peptide Mouse Cell Signalling 1:1000 Glucagon Mouse Sigma 1:1000

(32) C-Peptide Release Studies.

(33) C-peptide levels were measured using a human glucagon Quantikine ELISA kit (R&D Systems, Abingdon, UK), a human C-peptide ELISA kit (Millipore, Livingston, UK), a human prosinsulin ELISA kit or a human insulin ELISA kit (both from Mercodia, Uppsala, Sweden).

(34) siRNA Based Knockdown.

(35) Knockdown of Arx in transdifferentiating cells was performed by transfection with a pool of specific targeting small inhibitory RNAs, or scrambled controls (Dharmacon, Loughborough, UK). 100 nM siRNA was transfected on day 6 of the transdifferentiation protocol using Dharmafect 1 (Dharmacon), according to the manufacturer's instructions.

(36) Results

(37) Combinations of Transcription Factors (TFs) Induce Reprogramming of Human Exocrine Enriched Fraction (EEF).

(38) We examined various combinations of 6 pancreatic transcription factors (TFs), namely PDX1, MAFA, PAX4, NGN3, NKX6.1 and NeuroD1 in our previously published (Lima et al., 2013) reprogramming protocol. The difference in insulin expression observed between mature islets and transdifferentiated cells suggested that the latter had not reached the same maturation status as adult islets. In order to improve the transdifferentiation outcome, thirty different combinations of pancreatic TFs were tested (FIG. 1C). Expression of insulin (FIG. 1A), glucagon and somatostatin (FIG. 9) was measured.

(39) EEF cells were plated directly from the low purity fractions obtained following islet isolation. EEF cells were cultured as a monolayer for two days and then treated with SB, Y2, Aza and NaBu for 3 days. The cells were then transduced with various combinations of adenoviruses containing TFs (AD-TFs) as indicated in FIG. 1A and further cultured in presence of betacellulin, exendin-4 and nicotinamide (BEN) for 6 days.

(40) Of the 29 combinations, 4 provided significant levels of insulin gene expression (combinations 20, 24, 25 and 29 in FIG. 1). All four combinations increased the level of endogenous TFs suggesting that reprogramming or transdifferentiation was taking place (FIG. 2).

(41) Condition 1 is untreated cells.

(42) Condition 30 is cells treated with soluble factors (SFs) in absence of Ad-TFs.

(43) Condition 20 is cells treated with PDX1/NGN3/NKX6.1/NeuroD1

(44) Condition 24 is cells treated with PDX1/MAFA/NKX6.1/NeuroD1

(45) Condition 25 is cells treated with PDX1/MAFA/NGN3/NeuroD1

(46) Condition 29 is cells treated with PDX1/MAFA/NGN3/PAX4

(47) RT-qPCR analysis was used to monitor expression of the endocrine hormones insulin, glucagon and somatostatin relative to glyceraldehyde 3-phosphate dehydrogenase. The combinations after treatment with each transcription factor combination shown in FIG. 1C. There seems to be a preference for addition of NKX6.1 for reprogramming to glucagon-expressing alpha cells and of NeuroD1 for reprogramming to somatostatin-expressing delta cells (FIG. 9)

(48) The cells in this experiment may be reprogrammed to endocrine cells directly from exocrine cells without fully entering a mesenchymal state. Although the process of attaching to the culture may initiate the epithelial to mesenchymal transition (EMT), in these experiments the cells where attached for only a limited period (48 h) before inhibition of EMT. Thus in these experiments the processes that further establish and sustain the EMT were generally inhibited or supressed.

(49) PAX4 is Essential for the Generation of Glucose Sensitive -Cells

(50) Of the combinations, only combination 29 was able to regenerate cells that secreted insulin (C-peptide) in response to glucose (FIG. 3). This is the only combination of the four that contains PAX4.

(51) Therefore, although similar levels of insulin expression were obtained when replacing PAX4 by either NKX6.1 or NeuroD, only in the presence of PAX4 were the transdifferentiated cells able to secrete insulin in a glucose dependent manner, indicating that PAX4 plays a crucial role in establishing the functionality of mature beta cells in humans.

(52) RT-QPCR of late beta cell markers further demonstrated that MAFA expression was only present in cells transdifferentiated with the combination 29 (PDX1/MAFA/NGN3/PAX4, also named the 4TF combination) (FIG. 2), indicating that its expression is a key factor for beta cell functionality. This leads to the conclusion that inclusion of PAX4 is essential for the generation of glucose sensitive -cells.

