Reprogramming human gallbladder cells into insulin-producing β-like cells

<div><p>The gallbladder and cystic duct (GBCs) are parts of the extrahepatic biliary tree and share a common developmental origin with the ventral pancreas. Here, we report on the very first genetic reprogramming of patient-derived human GBCs to β-like cells for potential autologous cell replacement therapy for type 1 diabetes. We developed a robust method for large-scale expansion of human GBCs <i>ex vivo</i>. GBCs were reprogrammed into insulin-producing pancreatic β-like cells by a combined adenoviral-mediated expression of hallmark pancreatic endocrine transcription factors <i>PDX1</i>, <i>MAFA</i>, <i>NEUROG3</i>, and <i>PAX6</i> and differentiation culture <i>in vitro</i>. The reprogrammed GBCs (rGBCs) strongly induced the production of insulin and pancreatic endocrine genes and these responded to glucose stimulation <i>in vitro</i>. rGBCs also expressed an islet-specific surface marker, which was used to enrich for the most highly reprogrammed cells. More importantly, global mRNA and microRNA expression profiles and protein immunostaining indicated that rGBCs adopted an overall β-like state and these rGBCs engrafted in immunodeficient mice. Furthermore, comparative global expression analyses identified putative regulators of human biliary to β cell fate conversion. In summary, we have developed, for the first time, a reliable and robust genetic reprogramming and culture expansion of primary human GBCs—derived from multiple unrelated donors—into pancreatic β-like cells <i>ex vivo</i>, thus showing that human gallbladder is a potentially rich source of reprogrammable cells for autologous cell therapy in diabetes.</p></div

Primary human gallbladder cells (GBCs) <i>in vitro</i> culture, expansion, and adenoviral transduction.

<p>(A) Two-week old culture of primary human GBCs (scale bar = 200 μm) in a tightly packed colony. Inset shows high nuclear to cytoplasmic ratio. (B) Cell growth of GBCs for the first 4 passages on irradiated LA7 feeder cells. (C) Comparing the gene expression of human primary GBCs with human pancreatic β cells showing differential expression of β cell-associated genes as determined by RNA-seq from duplicate samples. (D) Ad5 vectors encoding for <i>PAX6</i> (P6), <i>NEUROG3</i> (N), <i>PDX1</i> (P), and <i>MAFA</i> (M) driven by CMV promoter. The tricistronic vector Ad5-M6P also encoded for green fluorescent protein (GFP). Self-cleaving 2A connects transgenes in multicistronic cassettes. (E) Transduction efficiency of cultured human GBCs to GFP-expressing adenoviral vector (MOI = 500 vg/cell). (F) Insulin mRNA expression in reprogrammed GBCs (rGBCs) five days after transduction with RDAd5 vectors (MOI = 500 vg/cell).</p

Determination of microRNA expression in rGBC relative to human pancreatic β cells and primary GBC by Illumina sequencing.

<p>(A) Venn diagram of microRNAs differentially expressed in human pancreatic β cells (blue) or primary GBCs (brown) or no difference (yellow). (B) Heat map and dendogram of microRNAs in β cells, primary GBC, and rGBC. (C,D) Top twenty differentially expressed microRNAs enriched in β cells (C) and GBC (D). (E-G) Bland-Altman plots comparing the microRNA populations in β cells, primary GBC, and rGBC. Y-axes correspond to expression differences [log₂(rGBC–β, GBC–rGBC, GBC–β, respectively)] and the X-axes correspond to average value of each microRNA in each paired cell types. Selected microRNAs are annotated to illustrate differential expression among the three cell types. (H) Hierarchical cluster analysis of microRNAs in Hpi1 subpopulations relative to unsorted rGBC, primary GBC, and β cells. (I) Heat map distribution of the twenty most differentially expressed microRNAs enriched in β cells and downregulated or absent in primary GBC across clustered cell types.</p

The human pan-endocrine antibody Hpi1 enriched for β-like cells.

<p>(A) Flow cytometry dot plots showing frequencies of Hpi1+ cells in both GBCs and rGBCs. (B) Immunofluorescent images of C-peptide co-localizing with the antigen for Hpi1. (scale bar = 50 μm) (C) mRNA expression levels of selected β-associated genes in Hpi1-based FACS-sorted rGBCs showing enrichment for <i>NKX6-1</i>, <i>PCSK1</i>, <i>NEUROD1</i>, <i>and INS</i> in Hpi1+ rGBCs by RT-qPCR. (D) Heat map of differentially expressed genes in sorted rGBCs by RNA-sequencing. Venn diagrams showing overlapping and non-overlapping "Beta genes" (E) and "GB genes" (F) upregulated in Hpi1-/+ and unsorted rGBC as measured by RNA-seq. Differential expression of pancreatic endocrine genes (G), β cell-associated transcription factors (H), and genes involved in the regulation of insulin secretion (I).</p

Transplantation of rGBCs into NSG mouse.

