Successful strategies for treating type 1 diabetes need to restore the function of pancreatic beta cells that are destroyed by the immune system and overcome further destruction of insulin-producing cells. Here, we infused adeno-associated virus carrying Pdx1 and MafA expression cassettes through the pancreatic duct to reprogram alpha cells into functional beta cells and normalized blood glucose in both beta cell-toxin-induced diabetic mice and in autoimmune non-obese diabetic (NOD) mice. The euglycemia in toxin-induced diabetic mice and new insulin + cells persisted in the autoimmune NOD mice for 4 months prior to reestablishment of autoimmune diabetes. This gene therapy strategy also induced alpha to beta cell conversion in toxin-treated human islets, which restored blood glucose levels in NOD/SCID mice upon transplantation. Hence, this strategy could represent a new therapeutic approach, perhaps complemented by immunosuppression, to bolster endogenous insulin production. Our study thus provides a potential basis for further investigation in human type 1 diabetes.

Non-integrative adeno-associated viral (AAV) vectors can impart long-term expression of transgenes up to 4.5 kb in length. Moreover, AAV vectors have been found to be more efficient than adenoviral and lentiviral vectors in transducing pancreatic cells (). Among AAV vectors, serotype 8 has been shown to have the highest transduction efficiency for mouse islet endocrine cells (). Here, we used a transgenic mouse model that allows lineage tracing of alpha cells, and we delivered Pdx1 and MafA expression vector virus to the mouse pancreas through a recently developed pancreatic intraductal infusion system (). We saw glucose normalization in beta cell-toxin-treated mice, and the new INScells were found to derive almost exclusively from alpha cells. We then similarly delivered virus to autoimmune, hyperglycemic non-obese diabetic (NOD) mice, and saw durable euglycemia, typically for 4 months. The mechanisms underlying the prolonged survival of new INScells in the autoimmune environment were further investigated.

Pancreatic and duodenal homeobox 1 (Pdx1) is a transcription factor necessary for pancreatic development, including beta cell maturation, beta cell proliferation, and function (). MafA is a transcription factor that binds to the INS promoter to regulate INS expression and beta cell metabolism (). Ectopic expression of a combination of three key pancreatic beta cell transcription factors (Pdx1, neurogenin 3 [Ngn3], and MafA) has been shown to reprogram adult mouse pancreatic acinar cells into beta cell-like cells (). Moreover, co-overexpression of these three genes has been shown to convert Sox9liver cells into INS-producing cells (). However, alpha cells may be the ideal source for beta cell replacement for several reasons. First, as endocrine cells, alpha cells are developmentally similar to beta cells, which may facilitate reprogramming (). Second, alpha cells are already situated within the islet () so that a reprogrammed beta cell from an alpha cell would be well positioned for ideal beta cell function. Third, alpha cell hyperplasia is commonly seen in diabetic animals and patients and constitutes a potentially abundant source for reprogramming, and human islets in particular have a large percentage of alpha cells (). Fourth, according to recent reports, a significant decrease in the number of alpha cells did not appear to harm proper glucose metabolism (). Fifth, glucagon (GCG) signaling appears to be detrimental in diabetes, which suggests that a partial reduction in alpha cell mass due to their conversion to beta cells may be beneficial to blood glucose control (). Sixth, alpha-to-beta cell conversion is feasible since it has been reported to occur after an extreme beta cell loss () and in an insulinoma model with alpha cell-specific Men1 ablation (). Last, a recent ATAC sequencing (ATAC-seq) study showed that the alpha cell genome is remarkably accessible and thus likely to more easily undergo transdifferentiation (). Based on these concepts, we were prompted to examine whether forced expression of key beta cell transcription factors in alpha cells may trigger their reprogramming to generate beta or beta-like cells. We expected that Ngn3 would not be necessary to convert alpha cells to beta cells because Ngn3 is necessary for the formation of endocrine cells, but alpha cells are already endocrine cells.

Although great efforts have been made to identify, isolate, and purify beta cell progenitors in the adult pancreas (), accumulating evidence does not support a substantial contribution of beta cell neogenesis to a functional beta cell mass in the adult pancreas (), except for a few rare situations (). Thus, gene therapy may be required in order to generate new beta cells from other cell types ().

Sox9+ ductal cells are multipotent progenitors throughout development but do not produce new endocrine cells in the normal or injured adult pancreas.

Insulin (INS) is a key regulator of glucose homeostasis and is produced by pancreatic beta cells. Insufficient INS leads to diabetes mellitus, a metabolic disease that affects over 300 million people worldwide (). The fundamental objective of diabetes treatment is to preserve and restore a functional beta cell mass, perhaps through beta cell replacement therapy. However, beta cell replacement may fall short in autoimmune type 1 diabetes (T1D) due to persistent, recurrent autoimmunity against the new beta cells (). In fact, this form of renewed autoimmune attack has been found to be particularly aggressive (). Unfortunately, a clinically applicable strategy leading to a more durable beta cell mass has yet to be developed for T1D ().

