(A) Schematic representation of excisable lentiviral vector used to produce the EndoC-βH2 reversely immortalized cell line. (B) Immunofluorescence analysis of EndoC-βH2 cells. EndoC-βH2 cells stained positive for insulin (INS) and coexpressed C-peptide (C-PEPT), CHGA, PDX1, NKX6.1, SV40 LT, and Ki67. The cells rarely express somatostatin (SST) and do not express glucagon (GCG), amylase (AMY), carboxipeptidase-A (CPA), or SOX9. Nuclei were stained with Hoechst 33342 fluorescent stain (blue). Photographs of all insulin stainings were taken using a 700 ms acquisition time to obtain an unsaturated signal. Scale bars: 50 μm.

EndoC-βH2 cells were transduced with either Cre- or GFP-expressing lentiviral vectors and analyzed 21 days later. (A) SV40 LT and hTERT expression were analyzed by RT-QPCR. Results are presented normalized to cyclophilin and relative to control nontransduced EndoC-βH2 cells. Results are shown as mean ± SD of 3 independent RNA preparations. The experiment was replicated 2 times. (B) Immunofluorescence analysis of SV40 LT (green) and insulin (red) in nonexcised cells (–CRE) and excised EndoC-βH2 cells (+CRE). Nuclei were stained with Hoechst 33342 fluorescent stain (blue). The settings of confocal images were used to obtain an unsaturated insulin signal for excised EndoC-βH2 cells. The same settings were used to generate the insulin image for nonexcised EndoC-βH2 cells. Scale bars: 50 μm. (C) Ki67 expression was analyzed by RT-QPCR. Results are presented normalized to cyclophilin and relative to control nontransduced EndoC-βH2 cells. Results are shown as mean ± SD of 3 independent RNA preparations. (D). BrdU incorporation by nonexcised and excised EndoC-βH2 cells following a 1-hour BrdU pulse. Results are presented as a mean percentage ± SD.

Heat map comparisons of the expression of a set of cell-cycle–related genes in EndoC-βH2 nonexcised cells and excised cells versus human islets. The heat map shows the log intensities in 3 sample sets of EndoC-βH2 nonexcised or excised cells and human islets (). For genes represented by several probe sets, the probe set with the highest fold change was used. Gene list is ordered from low to high expression relative to human islet sample. Gene lists have been generated using published transcriptomic data (), literature data, and manual curation.

(A) Expression of genes relevant for β cell function in heat map visualization. The heat map shows the log intensities in 3 sample sets of EndoC-βH2 nonexcised or excised cells, human islets (), and pancreas exocrine cell line SKPC. For genes represented by several probe sets, the probe set with the highest fold change was used. Most genes are highly expressed in the human β cell line EndoC-βH2 and in islet samples, but not in exocrine cell line SKPC. (B) Expression of exocrine marker genes in heat map visualization. The heat map shows the log intensities in 3 sample sets of EndoC-βH2 nonexcised or excised cells, human islets (), and pancreas exocrine cell line SKPC. Gene lists have been generated using published transcriptomics data (), literature data, and manual curation. For genes represented by several probe sets, the probe set with the highest fold change was used. Most genes are not expressed in human β cell line EndoC-βH2 samples (excised or not), but in islet samples.

EndoC-βH2 cells were transduced with either Cre- or GFP-expressing lentiviral vectors and analyzed 21 days later. (A) Insulin expression was analyzed by RT-QPCR and compared with its expression in EndoC-βH1 and SKPC cells. Results are presented normalized to cyclophilin and relative to control nontransduced EndoC-βH2 cells. Results are mean ± SD of 3 independent RNA preparations. (B) Immunofluorescence analysis of insulin (red) in nonexcised cells (–CRE) and excised EndoC-βH2 cells (+CRE). Nuclei were stained with Hoechst 33342 fluorescent stain (blue). Scale bars: 100 μm. (C) Immunofluorescence analysis of insulin (red) and Ki67 (green) in nonexcised cells (–CRE) and excised EndoC-βH2 cells (+CRE). Nuclei were stained with Hoechst 33342 fluorescent stain (blue). Scale bars: 50 μm (left and middle panel); 25 μm (right panel). In B and C, specific settings were applied in order to obtain confocal images with un-saturated insulin signal for excised EndoC-βH2 cells. The same settings were used to generate the insulin images for nonexcised EndoC-βH2 cells. (D) Insulin content in nonexcised and excised EndoC-βH2 cells. Results are shown as mean ± SEM of 3 independent cultures. The experiment was replicated 2 times. (E) IAPP, SLC2A2, ABCC8, KCNJ11, and RAB3A expression was analyzed by RT-QPCR. Results are presented normalized to cyclophilin and relative to control nontransduced EndoC-βH2 cells. Results are shown as mean ± SD of 3 independent RNA preparations.

RT-QPCR was performed to compare the level of expression of a set of β cell transcription factors (MAFA, GLIS3, RFX6, NKX2-2, NKX6-1, PDX1, MYT1, MNX1, and PAX6) among EndoC-βH2 nonexcised control cells, Cre-transduced excised EndoC-βH2 cells, GFP-transduced EndoC-βH2 cells, and islets. QPCR was normalized relative to TBP expression, and the relative expression of each transcription factor was arbitrary set to 1 for the control nonexcised EndoC-βH2 cells. Three independent RNA extractions were performed for each culture condition (control, Cre, and GFP) and 1 for the human islet preparation. QPCR was performed in quadruplicate. Results are shown as mean ± SEM. *P < 0.0002, unpaired 2-tailed Student’s t test with Welch correction.

EndoC-βH2 cells were transduced with Cre-expressing lentiviral vectors and analyzed 21 days later. Control nonexcised and excised EndoC-βH2 cells were tested for static insulin secretion under various glucose concentrations in the presence or absence of IBMX. In A, results are expressed as ng of secreted insulin per hour. In B and C, insulin secretion is presented as percentage of insulin content. Results are presented as mean ± SEM of 3 independent wells per condition assayed by ELISA. The experiment was replicated 2 times.