Transient regulation of focal adhesion via Tensin3 is required for nascent oligodendrocyte differentiation.

The differentiation of oligodendroglia from oligodendrocyte precursor cells (OPCs) to complex and extensive myelinating oligodendrocytes (OLs) is a multistep process that involves large-scale morphological changes with significant strain on the cytoskeleton. While key chromatin and transcriptional regulators of differentiation have been identified, their target genes responsible for the morphological changes occurring during OL myelination are still largely unknown. Here, we show that the regulator of focal adhesion, Tensin3 (Tns3), is a direct target gene of Olig2, Chd7, and Chd8, transcriptional regulators of OL differentiation. Tns3 is transiently upregulated and localized to cell processes of immature OLs, together with integrin-ß1, a key mediator of survival at this transient stage. Constitutive <i>Tns3</i> loss of function leads to reduced viability in mouse and humans, with surviving knockout mice still expressing Tns3 in oligodendroglia. Acute deletion of <i>Tns3</i> in vivo, either in postnatal neural stem cells (NSCs) or in OPCs, leads to a twofold reduction in OL numbers. We find that the transient upregulation of Tns3 is required to protect differentiating OPCs and immature OLs from cell death by preventing the upregulation of p53, a key regulator of apoptosis. Altogether, our findings reveal a specific time window during which transcriptional upregulation of Tns3 in immature OLs is required for OL differentiation likely by mediating integrin-ß1 survival signaling to the actin cytoskeleton as OL undergo the large morphological changes required for their terminal differentiation.

The Host Protein Aquaporin-9 is Required for Efficient Plasmodium falciparum Sporozoite Entry into Human Hepatocytes.

Hepatocyte invasion by Plasmodium sporozoites represents a promising target for innovative antimalarial therapy, but the molecular events mediating this process are still largely uncharacterized. We previously showed that Plasmodium falciparum sporozoite entry into hepatocytes strictly requires CD81. However, CD81-overexpressing human hepatoma cells remain refractory to P. falciparum infection, suggesting the existence of additional host factors necessary for sporozoite entry. Here, through differential transcriptomic analysis of human hepatocytes and hepatoma HepG2-CD81 cells, the transmembrane protein Aquaporin-9 (AQP9) was found to be among the most downregulated genes in hepatoma cells. RNA silencing showed that sporozoite invasion of hepatocytes requires AQP9 expression. AQP9 overexpression in hepatocytes increased their permissiveness to P. falciparum. Moreover, chemical disruption with the AQP9 inhibitor phloretin markedly inhibited hepatocyte infection. Our findings identify AQP9 as a novel host factor required for P. falciparum sporozoite hepatocyte-entry and indicate that AQP9 could be a potential therapeutic target.

Culture, differentiation, and transduction of mouse E12.5 pancreatic spheres: an in vitro model for the secondary transition of pancreas development.

During the secondary transition of rodent pancreatic development, mainly between E12.5 and E15.5 in mice, exocrine and endocrine populations differentiate from pancreatic progenitors. Here we describe an experimental system for its study in vitro. First, we show that spheres derived from dissociated E12.5 mouse pancreases differentiate within 7 days into most pancreatic exocrine and endocrine cell types, including beta cells. The proportion and spatial repartition of the different endocrine populations mirror those observed during normal development. Thus, dissociation and culture do not impair the developmental events affecting pancreatic progenitors during the secondary transition. Moreover, dissociated cells from mouse E12.5 pancreas were transduced with ecotropic MLV-based retroviral vectors or, though less efficiently, with a mixture of ALV(A)-based retroviral vectors and gesicles containing the TVA (Tumor Virus A) receptor. As an additional improvement, we also created a transgenic mouse line expressing TVA under the control of the 4.5 kB pdx1 promoter (pdx1-TVA). We demonstrate that pancreatic progenitors from dissociated pdx1-TVA pancreas can be specifically transduced by ALV(A)-based retroviral vectors. Using this model, we expressed an activated mutant of the YAP transcriptional co-activator in pancreatic progenitors. These experiments indicate that deregulated YAP activity reduces endocrine and exocrine differentiation in the resulting spheres, confirming and extending previously published data. Thus, our experimental model recapitulates in vitro the crucial developmental decisions arising at the secondary transition and provides a convenient tool to study their genetic control.