Based on data from these mouse studies, we then tested whether human alpha cells may be reprogrammed into functional INScells through a similar strategy. Human islets are resistant to ALX, but high-dose STZ has been reported to ablate a large proportion of human beta cells in vitro (). Here, we treated human islets with 20 mmol/L STZ for 12 hr to destroy beta cells, after which the islets were treated in vitro with either AAV-PM or AAV-GFP for 24 hr to trigger alpha-to-beta cell conversion and then transplanted into ALX-treated hyperglycemic NOD/SCID mice ( Figure 7 A). The beta cell-toxic effect of STZ was confirmed by examining INS content per islet ( Figure 7 B). Cells double positive for both INS and GCG were detected 3 days after STZ and AAV-PM treatment in culture ( Figure 7 C). In addition, the alpha cell mass in human islets was decreased by 35% 3 days after STZ and AAV-PM treatment in culture. We found that within 1 week after transplantation, the ALX-treated NOD/SCID mice that received human islets treated with STZ and AAV-PM had significantly lower blood glucose levels ( Figure 7 D), and significantly better glucose tolerance curves, compared to mice transplanted with STZ and AAV-GFP-treated human islets ( Figure 7 E). The grafts were harvested 4 weeks after transplantation, and we found a significantly higher INS content ( Figure 7 F), greater beta cell mass ( Figure 7 G), and higher serum human C-peptide ( Figure 7 H) in the grafts of AAV-PM-treated human islets compared to the graft of AAV-GFP-treated human islets, which was further confirmed by immunohistochemistry ( Figure 7 I). One week of continuous BrdU labeling was performed after the islet transplantation, and only 1.5% ± 0.3% of INScells had incorporated BrdU ( Figure 7 J). Thus, any contribution of proliferating residual beta cells to the increase in beta cell mass should be minimal. Together, these data suggest that AAV-PM can induce the generation of functional INScells from alpha cells in human islets, although we cannot exclude that human non-alpha/non-beta cells may have contributed to the augmented INScell numbers.

Statistics were analyzed by one-way ANOVA with a Bonferroni correction, followed by Fisher’s exact test. Data are presented as mean ± SD. ∗ p < 0.05. ∗∗ p < 0.01. n = 5. White scale bars, 50 μM. Yellow scale bars, 20 μM.

(J) One week of continuous BrdU labeling was performed after the islet transplantation, and only 1.5% ± 0.3% of INS + cells had incorporated BrdU, shown by a representative image in the left panel. The right panel is an inset of the yellow rectangle region in the left panel.

(I) Representative images for INS (in green) and GCG (in red) in the graft under the kidney capsule.

(D and E) The ALX-NOD/SCID mice that received human islets treated with STZ and AAV-PM (in red) had significantly lower fasting blood glucose levels (D), and significantly better glucose tolerance (E), as early as 1 week after transplantation, compared to the ALX-NOD/SCID mice that received human islets treated with STZ and AAV-GFP (in green).

(A) Human islets were treated with 20 mmol/L STZ for 12 hr, after which the islets were treated with either AAV-PM or AAV-GFP for 24 hr and then transplanted into ALX-treated hyperglycemic NOD/SCID mice.

It is well known that islet transplants into an autoimmune diabetic environment can be quickly destroyed by the “rapid-recurrent” form of immune attack (), even if the patient is adequately immunosuppressed for an accompanying kidney transplant (). One possible explanation for the resistance of our neogenic INScells to such a rapid-recurrent autoimmune rejection is that the pancreatic ductal infusion with AAV-PM may somehow directly alter the autoimmunity in the NOD mouse, resulting in extended survival of the neogenic INScells. This virus-induced “bystander effect” has been well described in NOD mice (). This possibility seemed unlikely since the control AAV-GFP infusion gave no protection to the beta cells. However, to further examine this possibility, we isolated splenocytes from AAV-PM-treated NOD mice 4 weeks after viral infusion and performed an adoptive transfer into NOD/SCID mice. Splenocytes from untreated hyperglycemic NOD mice (UT) and from AAV-GFP-treated NOD mice were used as controls, the latter two showing no significant difference from each other ( Figure 6 A). We found that the development of diabetes in recipient NOD/SCID mice after delivery of splenocytes from AAV-PM-treated NOD mice was somewhat delayed, but still present ( Figure 6 A). The timing and kinetics of the hyperglycemia onset mirrored what might be seen if naive NOD splenocytes (harvested prior to the onset of hyperglycemia) had been used (). To further test the competence of the NOD autoimmunity, NOD/SCID mouse islets (300) were transplanted under the kidney capsule of AAV-PM-treated and AAV-GFP-treated NOD mice 4 weeks after viral infusion, as well as into undisturbed NOD/SCID mice as controls ( Figure 6 B). We detected slightly higher graft INS content ( Figure 6 C) and higher INScell numbers ( Figure 6 D) in AAV-PM-treated NOD mice compared with AAV-GFP-treated NOD mice (but still much lower than control grafts). This result was further confirmed by INS immunohistochemistry of the graft under the kidney capsule ( Figure 6 E). Also important here is that the presumed reactivation of the autoimmunity in the AAV-PM-treated mice, targeted against the transplanted islets, did not lead to an adjuvant effect with an autoimmune attack on the neogenic INScells (derived from alpha cells) in the pancreas, as the blood glucose remained normal. These latter data further support that the autoimmunity of the NOD mice is intact and is behaving as if it were not actively being exposed to beta cell antigens at the time of transplant and thus showed a slightly delayed response to the transplanted islets.

