Inhibition of activin/nodal signalling is necessary for pancreatic differentiation of human pluripotent stem cells

Peer reviewedPublisher PD

Impaired Spleen Formation Perturbs Morphogenesis of the Gastric Lobe of the Pancreas

Despite the extensive use of the mouse as a model for studies of pancreas development and disease, the development of the gastric pancreatic lobe has been largely overlooked. In this study we use optical projection tomography to provide a detailed three-dimensional and quantitative description of pancreatic growth dynamics in the mouse. Hereby, we describe the epithelial and mesenchymal events leading to the formation of the gastric lobe of the pancreas. We show that this structure forms by perpendicular growth from the dorsal pancreatic epithelium into a distinct lateral domain of the dorsal pancreatic mesenchyme. Our data support a role for spleen organogenesis in the establishment of this mesenchymal domain and in mice displaying perturbed spleen development, including Dh +/−, Bapx1−/− and Sox11−/−, gastric lobe development is disturbed. We further show that the expression profile of markers for multipotent progenitors is delayed in the gastric lobe as compared to the splenic and duodenal pancreatic lobes. Altogether, this study provides new information regarding the developmental dynamics underlying the formation of the gastric lobe of the pancreas and recognizes lobular heterogeneities regarding the time course of pancreatic cellular differentiation. Collectively, these data are likely to constitute important elements in future interpretations of the developing and/or diseased pancreas

Cyclic Expression of Lhx2 Regulates Hair Formation

Hair is important for thermoregulation, physical protection, sensory activity, seasonal camouflage, and social interactions. Hair is generated in hair follicles (HFs) and, following morphogenesis, HFs undergo cyclic phases of active growth (anagen), regression (catagen), and inactivity (telogen) throughout life. The transcriptional regulation of this process is not well understood. We show that the transcription factor Lhx2 is expressed in cells of the outer root sheath and a subpopulation of matrix cells during both morphogenesis and anagen. As the HFs enter telogen, expression becomes undetectable and reappears prior to initiation of anagen in the secondary hair germ. In contrast to previously published results, we find that Lhx2 is primarily expressed by precursor cells outside of the bulge region where the HF stem cells are located. This developmental, stage- and cell-specific expression suggests that Lhx2 regulates the generation and regeneration of hair. In support of this hypothesis, we show that Lhx2 is required for anagen progression and HF morphogenesis. Moreover, transgenic expression of Lhx2 in postnatal HFs is sufficient to induce anagen. Thus, our results reveal an alternative interpretation of Lhx2 function in HFs compared to previously published results, since Lhx2 is periodically expressed, primarily in precursor cells distinct from those in the bulge region, and is an essential positive regulator of hair formation

Potential Pathways to Restore β-Cell Mass: Pluripotent Stem Cells, Reprogramming, and Endogenous Regeneration

Currently available β-cell replacement therapies for patients with diabetes, including islet and pancreas transplantation, are largely successful in restoring normal glucose metabolism, but the scarcity of organ donors restricts their more widespread use. To solve this supply problem, several different strategies for achieving β-cell mass restoration are being pursued. These include the generation of β cells from stem cells and their subsequent transplantation, or regeneration-type approaches, such as stimulating endogenous regenerative mechanisms or inducing reprogramming of non-β cells into β cells. Because these strategies would ultimately generate allogeneic or syngeneic β cells in humans, the control of alloimmunity and/or autoimmunity in addition to replacing lost β cells will be of utmost importance. We briefly review the recent literature on these three promising strategies toward β-cell replacement or restoration and point out the major issues impacting their translation to treating human diabetes

Pdx1 and Ngn3 Overexpression Enhances Pancreatic Differentiation of Mouse ES Cell-Derived Endoderm Population

In order to define the molecular mechanisms regulating the specification and differentiation of pancreatic β-islet cells, we investigated the effect of upregulating Pdx1 and Ngn3 during the differentiation of the β-islet-like cells from murine embryonic stem (ES) cell-derived activin induced-endoderm. Induced overexpression of Pdx1 resulted in a significant upregulation of insulin (Ins1 and Ins2), and other pancreas-related genes. To enhance the developmental progression from the pancreatic bud to the formation of the endocrine lineages, we induced the overexpression express of Ngn3 together with Pdx1. This combination dramatically increased the level and timing of maximal Ins1 mRNA expression to approximately 100% of that found in the βTC6 insulinoma cell line. Insulin protein and C-peptide expression was confirmed by immunohistochemistry staining. These inductive effects were restricted to c-kit+ endoderm enriched EB-derived populations suggesting that Pdx1/Ngn3 functions after the specification of pancreatic endoderm. Although insulin secretion was stimulated by various insulin secretagogues, these cells had only limited glucose response. Microarray analysis was used to evaluate the expression of a broad spectrum of pancreatic endocrine cell-related genes as well as genes associated with glucose responses. Taken together, these findings demonstrate the utility of manipulating Pdx1 and Ngn3 expression in a stage-specific manner as an important new strategy for the efficient generation of functionally immature insulin-producing β-islet cells from ES cells

