Novel surface markers directed against adult human gallbladder

Novel cell surface-reactive monoclonal antibodies generated against extrahepatic biliary cells were developed for the isolation and characterization of different cell subsets from normal adult human gallbladder. Eleven antigenically distinct gallbladder subpopulations were isolated by fluorescence-activated cell sorting. They were classified into epithelial, mesenchymal, and pancreatobiliary (PDX1+SOX9+) subsets based on gene expression profiling. These antigenically distinct human gallbladder cell subsets could potentially also reflect different functional properties in regards to bile physiology, cell renewal and plasticity. Three of the novel monoclonal antibodies differentially labeled archival sections of primary carcinoma of human gallbladder relative to normal tissue. The novel monoclonal antibodies described herein enable the identification and characterization of antigenically diverse cell subsets within adult human gallbladder and are putative tumor biomarkers

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

Interference of CD40L-Mediated Tumor Immunotherapy by Oncolytic Vesicular Stomatitis Virus

Oncolytic virotherapy can be achieved in two ways: (1) by exploiting an innate ability of certain viruses to selectively replicate in tumor tissues, and (2) by using viruses to deliver toxic or immunostimulatory genes to tumors. Vesicular stomatitis virus (VSV) selectively replicates in tumors lacking adequate type I interferon response. The efficacy of oncolytic virotherapy using VSV against B16 melanomas in C57BL/6 mice is dependent on CD8 + T and natural killer cells. Because immunotherapies that prime specific CD8 + T cells against melanocyte/melanoma antigens can generate significant therapeutic responses, we hypothesized that engineering VSV to express the potent T cell costimulatory molecule CD40 ligand (VSV-CD40L) would enhance virotherapy with concomitant priming of melanoma-specific T cells. However, we observed no difference in antitumor efficacy between the parental VSV-GFP and VSV-CD40L. In contrast, intratumoral injection of a replication-defective adenovirus expressing CD40L (Ad-CD40L) consistently produced significantly greater therapy than either replication-competent VSV-GFP or VSV-CD40L. The Ad-CD40L-mediated tumor regressions were associated with specific T cell responses against tumor-associated antigens (TAAs), which took several days to develop, whereas VSV-CD40L rapidly induced high levels of T cell activation without specificity for TAAs. These data suggest that the high levels of VSV-associated immunogenicity distracted immune responses away from priming of tumor-specific T cells, even in the presence of potent costimulatory signals. In contrast, a replication-defective Ad-CD40L allowed significant priming of T cells directed against TAAs. These observations suggest that an efficiently primed antitumor T cell response can produce similar, if not better, therapy against an established melanoma compared with intratumoral injection of a replication-competent oncolytic virus

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

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

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

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

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

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