Statistics were analyzed by one-way ANOVA with a Bonferroni correction, followed by Fisher’s exact test. Data are presented as mean ± SD. ∗ p < 0.05. ∗∗ p < 0.01. n = 5. Scale bars, 50 μM.

(E) Representative images for INS (in green) and CD45 (in red) in the islet graft under the kidney capsule.

(B) NOD/SCID mouse islets (300) were transplanted under the kidney capsule of AAV-PM-treated and AAV-GFP-treated NOD mice 4 weeks after viral infusion, and into undisturbed NOD/SCID mice as a control.

(A) Splenocytes isolated from UT diabetic NOD mice (UT), and from AAV-PM-infused and AAV-GFP- infused NOD mice 4 weeks after viral infusion were adoptively transferred into NOD/SCID mice. The development of diabetes in recipient NOD/SCID mice was compared.

Next, we examined whether the newly formed INScells would be recognized by an autoimmune diabetic immune system. Thus, we gave NOD mice a single ductal infusion of the PM virus early after the onset of hyperglycemia, when the blood glucose of the mice had surpassed 200 mg/dL. We found that the glycemia in these mice normalized fairly rapidly, and for about 4 months. The mice receiving control AAV-GFP showed continuously increasing blood glucose and died within 5 weeks ( Figures 5 A and S5 A). Immunohistological analysis showed that the NOD mice that received AAV-PM had significantly greater INScell mass ( Figures 5 B and 5C) as a basis for their normalized blood glucose, although the insulitis (based on CD45 staining for immune cells) was still present ( Figure 5 C). Moreover, we found that early on, some INScells also expressed GCG ( Figure 5 D), possibly representing in-transit cells. Furthermore, EM images showed single cells with both INS and GCG granules ( Figure 5 E), suggesting that these INS-producing cells are reprogrammed from alpha cells. When the intraductal infusion with AAV-PM was instead performed later, after the blood glucose had reached 400 mg/dL, we found that the blood glucose was normalized in only 3 out of 7 mice, and for about 16 weeks ( Figure S5 B). This variable result may stem from glucose toxicity or from some alpha cell injury. At the 22 week time point, when the AAV-PM-treated mice were again hyperglycemic, histology showed that, in the islet region, there were many infiltrating CD3lymphocytes and F4/80macrophages, but very few INScells (but were still GFP), and still many of the surrounding non-islet (acinar) cells were GFP, confirming persistence of transgene expression ( Figures S6 A and S6B).

Statistics were analyzed by one-way ANOVA with a Bonferroni correction, followed by Fisher’s exact test. Data are presented as mean ± SD. ∗∗ p < 0.01. n = 10. Scale bars, 50 μm.

(E) EM image showing an islet cell with both INS (red arrow) and GCG (yellow arrow) granules in the left panel. The right panel is the inset of the blue rectangle region in the left panel.

(D) Confocal images for INS (in red) and GCG (in blue) 5 weeks after infusion of AAV-PM, along with direct green fluorescence (GFP) from viral infection, to show presence of double positive cells for both INS and GCG (arrows).

(C) Immunostaining for INS (in red) and CD45 (in white) 5 weeks after infusion of control AAV-GFP (upper panels) or AAV-PM (lower panels), along with direct GFP from viral infection. HO, Hoechst, nuclear stain.

(A) When the blood glucose of female NOD mice surpassed 200 mg/dL, the mice received an intraductal infusion of either AAV-PM or control AAV-GFP. Fasting blood glucose levels were measured, showing continuously increasing hyperglycemia in control mice (green line), but rapid stabilization and then, by 2–3 weeks, normalization of hyperglycemia in mice infused with AAV-PM (red line), lasting for about 4 months.

