EGFR Signaling Promotes beta-Cell Proliferation and Survivin Expression during Pregnancy

Placental lactogen (PL) induced serotonergic signaling is essential for gestational beta-cell mass expansion. We have previously shown that intact Epidermal growth factor -receptor (EGFR) function is a crucial component of this pathway. We now explored more specifically the link between EGFR and pregnancy-induced beta-cell mass compensation. Islets were isolated from wild-type and beta-cell-specific EGFR-dominant negative mice (E1-DN), stimulated with PL and analyzed for beta-cell proliferation and expression of genes involved in gestational beta-cell growth. beta-cell mass dynamics were analyzed both with traditional morphometrical methods and three dimensional optical projection tomography (OPT) of whole-mount insulin-stained pancreata. Insulin-positive volume analyzed with OPT increased 1.4-fold at gestational day 18.5 (GD18.5) when compared to non-pregnant mice. Number of islets peaked by GD13.5 (680 vs 1134 islets per pancreas, non-pregnant vs. GD13.5). PL stimulated beta cell proliferation in the wild-type islets, whereas the proliferative response was absent in the E1-DN mouse islets. Serotonin synthesizing enzymes were upregulated similarly in both the wild-type and E1-DN mice. However, while survivin (Birc5) mRNA was upregulated 5.5-fold during pregnancy in the wild-type islets, no change was seen in the E1-DN pregnant islets. PL induced survivin expression also in isolated islets and this was blocked by EGFR inhibitor gefitinib, mTOR inhibitor rapamycin and MEK inhibitor PD0325901. Our 3D-volumetric analysis of beta-cell mass expansion during murine pregnancy revealed that islet number increases during pregnancy. In addition, our results suggest that EGFR signaling is required for lactogen-induced survivin expression via MAPK and mTOR pathways.Peer reviewe

Distinct differentiation characteristics of individual human embryonic stem cell lines

BACKGROUND: Individual differences between human embryonic stem cell (hESC) lines are poorly understood. Here, we describe the derivation of five hESC lines (called FES 21, 22, 29, 30 and 61) from frozen-thawed human embryos and compare their individual differentiation characteristic. RESULTS: The cell lines were cultured either on human or mouse feeder cells. The cells grew significantly faster and could be passaged enzymatically only on mouse feeders. However, this was found to lead to chromosomal instability after prolonged culture. All hESC lines expressed the established markers of pluripotent cells as well as several primordial germ cell (PGC) marker genes in a uniform manner. However, the cell lines showed distinct features in their spontaneous differentiation patterns. The embryoid body (EB) formation frequency of FES 30 cell line was significantly lower than that of other lines and cells within the EBs differentiated less readily. Likewise, teratomas derived from FES 30 cells were constantly cystic and showed only minor solid tissue formation with a monotonous differentiation pattern as compared with the other lines. CONCLUSION: hESC lines may differ substantially in their differentiation properties although they appear similar in the undifferentiated state

A Novel Feeder-Free Culture System for Human Pluripotent Stem Cell Culture and Induced Pluripotent Stem Cell Derivation

Peer reviewe

Laminin isoforms in human embryonic stem cells : synthesis, receptor usage and growth support

Sanna Vuoristo, Ismo Virtanen, Minna Takkunen, Jaan Palgi, Yamato Kikkawa, Patricia Rousselle, Kiyotoshi Sekiguchi, Timo Tuuri, and Timo Otonkoski, "Laminin isoforms in human embryonic stem cells: synthesis, receptor usage and growth support", Journal of Cellular and Molecular Medicine, 13, 8b, pp. 2622-2633, Wiley, 200

Antioxidative CXXC Peptide Motif From Mesencephalic Astrocyte-Derived Neurotrophic Factor Antagonizes Programmed Cell Death