(53) Growth on Laminin-Coated Plates Improves the Efficiency of Reprogramming

(54) We next compared the effect of extracellular matrices on the efficiency of the reprogramming protocol using PDX1/MAFA/NGN3/PAX4. Laminin, fibronectin, poly-lysine, collagen type I and collagen type IV were compared and the relative expression of insulin was assessed (FIG. 4). The results show that growth on laminin-coated plated improves the efficiency of reprogramming.

(55) Culture in Media Containing Low Concentrations of Glucose Further Enhances Reprogramming Towards -Cells

(56) Cells were cultured in different concentrations of glucose, and the relative expression of insulin is shown in FIG. 5. The results show that culture in media containing low concentrations of glucose further enhances reprogramming of EEFs towards insulin-expressing -cells. Glucose was added to SFM and cells were cultured with glucose for 10 days.

(57) Knock-Down of Endogenous Transcription Factor (TF) ARX in the Presence of Exogenous PAX4 Enhances the Efficiency of Reprogramming Towards -Cells

(58) EEF cells were cultured as a monolayer for two days and then treated with SB, Y2, Aza and NaBu for 3 days. The cells were then transduced with PDX1/MAFA/NGN3/PAX4 (REP) or PDX1/MAFA/NGN3 (-PAX4) and further cultured in presence of BEN for 6 days. Silencing of endogenous transcription factor (TF) ARX by siRNA was carried out at day 6 post addition of SB, Y2, Aza and NaBu (FIG. 6A) and the results are shown in FIGS. 6B, C and D. ARX silencing in the presence but not in the absence of exogenous Pax4 clearly stimulates INS (FIG. 6B) and SST (FIG. 6C) production but has no effect on GCG expression (FIG. 6D).

(59) ARX expression was inhibited by siRNA at the late stages of the reprogramming protocol, resulting in a 100 fold increase in insulin expression levels (FIG. 6B). Accordingly, ARX inhibition has led to an enhanced release of C-peptide by the reprogrammed cells in response to a high glucose concentration in vitro (FIG. 7). Reprogrammed islets express insulin at levels around 15% of that in adult human islets (FIG. 8).

(60) Moreover, removal of PAX4 from the reprogramming cocktail has abolished C-peptide release in response to high glucose levels, indicating that the action of PAX4 is essential for the functionality and maturation of the reprogrammed beta-cells. These studies indicate that the regulatory loop between ARX and PAX4 during the final stages of pancreatic development is essential for the functionality of human beta cells generated in vitro. These experiments show that knock-down of endogenous ARX in the presence of exogenous PAX4 enhances the efficiency of reprogramming.

(61) ARX expression was inhibited by siRNA at the late stages of the transdifferentiation protocol, resulting in a 60 fold increase in insulin expression levels compared to cells treated with control siRNA, bringing insulin expression levels much closer to those of mature beta cells (FIG. 10B). Further, reprogrammed islets (where ARX expression was inhibited) were shown to process proinsulin in a manner similar to that of adult human islets as evidenced by ELISA data using antibodies specific to proinsulin, insulin and C-peptide. (FIG. 10B)

(62) Cells reprogrammed in the absence of ARX inhibition (Lima et al. 2013) express only 1% of the insulin levels found in mature adult islets. These cells are labelled REP in FIG. 10B).

(63) ARX Inhibition Enhances Beta Cell Maturation and Decreases Alpha Cell Differentiation

(64) RT-QPCR has shown that ARX is expressed during the differentiation protocol and may favour the development of alpha versus beta cells during reprogramming of the exocrine derived material.

(65) Glucagon protein levels were significantly down-regulated after inhibition of ARX expression (FIG. 100). Specific ELISAs for human insulin and C-Peptide demonstrated that the transdifferentiated cells were able to efficiently store and process insulin, secreting C-peptide in a regulated glucose-responsive manner, with levels comparable to those found in human islets (FIG. 10). To further support its role in the functionality and maturation of the reprogrammed beta cells, removal of PAX4 from the transdifferentiation protocol resulted in the abolishment of C-peptide release in response to an increased glucose concentration. (FIG. 10A).

(66) Reprogrammed Insulin Producing Cells Prevent STZ-Induced Diabetes In Vivo.

