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.

Intrastriatally Infused Exogenous CDNF Is Endocytosed and Retrogradely Transported to Substantia Nigra.

Cerebral dopamine neurotrophic factor (CDNF) protects the nigrostriatal dopaminergic (DA) neurons in rodent models of Parkinson's disease and restores DA circuitry when delivered after these neurons have begun to degenerate. These DA neurons have been suggested to transport striatal CDNF retrogradely to the substantia nigra (SN). However, in cultured cells the binding and internalization of extracellular CDNF has not been reported. The first aim of this study was to examine the cellular localization and pharmacokinetic properties of recombinant human CDNF (rhCDNF) protein after its infusion into rat brain parenchyma. Second, we aimed to study whether the transport of rhCDNF from the striatum to the SN results from its retrograde transport via DA neurons or from its anterograde transport via striatal GABAergic projection neurons. We show that after intrastriatal infusion, rhCDNF diffuses rapidly and broadly, and is cleared with a half-life of 5.5 h. Confocal microscopy analysis of brain sections at 2 and 6 h after infusion of rhCDNF revealed its widespread unspecific internalization by cortical and striatal neurons, exhibiting different patterns of subcellular rhCDNF distribution. Electron microscopy analysis showed that rhCDNF is present inside the endosomes and multivesicular bodies. In addition, we present data that after intrastriatal infusion the rhCDNF found in the SN is almost exclusively localized to the DA neurons, thus showing that it is retrogradely transported.

Microarray Analysis Reveals Increased Transcriptional Repression and Reduced Metabolic Activity but Not Major Changes in the Core Apoptotic Machinery during Maturation of Sympathetic Neurons.

Postnatal maturation of the neurons whose main phenotype and basic synaptic contacts are already established includes neuronal growth, refinement of synaptic contacts, final steps of differentiation, programmed cell death period (PCD) etc. In the sympathetic neurons, postnatal maturation includes permanent end of the PCD that occurs with the same time schedule in vivo and in vitro suggesting that the process could be genetically determined. Also many other changes in the neuronal maturation could be permanent and thus based on stable changes in the genome expression. However, postnatal maturation of the neurons is poorly studied. Here we compared the gene expression profiles of immature and mature sympathetic neurons using Affymetrix microarray assay. We found 1310 significantly up-regulated and 1151 significantly down-regulated genes in the mature neurons. Gene ontology analysis reveals up-regulation of genes related to neuronal differentiation, chromatin and epigenetic changes, extracellular factors and their receptors, and cell adhesion, whereas many down-regulated genes were related to metabolic and biosynthetic processes. We show that termination of PCD is not related to major changes in the expression of classical genes for apoptosis or cell survival. Our dataset is deposited to the ArrayExpress database and is a valuable source to select candidate genes in the studies of neuronal maturation. As an example, we studied the changes in the expression of selected genes Igf2bp3, Coro1A, Zfp57, Dcx, and Apaf1 in the young and mature sympathetic ganglia by quantitative PCR and show that these were strongly downregulated in the mature ganglia.

EGFR signaling promotes ß-cell proliferation and survivin expression during pregnancy.

Placental lactogen (PL) induced serotonergic signaling is essential for gestational ß-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 ß-cell mass compensation. Islets were isolated from wild-type and ß-cell-specific EGFR-dominant negative mice (E1-DN), stimulated with PL and analyzed for ß-cell proliferation and expression of genes involved in gestational ß-cell growth. ß-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 ß-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.

A novel feeder-free culture system for human pluripotent stem cell culture and induced pluripotent stem cell derivation.

Correct interactions with extracellular matrix are essential to human pluripotent stem cells (hPSC) to maintain their pluripotent self-renewal capacity during in vitro culture. hPSCs secrete laminin 511/521, one of the most important functional basement membrane components, and they can be maintained on human laminin 511 and 521 in defined culture conditions. However, large-scale production of purified or recombinant laminin 511 and 521 is difficult and expensive. Here we have tested whether a commonly available human choriocarcinoma cell line, JAR, which produces high quantities of laminins, supports the growth of undifferentiated hPSCs. We were able to maintain several human pluripotent stem cell lines on decellularized matrix produced by JAR cells using a defined culture medium. The JAR matrix also supported targeted differentiation of the cells into neuronal and hepatic directions. Importantly, we were able to derive new human induced pluripotent stem cell (hiPSC) lines on JAR matrix and show that adhesion of the early hiPSC colonies to JAR matrix is more efficient than to matrigel. In summary, JAR matrix provides a cost-effective and easy-to-prepare alternative for human pluripotent stem cell culture and differentiation. In addition, this matrix is ideal for the efficient generation of new hiPSC lines.

Activin A and Wnt-dependent specification of human definitive endoderm cells.

