Vincenzo Cirulli

Derivation of naïve human embryonic stem cells

Significance We report on generation of nontransgenic, naïve human pluripotent cells that represent the developmentally earliest state described for human established cells. Existing human ES cell lines in the later primed state can be toggled in reverse to naïve by exposure to histone deacetylase inhibitors prior to naïve culture. A new line was established directly from an eight-cell embryo under naïve culture conditions. We describe the naïve state in humans and show that naïve human ES cells have expanded endoderm developmental capacity. The naïve pluripotent state has been shown in mice to lead to broad and more robust developmental potential relative to primed mouse epiblast cells. The human naïve ES cell state has eluded derivation without the use of transgenes, and forced expression of OCT4, KLF4, and KLF2 allows maintenance of human cells in a naïve state [Hanna J, et al. (2010) Proc Natl Acad Sci USA 107(20):9222–9227]. We describe two routes to generate nontransgenic naïve human ES cells (hESCs). The first is by reverse toggling of preexisting primed hESC lines by preculture in the histone deacetylase inhibitors butyrate and suberoylanilide hydroxamic acid, followed by culture in MEK/ERK and GSK3 inhibitors (2i) with FGF2. The second route is by direct derivation from a human embryo in 2i with FGF2. We show that human naïve cells meet mouse criteria for the naïve state by growth characteristics, antibody labeling profile, gene expression, X-inactivation profile, mitochondrial morphology, microRNA profile and development in the context of teratomas. hESCs can exist in a naïve state without the need for transgenes. Direct derivation is an elusive, but attainable, process, leading to cells at the earliest stage of in vitro pluripotency described for humans. Reverse toggling of primed cells to naïve is efficient and reproducible.

Controlled induction of human pancreatic progenitors produces functional beta-like cells in vitro

Directed differentiation of human pluripotent stem cells into functional insulin‐producing beta‐like cells holds great promise for cell replacement therapy for patients suffering from diabetes. This approach also offers the unique opportunity to study otherwise inaccessible aspects of human beta cell development and function in vitro. Here, we show that current pancreatic progenitor differentiation protocols promote precocious endocrine commitment, ultimately resulting in the generation of non‐functional polyhormonal cells. Omission of commonly used BMP inhibitors during pancreatic specification prevents precocious endocrine formation while treatment with retinoic acid followed by combined EGF/KGF efficiently generates both PDX1+ and subsequent PDX1+/NKX6.1+ pancreatic progenitor populations, respectively. Precise temporal activation of endocrine differentiation in PDX1+/NKX6.1+ progenitors produces glucose‐responsive beta‐like cells in vitro that exhibit key features of bona fide human beta cells, remain functional after short‐term transplantation, and reduce blood glucose levels in diabetic mice. Thus, our simplified and scalable system accurately recapitulates key steps of human pancreas development and provides a fast and reproducible supply of functional human beta‐like cells.

EpCAM: structure and function in health and disease.

Injection of tumor cells in mice more than 30 years ago resulted in the discovery of an epithelial antigen, later defined as a cell adhesion molecule (EpCAM). Although EpCAM has since evoked significant interest as a target in cancer therapy, mechanistic insights on the functions of this glycoprotein have been emerging only very recently. This may have been caused by the multitude of functions attributed to the glycoprotein, its localization at different subcellular sites and complex posttranslational modifications. Here, we review how EpCAM modifies cell-cell contact adhesion strength and tissue plasticity, and how it regulates cell proliferation and differentiation. Major knowledge derived from human diseases will be highlighted: Mutant EpCAM that is absent from the cell surface leads to fatal intestinal abnormalities (congenital tufting enteropathy). EpCAM-mediated cell proliferation in cancer may result from signaling (i) via regulated intramembrane proteolysis and/or (ii) the localization and association with binding partners in specialized membrane microdomains. New insight in EpCAM signaling will help to develop optimized cancer therapies and open new avenues in the field of regenerative medicine.