(67) The in vivo function of the reprogrammed insulin producing cells was further determined by transplanting these cells under the kidney capsule of NOD/SCID mice that had been rendered diabetic with STZ 1 day before surgery (FIG. 11A).

(68) The cells used were from exocrine enriched tissue that had been plated in SFM and treated with SB431542, Y27632, 5-Aza-2deoxycytidine, sodium butyrate, 4TF and BEN (no ARX inhibitor).

(69) Animals that were transplanted with reprogrammed cells retained normal blood glucose levels and maintained body weight throughout the course of the experiment. Animals that were transplanted with non-reprogrammed exocrine cells, or those that were not transplanted with cells under the kidney capsule, exhibited markedly elevated blood glucose levels associated with weight loss (FIG. 11A).

(70) Removal of the transplanted kidney after 30 days resulted in an increase in the blood glucose levels of the animals transplanted with the reprogrammed cells (FIG. 11A). Human C-peptide was present only in the serum of fed mice that were transplanted with the reprogrammed insulin-producing cells (FIG. 11 B) but was absent from the blood when fasted, suggesting that the reprogrammed cells released insulin in a glucose-responsive manner in vivo. Immunostaining of the grafted kidneys showed that the transplanted cells formed a cluster-like structure under the kidney capsule, where the centre of the structure was mainly composed of strongly positive insulin positive cells, with the majority of the glucagon-positive cells localized in the periphery of the cluster (FIG. 11C). The majority of the cells in this structure also were positive for the pancreatic TF Pdx1 (FIG. 11D). Collectively, these data support the conclusion that the exocrine pancreatic cells of the adult human pancreas can be reprogrammed toward functional insulin-producing cells. The reprogrammed cells are able to ameliorate diabetes in a diabetic mouse model and generate a cluster-like structure reminiscent of islets of Langerhans.

(71) Transdifferentiated Pancreatic Mesenchymal Stem Cells (MSCs) were Shown to Release Glucagon in a Regulated Manner In Vivo.

(72) NOD/SCID were mice grafted with cells reprogrammed using A+Bu+4TFs+BEN (inhibitors of EMT were not used), and non grafted mice were used as a control. Glucagon was measured from the serum of grafted animals after a 4 h starvation period (fast) or under ad libitum feeding (fed) conditions, and was shown to present at a higher concentration in mice grated with treated cells.

(73) The NOD/SCID mice were rendered diabetic with one dose of streptozotocin, one day prior to surgery. Glucagon was measured from the serum of grafted animals after a 4 h starvation period (fast) or under ad libitum feeding (fed) conditions, and was shown to be present at a higher concentration in the fasting mice.

(74) The data show that the treated pancreatic mesenchymal stem cells (MSCs) released glucagon in a regulated manner in vivo.

Example 2

Methods

(75) Reprogramming of Human Exocrine Pancreatic Fractions

(76) Human exocrine fractions were thawed and plated on tissue culture 9 cm.sup.2 dishes (Greiner, Stonehouse, UK) and cultured for two days in RPMI 1640 (Gibco, Life Technologies) supplemented with 10% foetal bovine serum (FBS), 10 mM HEPES, 1 mM sodium pyruvate (all from Gibco) and 75 M -mercaptoethanol (Sigma Aldrich). After 48 h, the cells were incubated for another 72 h in serum free medium (SFM) prepared with RPMI 1640, insulin-transferrin-selenium (Gibco) and 1% bovine serum albumin (Sigma), supplemented with 10 M SB431542, 2 M Y27632, 1 M 5-Aza-2deoxycytidine and 10 mM sodium butyrate (all from Sigma). On the next day the cells were incubated for 4 h with the adenoviruses encoding pancreatic transcription factors PDX1, MAFA, NGN3 and PAX4. On the following day the medium was changed for SFM supplemented with 1 nM betacellulin (R&D systems, Abingdon, UK), 10 nM exendin-4 and 10 mM nicotinamide (both from Sigma). The medium was changed every two days for another 6 days before harvesting.

(77) Knockdown of ARX was performed by transfection with a pool of specific targeting small inhibitory RNAs, or scrambled controls (all from Dharmacon, Loughborough, UK). siRNA (100 nM) transfected on day 6 of the reprogramming protocol using Dharmafect 1 (Dharmacon), according to the manufacturer's instructions. ZnCl.sub.2 was used at a concentration of 10 M and was used in combination with the reprogramming adenoviruses and the siARX.