Next, we examined the differences in gene expression patterns between alpha cell-derived INScells and normal alpha and beta cells. We thus generated triple transgenic mice (GCG-Cre; R26R; MIP-GFP). In these mice, alpha cells are lineage tagged with tomato and beta cells express GFP. However, the alpha cell-derived INScells will express both tomato and GFP (and therefore will be yellow), to allow separate isolation by flow cytometry ( Figures 4 A and S2 D), similar to our previous study (). Tomatocells in untreated mice are used as a control for normal alpha cells, while GFPcells in untreated mice are used as a control for normal beta cells ( Figures 4 B and 4C). Importantly, because of the MIP-GFP, the AAV-PM virus here did not have the GFP sequence. We found that the alpha cell-derived INScells have a gene expression pattern very close to normal beta cells, but very different from the original alpha cells, by both RNA sequencing (RNA-seq) ( Figures 4 D–4G), and by direct RT-qPCR quantification of some cell-specific transcripts ( Figure S4 ). These data suggest that the alpha cell-derived INScells have undergone a near-complete conversion to beta cells.

(G) Volcano plot for the group B versus group C comparison, of –log 10 (p value) versus log 2 (fold change), for differential gene expression analysis using Cuffdiff output. The horizontal dashed line in both plots corresponds to an FDR adjusted p value of 0.01. All points displayed above this this line on the plot have an adjusted p value of less than 0.01. n = 3. For each sample, purified cells from four or five mouse pancreases were pooled together for RNA-seq.

(D) Pearson correlation plot showing the FPKM values of all genes generated by Cuffnorm for all samples followed by heatmap generation using the Pearson correlation R2 values.

(B) Alpha cell-derived INS + cells (yellow cells) were isolated 1 month after AAV-PM infusion from ALX-treated GCG-Cre; R26R Tomato ; MIP-GFP mice, based on expression of tomato red (alpha cell lineage) and GFP by flow cytometry. Sorted normal GFP + beta cells and normal TOM + alpha cells from GCG-Cre; R26R Tomato ; MIP-GFP mice without any treatment were used as controls. Representative flow cytometry chart for sorting is shown.

In order to determine whether alpha cells in older mice retain the ability to transdifferentiate into beta cells in this model, we treated GCG-Cre; R26Rmice at 4 months of age with ALX injection and viral infusion. Our data suggest that alpha cells in older mice seemed to retain the ability to transdifferentiate into beta cells in response to activation of Pdx1 and MafA expression ( Figure S3 H).

In order to determine whether acinar cells may also be reprogrammed into INScells by AAV-PM, we performed the same viral treatment in ALX-treated, Elastase (Ela)-CreERT; R26Rmice, in which essentially all acinar cells were lineage tagged with tomato (). We did not detect significant numbers of tomatoINScells, suggesting that here acinar cells are not a major contributor to the newly reprogrammed INScells ( Figure S3 E).

In the GCG; R26Rmice, we found that ALX-induced hyperglycemia was again corrected within 2 weeks by intraductal infusion with AAV-PM, but not with control AAV-GFP ( Figure 3 B). The findings on IPGTT and beta cell mass were also reproduced in these mice ( Figures 3 C and 3D). Based on counting of 5,000 INScells in each mouse, with 5 mice in each experimental group, we found that 95.6% ± 2.3% of INScells after AAV-PM infusion in ALX-treated mice were labeled with tomato ( Figure 3 E; no tomato/INScells were found in the control AAV-GFP infused mice), suggesting an alpha cell origin. Moreover, when these mice were followed for 24 weeks, the mice remained euglycemic and the re-established beta cell mass appeared to be sustained ( Figure 3 D), and the beta cells remained as tomato tagged ( Figure 3 F). Interestingly, when AAV-PM was given to GCG; R26Rmice that did not receive ALX, the conversion rate of alpha cells to beta cells (percentage of tomatocells that were INS) was only 18.5% ± 2.5%, suggesting that the alpha-to-beta conversion may be greatly decreased under normal blood glucose ( Figure S3 D).

In order to exclude the possibility that pre-existing beta cells may transiently de-differentiate and activate the GCG promoter and thus become TOM labeled after ALX and AAV-PM, we generated a GCGknockin mouse to create an inducible Cre system (GCG; R26R) to lineage-tag native alpha cells only prior to ALX and AAV-PM treatment ( Figure 3 A). Quantification showed that the baseline labeling of alpha cells in GCG; R26Rmice was robust at 93.5% ± 6.5% after tamoxifen treatment, with complete absence of off-target labeling ( Figure S2 B), perhaps due to the knockin strategy. Rare alpha cells were pre-labeled without tamoxifen. Moreover, infusion of AAV-GFP into mice without ALX treatment did not induce tomato labeling of non-alpha cells ( Figure S2 C).

Statistics were analyzed by one-way ANOVA with a Bonferroni correction, followed by Fisher’s exact test. Data are presented as mean ± SD. ∗ p < 0.05. ∗∗ p < 0.01. NS, non-significant. n = 5. Scale bars, 50 μm.

