Pancreatic Lineage Research Articles

An expression profile analysis of ES cell-derived definitive endodermal cells and Pdx1-expressing cells

BackgroundWe developed an efficient in vitro method to differentiate mouse ES cells into the definitive endoderm (DE) and then Pdx1-expressing pancreatic lineages using mesodermal-derived supporting cells, M15. Using this method, resulting ES cell-derived DE and Pdx1-expressing cells were isolated by cell sorting, and their gene expression profiles were investigated with DNA microarray. Genes that were specifically expressed in DE and/or in Pdx1-expressing cells were extracted and their expression patterns in normal embryonic development were studied.ResultsGenes whose expression increased in DE and Pdx1 positive cells compared to the undifferentiated ES cells were chosen and in situ hybridizations were performed. Out of 54 genes examined, 27 were expressed in the DE of E8.5 mouse embryos and 15 genes were expressed in distinct domains in the pancreatic buds of E14.5 embryos. Among those genes expressed were Foxq1, CpM, Foxp4, Pcdh1, and Zmiz1, which were previously reported in other endodermal tissues. Genes, such as Parm1, Tmem184a, Hipk2 and Sox4 were reported to be expressed during early pancreatic development. Nptx2, C2cd4b, Tcf7l2 and Kiss1r were reported to be associated with beta cell or pancreatic functions in the adult. Akr1c19, Aebp2, Pbxip1 and Creb3l1, were novel and have not been described as being expressed either in DE or the pancreas.ConclusionsWe identified 27 genes, including 4 novel genes expressed in DE and pancreatic progenitor cells during normal development using an ES cell in vitro differentiation system. These results showed that DE cells and Pdx1/GFP-expressing cells obtained from our M15 based differentiation method mimic cells during the normal developmental processes. Additionally, ES cells are an excellent model for studies of early developmental processes.

In vitro models of pancreatic differentiation using embryonic stem or induced pluripotent stem cells

Embryonic stem (ES) cells or induced pluripotent stem (iPS) cells are expected as a surrogate cell source for regenerative medicine. Many researchers have reported the differentiation method of insulin-expressing pancreatic β cells from ES or iPS cells. However, the detailed molecular mechanisms underlying the differentiation of ES or iPS cells into pancreatic lineages are still unclear. We have established a feeder cell-based differentiation system into pancreatic progenitor cells, and revealed the signaling pathways that are involved in the differentiation of ES cells into mesendoderm, endoderm and pancreatic progenitor cells. Recently, we demonstrated that the extracellular environment, particularly the laminin-integrin signaling and heparan sulfate proteoglycan, is important for the regionalization of definitive endoderm cells into pancreatic lineages. These results provide new insights for the differentiation mechanism of pancreatic cell lineages.

Differentiation of Dental Pulp Stem Cells into Islet-like Aggregates

The post-natal dental pulp tissue contains a population of multipotent mesenchymal progenitor cells known as dental pulp stromal/stem cells (DPSCs), with high proliferative potential for self-renewal. In this investigation, we explored the potential of DPSCs to differentiate into pancreatic cell lineage resembling islet-like cell aggregates (ICAs). We isolated, propagated, and characterized DPSCs and demonstrated that these could be differentiated into adipogenic, chondrogenic, and osteogenic lineage upon exposure to an appropriate cocktail of differentiating agents. Using a three-step protocol reported previously by our group, we succeeded in obtaining ICAs from DPSCs. The identity of ICAs was confirmed as islets by dithiozone-positive staining, as well as by expression of C-peptide, Pdx-1, Pax4, Pax6, Ngn3, and Isl-1. There were several-fold up-regulations of these transcription factors proportional to days of differentiation as compared with undifferentiated DPSCs. Day 10 ICAs released insulin and C-peptide in a glucose-dependent manner, exhibiting in vitro functionality. Our results demonstrated for the first time that DPSCs could be differentiated into pancreatic cell lineage and offer an unconventional and non-controversial source of human tissue that could be used for autologous stem cell therapy in diabetes.