MiR-132 controls pancreatic beta cell proliferation and survival through Pten/Akt/Foxo3 signaling.

OBJECTIVE: MicroRNAs (miRNAs) play an integral role in maintaining beta cell function and identity. Deciphering their targets and precise role, however, remains challenging. In this study, we aimed to identify miRNAs and their downstream targets involved in the regeneration of islet beta cells following partial pancreatectomy in mice. METHODS: RNA from laser capture microdissected (LCM) islets of partially pancreatectomized and sham-operated mice were profiled with microarrays to identify putative miRNAs implicated in beta cell regeneration. Altered expression of the selected miRNAs, including miR-132, was verified by RT-PCR. Potential targets of miR-132 were selected through bioinformatic data mining. Predicted miR-132 targets were validated for their changed RNA, protein expression levels, and signaling upon miR-132 knockdown and/or overexpression in mouse MIN6 and human EndoC-ßH1 insulinoma cells. The ability of miR-132 to foster beta cell proliferation in vivo was further assessed in pancreatectomized miR-132-/- and control mice. RESULTS: Partial pancreatectomy significantly increased the number of BrdU+/insulin+ islet cells. Microarray profiling revealed that 14 miRNAs, including miR-132 and -141, were significantly upregulated in the LCM islets of the partially pancreatectomized mice compared to the LCM islets of the control mice. In the same comparison, miR-760 was the only downregulated miRNA. The changed expression of these miRNAs in the islets of the partially pancreatectomized mice was confirmed by RT-PCR only in the case of miR-132 and -141. Based on previous knowledge of its function, we focused our attention on miR-132. Downregulation of miR-132 reduced the proliferation of MIN6 cells while enhancing the levels of pro-apoptotic cleaved caspase-9. The opposite was observed in miR-132 overexpressing MIN6 cells. Microarray profiling, RT-PCR, and immunoblotting of the latter cells demonstrated their downregulated expression of Pten with concomitant increased levels of pro-proliferative factors phospho-Akt and phospho-Creb and inactivation of pro-apoptotic Foxo3a via its phosphorylation. Downregulation of Pten was further confirmed in the LCM islets of pancreatectomized mice compared to the sham-operated mice. Moreover, overexpression of miR-132 correlated with increased proliferation of EndoC-ßH1 cells. The regeneration of beta cells following partial pancreatectomy was lower in the miR-132/212-/- mice than the control littermates. CONCLUSIONS: This study provides compelling evidence about the critical role of miR-132 for the regeneration of mouse islet beta cells through the downregulation of its target Pten. Hence, the miR-132/Pten/Akt/Foxo3 signaling pathway may represent a suitable target to enhance beta cell mass.

Differentiation of Sendai Virus-Reprogrammed iPSC into ß Cells, Compared with Human Pancreatic Islets and Immortalized ß Cell Line.

BACKGROUND: New sources of insulin-secreting cells are strongly in demand for treatment of diabetes. Induced pluripotent stem cells (iPSCs) have the potential to generate insulin-producing cells (iß). However, the gene expression profile and secretory function of iß still need to be validated in comparison with native ß cells. METHODS: Two clones of human iPSCs, reprogrammed from adult fibroblasts through integration-free Sendai virus, were differentiated into iß and compared with donor pancreatic islets and EndoC-ßH1, an immortalized human ß cell line. RESULTS: Both clones of iPSCs differentiated into insulin+ cells with high efficiency (up to 20%). iß were negative for pluripotency markers (Oct4, Sox2, Ssea4) and positive for Pdx1, Nkx6.1, Chromogranin A, PC1/3, insulin, glucagon and somatostatin. iß basally secreted C-peptide, glucagon and ghrelin and released insulin in response either to increasing concentration of glucose or a depolarizing stimulus. The comparison revealed that iß are remarkably similar to donor derived islets in terms of gene and protein expression profile and similar level of heterogeneity. The ability of iß to respond to glucose instead was more related to that of EndoC-ßH1. DISCUSSION: We demonstrated that insulin-producing cells generated from iPSCs recapitulate fundamental gene expression profiles and secretory function of native human ß cells.