(E and F) Immunostaining for INS after infusion of AAV-PM in ALX-treated GCG CreERT ; R26R Tomato mice, along with direct fluorescence for tomato (Cre-activity) and for green fluorescence (GFP, from viral infection) at 4 weeks (E) and 24 weeks (F) after virus infusion.

(B) One week after tamoxifen administration, hyperglycemia was induced in GCG CreERT ; R26R Tomato mice by ALX injection. One week after ALX treatment, mice received a pancreatic intraductal infusion of either AAV-PM (red line) or control AAV-GFP (green line). Fasting blood glucose levels were measured.

We quantified tomatocells in the INScell population, based on counting 5,000 INScells in each mouse, with 5 mice in each experimental group. In pancreases of GCG-Cre; R26Rmice with infusion of control AAV-GFP, no INScells were found to be labeled with tomato ( Figure 2 A). However, about 78.9% ± 5.6% of the INScells after AAV-PM infusion in ALX-treated mice were labeled with tomato ( Figures 1 D, 2 A, and 2B), suggesting an alpha cell origin. Very few INScells were not tagged with tomato red ( Figures 1 D and 2 A), presumably representing those cells that either derived from the few surviving beta cells after ALX treatment or else derived mainly from unlabeled reprogrammed alpha cells. We gave the mice continuous bromodeoxyuridine (BrdU) in the drinking water starting immediately after the viral infusion for 4 weeks. We found that 78.5% ± 6.6% of the tomatoINScells had incorporated BrdU ( Figure 2 C). Meanwhile, 13.4% ± 1.6% Ki-67INScells were detected ( Figure 2 D). Islets isolated from the ALX/AAV-PM-treated mice expressed higher levels of the cell-cycle activators cyclinD1 (CCND1) and CDK4 and lower levels of the cell-cycle suppressor p27 ( Figures 2 E–2G). Together, these data suggest that significant proliferation may occur in the neogenic INScells reprogrammed from alpha cells. Moreover, the islets from the ALX/AAV-PM-treated mice showed no difference in glucose-stimulated INS release, compared to normal islets (from untreated mice; Figure 2 H).

Statistics were analyzed by one-way ANOVA with a Bonferroni correction, followed by Fisher’s exact test. Data are presented as mean ± SD. NS, non-significant. n = 5. Scale bars, 20 μm.

(C and D) BrdU was continuously provided in the drinking water during the 4 weeks after viral infusion. Immunostaining for BrdU (C) or Ki-67 (D), INS and TOM in ALX-treated, AAV-PM-infused GCG-Cre; R26R Tomato mice.

(B) Immunostaining for GCG from a region nearby to (A) after infusion of control AAV-GFP (upper panels) or AAV-PM (lower panels) in ALX-treated GCG-Cre; R26R Tomato mice, along with direct fluorescence for tomato (TOM, from GCG-Cre activity) and for green fluorescence (GFP, from viral infection).

(A) Immunostaining for INS after infusion of control AAV-GFP (upper panels) or AAV-PM (lower panels) in ALX-treated GCG-Cre; R26R Tomato mice, along with direct fluorescence for tomato (TOM, from GCG-Cre activity) and for green fluorescence (GFP, from viral infection). Both AAV-GFP and AAV-PM viruses carry a GFP cassette.

To allow lineage tracing of alpha cells, we first generated GCG-Cre; R26Rreporter mice (), in which tomato red fluorescence specifically labels the GCGalpha cell lineage in the pancreas ( Figure 1 A). Quantification showed that the baseline labeling of alpha cells in GCG-Cre; R26Rmice was 71.5% ± 5.5%, with absence of detectable off-target labeling ( Figure S2 A). Thus, we then gave ALX to destroy the majority of beta cells in these GCG-Cre; R26Rmice. One week later, AAV-PM or control AAV-GFP was directly introduced into the mouse pancreas, using a recently developed pancreatic intraductal viral infusion technique, in which infusion of 150 μL of AAV serotype 8 efficiently transduces endocrine cells (). The intrapancreatic ductal delivery of virus did not result in appreciable expression of GFP in the liver. More importantly, no significant levels of INS gene expression were detected in the liver ( Figures S3 A and S3B). As a quality control, we stained for Pdx1 and MafA in untreated mice (no ALX, no virus), ALX/AAV-GFP-treated mice, and ALX/AAV-PM-treated mice to confirm the expression of these transgenes in the transduced pancreatic cells ( Figure S3 C). We found that ALX-induced hyperglycemia was corrected within 2 weeks by intraductal infusion of AAV-PM, but not with control AAV-GFP mice ( Figure 1 B). We also saw a significant improvement in the glucose response (intraperitoneal glucose tolerance test, IPGTT) in ALX-treated, AAV-PM-infused mice at 4 weeks after viral infusion ( Figure 1 C). Moreover, beta cell mass significantly increased in ALX-mice receiving AAV-PM (0.84 ± 0.06 mg), compared to mice receiving AAV-GFP (0.09 ± 0.01 mg) at 4 weeks after viral infusion, reaching more than 60% of the beta cell mass of untreated mice (UT, no ALX, no virus; 1.35 ± 0.11 mg) ( Figure 1 D). Alpha cell mass was quantified, showing decreases in ALXAAV-PM mice, compared to those in ALXAAV-GFP mice ( Figure S3 D). Transgene (Pdx1 and MafA) expression was then analyzed in purified tomatoalpha cells 2 days after viral infusion, confirming the expression of these transgenes in alpha cells ( Figure S3 E). GFP could be detected in ALXAAV-GFP mouse pancreas 5 weeks after viral infusion, suggesting sustained expression of the transgene (black bar, Figure S3 A). Thus, intraductal infusion of AAV-PM reversed ALX-induced diabetes in mice.