(78) Transmission Electron Microscopy

(79) Cells were detached from plates using Accutase (BD Biosciences, Oxford, UK) and subsequently fixed in 2.5% glutaraldehyde in 0.1M sodium cacodylate buffer at 4 C. overnight. The cells were subsequently post-fixed with 1% osmium tetroxide for 1 h followed by embedding in epoxy resin. The samples were then dehydrated in a series of ethanol washes for 20 min each starting at 70%, 95% and 100%. The samples were then embedded in epoxy resin, placed into moulds, and left to polymerise at 65 C. for 48 h. Sections were taken between 75 and 90 nm on a Leica Ultracut E (Leica, Wetzlar, Germany) and placed on formvarlcarbon coated slot grids. Images were observed on a JEOL JEM-1400 Plus TEM, and captured using an AMT UltraVue camera (Woburn, Mass., USA).

(80) Results

(81) Electron microscopy of human exocrine cells reprogrammed according to the protocol containing siARX revealed the presence of dense core granules that were polarised towards one side of the cell (FIG. 13A), a pattern that is typical of islet beta cells.

(82) Higher magnification (FIGS. 13B and 13C) showed the presence of granules, with in some instances a clear dense core surrounded by a non-opaque halo, properties that are characteristic of insulin secretory granules. The dense core of these granules is due to the presence of insulin-zinc hexameric crystalline structures. However, there were also granules that had a less dense core and lacked a halo.

(83) It was hypothesised that the lack of zinc in the media could contribute to these intermediate granule forms. This suggested that inclusion of zinc in the media would not only lead to the formation of more dense core secretory granules, but would also enhance the insulin secretory response to glucose and the insulin content of the reprogrammed cells.

(84) Zinc Increases the Level of Insulin mRNA in Reprogramed Cells, Possibly Through a Mechanism that Involves PAX4

(85) To test this hypothesis cells were reprogrammed in the presence or absence of zinc and analysed by RT/QPCR. Cells were reprogrammed (siARX) using the transcription factors and siARX as set out under Methods. The results demonstrated a significant effect of zinc on insulin gene expression that could in part be attributed to increased levels of mRNA encoding PAX4 (FIG. 14).

(86) Zinc Increases the Insulin (C-Peptide) Content of the Reprogrammed Cells

(87) Further studies showed that Zinc (ZnCl.sub.2) had a stimulatory effect on the insulin (C-peptide) protein content of the reprogrammed (siARX) cells (FIG. 15).

