SDF-1 stimulates proliferation of epithelial cells in ICCs from early gestational human fetal pancreas.

<p>ICCs isolated from fetal pancreas of 12 to 14 weeks were grown in suspension culture, treated with 10% human serum alone, with HGF (10 ng/ml) or with SDF-1α (100 ng/ml) for six days. ICCs were fixed and analyzed by immunofluorescence staining. PanCytokeratin (green) is an epithelial marker. Ki67 (red) is used as a marker of proliferation.</p

Expression of insulin and glucagon in grafts from control and AMD3100 treated ICCs transplanted into nu/nu mice.

<p>Double immunofluorescent staining of insulin (green) and glucagon (red) in ICCs transplanted under the kidney capsule of nu/nu athymic mice treated with saline (A, B) or AMD3100 every two days during two weeks following transplantation. Insulin and glucagon expression is extensive in the control ICCs. C. Insulin or glucagon was not detectable in the grafts from the mice treated with AMD3100. Note the presence of the round beads that were inserted to identify the transplant site.</p

Serum Human C-peptide levels in nu/nu mice transplanted with human fetal ICCs treated with AMD3100.

<p>Sixteen weeks after transplantation, circulating human C-peptide levels were measured 30 minutes after a glucose challenge in fasted nu/nu mice that had been treated with saline or AMD3100. For both the saline and AMD3100 groups n = 5. For the control group, C-peptide levels were 517.3±198.83 (mean±SEM). C-peptide levels in the AMD3100 treatment group were undetectable. P<0.05 by Student’s t-test.</p

HGF or SDF-1 treatment of human ICCs <i>in vitro</i> does not alter insulin content.

<p>Insulin content expressed as picomoles/µg DNA of fetal ICCs after 5-day culture in medium containing 10% human serum or serum plus HGF or SDF-1α was assessed. Data represents the average of five separate sets of experiments. Values are expressed as mean ± SEM and were not significant by Student’s t-test.</p

SDF-1α stimulates Akt but not MAPK phosphorylation in CFPAC-1 cells and fetal ICCs.

<p>Following 48 hr serum starvation, CFPAC-1 cells were stimulated with 100 ng/ml or 300 ng/ml human recombinant SDF-1α for 10 min at 37°C. Whole cell lystes were analyzed by western blot, using antibodies raised against dually phosphorylated phospho-MAPK(ERK1/ERK2)(A) or phospho-Akt (Ser473)(B). Following overnight serum starvation, ICCs from human fetal pancreas of 15 weeks gestation were stimulated with 100 ng/ml or 300 ng/ml SDF-1α for 10 min at 37°C. Whole cell lystes were analyzed by western blot, using an antibody raised against phospho-MAPK(C) or phospho-Akt (Ser473) (D). All blots were stripped and reblotted with antibodies to total Akt, Erk, and Hsp90 sequentially to confirm equal loading.</p

The effect of the inhibitors of PI 3-kinase, MAPK, PLC, and PKA on SDF-1α stimulated proliferation in CFPAC-1 cells.

<p>CFPAC-1 cells were seeded in 12 well plates and grown to 50–70% confluence. Following 24 hr serum starvation, cells were pre-treated with LY294002 (30 µM), U0126 (30 µM), Edelfosine (10 µM), or H89 (10 µM) for 30 minutes and stimulated with SDF-1α for 16 hrs. BrdU was added 4 hrs before fixation. BrdU incorporation was determined as described under <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0038721#s4" target="_blank">Materials and Methods</a>. (*, P<0.05 SDF-1 versus basal); (**, p<0.02 SDF-1/Edelfosine versus SDF-1 alone); (***, p<0.0001 SDF-1/LY294002 and SDF-1/U0216 versus SDF-1 alone) by Student’s t-test. N.S.  =  not significant by Student’s t-test.</p

SDF-1 stimulates proliferation in CFPAC-1 cells.

<p>CFPAC-1 cells were seeded in 12 well plates and grown to 50–70% confluence. Following 24 hr serum starvation, cells were stimulated with SDF-1α (100 ng/ml or 300 ng/ml) for 16 hrs. BrdU was added 4 hrs before fixation. BrdU incorporation was determined as described under <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0038721#s4" target="_blank">Materials and Methods</a>. BrdU incorporation was stimulated 26.6% and 71.6% by 100 ng/ml and 300 ng/ml SDF-1α, respectively. Each bar represents the average of three experiments (mean ± SEM); P<0.03 by analysis of variance.</p

Comparison of mRNA levels for endocrine markers in control and AMD3100 treated ICCs derived from human fetal pancreas by RT-PCR.

<p>The values of mRNA for endocrine markers are expressed as mean ± SEM of ΔCTs of the transcription factors compared to Cyclophilin A. Ngn3 (Panel A), PAX-4 (Panel B) and MAF-A (data not shown) were detected at approximately 35 to 37 PCR cycles, PDX-1 (Panel C) and Nkx6.1 (Panel D) were detected at 30 to 31 PCR cycles both in control and AMD3100 treated ICCs. Four different preps from gestational ages 13 to 17 weeks were analyzed. N.S.  =  not significant by Student’s t-test.</p

The cytokine cocktail TNFα, IL1β and IFNγ and SDF-1α (300 ng/ml) stimulate CFPAC-1 apoptosis.

<p>CFPAC-1 cells were seeded in 12 well plates and grown to 50–70% confluence. Following 24 hr serum starvation, cells were stimulated with either the cytokine cocktail (TNF) consisting of IL-1β (2 ng/ml), IFN-γ (100 ng/ml) and TNF-α (100 ng/ml), or SDF-1α at 100 ng/ml, 300 ng/ml, or the TNF cocktail in combination with SDF-1α 100/ng/ml for 24 hrs. The cells were fixed at the end of the incubation apoptosis was quantitated using the TUNEL method as described in the <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0038721#s4" target="_blank">Materials and Methods</a>. The number of TUNEL positive nuclei was expressed as the percentage of the total number cells counted in the acquired images. Data is expressed as mean ± SEM. (*, p<0.03 versus basal); (**, p<0.01 versus basal) by Student’s t-test. N.S.  =  not significant by Student’s t-test.</p

CXCR4 is expressed in human adult islets and ducts.

<p>Panel A illustrates CXCR4 (green) expression in human adult islets; note the absence of CXCR4 staining in exocrine tissue surrounding the islet. Panel B reveals that in the human adult islet CXCR4 expressing cells coexpress insulin (yellow). Human adult ducts also exhibit CXCR4 (green) staining. C. RT-qPCR analysis of CXCR4 mRNA in human embryonic stem cells (hESCs), hESC derived definitive endoderm (DE), fetal islet cell clusters at 11, 12 and 18 weeks gestation (11 WK, 12 WK, 18 WK), and adult islets. Expression levels expressed as ΔCt values relative to Cyclophilin G. D. Due to extremely high expression in DE, CXCR4 mRNA levels shown comparing only hESCs, fetal ICCs, and adult islets.</p