Introduction

Pluripotent stem cells can provide an unlimited source of human islet-like clusters by directed differentiation. Generating pure high-quality pancreatic progenitor (PP, stage-4) cells and preventing off-target, non pancreatic derivatives in vivo remains critical for successful implementation of these therapies in the clinic. Despite advancements in the field, current differentiation protocols fail to produce pure PP cells. Herein, we show that the AKT inhibitor AT7867 has a significant effect on promoting PP cell differentiation from pluripotent stem cells.

Methods

Using a validated differentiation protocol, we differentiated human induced pluripotent stem cells (iPSCs) into PPs. We proceeded with stage-4 (PP) differentiation in the presence or absence of AT7867 to elucidate the role of AT7867-mediated AKT inhibition in PP cell differentiation. Proliferation (Ki67) and cell cycle markers were evaluated in control and AT7867-treated PPs. At the end of stage-4, control and AT7867-treated PPs were characterized and compared at protein (flow cytometry, immunohistochemistry and western blot) and transcriptional (RT-PCR) level. Control and AT7867-treated PPs were transplanted under the kidney capsule of streptozotocin-treated diabetic immunocompromised (SCID) mice.

Results

AT7867 treatment significantly increased the percentage of PDX1 expressing PP cells compared to control untreated cells (53.99% ± 3.86 vs 97.52% ± 1.35, p<0.0001). Flow cytometry analysis showed a higher frequency of Pdx1⁺Nkx6.1⁺ and Pdx1⁺GP2⁺ cells in PP cells treated with AT7867 compared to untreated controls (50.34% ± 7.18 vs 86.08% ± 7.73, p=0.087; and 15.82 ± 3.44 vs 84.81 ± 1.54, p<0.0001, respectively). AT7867 treated cells generated a similar cell mass as control untreated cells and there was no significant difference in the number of Ki67⁺ cells or frequency of cells at G1, S, and G2M phases of the cell cycle between control and treated PPs. AT7867 treatment upregulated Pdx1 (p=0.0001), Nkx6.1 (p=0.0005) and GP2 (p=0.002) transcript levels compared to untreated controls, while SOX17 (p<0.0001) and TBX2 (p<0.0001) were downregulated. Notably, transplantation of AT7867-treated PPs resulted in faster reversal of hyperglycemia (within 7 weeks) in diabetic mice compared to the untreated control group (11 weeks) (p<0.0001). Treatment with AT7867 resulted in the secretion of 3 ng/ml of human c-peptide, measured 60 minutes after intraperitoneal administration of glucose, 8 weeks post-transplantation; no c-peptide secretion was detected in the control group at this timepoint(p<0.0001).  

Conclusion

AT7867 improved differentiation efficiency of PPs at a protein expression and transcript level. Similarities in cell proliferation markers supports a role in differentiation, more than cell expansion.  Importantly, AT7867-treated cells enabled faster diabetes reversal upon transplantation as compared to controls, as well as improved glucose-stimulated insulin secretion.