REFERENCES

(88) Akinci, E., Banga, A., Greder, L. V., Dutton, J. R., and Slack, J. M. (2012). Reprogramming of pancreatic exocrine cells towards a beta (beta) cell character using PDX1, NGN3 and MAFA. Biochem. J. 442, 539-550. Alipio, Z., Liao, W., Roemer, E. J., Waner, M., Fink, L. M., Ward, D. C., and Ma, Y. (2010). Reversal of hyperglycemia in diabetic mouse models using induced-pluripotent stem (iPS)-derived pancreatic beta-like cells. Proc. Natl. Acad. Sci. U.S.A 107, 13426-13431. Baeyens, L., De Breuck, S., Lardon, J., Mfopou, J. K., Rooman, I., and Bouwens, L. (2005). In vitro generation of insulin-producing beta cells from adult exocrine pancreatic cells. Diabetologia 48, 49-57. Bar, Y., Russ, H. A., Sintov, E., Anker-Kitai, L., Knoller, S., and Efrat, S. (2012). Redifferentiation of Expanded Human Pancreatic beta-Cell-derived Cells by Inhibition of the NOTCH Pathway. J. Biol. Chem. 287, 17269-17280. Blum, B., Hrvatin, S. S., Schuetz, C., Bonal, C., Rezania, A., and Melton, D. A. (2012). Functional beta-cell maturation is marked by an increased glucose threshold and by expression of urocortin 3. Nat. Biotechnol. 30, 261-264. Chandra, V., Swetha, G., Muthyala, S., Jaiswal, A. K., Bellare, J. R., Nair, P. D., and Bhonde, R. R. (2011). Islet-like cell aggregates generated from human adipose tissue derived stem cells ameliorate experimental diabetes in mice. PLoS One 6, e20615. Cho, C-H, Hannan, N, Docherty, F M., Docherty, H M. Docherty, K. Vallier L. Activin/TGFb signalling pathway controls hepatic and pancreatic specification of human endoderm. Collombat, P., Mansouri, A., Hecksher-Sorensen, J., Serup, P., Krull, J., Gradwohl, G., and Gruss, P. (2003). Opposing actions of ARX and PAX4 in endocrine pancreas development. Genes Dev. 17, 2591-2603. D'Amour, K. A., Bang, A. G., Eliazer, S., Kelly, O. G., Agulnick, A. D., Smart, N. G., Moorman, M. A., Kroon, E., Carpenter, M. K., and Baetge, E. E. (2006). Production of pancreatic hormone-expressing endocrine cells from human embryonic stem cells. Nat. Biotechnol. 24, 1392-1401. Davani, B., Ikonomou, L., Raaka, B. M., Geras-Raaka, E., Morton, R. A., Marcus-Samuels, B., and Gershengorn, M. C. (2007). Human islet-derived precursor cells are mesenchymal stromal cells that differentiate and mature to hormone-expressing cells in vivo. Stem Cells 25, 3215-3222. Docherty, K. (2011). Reprogramming Toward Pancreas Beta Cells. Stem Cell Biology and Regenerative Medicine Docherty, K., Bernardo, A. S., and Vallier, L. (2007). Embryonic stem cell therapy for diabetes mellitus. Semin. Cell Dev. Biol. 18, 827-838. Ferber, S., Halkin, A., Cohen, H., Ber, I., Einav, Y., Goldberg, I., Barshack, I., Seijffers, R., Kopolovic, J., Kaiser, N., and Karasik, A. (2000). Pancreatic and duodenal homeobox gene 1 induces expression of insulin genes in liver and ameliorates streptozotocin-induced hyperglycemia 6. Nat. Med. 6, 568-572. Gershengorn, M. C., Hardikar, A. A., Wei, C., Geras-Raaka, E., Marcus-Samuels, B., and Raaka, B. M. (2004). Epithelial-to-mesenchymal transition generates proliferative human islet precursor cells. Science 306, 2261-2264. Hao, E., Tyrberg, B., Itkin-Ansari, P., Lakey, J. R., Geron, I., Monosov, E. Z., Barcova, M., Mercola, M., and Levine, F. (2006). Beta-cell differentiation from nonendocrine epithelial cells of the adult human pancreas. Nat. Med. 12, 310-316. Jiang, J., Au, M., Lu, K., Eshpeter, A., Korbutt, G., Fisk, G., and Majumdar, A. S. (2007). Generation of insulin-producing islet-like clusters from human embryonic stem cells. Stem Cells 25, 1940-1953. Jiang, W., Shi, Y., Zhao, D., Chen, S., Yong, J., Zhang, J., Qing, T., Sun, X., Zhang, P., Ding, M., Li, D., and Deng, H. (2007). In vitro derivation of functional insulin-producing cells from human embryonic stem cells. Cell Res. 17, 333-344. Karnieli, O., Izhar-Prato, Y., Bulvik, S., and Efrat, S. (2007). Generation of insulin-producing cells from human bone marrow mesenchymal stem cells by genetic manipulation. Stem Cells 25, 2837-2844. Kojima, H., Fujimiya, M., Matsumura, K., Younan, P., Imaeda, H., Maeda, M., and Chan, L. (2003). NeuroD-betacellulin gene therapy induces islet neogenesis in the liver and reverses diabetes in mice. Nat. Med. 9, 596-603. Kroon, E., Martinson, L. A., Kadoya, K., Bang, A. G., Kelly, O. G., Eliazer, S., Young, H., Richardson, M., Smart, N. G., Cunningham, J., et al. (2008). Pancreatic endoderm derived from human embryonic stem cells generates glucose-responsive insulin-secreting cells in vivo. Nat. Biotechnol. 