Activin/Nodal and Wnt signaling are known to play important roles in the regional specification of endoderm. Here we have investigated the effect of the length of stimulation with Activin A plus Wnt3a on the development of hepatic and pancreatic progenitors from the definitive endoderm (DE) cells derived from human pluripotent stem cells (hPSC). We show that DE-cells derived from hPSC with 3 days high Activin A and Wnt3a treatment were able to differentiate further into both tested endodermal lineages. When prolonging the DE-induction protocol from 3 to 5 or 7 days, almost pure DE-marker positive cell populations were obtained. However, these cells had an impaired pancreatic differentiation capacity, while they still developed into hepatocyte-like cells. Further propagation of the DE-cells in the presence of Wnt3a and Activin A led to the complete loss of differentiation capacity into hepatic or pancreatic lineages. When Wnt3a was removed after 24h from the initiation of the differentiation, the cells were able to differentiate into PDX1+/NKX6.1+ pancreatic progenitors even with longer DE induction time while efficiency of hepatic differentiation was lower. Our results suggest that both the length and the timing of Wnt3a treatment during DE induction are crucial for the final differentiation outcome. Although it is possible to derive apparently pure DE cells with prolonged Activin A/Wnt-stimulation, their progenitor capacity is restricted to a limited time window.

Comparative analysis of targeted differentiation of human induced pluripotent stem cells (hiPSCs) and human embryonic stem cells reveals variability associated with incomplete transgene silencing in retrovirally derived hiPSC lines.

Functional hepatocytes, cardiomyocytes, neurons, and retinal pigment epithelial (RPE) cells derived from human embryonic stem cells (hESCs) or human induced pluripotent stem cells (hiPSCs) could provide a defined and renewable source of human cells relevant for cell replacement therapies, drug discovery, toxicology testing, and disease modeling. In this study, we investigated the differences between the differentiation potentials of three hESC lines, four retrovirally derived hiPSC lines, and one hiPSC line derived with the nonintegrating Sendai virus technology. Four independent protocols were used for hepatocyte, cardiomyocyte, neuronal, and RPE cell differentiation. Overall, cells differentiated from hESCs and hiPSCs showed functional similarities and similar expression of genes characteristic of specific cell types, and differences between individual cell lines were also detected. Reactivation of transgenic OCT4 was detected specifically during RPE differentiation in the retrovirally derived lines, which may have affected the outcome of differentiation with these hiPSCs. One of the hiPSC lines was inferior in all directions, and it failed to produce hepatocytes. Exogenous KLF4 was incompletely silenced in this cell line. No transgene expression was detected in the Sendai virus-derived hiPSC line. These findings highlight the problems related to transgene expression in retrovirally derived hiPSC lines.

Proliferation and plasticity of human beta cells on physiologically occurring laminin isoforms.

We have previously characterized the molecular composition of human islet basement membranes and shown that human beta cells bind to laminin 511 (LM511) through integrin α3ß1 and Lutheran glycoprotein. We have now investigated the impact of physical contact between cultured human beta cells and the laminin isoforms occurring in their natural niche. Human islet preparations derived from 15 donors were used, beta cells and duct cells were purified by magnetic sorting. Overall beta-cell proliferation was low or undetectable. However, in many experiments the only proliferating beta cells were detected in contact with the laminin isoforms that are found in the human islets in vivo (511 and 411). Purified ductal and beta cells underwent epithelial-mesenchymal transition (EMT). LM511 partially blocked this dedifferentiation of purified beta cells, and did not affect purified duct cells. Interactions with the surrounding basement membrane are important for the growth and function of human beta cells. However, only a very limited level of beta-cell proliferation can be induced by exogenous factors. LM511 may be a useful substrate for human beta-cell maintenance in vitro.

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

To reveal the functional intrinsic niche of human embryonic stem cells (hESC) we examined the production of basement membrane (BM) proteins and the presence of their receptors in feeder-free cell culture conditions. In addition, we investigated binding of hESCs to purified human BM proteins and identified the receptors mediating these contacts. Also, we tested whether purified human laminin (Lm) isoforms have a role in hESC self-renewal and growth in short-term cultures. The results show that hESCs synthesize Lm alpha(1) and Lm alpha(5) chains together with Lm beta(1) and gamma(1) chains suggesting the production of Lms-111 and -511 into the culture medium and deposits on cells. hESCs contain functionally important integrin (Int) subunits, Int beta(1), alpha(3), alpha(6), alpha(5), beta(5) and alpha(V), as well as the Lm alpha(5) receptor, Lutheran (Lu) glycoprotein and its truncated form, basal cell adhesion molecule (B-CAM). In cell adhesion experiments, Int beta(1) was crucial for adhesion to most of the purified human BM proteins. Lu/B-CAM mediated adhesion to Lm-511 together with Int alpha(3)beta(1), and was essential for the adhesion of hESCs to embryonic feeder cells. Adhesion to Lm-411 was mediated by Int alpha(6)beta(1). Lm-511 supported hESC growth in defined medium equally well as Matrigel. These results provide consequential information of the biological role of BM in hESCs, warranting further investigation of BM biology of human pluripotent stem cells.

