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1.  Comparison Between Several Integrase-defective Lentiviral Vectors Reveals Increased Integration of an HIV Vector Bearing a D167H Mutant 
HIV-1 derived vectors are among the most efficient for gene transduction in mammalian tissues. As the parent virus, they carry out vector genome insertion into the host cell chromatin. Consequently, their preferential integration in transcribed genes raises several conceptual and safety issues. To address part of these questions, HIV-derived vectors have been engineered to be nonintegrating. This was mainly achieved by mutating HIV-1 integrase at functional hotspots of the enzyme enabling the development of streamlined nuclear DNA circles functional for transgene expression. Few integrase mutant vectors have been successfully tested so far for gene transfer. They are cleared with time in mitotic cells, but stable within nondividing retina cells or neurons. Here, we compared six HIV vectors carrying different integrases, either wild type or with different mutations (D64V, D167H, Q168A, K186Q+Q214L+Q216L, and RRK262-264AAH) shown to modify integrase enzymatic activity, oligomerization, or interaction with key cellular cofactor of HIV DNA integration as LEDGF/p75 or TNPO3. We show that these mutations differently affect the transduction efficiency as well as rates and patterns of integration of HIV-derived vectors suggesting their different processing in the nucleus. Surprisingly and most interestingly, we report that an integrase carrying the D167H substitution improves vector transduction efficiency and integration in both HEK-293T and primary CD34+ cells.
PMCID: PMC4272407  PMID: 25462529
CD34+ progenitors; integrase; integration sites; lentiviral vectors; residual integration; transduction
2.  Mouse Muscle As an Ectopic Permissive Site for Human Pancreatic Development 
Diabetes  2013;62(10):3479-3487.
While sporadic human genetic studies have permitted some comparisons between rodent and human pancreatic development, the lack of a robust experimental system has not permitted detailed examination of human pancreatic development. We previously developed a xenograft model of immature human fetal pancreas grafted under the kidney capsule of immune-incompetent mice, which allowed the development of human pancreatic β-cells. Here, we compared the development of human and murine fetal pancreatic grafts either under skeletal muscle epimysium or under the renal capsule. We demonstrated that human pancreatic β-cell development occurs more slowly (weeks) than murine pancreas (days) both by differentiation of pancreatic progenitors and by proliferation of developing β-cells. The superficial location of the skeletal muscle graft and its easier access permitted in vivo lentivirus-mediated gene transfer with a green fluorescent protein-labeled construct under control of the insulin or elastase gene promoter, which targeted β-cells and nonendocrine cells, respectively. This model of engraftment under the skeletal muscle epimysium is a new approach for longitudinal studies, which allows localized manipulation to determine the regulation of human pancreatic development.
PMCID: PMC3781474  PMID: 23835344
3.  Suppression of Epithelial-to-Mesenchymal Transitioning Enhances Ex Vivo Reprogramming of Human Exocrine Pancreatic Tissue Toward Functional Insulin-Producing β-Like Cells 
Diabetes  2013;62(8):2821-2833.
Because of the lack of tissue available for islet transplantation, new sources of β-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 β-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-β1 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.
PMCID: PMC3717833  PMID: 23610058
4.  Pancreatic islet enhancer clusters enriched in type 2 diabetes risk–associated variants 
Nature genetics  2014;46(2):136-143.
Type 2 diabetes affects over 300 million people, causing severe complications and premature death, yet the underlying molecular mechanisms are largely unknown. Pancreatic islet dysfunction is central for type 2 diabetes pathogenesis, and therefore understanding islet genome regulation could provide valuable mechanistic insights. We have now mapped and examined the function of human islet cis-regulatory networks. We identify genomic sequences that are targeted by islet transcription factors to drive islet-specific gene activity, and show that most such sequences reside in clusters of enhancers that form physical 3D chromatin domains. We find that sequence variants associated with type 2 diabetes and fasting glycemia are enriched in these clustered islet enhancers, and identify trait-associated variants that disrupt DNA-binding and islet enhancer activity. Our studies illustrate how islet transcription factors interact functionally with the epigenome, and provide systematic evidence that dysregulation of islet enhancers is relevant to the mechanisms underlying type 2 diabetes.