Oligodendrocyte precursor survival and differentiation requires chromatin remodeling by Chd7 and Chd8.

Oligodendrocyte precursor cells (OPCs) constitute the main proliferative cells in the adult brain, and deregulation of OPC proliferation-differentiation balance results in either glioma formation or defective adaptive (re)myelination. OPC differentiation requires significant genetic reprogramming, implicating chromatin remodeling. Mounting evidence indicates that chromatin remodelers play important roles during normal development and their mutations are associated with neurodevelopmental defects, with CHD7 haploinsuficiency being the cause of CHARGE syndrome and CHD8 being one of the strongest autism spectrum disorder (ASD) high-risk-associated genes. Herein, we report on uncharacterized functions of the chromatin remodelers Chd7 and Chd8 in OPCs. Their OPC-chromatin binding profile, combined with transcriptome and chromatin accessibility analyses of Chd7-deleted OPCs, demonstrates that Chd7 protects nonproliferative OPCs from apoptosis by chromatin closing and transcriptional repression of p53 Furthermore, Chd7 controls OPC differentiation through chromatin opening and transcriptional activation of key regulators, including Sox10, Nkx2.2, and Gpr17 However, Chd7 is dispensable for oligodendrocyte stage progression, consistent with Chd8 compensatory function, as suggested by their common chromatin-binding profiles and genetic interaction. Finally, CHD7 and CHD8 bind in OPCs to a majority of ASD risk-associated genes, suggesting an implication of oligodendrocyte lineage cells in ASD neurological defects. Our results thus offer new avenues to understand and modulate the CHD7 and CHD8 functions in normal development and disease.

The EndoC-ßH1 cell line is a valid model of human beta cells and applicable for screenings to identify novel drug target candidates.

OBJECTIVE: To characterize the EndoC-ßH1 cell line as a model for human beta cells and evaluate its beta cell functionality, focusing on insulin secretion, proliferation, apoptosis and ER stress, with the objective to assess its potential as a screening platform for identification of novel anti-diabetic drug candidates. METHODS: EndoC-ßH1 was transplanted into mice for validation of in vivo functionality. Insulin secretion was evaluated in cells cultured as monolayer and as pseudoislets, as well as in diabetic mice. Cytokine induced apoptosis, glucolipotoxicity, and ER stress responses were assessed. Beta cell relevant mRNA and protein expression were investigated by qPCR and antibody staining. Hundreds of proteins or peptides were tested for their effect on insulin secretion and proliferation. RESULTS: Transplantation of EndoC-ßH1 cells restored normoglycemia in streptozotocin induced diabetic mice. Both in vitro and in vivo, we observed a clear insulin response to glucose, and, in vitro, we found a significant increase in insulin secretion from EndoC-ßH1 pseudoislets compared to monolayer cultures for both glucose and incretins. Apoptosis and ER stress were inducible in the cells and caspase 3/7 activity was elevated in response to cytokines, but not affected by the saturated fatty acid palmitate. By screening of various proteins and peptides, we found Bombesin (BB) receptor agonists and Pituitary Adenylate Cyclase-Activating Polypeptides (PACAP) to significantly induce insulin secretion and the proteins SerpinA6, STC1, and APOH to significantly stimulate proliferation. ER stress was readily induced by Tunicamycin and resulted in a reduction of insulin mRNA. Somatostatin (SST) was found to be expressed by 1% of the cells and manipulation of the SST receptors was found to significantly affect insulin secretion. CONCLUSIONS: Overall, the EndoC-ßH1 cells strongly resemble human islet beta cells in terms of glucose and incretin stimulated insulin secretion capabilities. The cell line has an active cytokine induced caspase 3/7 apoptotic pathway and is responsive to ER stress initiation factors. The cells' ability to proliferate can be further increased by already known compounds as well as by novel peptides and proteins. Based on its robust performance during the functionality assessment assays, the EndoC-ßH1 cell line was successfully used as a screening platform for identification of novel anti-diabetic drug candidates.

miR-204 is associated with an endocrine phenotype in human pancreatic islets but does not regulate the insulin mRNA through MAFA.

