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

Immunohistochemical analysis of grafts developed in Scid mice.

<p>A–C: Staining for insulin (red), SV40T (green) and DAPI (blue); D–F: Staining for insulin (red), Pdx1 (green) and DAPI (blue); G–I: Staining for insulin (red), BrdU (green) and DAPI (blue). Scale bars: 25 µm</p

Transplanted RYAS41 cells restore normoglycemia in diabetic mice.

<p>Fourteen <i>scid</i> mice were injected with streptozotocin. Two days later, 3-weeks lasting insulin capsules were subcutaneously implanted to hyperglycemic mice. Two weeks later, half of the mice were transplanted under the kidney capsule with 10⁶ RYAS41 cells. Grafted cells were removed by nephrectomy at day 66. Values are means+/−S.E.M.</p

Immunocytochemical Characterization of RYAS41 cells.

<p>A: Schematic representation of the culture procedure used to derive the RYAS41 cell line. P represents passage number. Surface of the culture well is indicated below the time line. B: Coexpression of insulin (red) and SV40T (green); insulin (red) and c-peptide (green), insulin (red) and Pdx1 (green); and double staining for insulin (red) and BrdU (green). Scale bars: 25 µm.</p

Gene expression profile in RYAS41 and glucose-stimulated insulin secretion.

<p>A: Semi-quantitative RT PCR comparison between RYAS41, lung (negative control) and pancreas (positive control) from E17 rat embryos. PCR products after 40 amplification cycles are analyzed on a 2% agarose gel. B: CT (threshold cycle) value are normalized to cyclophilin and presented as fold increase compared to 832/13 INS-1 cells. Values are means+/−S.E.M. of Q-PCR performed in duplicates from 3 independent RNA extractions. C: RYAS41 secrete insulin in response to glucose stimulation. Insulin secreted into the medium is presented as % of insulin content secreted per hour. Values are means+/−S.E.M. of three independent cell cultures.</p

Scheme of phiC31-int mediated recombination in bacterial host.

<p>PhiC31 integrase performs precise recombination between an <i>attB</i> site located in the <i>Streptomyces</i> genome and an <i>attP</i> site located on the phiC31 phage genome. The outcome is integration of the phage into the host genome.</p