PMCID: PMC3935450  PMID: 24413736
5.  Tumor and Endothelial Cell Hybrids Participate in Glioblastoma Vasculature 
BioMed Research International  2014;2014:827327.
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.
PMCID: PMC4017715  PMID: 24868550
6.  Development of a conditionally immortalized human pancreatic β cell line 
The Journal of Clinical Investigation  2014;124(5):2087-2098.
Diabetic patients exhibit a reduction in β 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 β cells is limited and a challenge for both functional studies and drug discovery progress. We previously reported the generation of a human β cell line (EndoC-βH1) that was generated from human fetal pancreas by targeted oncogenesis followed by in vivo cell differentiation in mice. EndoC-βH1 cells display many functional properties of adult β cells, including expression of β cell markers and insulin secretion following glucose stimulation; however, unlike primary β cells, EndoC-βH1 cells continuously proliferate. Here, we devised a strategy to generate conditionally immortalized human β cell lines based on Cre-mediated excision of the immortalizing transgenes. The resulting cell line (EndoC-βH2) 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 β cell–specific features such as insulin expression, content, and secretion. Our data indicate that excised EndoC-βH2 cells are highly representative of human β cells and should be a valuable tool for further analysis of human β cells.
PMCID: PMC4001549  PMID: 24667639
7.  Concise Review: In Search of Unlimited Sources of Functional Human Pancreatic Beta Cells 
Rodent and human beta cells are not identical, and knowledge of human beta cells has not progressed as quickly as understanding of rodent beta cells because of the difficulty of accessing unlimited sources of functional human pancreatic beta cells. The main focus of this review concerns recent strategies to generate new sources of human pancreatic beta cells. This knowledge would provide a strong basis for developing new treatments for diabetic patients.
It is well-established that insulin-producing pancreatic beta cells are central in diabetes. In type 1 diabetes, beta cells are destroyed by an autoimmune mechanism, whereas in type 2 diabetes, there is a decrease in functional beta-cell mass. In this context, studying beta cells is of major importance. Beta cells represent only 1% of total pancreatic cells and are found dispersed in the pancreatic gland. During the past decades, many tools and approaches have been developed to study rodent beta cells that efficiently pushed the field forward. However, rodent and human beta cells are not identical, and our knowledge of human beta cells has not progressed as quickly as our understanding of rodent beta cells. We believe that one of the reasons for this inefficient progress is the difficulty of accessing unlimited sources of functional human pancreatic beta cells. The main focus of this review concerns recent strategies to generate new sources of human pancreatic beta cells.
PMCID: PMC3659747  PMID: 23283495
Diabetes; Differentiation; Pancreas; Beta cells
8.  The Inactivation of Arx in Pancreatic α-Cells Triggers Their Neogenesis and Conversion into Functional β-Like Cells 
PLoS Genetics  2013;9(10):e1003934.
Recently, it was demonstrated that pancreatic new-born glucagon-producing cells can regenerate and convert into insulin-producing β-like cells through the ectopic expression of a single gene, Pax4. Here, combining conditional loss-of-function and lineage tracing approaches, we show that the selective inhibition of the Arx gene in α-cells is sufficient to promote the conversion of adult α-cells into β-like cells at any age. Interestingly, this conversion induces the continuous mobilization of duct-lining precursor cells to adopt an endocrine cell fate, the glucagon+ cells thereby generated being subsequently converted into β-like cells upon Arx inhibition. Of interest, through the generation and analysis of Arx and Pax4 conditional double-mutants, we provide evidence that Pax4 is dispensable for these regeneration processes, indicating that Arx represents the main trigger of α-cell-mediated β-like cell neogenesis. Importantly, the loss of Arx in α-cells is sufficient to regenerate a functional β-cell mass and thereby reverse diabetes following toxin-induced β-cell depletion. Our data therefore suggest that strategies aiming at inhibiting the expression of Arx, or its molecular targets/co-factors, may pave new avenues for the treatment of diabetes.