Anchored phosphatases modulate glucose homeostasis

Endocrine release of insulin principally controls glucose homeostasis. Nutrient‐induced exocytosis of insulin granules from pancreatic β‐cells involves ion channels and mobilization of Ca2+ and cyclic AMP (cAMP) signalling pathways. Whole‐animal physiology, islet studies and live‐β‐cell imaging approaches reveal that ablation of the kinase/phosphatase anchoring protein AKAP150 impairs insulin secretion in mice. Loss of AKAP150 impacts L‐type Ca2+ currents, and attenuates cytoplasmic accumulation of Ca2+ and cAMP in β‐cells. Yet surprisingly AKAP150 null animals display improved glucose handling and heightened insulin sensitivity in skeletal muscle. More refined analyses of AKAP150 knock‐in mice unable to anchor protein kinase A or protein phosphatase 2B uncover an unexpected observation that tethering of phosphatases to a seven‐residue sequence of the anchoring protein is the predominant molecular event underlying these metabolic phenotypes. Thus anchored signalling events that facilitate insulin secretion and glucose homeostasis may be set by AKAP150 associated phosphatase activity.

β1 integrin is a crucial regulator of pancreatic β-cell expansion

Development of the endocrine compartment of the pancreas, as represented by the islets of Langerhans, occurs through a series of highly regulated events encompassing branching of the pancreatic epithelium, delamination and differentiation of islet progenitors from ductal domains, followed by expansion and three-dimensional organization into islet clusters. Cellular interactions with the extracellular matrix (ECM) mediated by receptors of the integrin family are postulated to regulate key functions in these processes. Yet, specific events regulated by these receptors in the developing pancreas remain unknown. Here, we show that ablation of the β1 integrin gene in developing pancreatic β-cells reduces their ability to expand during embryonic life, during the first week of postnatal life, and thereafter. Mice lacking β1 integrin in insulin-producing cells exhibit a dramatic reduction of the number of β-cells to only ∼18% of wild-type levels. Despite the significant reduction in β-cell mass, these mutant mice are not diabetic. A thorough phenotypic analysis of β-cells lacking β1 integrin revealed a normal expression repertoire of β-cell markers, normal architectural organization within islet clusters, and a normal ultrastructure. Global gene expression analysis revealed that ablation of this ECM receptor in β-cells inhibits the expression of genes regulating cell cycle progression. Collectively, our results demonstrate that β1 integrin receptors function as crucial positive regulators of β-cell expansion.

ECM Signaling Regulates Collective Cellular Dynamics to Control Pancreas Branching Morphogenesis

During pancreas development, epithelial buds undergo branching morphogenesis to form an exocrine and endocrine gland. Proper morphogenesis is necessary for correct lineage allocation of pancreatic progenitors; however, the cellular events underlying pancreas morphogenesis are unknown. Here, we employed time-lapse microscopy and fluorescent labeling of cells to analyze cell behaviors associated with pancreas morphogenesis. We observed that outer bud cells adjacent to the basement membrane are pleomorphic and rearrange frequently; additionally, they largely remain in the outer cell compartment even after mitosis. These cell behaviors and pancreas branching depend on cell contacts with the basement membrane, which induce actomyosin cytoskeleton remodeling via integrin-mediated activation of FAK/Src signaling. We show that integrin signaling reduces E-cadherin-mediated cell-cell adhesion in outer cells and provide genetic evidence that this regulation is necessary for initiation of branching. Our study suggests that regulation of cell motility and adhesion by local niche cues initiates pancreas branching morphogenesis.

Cadherins in Islet β-Cells: More Than Meets the Eye

In 1975, Orci et al. (1) reported that human islet cells contain specialized membrane domains that are compatible with the ultrastructural features of two types of intercellular junctions: tight junctions and gap junctions. Since then, numerous reports have demonstrated critical functions for these cell–cell junctional complexes in islet cells (2–8). Eventually, a number of proteins were identified that regulated cell aggregation, islet cell–type segregation, architectural organization within islets of Langerhans, and state of differentiation, cell growth, and hormone secretion (9–16). Hints that direct islet cell-to-islet cell interactions are required for proper insulin secretion were uncovered in the 1980s when it was observed that single (isolated) β-cells are unresponsive to glucose unless they are given the opportunity to reaggregate into small clusters (17). Even more interesting, it was observed that islet cell types harbor specific cell-to-cell recognition signatures that drive their reaggregation into organoids that have architectural organization indistinguishable from that of native islets (18). These earlier observations have inspired numerous investigations that have led to a more complete understanding of mechanisms regulating islet cell development, architectural organization, and function. In a time of considerable interest in the development of cell-based replacement therapies for diabetes, lessons learned over the past three decades on the function of cell adhesion molecules in islet cells harbor significant translational implications. Hence, promoting the function of select members of the cadherin and integrin families of adhesion receptors …