Differentiation of nestin-positive cells derived from bone marrow into pancreatic endocrine and ductal cells in vitro

Promising results of pancreatic islet transplantation to treat type 1 diabetes mellitus, combined with severe shortage of donor pancreata, have spurred efforts to generate pancreatic islet-like cells and insulin-producing β-cells from various progenitor populations. In this study, we show for the first time that multipotent nestin-positive stem cells selected from rat bone marrow can be differentiated into pancreatic ductal and insulin-producing β-cells in vitro. We report an effective multistep protocol in a serum-free system, which could efficiently induce β-cell differentiation from multipotent nestin-positive bone marrow stem cells. To enhance the induction and differentiation toward pancreatic lineage we used trichostatin A, an important regulator of chromatin remodeling, and 5-aza 2' deoxycytidine, an inhibitor of DNA methylase. All-trans retinoic acid was then utilized to promote pancreatic differentiation. We sequentially induced important transcription factor genes, such as Pdx1, Ngn3, and Pax6, following the in vivo development timeline of the pancreas in rats. Furthermore, in the final stage with the presence of nicotinamide, the induced cells expressed islet and ductal specific markers. The differentiated cells not only expressed insulin and glucose transporter 2, but also displayed a glucose-responsive secretion of the hormone. Our results delineate a new model system to study islet neogenesis and possible pharmaceutical targets. Nestin-positive bone marrow stem cells may be therapeutically relevant for β-cell replacement in type 1 diabetes.

Stage-specific signaling through TGFβ family members and WNT regulates patterning and pancreatic specification of human pluripotent stem cells

The generation of insulin-producing β-cells from human pluripotent stem cells is dependent on efficient endoderm induction and appropriate patterning and specification of this germ layer to a pancreatic fate. In this study, we elucidated the temporal requirements for TGFβ family members and canonical WNT signaling at these developmental stages and show that the duration of nodal/activin A signaling plays a pivotal role in establishing an appropriate definitive endoderm population for specification to the pancreatic lineage. WNT signaling was found to induce a posterior endoderm fate and at optimal concentrations enhanced the development of pancreatic lineage cells. Inhibition of the BMP signaling pathway at specific stages was essential for the generation of insulin-expressing cells and the extent of BMP inhibition required varied widely among the cell lines tested. Optimal stage-specific manipulation of these pathways resulted in a striking 250-fold increase in the levels of insulin expression and yielded populations containing up to 25% C-peptide+ cells.

Differentiation of mouse embryonic stem cells into insulin-secreting cells in vitro

Regenerative medicine, including cell-replacement strategies, may have an important role in the treatment of type 1 diabetes which is associated with decreased islet cell mass. To date, significant progress has been made in generating insulin-secreting β cells from pluripotent mouse embryonic stem cells (ESCs). The aim of this study is to explore the potential of regulating the differentiation of ESCs into pancreatic endocrine cells capable of synthesizing the pancreatic hormones including insulin, glucagon, somatostatin and pancreatic polypeptide under proper conditions. Undifferentiated ES cell line was stably transfected with mouse RIP-YFP plasmid construction in serum-free medium using Lipofectamine™ 2000 Reagents. We tested pancreatic specific gene expression and characterized these ESC-derived pancreatic endocrine cells. Most of these insulin-secreting cells co-expressed many of the phenotypic markers characteristic of β cells such as insulin1, insulin2, Islet1, MafA, insulinoma-associated antigen 1 (IA1) and so on, indicating a similar gene expression pattern to adult islet β cells in vivo. Characterization of this population revealed that it consisted predominantly of pancreatic endocrine cells that were able to undergo pancreatic specification under the appropriate conditions. We also demonstrated that zinc supplementation mediated up-regulation of insulin-secreting cells as an effective inducer promoted the development of ESC-derived diabetes therapy. In conclusion, this work not only established an efficient pancreatic differentiation strategy from ESCs to pancreatic endocrine lineage in vitro, but also leaded to the development of new strategies to derive transplantable islet-replacement β cells from embryonic stem cells for the future applications of a stem cell based therapy of diabetes.

MiRNAs control insulin content in pancreatic β-cells via downregulation of transcriptional repressors

MicroRNAs (miRNAs) were shown to be important for pancreas development, yet their roles in differentiated β-cells remain unclear. Here, we show that miRNA inactivation in β-cells of adult mice results in a striking diabetic phenotype. While islet architecture is intact and differentiation markers are maintained, Dicer1-deficient β-cells show a dramatic decrease in insulin content and insulin mRNA. As a consequence of the change in insulin content, the animals become diabetic. We provide evidence for involvement of a set of miRNAs in regulating insulin synthesis. The specific knockdown of miR-24, miR-26, miR-182 or miR-148 in cultured β-cells or in isolated primary islets downregulates insulin promoter activity and insulin mRNA levels. Further, miRNA-dependent regulation of insulin expression is associated with upregulation of transcriptional repressors, including Bhlhe22 and Sox6. Thus, miRNAs in the adult pancreas act in a new network that reinforces insulin expression by reducing the expression of insulin transcriptional repressors.