Author Summary
Type 1 diabetes is a condition that results from the loss of insulin-producing β-cells. Despite current therapies, diabetic patients are prone to vascular complications. Using the mouse as a model, we previously found that pancreatic glucagon-expressing cells can be regenerated and converted into β-like cells by the forced expression of a single gene, Pax4. Here, we generated transgenic mice allowing both the permanent labeling of α-cells and the inactivation of Arx solely in this cell subtype. Our results indicate that, upon Arx inactivation, α-cells can be continuously regenerated from duct-lining precursors and converted into β-like cells. Importantly, the additional loss of Pax4 does not impact these processes, suggesting that Arx is the main trigger of α-cell-mediated β-like cell neogenesis. Most interestingly, upon chemical induction of diabetes/β-cell loss, while control animals die or remain severely hyperglycemic, a normalization of the glycemia, a clear regeneration of the β-like cell mass, and an extended lifespan are noted in animals with the conditional inactivation of Arx. Our data therefore suggest that strategies aiming at inhibiting the expression of Arx, or its molecular targets/co-factors, may pave new avenues for the treatment of diabetes.
PMCID: PMC3814322  PMID: 24204325
9.  Human β Cell Transcriptome Analysis Uncovers lncRNAs That Are Tissue-Specific, Dynamically Regulated, and Abnormally Expressed in Type 2 Diabetes 
Cell metabolism  2012;16(4):435-448.
A significant portion of the genome is transcribed as long non-coding RNAs (lncRNAs), several of which are known to control gene expression. The repertoire and regulation of lncRNAs in disease-relevant tissues, however, has not been systematically explored. We report a comprehensive strand-specific transcriptome map of human pancreatic islets and β-cells, and uncover >1100 intergenic and antisense islet-cell lncRNA genes. We find islet lncRNAs that are dynamically regulated, and show that they are an integral component of the β-cell differentiation and maturation program. We sequenced the mouse islet transcriptome, and identify lncRNA orthologs that are regulated like their human counterparts. Depletion of HI-LNC25, a β-cell specific lncRNA, downregulated GLIS3 mRNA, thus exemplifying a gene regulatory function of islet lncRNAs. Finally, selected islet lncRNAs were dysregulated in type 2 diabetes or mapped to genetic loci underlying diabetes susceptibility. These findings reveal a new class of islet-cell genes relevant to β-cell programming and diabetes pathophysiology.
PMCID: PMC3475176  PMID: 23040067
10.  Directed Pancreatic Acinar Differentiation of Mouse Embryonic Stem Cells via Embryonic Signalling Molecules and Exocrine Transcription Factors 
PLoS ONE  2013;8(1):e54243.
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.
PMCID: PMC3547908  PMID: 23349836
11.  Specific Control of Pancreatic Endocrine β- and δ-Cell Mass by Class IIa Histone Deacetylases HDAC4, HDAC5, and HDAC9 
Diabetes  2011;60(11):2861-2871.
Class IIa histone deacetylases (HDACs) belong to a large family of enzymes involved in protein deacetylation and play a role in regulating gene expression and cell differentiation. Previously, we showed that HDAC inhibitors modify the timing and determination of pancreatic cell fate. The aim of this study was to determine the role of class IIa HDACs in pancreas development.
We took a genetic approach and analyzed the pancreatic phenotype of mice lacking HDAC4, -5, and -9. We also developed a novel method of lentiviral infection of pancreatic explants and performed gain-of-function experiments.
We show that class IIa HDAC4, -5, and -9 have an unexpected restricted expression in the endocrine β- and δ-cells of the pancreas. Analyses of the pancreas of class IIa HDAC mutant mice revealed an increased pool of insulin-producing β-cells in Hdac5−/− and Hdac9−/− mice and an increased pool of somatostatin-producing δ-cells in Hdac4−/− and Hdac5−/− mice. Conversely, HDAC4 and HDAC5 overexpression showed a decreased pool of insulin-producing β-cells and somatostatin-producing δ-cells. Finally, treatment of pancreatic explants with the selective class IIa HDAC inhibitor MC1568 enhances expression of Pax4, a key factor required for proper β-and δ-cell differentiation and amplifies endocrine β- and δ-cells.
We conclude that HDAC4, -5, and -9 are key regulators to control the pancreatic β/δ-cell lineage. These results highlight the epigenetic mechanisms underlying the regulation of endocrine cell development and suggest new strategies for β-cell differentiation-based therapies.
PMCID: PMC3198089  PMID: 21953612
12.  Gene delivery to pancreatic exocrine cells in vivo and in vitro 
BMC Biotechnology  2012;12:74.