miR-204 has been proposed to modulate insulin expression in human pancreatic islets by regulating the expression of the MAFA transcript, and in turn insulin transcription. We investigated miR-204 expression in pancreatic endocrine tumors (PET), a panel of human tissues, tissues derived from pancreatic islet purification, and in induced pluripotent stem cells (iPSCs) differentiated towards a pancreatic endocrine phenotype by quantitative real time RT-PCR or droplet digital PCR (ddPCR). In addition, we evaluated the effect of miR-204 up- or down-regulation in purified human islets and in the EndoC-ßH1 cell line, as an experimental model of human pancreatic ß cells. Our results confirm that miR-204 was enriched in insulin producing PET, in ß cells within healthy pancreatic islets, and highly expressed in EndoC-ßH1 cells. Moreover, in iPSCs miR-204 increased stepwise upon stimulated differentiation to insulin producing cells. However, up- or down-regulation of miR-204 in human islets and in EndoC-ßH1 cells resulted in modest and not significant changes of the MAFA and INS mRNAs measured by ddPCR or c-peptide release. Our data confirm the association of miR-204 with a ß cell endocrine phenotype in human pancreatic islets, but do not support its direct role in regulating the levels of insulin mRNA through MAFA.

Aggregation of Engineered Human ß-Cells Into Pseudoislets: Insulin Secretion and Gene Expression Profile in Normoxic and Hypoxic Milieu.

Innovative treatments to cure type 1 diabetes are being actively researched. Among the different strategies, the replacement of ß-cells has given promising results. Classically, islets from cadaveric donors are transplanted into diabetic patients, but recently phase I clinical trials that use stem cell-derived ß-cells have been started. Such protocols require either an immunosuppressive treatment or the macroencapsulation of the ß-cells. They involve cell aggregation and the exposure of the cells to hypoxia. Using an engineered human ß-cell, we have addressed these two problems: a novel human ß-cell line called EndoC-ßH3 was cultured as single cells or aggregated clusters. EndoC-ßH3 cells were also cultured at normal atmospheric oxygen tension (pO2 = 21%) or hypoxia (pO2 = 3%) in the presence or absence of modulators of the hypoxia-inducible factor 1α (HIF1α) pathway. Cell aggregation improved glucose-stimulated insulin secretion, demonstrating the benefit of cell-cell contacts. Low oxygen tension decreased ß-cell viability and their sensitivity to glucose, but did not alter insulin production nor the insulin secretion capacity of the remaining cells. To investigate the role of HIF1α, we first used a HIF stabilizer at pO2 = 21%. This led to a mild decrease in cell viability, impaired glucose sensitivity, and altered insulin secretion. Finally, we used a HIF inhibitor on EndoC-ßH3 pseudoislets exposed to hypoxia. Such treatment considerably decreased cell viability. In conclusion, aggregation of the EndoC-ßH3 cells seems to be important to improve their function. A fraction of the EndoC-ßH3 cells are resistant to hypoxia, depending on the level of activity of HIF1α. Thus, these cells represent a good human cell model for future investigations on islet cell transplantation analysis.

Regulating the expression of therapeutic transgenes by controlled intake of dietary essential amino acids.

Widespread application of gene therapy will depend on the development of simple methods to regulate the expression of therapeutic genes. Here we harness an endogenous signaling pathway to regulate therapeutic gene expression through diet. The GCN2-eIF2α signaling pathway is specifically activated by deficiencies in any essential amino acid (EAA); EAA deficiency leads to rapid expression of genes regulated by ATF4-binding cis elements. We found that therapeutic genes under the control of optimized amino acid response elements (AAREs) had low basal expression and high induced expression. We applied our system to regulate the expression of TNFSF10 (TRAIL) in the context of glioma therapy and found that intermittent activation of this gene by EEA-deficient meals retained its therapeutic efficacy while abrogating its toxic effects on normal tissue. The GCN2-eIF2α pathway is expressed in many tissues, including the brain, and is highly specific to EAA deficiency. Our system may be particularly well suited for intermittent regulation of therapeutic transgenes over short or long time periods.

Xenotropic retrovirus Bxv1 in human pancreatic ß cell lines.