Stage-specific signaling through TGFβ family members and WNT regulates patterning and pancreatic specification of human pluripotent stem cells

The generation of insulin-producing β-cells from human pluripotent stem cells is dependent on efficient endoderm induction and appropriate patterning and specification of this germ layer to a pancreatic fate. In this study, we elucidated the temporal requirements for TGFβ family members and canonical WNT signaling at these developmental stages and show that the duration of nodal/activin A signaling plays a pivotal role in establishing an appropriate definitive endoderm population for specification to the pancreatic lineage. WNT signaling was found to induce a posterior endoderm fate and at optimal concentrations enhanced the development of pancreatic lineage cells. Inhibition of the BMP signaling pathway at specific stages was essential for the generation of insulin-expressing cells and the extent of BMP inhibition required varied widely among the cell lines tested. Optimal stage-specific manipulation of these pathways resulted in a striking 250-fold increase in the levels of insulin expression and yielded populations containing up to 25% C-peptide+ cells.

Human Liver Cells Expressing Albumin and Mesenchymal Characteristics Give Rise to Insulin-Producing Cells

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.

Endodermal progenitor cells isolated from mouse pancreas

In this study we have isolated and identified a progenitor cell population located in murine pancreatic tissue. This cell population has specific properties that make it different from previously described candidate pancreatic progenytor cell populations. It is located in the periductal region outside the islet architecture yet the cells have features consistent with endodermal development and endocrine functions within the islet. Cells are proliferative and capable of differentiation into pancreatic lineages expressing pancreatic associated transcription factors and proteins and maintaining the ability to produce insulin.

Musculoskeletal Tissue Engineering with Human Umbilical Cord mesenchymal Stromal Cells

Multipotent mesenchymal stromal cells (MSCs) hold tremendous promise for tissue engineering and regenerative medicine, yet with so many sources of MSCs, what are the primary criteria for selecting leading candidates? Ideally, the cells will be multipotent, inexpensive, lack donor site morbidity, donor materials should be readily available in large numbers, immunocompatible, politically benign and expandable in vitro for several passages. Bone marrow MSCs do not meet all of these criteria and neither do embryonic stem cells. However, a promising new cell source is emerging in tissue engineering that appears to meet these criteria: MSCs derived from Wharton's jelly of umbilical cord MSCs. Exposed to appropriate conditions, umbilical cord MSCs can differentiate in vitro along several cell lineages such as the chondrocyte, osteoblast, adipocyte, myocyte, neuronal, pancreatic or hepatocyte lineages. In animal models, umbilical cord MSCs have demonstrated in vivo differentiation ability and promising immunocompatibility with host organs/tissues, even in xenotransplantation. In this article, we address their cellular characteristics, multipotent differentiation ability and potential for tissue engineering with an emphasis on musculoskeletal tissue engineering.

NKX6.1 Promotes PDX-1-Induced Liver to Pancreatic β-Cells Reprogramming

Reprogramming adult mammalian cells is an attractive approach for generating cell-based therapies for degenerative diseases, such as diabetes. Adult human liver cells exhibit a high level of developmental plasticity and have been suggested as a potential source of pancreatic progenitor tissue. An instructive role for dominant pancreatic transcription factors in altering the hepatic developmental fate along the pancreatic lineage and function has been demonstrated. Here we analyze whether transcription factors expressed in mature pancreatic β-cells preferentially activate β-cell lineage differentiation in liver. NKX6.1 is a transcription factor uniquely expressed in β-cells of the adult pancreas, its potential role in reprogramming liver cells to pancreatic lineages has never been analyzed. Our results suggest that NKX6.1 activates immature pancreatic markers such as NGN-3 and ISL-1 but not pancreatic hormones gene expression in human liver cells. We hypothesized that its restricted capacity to activate a wide pancreatic repertoire in liver could be related to its incapacity to activate endogenous PDX-1 expression in liver cells. Indeed, the complementation of NKX6.1 by ectopic PDX-1 expression substantially and specifically promoted insulin expression and glucose regulated processed hormone secretion to a higher extent than that of PDX-1 alone, without increasing the reprogrammed cells. This may suggest a potential role for NKX6.1 in promoting PDX-1 reprogrammed cells maturation along the β-cell-like lineage. By contrast, NKX6.1 repressed PDX-1 induced proglucagon gene expression. The individual and concerted effects of pancreatic transcription factors in adult extra-pancreatic cells, is expected to facilitate developing regenerative medicine approaches for cell replacement therapy in diabetics.