Statistics were analyzed by one-way ANOVA with a Bonferroni correction, followed by Fisher’s exact test. Data are presented as mean ± SD. ∗ p < 0.05. ∗∗ p < 0.01. n = 10. Scale bars, 50 μm.

(D) Beta cell mass at 4 weeks after virus infusion. The contribution of INS + cells without tomato red fluorescence is shown by the hatched bar contained within the red bar, compared to the beta cell mass in mice that received AAV-GFP viral infusion (green bar), and the beta cell mass of untreated mice (UT, no ALX, no virus, blue bar).

(C) IPGTT was performed in these mice 4 weeks after viral infusion. Untreated mice (no ALX, no virus, in blue) were used as an additional control.

(B) Hyperglycemia was induced in GCG-Cre; R26R Tomato mice by ALX injection. One week after ALX treatment, mice received a pancreatic intraductal infusion of either AAV-PM (red line) or control AAV-GFP (green line). Fasting blood glucose levels were measured.

Here, we examined the potential reprogramming of alpha cells into beta cells by inducing the expression of Pdx1 and MafA (PM) using AAV (AAV-PM) in mice. We first administered a single dose of either alloxan (ALX) or streptotozocin (STZ) to induce a sustained hyperglycemia in C57BL/6 mice ( Figure S1 A) () due to a significant decrease in beta cell mass ( Figures S1 B–S1E, ALX: decreased to 3.8% ± 0.2%, STZ: decreased to 6.5% ± 0.9%). Moreover, both ALX and STZ induced a modest but significant increase in alpha cell mass ( Figures S1 B–S1E), similar to that seen in some diabetic patients (). No significant increase in INScell proliferation was detected after ALX treatment ( Figures S1 F and S1G). We thus chose to use ALX in further studies to examine alpha-to-beta cell conversion in vivo, due to the higher and more consistent degree of beta cell ablation at the dosages used.

Discussion

Xiao et al., 2014b Xiao X.

Guo P.

Prasadan K.

Shiota C.

Peirish L.

Fischbach S.

Song Z.

Gaffar I.

Wiersch J.

El-Gohary Y.

et al. Pancreatic cell tracing, lineage tagging and targeted genetic manipulations in multiple cell types using pancreatic ductal infusion of adeno-associated viral vectors and/or cell-tagging dyes. Xiao et al., 2014b Xiao X.

Guo P.

Prasadan K.

Shiota C.

Peirish L.

Fischbach S.

Song Z.

Gaffar I.

Wiersch J.

El-Gohary Y.

et al. Pancreatic cell tracing, lineage tagging and targeted genetic manipulations in multiple cell types using pancreatic ductal infusion of adeno-associated viral vectors and/or cell-tagging dyes. Xiao et al., 2014b Xiao X.

Guo P.

Prasadan K.

Shiota C.

Peirish L.

Fischbach S.

Song Z.

Gaffar I.

Wiersch J.

El-Gohary Y.

et al. Pancreatic cell tracing, lineage tagging and targeted genetic manipulations in multiple cell types using pancreatic ductal infusion of adeno-associated viral vectors and/or cell-tagging dyes. Xiao et al., 2014b Xiao X.

Guo P.

Prasadan K.

Shiota C.

Peirish L.

Fischbach S.

Song Z.

Gaffar I.

Wiersch J.

El-Gohary Y.

et al. Pancreatic cell tracing, lineage tagging and targeted genetic manipulations in multiple cell types using pancreatic ductal infusion of adeno-associated viral vectors and/or cell-tagging dyes. Here, we showed that PM overexpression in vivo was able to correct hyperglycemia in both ALX-induced diabetes and in autoimmune diabetic NOD mice, suggesting that a true beta cell-like reprogramming was occurring, rather than simply activation of the INS promoter and suppression of the GCG promoter in alpha cells. Among all AAV serotypes, we found that 8 and 6 were the best for infecting mouse pancreatic cells (). We chose AAV serotype 8 vectors, since we found that serotype 8 had a better infection efficiency in mouse islet cells than serotype 6 (). Serotype 6 infects mouse pancreatic ducts cells, while serotype 8 does not, but duct cells were not the focus of the current study (). Both serotypes infect mouse acinar cells well ().