Mesencephalic astrocyte-derived neurotrophic factor (MANF) is a potent survival-promoting protein with neurorestorative effect for neurodegenerative diseases. Its mechanism of action, albeit poorly known, depends strongly on the CXXC motif (CKGC). Here we studied the survival-promoting properties of the CKGC tetrapeptide from MANF. In the Jurkat T lymphocytic cell line, CKGC potently inhibits death receptor Fas-induced apoptosis and mildly counteracts mitochondrial apoptosis and necroptosis. The peptide with serines instead of cysteines (SKGS) has no survival-promoting activity. The cytoprotective efficiency of the peptide against Fas-induced apoptosis is significantly improved by reduction of its cysteines by dithiotreitol, suggesting that it protects the cells via cysteine thiol groups, partially as an antioxidant. CKGC neutralizes the reactive oxygen species, maintains the mitochondrial membrane potential and prevents activation of the effector caspases in the Jurkat cells with activated Fas. The peptide does not require intracellular administration, as it is endocytosed and resides mainly in the Golgi. Finally, the peptide also potently promotes survival of cultured primary dopaminergic neurons.Peer reviewe

E1-DN mice do not increase β-cell mass during pregnancy.

<p>A: The β-cell mass of wild-type mice increases during pregnancy at GD14.5. There is no difference between groups of E1-DN mice, n = 8–13. B: Isolated islets from wild-type (WT) and E1-DN (E1) female virgin mice were cultured for 96 h either with PL 500 ng/ml or in control media (con). BrdU was added to the media for the last 48 h. Quantification of proliferative β-cells, n = 3 in each group. C: Total number of islets per pancreatic area from WT and E1-DN (E1) control and pregnant gestational day 14.5 (GD14.5) mice, n = 4–6 per group. D: The distribution of islet sizes relative to total pancreatic area. Islets were classified into small <12076 μm², medium 12076–35033 μm² and large islets >35033 μm² from wild-type (WT) and E1-DN (E1) control (con) and gestational day 14.5 (GD14.5) pancreata, n = 4–6 per group. E: The islet size distribution corrected for pancreatic weight. AU, arbitrary unit. Bars represent the mean ±SEM for each group. *p<0.05, **p<0.01,***p<0.001, ****p<0.0001 ns = no statistical significance.</p

OPT analysis of insulin-positive volume and islet number during pregnancy.

<p>A–C: Isosurface rendered OPT images of insulin labeled (red) representative pancreata from control (A), gestational day 13.5 (GD13.5) (B) and gestational day 18.5 (GD18.5) (C) female mice. The pancreas outline is based on autofluorescence of the tissue. D: Graph illustrating the average insulin-positive volume in control (white), GD13.5 (gray) and GD18.5 (black) pancreata, n = 7–8 per group. E: Graph illustrating the average number of islets per pancreas in control (white), GD13.5 (gray) and GD18.5 (black) pancreata, n = 7–8 per group. F–G: The distribution of islet sizes (G) and contribution to total islet volume (F) n = 7–8 per group. Islets were classified into small 0–1000 um³×10³, intermediate 1000–5000 um³×10³ and large islets >5000 um³×10³ from control (white), GD13.5 (gray) and GD18.5 (black) pancreata. Bars represent the mean ±SEM for each group. *p<0.05, **p<0.01, ***p<0.001 ns = no statistical significance.</p

PL-induced survivin upregulation is dependent of EGFR, mTOR and MEK.

<p>A–B: Islets were isolated from wild-type mice, stimulated with or without PL (500 ng/ml), gefitinib (2 μM), rapamycin (10 nM), MEK inhibitor PD0325901 (0,5 μM) or EGF (50 ng/ml) and BTC (50 ng/ml) for 96 h and RT-qPCR performed. Graph is showing the relative mRNA level of survivin (A) and <i>Tph1</i> (B). n = 6–9 per group. Bars represent the mean ±SEM for each group. *p<0.05, **p<0.01 ***p<0.001. C: Proposed model for PL-induced survivin upregulation. Prl-R activation leads to transactivation of EGFR, which activates MAPK and PI3K-Akt-mTOR pathways, that together stimulate survivin gene expression.</p

Reduced EGFR signaling leads to decreased survivin expression in islets during pregnancy in mice.

<p>A–G: Islets were isolated from wild-type (WT) and E1-DN (E1) virgin (con) and gestational day 13.5 (GD13.5) mice and RT-qPCR performed. Graphs are showing the relative mRNA level of survivin (A), <i>Tph1</i> (B), <i>Tph2</i> (C), serotonin receptor <i>Htr2B</i> (D), <i>FoxM1</i> (E), endogenous mouse <i>Egfr</i> (F) and prolactin receptor (G) n = 4–5 per group. *p<0.05, **p<0.01, ns = no statistical significance.</p