Reversal of experimental diabetes in mice by transplantation of neo-islets generated from human amnion-derived mesenchymal stromal cells using immuno-isolatory macrocapsules

The ethical and biologic limitations with current sources of stem cells have resulted in a quest to look for alternative sources of multipotent stem cells of human origin. Amniotic membrane is of interest as a source of cells for regenerative medicine because of its ease of availability, plasticity and inexhaustible source that does not violate the sanctity of independent life. Although researchers have shown the stem cell-like potential of human amniotic epithelial cells, the mesenchymal part of amnion has remained less explored. We established a long-term culture of mesenchymal-like stem cells derived from full-term human amniotic membrane and their differentiation into functional pancreatic lineage. The amnion-derived mesenchymal-like stem cells expressed various mesenchymal markers and demonstrated multilineage differentiation capacity. We also observed that these cells could form islet-like clusters (ILC) on exposure to serum-free defined media containing specific growth factor and differentiating agents. Differentiated ILC showed expression of human insulin, glucagon and somatostatin by immunocytochemistry, while quantitative reverse transcription/real-time-polymerase chain reaction (qRT-PCR) data demonstrated the expression of insulin, glucagon, somatostatin, Ngn3 and Isl1. Moreover, encapsulation of the ILC in polyurethane-polyvinyl pyrrolidone macrocapsules and their subsequent transplantation in experimental diabetic mice resulted in restoration of normoglycemia, indicating their ability to respond to high glucose without immunorejection. Our results demonstrate that amnion-derived mesenchymal stromal cells can undergo islet neogenesis, indicating amnion as an alternative source of islets for cell replacement therapy in diabetes.

Taurine Supports Preservation of Proendocrine Cell Types in Human Umbilical Cord Blood-Derived Mononuclear Cells During Cryostorage

Background We demonstrated earlier that a subset of human umbilical cord-blood (hUCB)-derived mononuclear cells (MNCs) express proendocrine transcription factors and exhibit potential to differentiate into endocrine pancreatic lineage. The growing interest in the use of MNCs for diabetes has promoted cryopreservation of these cells for future use in translational research. Development of optimal cryopreservation media is critical to the success of translational research. Methods We explored protective effects of taurine in cryopreservation of hUCB-derived MNCs. MNCs were isolated using Histopaque and 3 million viable cells were cryofrozen using 10% dimethyl sulfoxide (v/v) in fetal bovine serum or supplemented with taurine (0.3/3.0 mmol/L). Cryopreservation conditions were assessed based on their viability, growth, and ability to retain endocrine pancreatic transcription factor expressing cells. Results UCB-derived MNCs adhered and grew as mesenchymal-like cells in culture following revival from various cryopreservation conditions. Interestingly, MNCs expressed threefold more ngn3, 2-fold more nkx6.1, and 15-fold more isl1 transcripts in taurine-supplemented cryo-medium compared to the conventional cryomix. Conclusion The present study demonstrates that taurine supplementation to cryopreservation media improved retention of endocrine pancreatic transcription factor-expressing MNCs.

Differentiation of Mouse Embryonic Stem Cells into Endoderm without Embryoid Body Formation

Pluripotent embryonic stem cells hold a great promise as an unlimited source of tissue for treatment of chronic diseases such as Type 1 diabetes. Herein, we describe a protocol using all-trans-retinoic acid, basic fibroblast growth factor and dibutyryl cAMP (DBcAMP) in the absence of embryoid body formation, for differentiation of murine embryonic stem cells into definitive endoderm that may serve as pancreatic precursors. The produced cells were analyzed by quantitative PCR, immunohistochemistry and static insulin release assay for markers of trilaminar embryo, and pancreas. Differentiated cells displayed increased Sox17 and Foxa2 expression consistent with definitive endoderm production. There was minimal production of Sox7, an extraembryonic endoderm marker, and Oct4, a marker of pluripotency. There was minimal mesoderm or neuroectoderm formation based on expression levels of the markers brachyury and Sox1, respectively. Various assays revealed that the cell clusters generated by this protocol express markers of the pancreatic lineage including insulin I, insulin II, C-peptide, PDX-1, carboxypeptidase E, pan-cytokeratin, amylase, glucagon, PAX6, Ngn3 and Nkx6.1. This protocol using all-trans-retinoic acid, DBcAMP, in the absence of embryoid bodies, generated cells that have features of definitive endoderm that may serve as pancreatic endocrine precursors.