Tomato reporter mice and GCGCreERT; R26RTomato reporter mice to lineage-trace alpha cells. In GCG-Cre; R26RTomato reporter mice, although this transgenic GCG promoter that drives Cre is not strong ( Herrera, 2000 Herrera P.L. Adult insulin- and glucagon-producing cells differentiate from two independent cell lineages. Shiota et al., 2013 Shiota C.

Prasadan K.

Guo P.

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Xiao X.

Esni F.

Gittes G.K. α-Cells are dispensable in postnatal morphogenesis and maturation of mouse pancreatic islets. + cells were detected, and none of them were tagged with tomato red, suggesting that neither the hyperglycemia, nor the viral infection alone, without overexpression of Pdx1 and MafA in target cells, is sufficient to trigger alpha-to-beta cell conversion. Of note, these findings were confirmed in our newly created GCGCreERT; R26RTomato reporter knockin mice, in which nearly all INS+ cells were tagged with tomato in the ALX and AAV-PM-treated mice. Since leakiness may occur in some creERT mice, in which creERT can go into nuclei of the cells and cause recombination without induction by tamoxifen, we examined this issue in this new strain. We found that the pre-labeling of the alpha cells without tamoxifen in these mice is very rare (none of the 2,000 examined alpha cells) and thus should not affect the interpretation of the data in the current study. We used both GCG-Cre; R26Rreporter mice and GCG; R26Rreporter mice to lineage-trace alpha cells. In GCG-Cre; R26Rreporter mice, although this transgenic GCG promoter that drives Cre is not strong (), the highly sensitive tomato reporter allowed for more than 70% of the GCG-lineage cells to be successfully labeled. In the control diabetic AAV-GFP-infused mice, very few INScells were detected, and none of them were tagged with tomato red, suggesting that neither the hyperglycemia, nor the viral infection alone, without overexpression of Pdx1 and MafA in target cells, is sufficient to trigger alpha-to-beta cell conversion. Of note, these findings were confirmed in our newly created GCG; R26Rreporter knockin mice, in which nearly all INScells were tagged with tomato in the ALX and AAV-PM-treated mice. Since leakiness may occur in some creERT mice, in which creERT can go into nuclei of the cells and cause recombination without induction by tamoxifen, we examined this issue in this new strain. We found that the pre-labeling of the alpha cells without tamoxifen in these mice is very rare (none of the 2,000 examined alpha cells) and thus should not affect the interpretation of the data in the current study.

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et al. Phenotypic characterization of MIP-CreERT1Lphi mice with transgene-driven islet expression of human growth hormone. A human growth hormone (hGH) minigene has been shown to affect the interpretation of beta cell proliferation and survival studies (). However, this problem should not affect the current study, none of the constructs here contained this hGH minigene.

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Kanarek A.

Rajagopal J.

Melton D.A. In vivo reprogramming of adult pancreatic exocrine cells to beta-cells. In a previous report, Zhou et al. found conversion of acinar cells into INS-producing cells by overexpression of Pdx1, Ngn3, and MafA (). Here, we did not detect acinar-to-beta cell conversion with overexpression of Pdx1 and MafA. Thus, Ngn3 may be specifically necessary for acinar-to-endocrine conversion but may not to be required for reprogramming between different endocrine cell types, as in the conversion from alpha cells to beta-like cells here.

The normal blood glucose restoration and beta cell survival in NOD mice by PM appear to last 4 months prior to re-establishment of autoimmunity. It is impossible to predict how the immune protection in mice might translate to humans. It is conceivable that any immune protection might last a length of time equivalent to the 4 months as a percentage of the mouse lifespan. It is also possible, however, that little or no immune protection may occur in humans. Islet transplants into untreated diabetic normal mice are rapidly rejected within days to weeks, whereas the newly formed beta cells in our system survive for months. Thus, the window of time we report showing a reversal of hyperglycemia in NOD mice without direct immunomodulation or exogenous INS is a key difference between our approach and similar previously reported strategies. It is unlikely that the return of hyperglycemia in the normalized NOD mice is due to loss of transgene expression, since the few INS+ cells present, as well as the surrounding acinar cells were GFP+, and also because in ALX-treated GCG-CreERT; ROSAtomato mice at 24 weeks, the GFP+ neogenic beta cells persisted and the mice remained euglycemic. Instead, it appears that a return of autoimmunity caused the loss of beta cells, consistent with the presence of many inflammatory cells in the islet region. Thus, the window of protection from autoimmunity may stem from two possible mechanisms. The first possibility is that the ductal viral infusion directly altered the autoimmunity of the NOD mouse, leading to extended survival of the neogenic INS+ cells (viral-induced bystander effect). However, the fact that the control virus infusion had no protective effect argues against this possibility. The delayed, but eventually effective attack on beta cells in both the adoptive transfer experiments and the NOD/SCID islet transplants, suggests that the autoimmune NOD immune system is intact, but there is a delayed response to the newly formed beta cells. Moreover, we transplanted intact islets into the AAV-GFP-treated NOD mice, and the beta cells were completely ablated (no difference from untreated NOD mice), again consistent with no viral bystander effect.