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.
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.
In summary, our study remarkably demonstrates that transduction of pancreatic exocrine cells requires lentiviral vectors in vitro but adenoviral vectors in vivo.
PMCID: PMC3487942  PMID: 23088534
Lentiviral vector; Adenoviral vector; Lipofection; Gene transfer; Pancreas; Acinar cell
13.  Plasticity of Adult Human Pancreatic Duct Cells by Neurogenin3-Mediated Reprogramming 
PLoS ONE  2012;7(5):e37055.
Duct cells isolated from adult human pancreas can be reprogrammed to express islet beta cell genes by adenoviral transduction of the developmental transcription factor neurogenin3 (Ngn3). In this study we aimed to fully characterize the extent of this reprogramming and intended to improve it.
The extent of the Ngn3-mediated duct-to-endocrine cell reprogramming was measured employing genome wide mRNA profiling. By modulation of the Delta-Notch signaling or addition of pancreatic endocrine transcription factors Myt1, MafA and Pdx1 we intended to improve the reprogramming.
Ngn3 stimulates duct cells to express a focused set of genes that are characteristic for islet endocrine cells and/or neural tissues. This neuro-endocrine shift however, is incomplete with less than 10% of full duct-to-endocrine reprogramming achieved. Transduction of exogenous Ngn3 activates endogenous Ngn3 suggesting auto-activation of this gene. Furthermore, pancreatic endocrine reprogramming of human duct cells can be moderately enhanced by inhibition of Delta-Notch signaling as well as by co-expressing the transcription factor Myt1, but not MafA and Pdx1.
The results provide further insight into the plasticity of adult human duct cells and suggest measurable routes to enhance Ngn3-mediated in vitro reprogramming protocols for regenerative beta cell therapy in diabetes.
PMCID: PMC3351393  PMID: 22606327
14.  Metabolic acidosis induced by Plasmodium falciparum intraerythrocytic stages alters blood–brain barrier integrity 
The pathogenesis of cerebral malaria (CM) remains largely unknown. There is growing evidence that combination of both parasite and host factors could be involved in blood–brain barrier (BBB) breakdown. However, lack of adequate in vitro model of human BBB so far hampered molecular studies. In this article, we propose the use of hCMEC/D3 cells, a well-established human cerebral microvascular endothelial cell (EC) line, to study BBB breakdown induced by Plasmodium falciparum-parasitized red blood cells and environmental conditions. We show that coculture of parasitized erythrocytes with hCMEC/D3 cells induces cell adhesion and paracellular permeability increase, which correlates with disorganization of zonula occludens protein 1 expression pattern. Permeability increase and modification of tight junction proteins distribution are cytoadhesion independent. Finally, we show that permeability of hCMEC/D3 cell monolayers is mediated through parasite induced metabolic acidosis, which in turns correlates with apoptosis of parasitized erythrocytes. This new coculture model represents a very useful tool, which will improve the knowledge of BBB breakdown and the development of adjuvant therapies, together with antiparasitic drugs.
PMCID: PMC3049507  PMID: 20683453
brain endothelial cell line; cerebral malaria; human ICAM-1; metabolic acidosis; permeability; Plasmodium falciparum
15.  Ectopic PDX-1 Expression Directly Reprograms Human Keratinocytes along Pancreatic Insulin-Producing Cells Fate 
PLoS ONE  2011;6(10):e26298.
Cellular differentiation and lineage commitment have previously been considered irreversible processes. However, recent studies have indicated that differentiated adult cells can be reprogrammed to pluripotency and, in some cases, directly into alternate committed lineages. However, although pluripotent cells can be induced in numerous somatic cell sources, it was thought that inducing alternate committed lineages is primarily only possible in cells of developmentally related tissues. Here, we challenge this view and analyze whether direct adult cell reprogramming to alternate committed lineages can cross the boundaries of distinct developmental germ layers.
Methodology/Principal Findings
We ectopically expressed non-integrating pancreatic differentiation factors in ectoderm-derived human keratinocytes to determine whether these factors could directly induce endoderm-derived pancreatic lineage and β-cell-like function. We found that PDX-1 and to a lesser extent other pancreatic transcription factors, could rapidly and specifically activate pancreatic lineage and β-cell-like functional characteristics in ectoderm-derived human keratinocytes. Human keratinocytes transdifferentiated along the β cell lineage produced processed and secreted insulin in response to elevated glucose concentrations. Using irreversible lineage tracing for KRT-5 promoter activity, we present supporting evidence that insulin-positive cells induced by ectopic PDX-1 expression are generated in ectoderm derived keratinocytes.