Downregulation of EGF receptor signaling in pancreatic islets causes diabetes due to impaired postnatal beta-cell growth.

Epidermal growth factor receptor (EGF-R) signaling is essential for proper fetal development and growth of pancreatic islets, and there is also evidence for its involvement in beta-cell signal transduction in the adult. To study the functional roles of EGF-R in beta-cell physiology in postnatal life, we have generated transgenic mice that carry a mutated EGF-R under the pancreatic duodenal homeobox-1 promoter (E1-DN mice). The transgene was expressed in islet beta- and delta-cells but not in alpha-cells, as expected, and it resulted in an approximately 40% reduction in pancreatic EGF-R, extracellular signal-related kinase, and Akt phosphorylation. Homozygous E1-DN mice were overtly diabetic after the age of 2 weeks. The hyperglycemia was more pronounced in male than in female mice. The relative beta-cell surface area of E1-DN mice was highly reduced at the age of 2 months, while alpha-cell surface area was not changed. This defect was essentially postnatal, since the differences in beta-cell area of newborn mice were much smaller. An apparent explanation for this is impaired postnatal beta-cell proliferation; the normal surge of beta-cell proliferation during 2 weeks after birth was totally abolished in the transgenic mice. Heterozygous E1-DN mice were glucose intolerant in intraperitoneal glucose tests. This was associated with a reduced insulin response. However, downregulation of EGF-R signaling had no influence on the insulinotropic effect of glucagon-like peptide-1 analog exendin-4. In summary, our results show that even a modest attenuation of EGF-R signaling leads to a severe defect in postnatal growth of the beta-cells, which leads to the development of diabetes.

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.

Transcription factor GATA-6 is expressed in the endocrine and GATA-4 in the exocrine pancreas.

GATA-4 and GATA-6 are zinc finger transcription factors that regulate gene expression, differentiation, and cell proliferation in various tissues. These factors have been implicated in the development of endodermal derivatives, including epithelial cells in the yolk sac, lung, and stomach. In the present study, we have characterized the expression of GATA-4 and GATA-6 during development of another endodermal derivative, the mouse pancreas, using a combination of in situ hybridization and immunohistochemistry. Neither GATA-4 nor GATA-6 antigen was detected in E10.5 pancreatic epithelial buds expressing Pdx-1. By E15.5, GATA-4 mRNA and protein were evident in developing pancreatic acini, but not in ductal or endocrine cells of the pancreas; GATA-6 mRNA and protein were present in both endocrine and exocrine cell precursors. In the newborn and adult pancreas, GATA-4 protein was seen in acinar cells, while GATA-6 antigen was found mainly in islet beta-cells. The amphicrine pancreatic AR42J-B13 cell line was used to study the expression of GATA-4 and GATA-6 during the differentiation of these cells towards an endocrine phenotype. Endocrine differentiation was associated with marked increase in GATA-6 but not GATA-4 mRNA levels. We conclude that GATA-4 is a marker of exocrine pancreatic differentiation, whereas GATA-6 is a marker of endocrine pancreatic development.

ErbB signaling regulates lineage determination of developing pancreatic islet cells in embryonic organ culture.

The neuregulin (NRG)/epidermal growth factor (EGF) family of growth factors consists of several ligands that specifically activate four erbB receptor-tyrosine kinases, namely erbB-1 (EGF-R), erbB-2 (neu), erbB-3, and erbB-4. We have previously shown that islet morphogenesis is impaired and beta-cell differentiation delayed in mice lacking functional EGF-R [EGF-R (-/-)]. The present study aims to clarify which erbB ligands are important for islet development. Pancreatic expression of EGF, TGF-alpha, heparin-binding EGF, betacellulin (BTC), and NRG-4 was detected as early as embryonic d 13 (E13). Effects of these ligands were studied in E12.5 pancreatic explant cultures grown for 5 d ex vivo. None of the growth factors affected the ratio of endocrine to exocrine cells. However, significant effects within the endocrine cell populations were induced by EGF, BTC, and NRG-4. beta-Cell development was augmented by BTC, whereas the development of somatostatin-expressing delta-cells was stimulated by NRG-4. Both ligands decreased the numbers of glucagon-containing alpha-cells. The effect of BTC was abolished in the EGF-R (-/-) mice. A soluble erbB-4 binding fusion protein totally inhibited the effects of NRG-4 but not of BTC. Neutralization of endogenous NRG-4 activity in the model system effectively inhibited delta-cell development, indicating that this erbB4-ligand is an essential factor for delineation of the somatostatin-producing delta-cells. Our results suggest that ligands of the EGF-R/erbB-1 and erbB-4 receptors regulate the lineage determination of islet cells during pancreatic development. BTC, acting through EGF-R/erbB-1, is important for the differentiation of beta-cells. This could be applied in the targeted differentiation of stem cells into insulin-producing cells.