Juan Domínguez-Bendala

Multipotent stem/progenitor cells in human biliary tree give rise to hepatocytes, cholangiocytes, and pancreatic islets

Multipotent stem/progenitors are present in peribiliary glands of extrahepatic biliary trees from humans of all ages and in high numbers in hepato‐pancreatic common duct, cystic duct, and hilum. They express endodermal transcription factors (e.g., Sox9, SOX17, FOXA2, PDX1, HES1, NGN3, PROX1) intranuclearly, stem/progenitor surface markers (EpCAM, NCAM, CD133, CXCR4), and sometimes weakly adult liver, bile duct, and pancreatic genes (albumin, cystic fibrosis transmembrane conductance regulator [CFTR], and insulin). They clonogenically expand on plastic and in serum‐free medium, tailored for endodermal progenitors, remaining phenotypically stable as undifferentiated cells for months with a cell division initially every ≈36 hours and slowing to one every 2‐3 days. Transfer into distinct culture conditions, each comprised of a specific mix of hormones and matrix components, yields either cords of hepatocytes (express albumin, CYP3A4, and transferrin), branching ducts of cholangiocytes (expressing anion exchanger‐2‐AE2 and CFTR), or regulatable C‐peptide secreting neoislet‐like clusters (expressing glucagon, insulin) and accompanied by changes in gene expression correlating with the adult fate. Transplantation into quiescent livers of immunocompromised mice results in functional human hepatocytes and cholangiocytes, whereas if into fat pads of streptozocin‐induced diabetic mice, results in functional islets secreting glucose‐regulatable human C‐peptide. Conclusion: The phenotypes and availability from all age donors suggest that these stem/progenitors have considerable potential for regenerative therapies of liver, bile duct, and pancreatic diseases including diabetes. (HEPATOLOGY2011;)

MicroRNA expression in alpha and beta cells of human pancreatic islets.

microRNAs (miRNAs) play an important role in pancreatic development and adult β-cell physiology. Our hypothesis is based on the assumption that each islet cell type has a specific pattern of miRNA expression. We sought to determine the profile of miRNA expression in α-and β-cells, the main components of pancreatic islets, because this analysis may lead to a better understanding of islet gene regulatory pathways. Highly enriched (>98%) subsets of human α-and β-cells were obtained by flow cytometric sorting after intracellular staining with c-peptide and glucagon antibody. The method of sorting based on intracellular staining is possible because miRNAs are stable after fixation. MiRNA expression levels were determined by quantitative high throughput PCR-based miRNA array platform screening. Most of the miRNAs were preferentially expressed in β-cells. From the total of 667 miRNAs screened, the Significant Analysis of Microarray identified 141 miRNAs, of which only 7 were expressed more in α-cells (α-miRNAs) and 134 were expressed more in β-cells (β-miRNAs). Bioinformatic analysis identified potential targets of β-miRNAs analyzing the Beta Cell Gene Atlas, described in the T1Dbase, the web platform, supporting the type 1 diabetes (T1D) community. cMaf, a transcription factor regulating glucagon expression expressed selectively in α-cells (TFα) is targeted by β-miRNAs; miR-200c, miR-125b and miR-182. Min6 cells treated with inhibitors of these miRNAs show an increased expression of cMaf RNA. Conversely, over expression of miR-200c, miR-125b or miR-182 in the mouse alpha cell line αTC6 decreases the level of cMAF mRNA and protein. MiR-200c also inhibits the expression of Zfpm2, a TFα that inhibits the PI3K signaling pathway, at both RNA and protein levels. In conclusion, we identified miRNAs differentially expressed in pancreatic α- and β-cells and their potential transcription factor targets that could add new insights into different aspects of islet biology and pathophysiology.

Concise Review: Mesenchymal Stem Cells for Diabetes

Mesenchymal stem cells (MSCs) have already made their mark in the young field of regenerative medicine. Easily derived from many adult tissues, their therapeutic worth has already been validated for a number of conditions. Unlike embryonic stem cells, neither their procurement nor their use is deemed controversial. Here we review the potential use of MSCs for the treatment of type 1 diabetes mellitus, a devastating chronic disease in which the insulin‐producing cells of the pancreas (the β‐cells) are the target of an autoimmune process. It has been hypothesized that stem cell‐derived β‐cells may be used to replenish the islet mass in diabetic patients, making islet transplantation (a form of cell therapy that has already proven effective at clinically restoring normoglycemia) available to millions of prospective patients. Here we review the most current advances in the design and application of protocols for the differentiation of transplantable β‐cells, with a special emphasis in analyzing MSC potency according to their tissue of origin. Although no single method appears to be ripe enough for clinical trials yet, recent progress in reprogramming (a biotechnological breakthrough that relativizes the thus far insurmountable barriers between embryonal germ layers) bodes well for the rise of MSCs as a potential weapon of choice to develop personalized therapies for type 1 diabetes.

