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Fig. 3 | Stem Cell Research & Therapy

Fig. 3

From: Improvement of the therapeutic capacity of insulin-producing cells trans-differentiated from human liver cells using engineered cell sheet

Fig. 3

Ectopic co-expression of transcription factors in the liver in vitro promotes endocrine differentiation. a GFP expression in liver cells (passage 6) after Ad-CMV-GFP transduction for 2 days with various MOIs analyzed by flow cytometry and fluorescence imaging (n = 3). Scale bars represent 400 μm. b Multiple immunofluorescence staining of human liver cells. Human liver cells (passage 6) were simultaneously treated with Ad-CMV-PDX1 (500 MOI) and Ad-CMV-NEUROD1 (250 MOI) for 2 days and then re-plated with Ad-CMV-hPDX1 (MOI 50) for 3 days. Green, PDX1 or MAFA; red, NEUROD1; blue, nucleus. Scale bars represent 200 μm. Flow cytometry data of PDX1, NEUROD1, and MAFA transduced liver cells with various MOIs (lower panel). c Cultures were co-infected with the combined transcription factors and with adenovirus carrying a luciferase reporter gene under control of the insulin promoter (Ad-RIP-luciferase: 200 MOI), and luciferase activity was measured. The results are expressed as relative light units (RLU)/mg protein (n = 3). d Real-time qPCR analysis of insulin gene expression in liver cells, insulin-producing cells (IPCs) treated with various combinations of transcription factors and human pancreatic islets (n = 3). The results normalized to the GAPDH gene expression for the same cDNA sample are represented as the relative levels of the mean ± SD; P < 0.05 indicates a significant difference. e Production of insulin by IPCs. Each data point represents the mean ± SD in cells isolated from four different donors compared to that in control virus-treated cells. P < 0.05 compared to liver. f Immunocytochemistry of insulin and glucagon in the liver cell, IPCs, and IPC sheet. Representative insulin and glucagon staining using immunocytochemistry (left). Red, insulin or glucagon; blue, nuclei. Scale bars represent 200 μm. Percentage of insulin- or glucagon-positive cells in IPCs and IPC sheet (right). g TEM images of IPCs. The red arrow indicates an insulin immunogold aggregate (9–11 nm), and the white arrow indicates glucagon immunogold aggregate dot (5 nm)

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