26, 443-452. Lima, M. J., Docherty, H. M., Chen, Y., and Docherty, K. (2012). Efficient differentiation of AR42J cells towards insulin-producing cells using pancreatic transcription factors in combination with growth factors. Mol. Cell. Endocrinol. 358, 69-80. Lima, M. J., Muir, K. R., Docherty, H. M., Drummond, R., McGowan, N. W., Forbes, S., Heremans, Y., Houbracken, I., Ross, J. A., Forbes, S. J., et al. (2013). Suppression of epithelial-to-mesenchymal transitioning enhances ex vivo reprogramming of human exocrine pancreatic tissue toward functional insulin-producing beta-like cells. Diabetes 62, 2821-2833. Montgomery, A. M., and Yebra, M. (2011). The Epithelial-to-Mesenchymal Transition of Human Pancreatic beta Cells: Inductive Mechanisms and Implications for the Cell-Based Therapy of Type I Diabetes. Curr. Diabetes Rev. Ogihara, T., Fujitani, Y., Uchida, T., Kanno, R., Choi, J. B., Hirose, T., Kawamori, R., and Watada, H. (2008). Combined expression of transcription factors induces AR42J-B13 cells to differentiate into insulin-producing cells. Endocr. J. 55, 691-698. Ouziel-Yahalom, L., Zalzman, M., Anker-Kitai, L., Knoller, S., Bar, Y., Glandt, M., Herold, K., and Efrat, S. (2006). Expansion and redifferentiation of adult human pancreatic islet cells. Biochem. Biophys. Res. Commun. 341, 291-298. Porat, S., Weinberg-Corem, N., Tornovsky-Babaey, S., Schyr-Ben-Haroush, R., Hija, A., Stolovich-Rain, M., Dadon, D., Granot, Z., Ben-Hur, V., White, P., et al. (2011). Control of pancreatic beta cell regeneration by glucose metabolism. Cell. Metab. 13, 440-449. Rezania, A., Bruin, J. E., Riedel, M. J., Mojibian, M., Asadi, A., Xu, J., Gauvin, R., Narayan, K., Karanu, F., O'Neil, J. J., et al. (2012). Maturation of Human Embryonic Stem Cell-Derived Pancreatic Progenitors into Functional Islets Capable of Treating Pre-existing Diabetes in Mice. Diabetes Rezania, A., Riedel, M. J., Wideman, R. D., Karanu, F., Ao, Z., Warnock, G. L., and Kieffer, T. J. (2011). Production of functional glucagon-secreting alpha-cells from human embryonic stem cells. Diabetes 60, 239-247. Schulz, T. C., Young, H. Y., Agulnick, A. D., Babin, M. J., Baetge, E. E., Bang, A. G., Bhoumik, A., Cepa, I., Cesario, R. M., Haakmeester, C., et al. (2012). A scalable system for production of functional pancreatic progenitors from human embryonic stem cells. PLoS One 7, e37004. Shapiro, A. M., Lakey, J. R., Ryan, E. A., Korbutt, G. S., Toth, E., Warnock, G. L., Kneteman, N. M., and Rajotte, R. V. (2000). Islet transplantation in seven patients with type 1 diabetes mellitus using a glucocorticoid-free immunosuppressive regimen 4. N. Engl. J. Med. 343, 230-238. Swales, N., Martens, G. A., Bonne, S., Heremans, Y., Borup, R., Van de Casteele, M., Ling, Z., Pipeleers, D., Ravassard, P., Nielsen, F., Ferrer, J., and Heimberg, H. (2012). Plasticity of adult human pancreatic duct cells by neurogenin3-mediated reprogramming. PLoS One 7, e37055. Tateishi, K., He, J., Taranova, O., Liang, G., D'Alessio, A. C., and Zhang, Y. (2008). Generation of insulin-secreting islet-like clusters from human skin fibroblasts. J. Biol. Chem. 283, 31601-31607. Wang, H. S., Shyu, J. F., Shen, W. S., Hsu, H. C., Chi, T. C., Chen, C. P., Huang, S. W., Shyr, Y. M., Tang, K. T., and Chen, T. H. (2011). Transplantation of insulin-producing cells derived from umbilical cord stromal mesenchymal stem cells to treat NOD mice. Cell Transplant. 20, 455-466. Yechoor, V., Liu, V., Espiritu, C., Paul, A., Oka, K., Kojima, H., and Chan, L. (2009). Neurogenin3 is sufficient for transdetermination of hepatic progenitor cells into neo-islets in vivo but not transdifferentiation of hepatocytes. Dev. Cell. 16, 358-373. Zhang, T., Saunee, N. A., Breslin, M. B., Song, K., and Lan, M. S. (2012). Functional role of an islet transcription factor, INSM1/1A-1, on pancreatic acinar cell trans-differentiation. J. Cell. Physiol. 227, 2470-2479. Zhou, Q., Brown, J., Kanarek, A., Rajagopal, J., and Melton, D. A. (2008). In vivo reprogramming of adult pancreatic exocrine cells to beta-cells. Nature