Enhanced oxygenation promotes β-cell differentiation in vitro

Despite progress in our knowledge about pancreatic islet specification, most attempts at differentiating stem/progenitor cells into functional, transplantable β cells have met only with moderate success thus far. A major challenge is the intrinsic simplicity of in vitro culture systems, which cannot approximate the physiological complexity of in vivo microenvironments. Oxygenation is a critical limitation of standard culture methods, and one of special relevance for the development of β cells, known for their high O2 requirements. Based on our understanding of islet physiology, we have tested the hypothesis that enhanced O2 delivery (as provided by novel perfluorocarbon‐based culture devices) may result in higher levels of β‐cell differentiation from progenitor cells in vitro. Using a mouse model of pancreatic development, we demonstrate that a physiological‐like mode of O2 delivery results in a very significant upregulation of endocrine differentiation markers (up to 30‐fold for insulin one and 2), comparable to relevant in vivo controls. This effect was not observed by merely increasing environmental O2 concentrations in conventional settings. Our findings indicate that O2 plays an important role in the differentiation of β cells from their progenitors and may open the door to more efficient islet differentiation protocols from embryonic and/or adult stem cells.

Antisense miR-7 Impairs Insulin Expression in Developing Pancreas and in Cultured Pancreatic Buds

MicroRNAs regulate gene expression by inhibiting translation or inducing target mRNA degradation. MicroRNAs regulate organ differentiation and embryonic development, including pancreatic specification and islet function. We showed previously that miR-7 is highly expressed in human pancreatic fetal and adult endocrine cells. Here we determined the expression profile of miR-7 in the mouse-developing pancreas by RT-PCR and in situ hybridization. MiR-7 expression was low between embryonic days e10.5 and e11.5, then began to increase at e13.5 through e14.5, and eventually decreased by e18. In situ hybridization and immunostaining analysis showed that miR-7 colocalizes with endocrine marker Isl1, suggesting that miR-7 is expressed preferentially in endocrine cells. Whole-mount in situ hybridization shows miR-7 highly expressed in the embryonic neural tube. To investigate the role of miR-7 in development of the mouse endocrine pancreas, antisense miR-7 morpholinos (MO) were delivered to the embryo at an early developmental stage (e10.5 days) via intrauterine fetal heart injection. Inhibition of miR-7 during early embryonic life results in an overall downregulation of insulin production, decreased β-cell numbers, and glucose intolerance in the postnatal period. This phenomenon is specific for miR-7 and possibly due to a systemic effect on pancreatic development. On the other hand, the in vitro inhibition of miR-7 in explanted pancreatic buds leads to β-cell death and generation of β-cells expressing less insulin than those in MO control. Therefore, in addition to the potential indirect effects on pancreatic differentiation derived from its systemic downregulation, the knockdown of miR-7 appears to have a β-cell-specific effect as well. These findings suggest that modulation of miR-7 expression could be utilized in the development of stem cell therapies to cure diabetes.

Generation of Glucose-Responsive, Insulin-Producing Cells from Human Umbilical Cord Blood-Derived Mesenchymal Stem Cells

We sought to assess the potential of human cord blood-derived mesenchymal stem cells (CB-MSCs) to derive insulin-producing, glucose-responsive cells. We show here that differentiation protocols based on stepwise culture conditions initially described for human embryonic stem cells (hESCs) lead to differentiation of cord blood-derived precursors towards a pancreatic endocrine phenotype, as assessed by marker expression and in vitro glucose-regulated insulin secretion. Transplantation of these cells in immune-deficient animals shows human C-peptide production in response to a glucose challenge. These data suggest that human cord blood may be a promising source for regenerative medicine approaches for the treatment of diabetes mellitus.