These findings constitute the first demonstration of human ectoderm cells to endoderm derived pancreatic cells transdifferentiation. The study represents a proof of concept which suggests that transcription factors induced reprogramming is wider and more general developmental process than initially considered. These results expanded the arsenal of adult cells that can be used as a cell source for generating functional endocrine pancreatic cells. Directly reprogramming somatic cells into alternate desired tissues has important implications in developing patient-specific, regenerative medicine approaches.
PMCID: PMC3196540  PMID: 22028850
16.  Insulin-Producing Cells Generated from Dedifferentiated Human Pancreatic Beta Cells Expanded In Vitro 
PLoS ONE  2011;6(9):e25566.
Expansion of beta cells from the limited number of adult human islet donors is an attractive prospect for increasing cell availability for cell therapy of diabetes. However, attempts at expanding human islet cells in tissue culture result in loss of beta-cell phenotype. Using a lineage-tracing approach we provided evidence for massive proliferation of beta-cell-derived (BCD) cells within these cultures. Expansion involves dedifferentiation resembling epithelial-mesenchymal transition (EMT). Epigenetic analyses indicate that key beta-cell genes maintain open chromatin structure in expanded BCD cells, although they are not transcribed. Here we investigated whether BCD cells can be redifferentiated into beta-like cells.
Methodology/Principal Finding
Redifferentiation conditions were screened by following activation of an insulin-DsRed2 reporter gene. Redifferentiated cells were characterized for gene expression, insulin content and secretion assays, and presence of secretory vesicles by electron microscopy. BCD cells were induced to redifferentiate by a combination of soluble factors. The redifferentiated cells expressed beta-cell genes, stored insulin in typical secretory vesicles, and released it in response to glucose. The redifferentiation process involved mesenchymal-epithelial transition, as judged by changes in gene expression. Moreover, inhibition of the EMT effector SLUG (SNAI2) using shRNA resulted in stimulation of redifferentiation. Lineage-traced cells also gave rise at a low rate to cells expressing other islet hormones, suggesting transition of BCD cells through an islet progenitor-like stage during redifferentiation.
These findings demonstrate for the first time that expanded dedifferentiated beta cells can be induced to redifferentiate in culture. The findings suggest that ex-vivo expansion of adult human islet cells is a promising approach for generation of insulin-producing cells for transplantation, as well as basic research, toxicology studies, and drug screening.
PMCID: PMC3184150  PMID: 21984932
17.  A genetically engineered human pancreatic β cell line exhibiting glucose-inducible insulin secretion 
The Journal of Clinical Investigation  2011;121(9):3589-3597.
Despite intense efforts over the past 30 years, human pancreatic β cell lines have not been available. Here, we describe a robust technology for producing a functional human β cell line using targeted oncogenesis in human fetal tissue. Human fetal pancreatic buds were transduced with a lentiviral vector that expressed SV40LT under the control of the insulin promoter. The transduced buds were then grafted into SCID mice so that they could develop into mature pancreatic tissue. Upon differentiation, the newly formed SV40LT-expressing β cells proliferated and formed insulinomas. The resulting β cells were then transduced with human telomerase reverse transcriptase (hTERT), grafted into other SCID mice, and finally expanded in vitro to generate cell lines. One of these cell lines, EndoC-βH1, expressed many β cell–specific markers without any substantial expression of markers of other pancreatic cell types. The cells secreted insulin when stimulated by glucose or other insulin secretagogues, and cell transplantation reversed chemically induced diabetes in mice. These cells represent a unique tool for large-scale drug discovery and provide a preclinical model for cell replacement therapy in diabetes. This technology could be generalized to generate other human cell lines when the cell type–specific promoter is available.
PMCID: PMC3163974  PMID: 21865645
18.  Human Liver Cells Expressing Albumin and Mesenchymal Characteristics Give Rise to Insulin-Producing Cells 
Journal of Transplantation  2011;2011:252387.