It has been reported that endogenous retroviruses can contaminate human cell lines that have been passaged as xenotransplants in immunocompromised mice. We previously developed and described 2 human pancreatic ß cell lines (EndoC-ßH1 and EndoC-ßH2) that were generated in this way. Here, we have shown that B10 xenotropic virus 1 (Bxv1), a xenotropic endogenous murine leukemia virus (MuLV), is present in these 2 recently described cell lines. We determined that Bxv1 was also present in SCID mice that were used for in vivo propagation of EndoC-ßH1/2 cells, suggesting that contamination occurred during xenotransplantation. EndoC-ßH1/2 cells released Bxv1 particles that propagated to human 293T and Mus dunni cells. Mobilization assays demonstrated that Bxv1 transcomplements defective MuLV-based retrovectors. In contrast, common rodent ß cell lines, rat INS-1E and RIN-5F cells and mouse MIN6 and ßTC3 cells, displayed either no or extremely weak xenotropic helper activity toward MuLV-based retrovectors, although xenotropic retrovirus sequences and transcripts were detected in both mouse cell lines. Bxv1 propagation from EndoC-ßH1/2 to 293T cells occurred only under optimized conditions and was overall poorly efficient. Thus, although our data imply that MuLV-based retrovectors should be cautiously used in EndoC-ßH1/2 cells, our results indicate that an involuntary propagation of Bxv1 from these cells can be easily avoided with good laboratory practices.

Innate and adaptive immunity to human beta cell lines: implications for beta cell therapy.

AIMS/HYPOTHESIS: Genetically engineered human beta cell lines provide a novel source of human beta cells to study metabolism, pharmacology and beta cell replacement therapy. Since the immune system is essentially involved in beta cell destruction in type 1 diabetes and after beta cell transplantation, we investigated the interaction of human beta cell lineswith the immune system to resolve their potential for immune intervention protocol studies. METHODS: Human pancreatic beta cell lines (EndoC-ßH1 and ECi50) generated by targeted oncogenesis in fetal pancreas were assessed for viability after innate and adaptive immune challenges. Beta cell lines were pre-conditioned with T helper type 1 (Th1) cytokines or high glucose to mimic inflammatory and hyperglycaemia-stressed conditions. Beta cells were then co-cultured with auto- and alloreactive cytotoxic T cells (CTL), natural killer (NK) cells, supernatant fraction from activated autoreactive Th1 cells, or alloantibodies in the presence of complement or effector cells. RESULTS: Low HLA expression protected human beta cell lines from adaptive immune destruction, but it was associated with direct killing by activated NK cells. Autoreactive Th1 cell inflammation, rather than glucose stress, induced increased beta cell apoptosis and upregulation of HLA, increasing beta cell vulnerability to killing by auto- and alloreactive CTL and alloreactive antibodies. CONCLUSIONS/INTERPRETATION: We demonstrate that genetically engineered human beta cell lines can be used in vitro to assess diverse immune responses that may be involved in the pathogenesis of type 1 diabetes in humans and beta cell transplantation, enabling preclinical evaluation of novel immune intervention strategies protecting beta cells from immune destruction.

Characterization of stimulus-secretion coupling in the human pancreatic EndoC-ßH1 beta cell line.