MicroRNA signature of the human developing pancreas

BackgroundMicroRNAs are non-coding RNAs that regulate gene expression including differentiation and development by either inhibiting translation or inducing target degradation. The aim of this study is to determine the microRNA expression signature during human pancreatic development and to identify potential microRNA gene targets calculating correlations between the signature microRNAs and their corresponding mRNA targets, predicted by bioinformatics, in genome-wide RNA microarray study.ResultsThe microRNA signature of human fetal pancreatic samples 10-22 weeks of gestational age (wga), was obtained by PCR-based high throughput screening with Taqman Low Density Arrays. This method led to identification of 212 microRNAs. The microRNAs were classified in 3 groups: Group number I contains 4 microRNAs with the increasing profile; II, 35 microRNAs with decreasing profile and III with 173 microRNAs, which remain unchanged. We calculated Pearson correlations between the expression profile of microRNAs and target mRNAs, predicted by TargetScan 5.1 and miRBase altgorithms, using genome-wide mRNA expression data. Group I correlated with the decreasing expression of 142 target mRNAs and Group II with the increasing expression of 876 target mRNAs. Most microRNAs correlate with multiple targets, just as mRNAs are targeted by multiple microRNAs. Among the identified targets are the genes and transcription factors known to play an essential role in pancreatic development.ConclusionsWe have determined specific groups of microRNAs in human fetal pancreas that change the degree of their expression throughout the development. A negative correlative analysis suggests an intertwined network of microRNAs and mRNAs collaborating with each other. This study provides information leading to potential two-way level of combinatorial control regulating gene expression through microRNAs targeting multiple mRNAs and, conversely, target mRNAs regulated in parallel by other microRNAs as well. This study may further the understanding of gene expression regulation in the human developing pancreas.

Cell Replacement Strategies Aimed at Reconstitution of the β-Cell Compartment in Type 1 Diabetes

Emerging technologies in regenerative medicine have the potential to restore the β-cell compartment in diabetic patients, thereby overcoming the inadequacies of current treatment strategies and organ supply. Novel approaches include: 1) Encapsulation technology that protects islet transplants from host immune surveillance; 2) stem cell therapies and cellular reprogramming, which seek to regenerate the depleted β-cell compartment; and 3) whole-organ bioengineering, which capitalizes on the innate properties of the pancreas extracellular matrix to drive cellular repopulation. Collaborative efforts across these subfields of regenerative medicine seek to ultimately produce a bioengineered pancreas capable of restoring endocrine function in patients with insulin-dependent diabetes.

Regeneration of pancreatic beta-cell mass for the treatment of diabetes

Introduction: The study of the endocrine compartment of the pancreas (the islets of Langerhans) is of great translational interest, as strategies aimed at restoring its mass could become therapies for glycemic dysregulation in type 1 and 2 diabetes mellitus, drug-related diabetes following diabetogenic therapies or hyperglycemic disturbances following the treatment of cancer and nesidioblastosis. Such strategies generally fall under one of the ‘three Rs,’ namely, replacement (islet transplantation and stem cell differentiation), reprogramming (chiefly from the exocrine compartment of the pancreas) and regeneration (replication and induction of endogenous stem cells). Areas covered: This expert opinion focuses on the latter, as islets are known to regenerate under specific circumstances of physiological (e.g., pregnancy), pathological (e.g., obesity, hyperglycemia, mutations in the glucose-sensing pathway) or experimental (e.g., partial pancreatectomy, cellophane wrapping, partial duct ligation) nature. This review presents the different models of pancreatic regeneration, which encompass the replication of existing beta-cells, reversible epithelial-to-mesenchymal transition and the reactivation of resident stem cells. Expert opinion: Rather than a set mechanism, the pancreas appears to possess a wide range of facultative regeneration pathways. These are discussed in the context of the development of potential strategies aimed at restoring beta-cell function in insulin-dependent diabetes.

Oxygen: a master regulator of pancreatic development?

Beyond its role as an electron acceptor in aerobic respiration, oxygen is also a key effector of many developmental events. The oxygen‐sensing machinery and the very fabric of cell identity and function have been shown to be deeply intertwined. Here we take a first look at how oxygen might lie at the crossroads of at least two of the major molecular pathways that shape pancreatic development. Based on recent evidence and a thorough review of the literature, we present a theoretical model whereby evolving oxygen tensions might choreograph to a large extent the sequence of molecular events resulting in the development of the organ. In particular, we propose that lower oxygenation prior to the expansion of the vasculature may favour HIF (hypoxia inducible factor)‐mediated activation of Notch and repression of Wnt/β‐catenin signalling, limiting endocrine cell differentiation. With the development of vasculature and improved oxygen delivery to the developing organ, HIF‐mediated support for Notch signalling may decline while the β‐catenin‐directed Wnt signalling is favoured, which would support endocrine cell differentiation and perhaps exocrine cell proliferation/differentiation.