274. Expansion and Conversion of Patient-Derived Human Gallbladder Cells Into Insulin-Producing Pancreatic Endocrine-Like Type Cells
Authors: Feorillo Galivo, Eric Benedetti, Yuhan Wang, Jonghyeob Lee, Jonathan Schug, Seung Kim, Klaus Kaestner, Markus Grompe
Journal: Molecular Therapy (2015)

Islet transplantation is an efficacious therapeutic option for T1D, but, supply of donor pancreata does not meet the demand. Variable successes in generating glucose-responsive insulin-producing beta-like cells from pluripotent and differentiated cells have been reported. We report on genetic reprogramming of highly expandable patient-derived human gallbladder cells (GBCs) to produce insulin for autologous transplant. Using feeder-free and feeder-type culture methods, we expanded GBCs in vitro in the order of 10^8 total cells after 4 passages starting from 10^4 GBCs. Cultures can be maintained for at least 15 passages, albeit with slowdown in growth. Transcriptome profiling revealed GBCs to be deficient in factors important for beta-cell development, specification, and maintenance such as NEUROG3, MAFA, NKX6-1, NKX2-2, PAX4, PAX6, NEUROD1, INSM1, RFX6, GCK, and INS. MNX1 and PDX1 are moderately expressed in cultures but at significantly lower levels found in beta-cells. Viral transduction of PDX1, MAFA, and NEUROG3 (PNM) induced expression of INS at 1/10,000th compared to that of human islet cells. Transduced GBCs began to show morphological changes from a monolayer into 3D clusters as early as 2 days after transduction. Retinoic acid, GLP-1, FGF10, DAPT, T3, Alk5 inhibitor, and B27 in the reprogramming medium and the addition of PAX6 to PNM transduction highly improved both INS and NKX6-1 induction reaching 10% of islet cells. These reprogrammed GBCs also express high levels of NEUROD1, NKX2-2, RFX6, PAX4, MAFB, HOPX, SST, GHRL, CHGA, TMEM27, SYP, KCNJ11, and ABCC8 comparable to islet cells. Average transduction efficiency was 50% yielding 9% C-peptide+ cells by immunostaining. Cellular C-peptide content was equivalent to 0.3% of that of purified beta-cells. At best, we observed 5-fold increase in C-peptide concentration in response to high glucose. We further enriched for INS+ reprogrammed GBCs using a cell surface mAb (HPi1) shown to isolate human pancreatic islet cells. Immunolabeling showed that reprogramming significantly increased the percentage of Hpi1+ GBCs. RT-qPCR of Hpi1+ GBCs showed greater than 1000-fold enrichment in gene expression of INS, NKX6-1, and PCSK1 compared to Hpi1- cells. Likewise, we observed greater than 100-fold enrichment for NEUROD1, SST, GHRL, and RFX6. In addition, we saw significant increase in reprogramming efficiency by adding an isoxazole to the reprogramming medium resulting to 10-fold higher INS expression, 2.5-fold increase in the frequency of C-peptide+ cells, and significant drop in SST and GHRL expression. Reprogrammed GBCs can engraft, survive, and produce C-peptide for at least 2 weeks in under the kidney capsule, mammary fat pad, and dorsal subcutaneous fat in NSG mice. Transplant experiments in diabetic mice are ongoing. In summary, we have transdifferentiated patient-derived adult GBCs into beta-like cells ex vivo, thus showing that GBCs are potential replacement sources of autologous insulin-producing cells.