Lentivirus-mediated gene transfer in human fetal pancreas.

<p>Human fetal pancreases were partially dissociated, transduced with lentiviruses expressing GFP under the control of the CMV promoter, grafted into scid mice and analyzed 10 days later. A double staining for Pdx1 (red) and GFP (green); B represent enlargements of A. Dotted arrows: infected fibroblastic-like cells. Arrows: Pdx1-positive cells. Arrow head: Pdx1–negative cells. C double staining for insulin (red) and GFP. Scale bars: A, C: 25 µm, B: 10 µm.</p

Human beta cells within a single islet are derived from more than one progenitor.

<p>Human fetal pancreases were dissociated, transduced with lentiviruses expressing GFP under the control of the insulin promoter, grafted and analyzed 4.5 months later. A: double staining for insulin (red) and GFP (green) showing that GFP is only found in islets; B: double staining for glucagon (red) and GFP (green) showing that GFP is not found in alpha cells; C: double staining for insulin (red) and GFP (green) showing that GFP is only found in a subpopulation of beta cells. D is an enlargement of panel C (dotted square). Scale bars: A: 50 µm; B–D: 25 µm.</p

Cell type specificity of the rat insulin II promoter in human adult pancreas.

<p>Crude human islet preparations were transduced with lentiviruses expressing eGFP under the control of the rat insulin II promoter and analyzed 72 hours after infection. Cultures were photographed under a fluorescent inverted microscope (panels A and B), fixed and sectioned. C–D: staining for GFP (green) and insulin (red). E staining for GFP (green) and amylase (red). F: staining for GFP (green) and CK19 (red). Scale bars: 20 µm.</p

Cytoarchitecture of newly formed islets from human fetal pancreas.

<p>A–C: Section of adult mouse pancreas stained for glucagon (green) and insulin (red). D–F: Section of an adult human pancreas stained for glucagon (green) and insulin (red). G–I: Section of a human fetal pancreas analyzed 4.5 months after transplantation and stained for glucagon (green) and insulin (red). Nuclear staining (blue) was performed with DAPI. Scale bars: 25 µm.</p

Both human pancreatic progenitors and beta cells proliferate.

<p>Human embryonic pancreases were transplanted into <i>scid</i> mice. Ten days and 4.5 months later, the grafts were removed and analyzed. A–D: Representative section of a fetal pancreas analyzed 10 days after transplantation and double stained for Pdx1 (green) and Ki67 (red). E–H: Representative section of a fetal pancreas analyzed 4.5 months after transplantation and double stained for insulin (red) and Ki67 (green). Scale bars: A–C and E–G: 25 µm; D and H: 10 µm.</p

Expression of mesenchymal genes in eGFP⁺ cells.

<p>Cells at passage 4 were stained with antibodies to eGFP and the indicated mesenchymal marker. Nuclei were stained blue with DAPI. Bar = 20 µm. The percentages indicate the fraction of cells positive for each mesenchymal marker among eGFP⁺ cells (green digits) and among all the cells (white digits). Data are mean±SD of >400 cells scored from each donor (n = 3 donors).</p

Differentiation of cells expanded from human islet cells into adipocytes and osteocytes.

<p>A, Islet cells at the indicated passage number were incubated in Lonza induction medium and stained with Oil Red O for adipocytes and Alizarin Red for osteocytes. Human BM-MSC served as positive control. Bar = 100 µm. B, Islet cells at passage 5 and BM-MSC were incubated in Lonza adipogenesis induction medium and stained with antibodies to eGFP and FABP4. Nuclei were stained blue with DAPI. Bar = 30 µm. The eGFP⁺ cells shown do not stain for FABP4. The single FABP4⁺ cell shown is not eGFP⁺. C, Quantitation of the staining in B, based on counting >500 cells in cultures derived from each donor. Data represent percent of FABP4⁺ among eGFP⁺ cells (green bars) and among all the cells (black bars) and are mean±SD (n = 6 donors for islet cells and 2 donors for BM-MSC). p = 4.18E-14.</p

Changes in expression of epithelial and mesenchymal genes in human islet cells during the first 3 weeks in culture.

<p>RNA was extracted from cells at the indicated passage number (each passage is equivalent to one week) and analyzed by qRT-PCR. A, analysis of epithelial genes. B, analysis of mesenchymal genes. Data represent relative quantification (RQ) (compared to passage 0) and are mean±SE (n = 6 donors). *p<0.05; ** p<0.005; ***p<0.0005 (compared to passage 0).</p

Changes in expression of epithelial and mesenchymal genes in sorted eGFP⁺ and eGFP⁻ cells following 2 weeks in culture.

<p>RNA was extracted from cells at the indicated passage number and analyzed by qRT-PCR. A, analysis of epithelial genes. B, analysis of mesenchymal genes. Data represent relative quantification (RQ) (compared to passage 0) and are mean±SE (n = 3 donors). *p<0.05; ** p<0.005.</p

Inducible labeling of human islet cells.

<p>A, Schematic representation of the two lentivirus vectors. B, Labeling efficiency and leakiness. Adult human islet cells from 5 donors were infected one day after plating with the reporter virus alone, or with both viruses, and cultured overnight in the absence or presence of tamoxifen. Five days later 10⁴ cells from each donor were analyzed by flow cytometry for eGFP expression. Data are mean±SD (n = 5).</p