AIMS/HYPOTHESIS: Studies on beta cell metabolism are often conducted in rodent beta cell lines due to the lack of stable human beta cell lines. Recently, a human cell line, EndoC-ßH1, was generated. Here we investigate stimulus-secretion coupling in this cell line, and compare it with that in the rat beta cell line, INS-1 832/13, and human islets. METHODS: Cells were exposed to glucose and pyruvate. Insulin secretion and content (radioimmunoassay), gene expression (Gene Chip array), metabolite levels (GC/MS), respiration (Seahorse XF24 Extracellular Flux Analyzer), glucose utilization (radiometric), lactate release (enzymatic colorimetric), ATP levels (enzymatic bioluminescence) and plasma membrane potential and cytoplasmic Ca2+ responses (microfluorometry) were measured. Metabolite levels, respiration and insulin secretion were examined in human islets. RESULTS: Glucose increased insulin release, glucose utilization, raised ATP production and respiratory rates in both lines, and pyruvate increased insulin secretion and respiration. EndoC-ßH1 cells exhibited higher insulin secretion, while plasma membrane depolarization was attenuated, and neither glucose nor pyruvate induced oscillations in intracellular calcium concentration or plasma membrane potential. Metabolite profiling revealed that glycolytic and TCA-cycle intermediate levels increased in response to glucose in both cell lines, but responses were weaker in EndoC-ßH1 cells, similar to those observed in human islets. Respiration in EndoC-ßH1 cells was more similar to that in human islets than in INS-1 832/13 cells. CONCLUSIONS/INTERPRETATION: Functions associated with early stimulus-secretion coupling, with the exception of plasma membrane potential and Ca2+ oscillations, were similar in the two cell lines; insulin secretion, respiration and metabolite responses were similar in EndoC-ßH1 cells and human islets. While both cell lines are suitable in vitro models, with the caveat of replicating key findings in isolated islets, EndoC-ßH1 cells have the advantage of carrying the human genome, allowing studies of human genetic variants, epigenetics and regulatory RNA molecules.

Human Bone Marrow Subpopulations Sustain Human Islet Function and Viability In vitro.

AIMS: Allogeneic bone marrow (BM) has been shown to support human islet survival and function in long-term culture by initiating human islet vascularization and ß-cell regeneration. Various BM subpopulations may play different roles in human islet functions and survival. In this paper we investigated the effects of BM and its subpopulations, endothelial progenitor cells (E) and mesenchymal (M) cells on human islet's ß-cell function and regeneration. STUDY DESIGN: Isolation and identification of subpopulations from human bone marrow and culture with allogeneic human islet to investigate effects of different cell population on human islet function and regeneration. PLACE AND DURATION OF STUDY: Department of Medicine, Center for Stem Cell & Diabetes Research, RWMC, Providence, RI, USA, between 2010 - 2014. METHODOLOGY: Human islets were distributed from Integrated Islet Distribution Program (IIDP) and human bone marrow (BM) was harvested by Bone marrow transplantation center at Roger Williams Hospital. BM subpopulation was identified cell surface markers through Fluorescence-activated cell sorting, applied in flow cytometry (FACS), islet function was evaluated by human ELISA kit and ß cell regeneration was evaluated by three methods of Cre-Loxp cell tracing, ß cell sorting and RT-PCR for gene expression. RESULTS: Four different BM and seven different islet donates contributed human tissues. We observed islet ß-cell having self regeneration capability in short term culture (3∼5 days) using a Cre-Loxp cell tracing. BM and its subtype E, M have similar benefits on ß cell function during co-culture with human islet comparison to islet only. However, only whole BM enables to sustain the capability of islet ß-cell self regeneration resulting in increasing ß cell population while single E and M individual do not significantly affect on that. Mechanism approach to explore ß-cell self regeneration by evaluating transcription factor expressions, we found that BM significantly increases the activations of ß-cell regeneration relative transcription factors, the LIM homeodomain protein (Isl1), homologue to zebrafish somite MAF1 (MAFa), the NK-homeodomain factor 6.1 (NKX6.1), the paired box family factors 6 (PAX6), insulin promoter factor 1 (IPF1) and kinesin family member 4A (KIF4a). CONCLUSION: These results suggest that BM and its derived M and E cells enable to support human islet ß-cell function. However, only BM can sustain the capability of ß-cell self regeneration through initiating ß-cell transcriptional factors but not individual E and M cells suggesting pure E and M cells less supportive for islet long-term survival in vitro.

Early netrin-1 expression impairs central nervous system remyelination.