SEL1L regulates adhesion, proliferation and secretion of insulin by affecting integrin signaling

SEL1L, a component of the endoplasmic reticulum associated degradation (ERAD) pathway, has been reported to regulate the (i) differentiation of the pancreatic endocrine and exocrine tissue during the second transition of mouse embryonic development, (ii) neural stem cell self-renewal and lineage commitment and (iii) cell cycle progression through regulation of genes related to cell-matrix interaction. Here we show that in the pancreas the expression of SEL1L is developmentally regulated, such that it is readily detected in developing islet cells and in nascent acinar clusters adjacent to basement membranes, and becomes progressively restricted to the islets of Langherans in post-natal life. This peculiar expression pattern and the presence of two inverse RGD motifs in the fibronectin type II domain of SEL1L protein indicate a possible interaction with cell adhesion molecules to regulate islets architecture. Co-immunoprecipitation studies revealed SEL1L and ß1-integrin interaction and, down-modulation of SEL1L in pancreatic ß-cells, negatively influences both cell adhesion on selected matrix components and cell proliferation likely due to altered ERK signaling. Furthermore, the absence of SEL1L protein strongly inhibits glucose-stimulated insulin secretion in isolated mouse pancreatic islets unveiling an important role of SEL1L in insulin trafficking. This phenotype can be rescued by the ectopic expression of the ß1-integrin subunit confirming the close interaction of these two proteins in regulating the cross-talk between extracellular matrix and insulin signalling to create a favourable micro-environment for ß-cell development and function.

αE-Catenin Is a Positive Regulator of Pancreatic Islet Cell Lineage Differentiation

SUMMARY The development and function of epithelia depend on the establishment and maintenance of cell-cell adhesion and intercellular junctions, which operate as mechanosensor hubs for the transduction of biochemical signals regulating cell proliferation, differentiation, survival, and regeneration. Here, we show that αE-catenin, a key component of adherens junctions, functions as a positive regulator of pancreatic islet cell lineage differentiation by repressing the sonic hedgehog pathway (SHH). Thus, deletion of αE-catenin in multipotent pancreatic progenitors resulted in (1) loss of adherens junctions, (2) constitutive activation of SHH, (3) decrease in islet cell lineage differentiation, and (4) accumulation of immature Sox9+ progenitors. Pharmacological blockade of SHH signaling in pancreatic organ cultures and in vivo rescued this defect, allowing αE-catenin-null Sox9+ pancreatic progenitors to differentiate into endocrine cells. The results uncover crucial functions of αE-catenin in pancreatic islet development and harbor significant implications for the design of β cell replacement and regeneration therapies in diabetes.

Essential Role of Lyn in Fibrosis

Fibrotic disorders involve replacement of normal parenchyma with myofibroblasts, which deposit connective tissue, leading to obliteration of the function of the underlying organ. The treatment options are inadequate and reflect the fact that signaling targets in myofibroblasts are unknown. Here we identify the hyperactive Lyn signaling in myofibroblasts of patients with chronic pancreatitis-induced fibrosis. Lyn activation coexpress with markers of activated myofibroblasts, and is increased ~11-fold in chronic pancreatitis compared to normal tissue. Inhibition of Lyn with siRNA or INNO-406 leads to the substantial decrease of migration and proliferation of human chronic pancreatitis myofibroblasts in vitro, while leaving migration and proliferation of normal myofibroblasts only slightly affected. Furthermore, inhibition of Lyn prevents synthesis of procollagen and collagen in myofibroblasts in a mouse model of chronic pancreatitis-induced fibrosis. We conclude that Lyn, as a positive regulator of myofibroblast migration, proliferation, and collagen production, is a key target for preventing fibrosis.