Activation of the pancreatic lineage in the liver has been suggested as a potential autologous cell replacement therapy for diabetic patients. Transcription factors-induced liver-to-pancreas reprogramming has been demonstrated in numerous species both in vivo and in vitro. However, human-derived liver cells capable of acquiring the alternate pancreatic repertoire have never been characterized. It is yet unknown whether hepatic-like stem cells or rather adult liver cells give rise to insulin-producing cells. Using an in vitro experimental system, we demonstrate that proliferating adherent human liver cells acquire mesenchymal-like characteristics and a considerable level of cellular plasticity. However, using a lineage-tracing approach, we demonstrate that insulin-producing cells are primarily generated in cells enriched for adult hepatic markers that coexpress both albumin and mesenchymal markers. Taken together, our data suggest that adult human hepatic tissue retains a substantial level of developmental plasticity, which could be exploited in regenerative medicine approaches.
PMCID: PMC3163017  PMID: 21876779
19.  Mesenchymal Bone Morphogenetic Protein Signaling Is Required for Normal Pancreas Development 
Diabetes  2010;59(8):1948-1956.
Pancreas organogenesis is orchestrated by interactions between the epithelium and the mesenchyme, but these interactions are not completely understood. Here we investigated a role for bone morphogenetic protein (BMP) signaling within the pancreas mesenchyme and found it to be required for the normal development of the mesenchyme as well as for the pancreatic epithelium.
We analyzed active BMP signaling by immunostaining for phospho-Smad1,5,8 and tested whether pancreas development was affected by BMP inhibition after expression of Noggin and dominant negative BMP receptors in chicken and mouse pancreas.
Endogenous BMP signaling is confined to the mesenchyme in the early pancreas and inhibition of BMP signaling results in severe pancreatic hypoplasia with reduced epithelial branching. Notably, we also observed an excessive endocrine differentiation when mesenchymal BMP signaling is blocked, presumably secondary to defective mesenchyme to epithelium signaling.
We conclude that BMP signaling plays a previously unsuspected role in the mesenchyme, required for normal development of the mesenchyme as well as for the epithelium.
PMCID: PMC2911072  PMID: 20522595
21.  Insulin Storage and Glucose Homeostasis in Mice Null for the Granule Zinc Transporter ZnT8 and Studies of the Type 2 Diabetes–Associated Variants 
Diabetes  2009;58(9):2070-2083.
Zinc ions are essential for the formation of hexameric insulin and hormone crystallization. A nonsynonymous single nucleotide polymorphism rs13266634 in the SLC30A8 gene, encoding the secretory granule zinc transporter ZnT8, is associated with type 2 diabetes. We describe the effects of deleting the ZnT8 gene in mice and explore the action of the at-risk allele.
Slc30a8 null mice were generated and backcrossed at least twice onto a C57BL/6J background. Glucose and insulin tolerance were measured by intraperitoneal injection or euglycemic clamp, respectively. Insulin secretion, electrophysiology, imaging, and the generation of adenoviruses encoding the low- (W325) or elevated- (R325) risk ZnT8 alleles were undertaken using standard protocols.
ZnT8−/− mice displayed age-, sex-, and diet-dependent abnormalities in glucose tolerance, insulin secretion, and body weight. Islets isolated from null mice had reduced granule zinc content and showed age-dependent changes in granule morphology, with markedly fewer dense cores but more rod-like crystals. Glucose-stimulated insulin secretion, granule fusion, and insulin crystal dissolution, assessed by total internal reflection fluorescence microscopy, were unchanged or enhanced in ZnT8−/− islets. Insulin processing was normal. Molecular modeling revealed that residue-325 was located at the interface between ZnT8 monomers. Correspondingly, the R325 variant displayed lower apparent Zn2+ transport activity than W325 ZnT8 by fluorescence-based assay.
ZnT8 is required for normal insulin crystallization and insulin release in vivo but not, remarkably, in vitro. Defects in the former processes in carriers of the R allele may increase type 2 diabetes risks.
PMCID: PMC2731533  PMID: 19542200
22.  Quantitative Comparison of Constitutive Promoters in Human ES cells 
PLoS ONE  2010;5(8):e12413.