OBJECTIVE: Chronically demyelinated multiple sclerosis (MS) lesions are frequently characterized by scarce undifferentiated oligodendrocyte progenitor cells (OPCs), suggesting the exhaustion of a local OPC pool followed by failure of recruitment and differentiation. Stimulating prompt OPC recruitment following demyelination could improve myelin repair by providing sufficient numbers of remyelinating cells during the repair-permissive period. Understanding mechanisms that determine this process may have important therapeutic implications. We therefore investigated the role of the guidance molecule netrin-1 in OPC recruitment and central nervous system (CNS) remyelination. METHODS: Netrin-1 expression was analyzed immunohistochemically in different types of MS lesions and in the murine lysolecithin model of demyelination. The influence of netrin-1 on CNS remyelination was examined using gain and loss of function experiments. RESULTS: We show that in MS lesions, astrocytes upregulate netrin-1 expression early during demyelination and netrin-1 receptors are expressed by OPCs. In contrast, in the efficiently repairing lysolecithin model of demyelination (astrocyte-free), netrin-1 expression is absent during early phases and detected concomitant with completion of OPC recruitment. In vitro migration assays demonstrated that netrin-1 is a chemorepellent for migrating adult OPCs. In mouse lesions, antibody-mediated disruption of netrin-1 function at the peak phase of recruitment increased OPC numbers. Conversely, lentiviral-mediated induction of netrin-1 expression prior to OPC recruitment reduced the number of cells recruited and impaired remyelination. INTERPRETATION: Our findings support the conclusion that netrin-1 expression within demyelinating MS plaques blocks OPC recruitment, which with repeated demyelinating episodes contributes to permanent remyelination failure.

Tumor and endothelial cell hybrids participate in glioblastoma vasculature.

BACKGROUND: Recently antiangiogenic therapy with bevacizumab has shown a high but transient efficacy in glioblastoma (GBM). Indeed, GBM is one of the most angiogenic human tumors and endothelial proliferation is a hallmark of the disease. We therefore hypothesized that tumor cells may participate in endothelial proliferation of GBM. MATERIALS AND METHODS: We used EGFR FISH Probe to detect EGFR amplification and anti-CD31, CD105, VE-cadherin, and vWF to identify endothelial cells. Endothelial and GBM cells were grown separately, labeled with GFP and DsRed lentiviruses, and then cocultured with or without contact. RESULTS: In a subset of GBM tissues, we found that several tumor endothelial cells carry EGFR amplification, characteristic of GBM tumor cells. This observation was reproduced in vitro: when tumor stem cells derived from GBM were grown in the presence of human endothelial cells, a fraction of them acquired endothelial markers (CD31, CD105, VE-cadherin, and vWF). By transduction with GFP and DsRed expressing lentiviral vectors, we demonstrate that this phenomenon is due to cell fusion and not transdifferentiation. CONCLUSION: A fraction of GBM stem cells thus has the capacity to fuse with endothelial cells and the resulting hybrids may participate in tumor microvascular proliferation and in treatment resistance.

Development of a conditionally immortalized human pancreatic ß cell line.

Diabetic patients exhibit a reduction in ß cells, which secrete insulin to help regulate glucose homeostasis; however, little is known about the factors that regulate proliferation of these cells in human pancreas. Access to primary human ß cells is limited and a challenge for both functional studies and drug discovery progress. We previously reported the generation of a human ß cell line (EndoC-ßH1) that was generated from human fetal pancreas by targeted oncogenesis followed by in vivo cell differentiation in mice. EndoC-ßH1 cells display many functional properties of adult ß cells, including expression of ß cell markers and insulin secretion following glucose stimulation; however, unlike primary ß cells, EndoC-ßH1 cells continuously proliferate. Here, we devised a strategy to generate conditionally immortalized human ß cell lines based on Cre-mediated excision of the immortalizing transgenes. The resulting cell line (EndoC-ßH2) could be massively amplified in vitro. After expansion, transgenes were efficiently excised upon Cre expression, leading to an arrest of cell proliferation and pronounced enhancement of ß cell-specific features such as insulin expression, content, and secretion. Our data indicate that excised EndoC-ßH2 cells are highly representative of human ß cells and should be a valuable tool for further analysis of human ß cells.

Suppression of epithelial-to-mesenchymal transitioning enhances ex vivo reprogramming of human exocrine pancreatic tissue toward functional insulin-producing ß-like cells.