<p>(A) H&E sections of engrafted rGBCs (Xeno) into the epididymal fat (n = 17) surrounded by mouse tissue (Mu) (scale bar = 200 μm). (B) Immunofluorescent staining of human EPCAM marking the location of rGBCs in the epididymal fat (scale bar = 200 μm). (C) Human mitochondria and C-peptide immunofluorescence of rGBC grafts at three time points after transplantation into the epididymal fat. (D) C-peptide immunofluorescence of rGBC clusters 2 weeks after transplant into mammary fat pad (left) (n = 17), upper back subcutaneous area (middle) (n = 12), and kidney capsule (right) (n = 17) (scale bar = 20 μm).</p

List of β cell-enriched and GBC-enriched microRNAs that were differentially represented in rGBC.

<p>List of β cell-enriched and GBC-enriched microRNAs that were differentially represented in rGBC.</p

Expression of islet-associated genes in transduced GBCs.

<p>(A) Insulin mRNA level expression in GBCs after adenoviral transduction with <i>NEUROG3</i> (NGN3), <i>MAFA</i> (M), <i>PAX6</i> (6), <i>PDX1</i> (P) as measured by RT-qPCR. The dashed line denotes insulin mRNA level in human pancreatic islets. (B) Expression levels of pancreatic genes on day 14 in rGBCs as measured by RT-qPCR. <i>MUC5B</i> served as a GBC marker. (C-D) M6PN-transduced GBCs were harvested on days 5, 9, 14 and 19 post-transduction. Gene expression levels of pancreatic endocrine factors were measured by RT-qPCR. Relative expression levels were calculated using the formula: [2^(-ΔCq rGBC)]/[2^(-ΔCq human islet)]. The dashed line marks the point where gene expression level is equivalent to human islets. (E) Heat map from clustering of differentially expressed genes in rGBCs in comparison to GBCs and human β cells as determined by RNA-sequencing. (F) Venn diagram depicting non-differential genes (light green) and differentially expressed genes upregulated in GBC ("GBC genes") and β cells ("Beta genes"). (G) Break-down of the frequencies of "GBC genes" and "Beta genes" into induced or uninduced genes in rGBC. (H) Pie chart distribution of the 9254 non-differentially expressed genes in both unreprogrammed GBC and β cells into 4 groups based on global gene expression profile in rGBC.</p

Upregulation of trophic factors.

<p>A. Semi-quantitative RT-PCR for CNTF, bFGF, BDNF and beta actin. Lane 1: RNA isolated from MSC prior to injection; Lane 2–4: RNA isolated from retinas treated with MSC; Lane 5–7: RNA isolated from non-treated control retinas. B. Densitometry analysis of CNTF, BDNF and bFGF in treated versus untreated samples. Beta actin was used to normalize the data for comparison. Level of CNTF and BDNF in the treated retinas were significantly higher than non-treated controls (p<0.05), while the level of bFGF in MSC treated retina did not increase significantly. C–J: confocal images of retinal sections double stained with antibodies to CNTF (green) and GFAP (red), counterstained with DAPI (blue in C and G) from MSC treated and controls. Strong CNTF staining in MSC treated retina (D) compared with untreated control (H); E&I: retinal sections stained with GFAP (red) showing upregulation of GFAP in Müller glia in both MSC treated and untreated control; F&J: merged images showing colocalization of CNTF and GFAP in MSC treated retina (F), which was not observed in untreated control (J) (Scale bar equals 50 µm).</p

Vascular protection.

<p>A–F: Retinal whole mount was stained with NADPH-diaphorase: A. typical vascular pathology in the eye at P90 in untreated RCS rat: vascular complexes (abnormal vessels associated with RPE cells) were mainly located around the optic nerve disc (arrows) and spread peripheral with age. B. vascular complexes in the middle to peripheral retina (arrows). C. high power image showing vascular complexes (arrows) from B. D. RCS retina treated with MSCs at P90: the vascular complexes were dramatically reduced around the optic nerve disc. E. two vascular complexes (arrows) in the middle field of the retina. F. high power image from E showing vascular complexes (arrow). G–L. animal was perfused with FITC-dextran, whole mount was prepared: G. typical vascular leakage, mainly around the optic disc in untreated eye at P90. H–K. high power images from G showing vascular leakage (arrows in H) and abnormal vessels (arrows in I–K). L. MSC treated retina, the vascular leakage around the optic nerve disc was greatly reduced. M&N. high power images from L showing much reduced leakage (arrows in M) and small abnormal vessels (arrow in N) (Scale bars equal 250 µm for A, D, G &L; 100 µm for F).</p

Distribution of MSCs.

<p>A. phase contrast microphotograph of bone marrow derived mesenchymal stem cells at passage 2. B. MSCs were preincubated with PKH26 before intravenous injection. C. PKH26 labeled MSCs in the retina two weeks after intravenous injection (arrows); blood vessels were perfused with FITC-dextran (green). D–F. showing PKH26 labeled MSCs in the retinal section (D, arrows pointing PKH26 labeled MSCs; double arrows indicating background staining in debris zone); sections counterstained with DAPI (E); F. merged image from D&E showing PKH26 labeled MSCs counterstained with DAPI (scale bar equals 100 µm).</p