Constitutive promoters that ensure sustained and high level gene expression are basic research tools that have a wide range of applications, including studies of human embryology and drug discovery in human embryonic stem cells (hESCs). Numerous cellular/viral promoters that ensure sustained gene expression in various cell types have been identified but systematic comparison of their activities in hESCs is still lacking.
Methodology/Principal Findings
We have quantitatively compared promoter activities of five commonly used constitutive promoters, including the human β-actin promoter (ACTB), cytomegalovirus (CMV), elongation factor-1α, (EF1α), phosphoglycerate kinase (PGK) and ubiquitinC (UbC) in hESCs. Lentiviral gene transfer was used to ensure stable integration of promoter-eGFP constructs into the hESCs genome. Promoter activities were quantitatively compared in long term culture of undifferentiated hESCs and in their differentiated progenies.
The ACTB, EF1α and PGK promoters showed stable activities during long term culture of undifferentiated hESCs. The ACTB promoter was superior by maintaining expression in 75–80% of the cells after 50 days in culture. During embryoid body (EB) differentiation, promoter activities of all five promoters decreased. Although the EF1α promoter was downregulated in approximately 50% of the cells, it was the most stable promoter during differentiation. Gene expression analysis of differentiated eGFP+ and eGFP- cells indicate that promoter activities might be restricted to specific cell lineages, suggesting the need to carefully select optimal promoters for constitutive gene expression in differentiated hESCs.
PMCID: PMC2928720  PMID: 20865032
23.  The ectopic expression of Pax4 in the mouse pancreas converts progenitor cells into α- and subsequently β-cells 
Cell  2009;138(3):449-462.
PMCID: PMC2792203  PMID: 19665969
Facultative endocrine progenitor cell; Endocrine pancreas development; Arx; Pax4; Mouse; Diabetes; Fate specification
24.  ASPM-associated stem cell proliferation is involved in malignant progression of gliomas and constitutes an attractive therapeutic target 
ASPM (Abnormal Spindle-like Microcephaly associated) over-expression was recently implicated in the development of malignant gliomas.
To better characterize the involvement of ASPM in gliomas, we investigated the mRNA expression in 175 samples, including 8 WHO Grade II, 75 WHO Grade III and 92 WHO Grade IV tumors. Aspm expression was strongly correlated with tumor grade and increased at recurrence when compared to the initial lesion, whatever the initial grade of the primary tumor. ASPM expression also increased over serial passages in gliomaspheres in vitro and in mouse xenografts in vivo. Lentivirus-mediated shRNA silencing of ASPM resulted in dramatic proliferation arrest and cell death in two different gliomasphere models.
These data suggest that ASPM is involved in the malignant progression of gliomas, possibly through expansion of a cancer stem cell compartment, and is an attractive therapeutic target in glioblastoma multiforme.
PMCID: PMC2817685  PMID: 20142996
25.  Epithelial-Mesenchymal Transition in Cells Expanded In Vitro from Lineage-Traced Adult Human Pancreatic Beta Cells 
PLoS ONE  2009;4(7):e6417.
In-vitro expansion of functional beta cells from adult human islets is an attractive approach for generating an abundant source of cells for beta-cell replacement therapy of diabetes. Using genetic cell-lineage tracing we have recently shown that beta cells cultured from adult human islets undergo rapid dedifferentiation and proliferate for up to 16 population doublings. These cells have raised interest as potential candidates for redifferentiation into functional insulin-producing cells. Previous work has associated dedifferentiation of cultured epithelial cells with epithelial-mesenchymal transition (EMT), and suggested that EMT generates cells with stem cell properties. Here we investigated the occurrence of EMT in these cultures and assessed their stem cell potential.
Methodology/Principal Findings
Using cell-lineage tracing we provide direct evidence for occurrence of EMT in cells originating from beta cells in cultures of adult human islet cells. These cells express multiple mesenchymal markers, as well as markers associated with mesenchymal stem cells (MSC). However, we do not find evidence for the ability of such cells, nor of cells in these cultures derived from a non-beta-cell origin, to significantly differentiate into mesodermal cell types.
These findings constitute the first demonstration based on genetic lineage-tracing of EMT in cultured adult primary human cells, and show that EMT does not induce multipotency in cells derived from human beta cells.
PMCID: PMC2712769  PMID: 19641613

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