Mouse Transgenesis Identifies Conserved Functional Enhancers and cis-Regulatory Motif in the Vertebrate LIM Homeobox Gene Lhx2 Locus

The vertebrate Lhx2 is a member of the LIM homeobox family of transcription factors. It is essential for the normal development of the forebrain, eye, olfactory system and liver as well for the differentiation of lymphoid cells. However, despite the highly restricted spatio-temporal expression pattern of Lhx2, nothing is known about its transcriptional regulation. In mammals and chicken, Crb2, Dennd1a and Lhx2 constitute a conserved linkage block, while the intervening Dennd1a is lost in the fugu Lhx2 locus. To identify functional enhancers of Lhx2, we predicted conserved noncoding elements (CNEs) in the human, mouse and fugu Crb2-Lhx2 loci and assayed their function in transgenic mouse at E11.5. Four of the eight CNE constructs tested functioned as tissue-specific enhancers in specific regions of the central nervous system and the dorsal root ganglia (DRG), recapitulating partial and overlapping expression patterns of Lhx2 and Crb2 genes. There was considerable overlap in the expression domains of the CNEs, which suggests that the CNEs are either redundant enhancers or regulating different genes in the locus. Using a large set of CNEs (810 CNEs) associated with transcription factor-encoding genes that express predominantly in the central nervous system, we predicted four over-represented 8-mer motifs that are likely to be associated with expression in the central nervous system. Mutation of one of them in a CNE that drove reporter expression in the neural tube and DRG abolished expression in both domains indicating that this motif is essential for expression in these domains. The failure of the four functional enhancers to recapitulate the complete expression pattern of Lhx2 at E11.5 indicates that there must be other Lhx2 enhancers that are either located outside the region investigated or divergent in mammals and fishes. Other approaches such as sequence comparison between multiple mammals are required to identify and characterize such enhancers

New Approaches in the Differentiation of Human Embryonic Stem Cells and Induced Pluripotent Stem Cells toward Hepatocytes

Orthotropic liver transplantation is the only established treatment for end-stage liver diseases. Utilization of hepatocyte transplantation and bio-artificial liver devices as alternative therapeutic approaches requires an unlimited source of hepatocytes. Stem cells, especially embryonic stem cells, possessing the ability to produce functional hepatocytes for clinical applications and drug development, may provide the answer to this problem. New discoveries in the mechanisms of liver development and the emergence of induced pluripotent stem cells in 2006 have provided novel insights into hepatocyte differentiation and the use of stem cells for therapeutic applications. This review is aimed towards providing scientists and physicians with the latest advancements in this rapidly progressing field

Investigating the bona fide differentiation capacity of human pluripotent stem cells

10.1038/cr.2011.142Cell Research2216-

The polarity protein Scrib is essential for directed endothelial cell migration

RATIONALE: Polarity proteins are involved in the apico-basal orientation of epithelial cells, but relatively little is known regarding their function in mesenchymal cells. OBJECTIVE: We hypothesized that polarity proteins also contribute to endothelial processes like angiogenesis. METHODS AND RESULTS: Screening of endothelial cells revealed high expression of the polarity protein Scribble (Scrib). On fibronectin-coated carriers Scrib siRNA (siScrib) blocked directed but not random migration of human umbilical vein endothelial cells and led to an increased number and disturbed orientation of cellular lamellipodia. Coimmunoprecipitation/mass spectrometry and glutathione S-transferase (GST) pulldown assays identified integrin α5 as a novel Scrib interacting protein. By total internal reflection fluorescence (TIRF) microscopy, Scrib and integrin α5 colocalize at the basal plasma membrane of endothelial cells. Western blot and fluorescence activated cell sorting (FACS) analysis revealed that silencing of Scrib reduced the protein amount and surface expression of integrin α5 whereas surface expression of integrin αV was unaffected. Moreover, in contrast to fibronectin, the ligand of integrin α5, directional migration on collagen mediated by collagen-binding integrins was unaffected by siScrib. Mechanistically, Scrib supported integrin α5 recycling and protein stability by blocking its interaction with Rab7a, its translocation into lysosomes, and its subsequent degradation by pepstatin-sensitive proteases. In siScrib-treated cells, reinduction of the wild-type protein but not of PSD95, Dlg, ZO-1 (PDZ), or leucine rich repeat domain mutants restored integrin α5 abundance and directional cell migration. The downregulation of Scrib function in Tg(kdrl:EGFP)(s843) transgenic zebrafish embryos delayed the angiogenesis of intersegmental vessels. CONCLUSIONS: Scrib is a novel regulator of integrin α5 turnover and sorting, which is required for oriented cell migration and sprouting angiogenesis