+ cells are not recognized well by the autoimmune system. The more variable effectiveness of the AAV-PM treatment at late stages of hyperglycemia may reflect glucotoxicity or alpha cell injury. By RT-qPCR and RNA-seq, we found that expression levels of beta cell-specific genes in these new INS+ cells were similar, but not identical to true beta cells. Interestingly, a recent study showed that alpha cells could be converted to beta cells upon loss of both Arx and DNA-methyltransferase 1 (Dnmt1) ( Chakravarthy et al., 2017 Chakravarthy H.

Gu X.

Enge M.

Dai X.

Wang Y.

Damond N.

Downie C.

Liu K.

Wang J.

Xing Y.

et al. Converting adult pancreatic islet α cells into β cells by targeting both Dnmt1 and Arx. The second possible explanation is that the alpha cell-derived INScells are not recognized well by the autoimmune system. The more variable effectiveness of the AAV-PM treatment at late stages of hyperglycemia may reflect glucotoxicity or alpha cell injury. By RT-qPCR and RNA-seq, we found that expression levels of beta cell-specific genes in these new INScells were similar, but not identical to true beta cells. Interestingly, a recent study showed that alpha cells could be converted to beta cells upon loss of both Arx and DNA-methyltransferase 1 (Dnmt1) (). Here, we also found that DNMT1 was downregulated in the reprogrammed beta cells, suggesting that the two experimental paradigms may share some molecular signaling pathways.

Tomato; MIP-GFP rather than GCGCreERT; R26RTomato; MIP-GFP mice to characterize newly formed INS+ cells by RNA-seq since tamoxifen binding to the estrogen receptor in INS+ cells may result in alterations in gene expression ( Liu et al., 2009 Liu S.

Le May C.

Wong W.P.

Ward R.D.

Clegg D.J.

Marcelli M.

Korach K.S.

Mauvais-Jarvis F. Importance of extranuclear estrogen receptor-alpha and membrane G protein-coupled estrogen receptor in pancreatic islet survival. Ropero et al., 2002 Ropero A.B.

Soria B.

Nadal A. A nonclassical estrogen membrane receptor triggers rapid differential actions in the endocrine pancreas. Tiano et al., 2011 Tiano J.P.

Delghingaro-Augusto V.

Le May C.

Liu S.

Kaw M.K.

Khuder S.S.

Latour M.G.

Bhatt S.A.

Korach K.S.

Najjar S.M.

et al. Estrogen receptor activation reduces lipid synthesis in pancreatic islets and prevents β cell failure in rodent models of type 2 diabetes. + cells either lack key antigen(s) to induce an autoimmune attack, or because of their localization to the islet microenvironment, they may somehow avoid a rapid-recurrent autoimmune attack. If the islet niche is critical to the survival of newly introduced beta cells in autoimmune diabetes, it would have important ramifications for any future clinical strategy to deliver exogenous beta cells to T1D patients. We used GCG-Cre; R26R; MIP-GFP rather than GCG; R26R; MIP-GFP mice to characterize newly formed INScells by RNA-seq since tamoxifen binding to the estrogen receptor in INScells may result in alterations in gene expression (). Thus, it may be that these new INScells either lack key antigen(s) to induce an autoimmune attack, or because of their localization to the islet microenvironment, they may somehow avoid a rapid-recurrent autoimmune attack. If the islet niche is critical to the survival of newly introduced beta cells in autoimmune diabetes, it would have important ramifications for any future clinical strategy to deliver exogenous beta cells to T1D patients.

It is also important to note here that for human therapy we would strive to use a GCG promoter to drive the PM construct to avoid the risks of prolonged transgene expression, since, once the alpha cells had converted to beta cells, they would presumably transition to express Pdx1 and MafA from the endogenous locus, not the viral transgene since the GCG promoter should become inactive in beta cells. However, additional work is needed to optimize the generation of an effective, highly specific GCG promoter construct of reduced size to fit into the AAV vector. Moreover, augmentation of the strength of this GCG promoter may be required to allow sufficient expression of the transgene. In addition, the delivery of gene therapeutic virus through the pancreatic duct is potentially easily translatable to humans, since such pancreatic injections are routinely performed in humans through a non-surgical endoscopic procedure known as endoscopic retrograde cholangiopancreatography (ERCP).