Because of the lack of tissue available for islet transplantation, new sources of ß-cells have been sought for the treatment of type 1 diabetes. The aim of this study was to determine whether the human exocrine-enriched fraction from the islet isolation procedure could be reprogrammed to provide additional islet tissue for transplantation. The exocrine-enriched cells rapidly dedifferentiated in culture and grew as a mesenchymal monolayer. Genetic lineage tracing confirmed that these mesenchymal cells arose, in part, through a process of epithelial-to-mesenchymal transitioning (EMT). A protocol was developed whereby transduction of these mesenchymal cells with adenoviruses containing Pdx1, Ngn3, MafA, and Pax4 generated a population of cells that were enriched in glucagon-secreting α-like cells. Transdifferentiation or reprogramming toward insulin-secreting ß-cells was enhanced, however, when using unpassaged cells in combination with inhibition of EMT by inclusion of Rho-associated kinase (ROCK) and transforming growth factor-ß1 inhibitors. Resultant cells were able to secrete insulin in response to glucose and on transplantation were able to normalize blood glucose levels in streptozotocin diabetic NOD/SCID mice. In conclusion, reprogramming of human exocrine-enriched tissue can be best achieved using fresh material under conditions whereby EMT is inhibited, rather than allowing the culture to expand as a mesenchymal monolayer.

Directed pancreatic acinar differentiation of mouse embryonic stem cells via embryonic signalling molecules and exocrine transcription factors.

Pluripotent embryonic stem cells (ESC) are a promising cellular system for generating an unlimited source of tissue for the treatment of chronic diseases and valuable in vitro differentiation models for drug testing. Our aim was to direct differentiation of mouse ESC into pancreatic acinar cells, which play key roles in pancreatitis and pancreatic cancer. To that end, ESC were first differentiated as embryoid bodies and sequentially incubated with activin A, inhibitors of Sonic hedgehog (Shh) and bone morphogenetic protein (BMP) pathways, fibroblast growth factors (FGF) and retinoic acid (RA) in order to achieve a stepwise increase in the expression of mRNA transcripts encoding for endodermal and pancreatic progenitor markers. Subsequent plating in Matrigel® and concomitant modulation of FGF, glucocorticoid, and folllistatin signalling pathways involved in exocrine differentiation resulted in a significant increase of mRNAs encoding secretory enzymes and in the number of cells co-expressing their protein products. Also, pancreatic endocrine marker expression was down-regulated and accompanied by a significant reduction in the number of hormone-expressing cells with a limited presence of hepatic marker expressing-cells. These findings suggest a selective activation of the acinar differentiation program. The newly differentiated cells were able to release α-amylase and this feature was greatly improved by lentiviral-mediated expression of Rbpjl and Ptf1a, two transcription factors involved in the maximal production of digestive enzymes. This study provides a novel method to produce functional pancreatic exocrine cells from ESC.

Gene delivery to pancreatic exocrine cells in vivo and in vitro.

BACKGROUND: Effective gene transfer to the pancreas or to pancreatic cells has remained elusive although it is essential for studies of genetic lineage tracing and modulation of gene expression. Different transduction methods and viral vectors were tested in vitro and in vivo, in rat and mouse pancreas. RESULTS: For in vitro transfection/transduction of rat exocrine cells lipofection reagents, adenoviral vectors, and Mokola- and VSV-G pseudotyped lentiviral vectors were used. For in vivo transduction of mouse and rat pancreas adenoviral vectors and VSV-G lentiviral vectors were injected into the parenchymal tissue. Both lipofection of rat exocrine cell cultures and transduction with Mokola pseudotyped lentiviral vectors were inefficient and resulted in less than 4% EGFP expressing cells. Adenoviral transduction was highly efficient but its usefulness for gene delivery to rat exocrine cells in vitro was hampered by a drastic increase in cell death. In vitro transduction of rat exocrine cells was most optimal with VSV-G pseudotyped lentiviral vectors, with stable transgene expression, no significant effect on cell survival and about 40% transduced cells. In vivo, pancreatic cells could not be transduced by intra-parenchymal administration of lentiviral vectors in mouse and rat pancreas. However, a high efficiency could be obtained by adenoviral vectors, resulting in transient transduction of mainly exocrine acinar cells. Injection in immune-deficient animals diminished leukocyte infiltration and prolonged transgene expression. CONCLUSIONS: In summary, our study remarkably demonstrates that transduction of pancreatic exocrine cells requires lentiviral vectors in vitro but adenoviral vectors in vivo.