Human bone marrow-derived mesenchymal cells differentiate and mature into endocrine pancreatic lineage in vivo

The scarcity of human islets for transplantation remains a major limitation of cell replacement therapy for diabetes. Bone marrow-derived progenitor cells are of interest because they can be isolated, expanded and offered for such therapy under autologous/allogeneic settings. We characterized and compared human bone marrow-derived mesenchymal cells (hBMC) obtained from (second trimester), young (1-24 years) and adult (34-81 years) donors. We propose a novel protocol that involves assessment of paracrine factors from regenerating pancreas in differentiation and maturation of hBMC into endocrine pancreatic lineage in vivo. We observed that donor age was inversely related to growth potential of hBMC. Following in vitro expansion and exposure to specific growth factors involved in pancreatic development, hBMC migrated and formed islet-like cell aggregates (ICA). ICA show increased abundance of pancreatic transcription factors (Ngn3, Brn4, Nkx6.1, Pax6 and Isl1). Although efficient differentiation was not achieved in vitro, we observed significant maturation and secretion of human c-peptide (insulin) upon transplantation into pancreactomized and Streptozotocin (STZ)-induced diabetic mice. Transplanted ICA responded to glucose and maintained normoglycemia in diabetic mice. Our data demonstrate that hBMC have tremendous in vitro expansion potential and can be differentiated into multiple lineages, including the endocrine pancreatic lineage. Paracrine factors secreted from regenerating pancreas help in efficient differentiation and maturation of hBMC, possibly via recruiting chromatin modulators, to generate glucose-responsive insulin-secreting cells.

O16. Pancreatic stem cells

The search for putative precursor cells within the pancreas has been the focus of extensive research. Rare adult mouse pancreas-derived multipotent precursor (PMP) cells, possessing the intriguing capacity to generate cross-germ layer progeny in the pancreatic and neural lineages, have been identified. Here, genetic lineage-labeling experiments excluded the neural crest, and established the embryonic pancreas, as the developmental source of PMPs. The PMP cell is shown to express insulin in vivo, providing reconciliation with reports that new adult β-cells are formed exclusively by self-replication. These insulin+stem cells were found to contribute to multiple pancreatic and neural cell populations in vivo. Further, PMP cells were isolated from adult human islet tissue, each capable of extensive proliferation, self-renewal, and generation of multiple differentiated pancreatic and neural cell types, including≤cell progeny displaying regulated insulin secretion. Finally, both mouse and human PMP-derived cells were shown to ameliorate diabetes in transplanted mice. These findings demonstrate that the adult mammalian pancreas contains a population of insulin+multipotent stem cells.

Dachshund homologues play a conserved role in islet cell development

All metazoans use insulin to control energy metabolism, but they secrete it from different cells: neurons in the central nervous system in invertebrates and endocrine cells in the gut or pancreas in vertebrates. Despite their origins in different germ layers, all of these insulin-producing cells share common functional features and gene expression patterns. In this study, we tested the role in insulin-producing cells of the vertebrate homologues of Dachshund, a transcriptional regulator that marks the earliest committed progenitors of the neural insulin-producing cells in Drosophila. Both zebrafish and mice expressed a single dominant Dachshund homologue in the pancreatic endocrine lineage, and in both species loss of this homologue reduced the numbers of all islet cell types including the insulin-producing β-cells. In mice, Dach1 gene deletion left the pancreatic progenitor cells unaltered, but blocked the perinatal burst of proliferation of differentiated β-cells that normally generates most of the β-cell mass. In β-cells, Dach1 bound to the promoter of the cell cycle inhibitor p27Kip1, which constrains β-cell proliferation. Taken together, these data demonstrate a conserved role for Dachshund homologues in the production of insulin-producing cells.

A factor(s) secreted from MIN-6 β-cells stimulates differentiation of definitive endoderm enriched embryonic stem cells towards a pancreatic lineage

In the mouse the developing pancreas is controlled by contact with, and signalling molecules secreted from, surrounding cells. These factors are best studied using explant cultures of embryonic tissue. The present study was undertaken to determine whether embryonic stem (ES) cells could be used as an alternative model in vitro system to investigate the role of cell–cell interactions in the developing pancreas. Transwell culture experiments showed that MIN-6 β-cells secreted a factor or factors that promoted differentiation of ES cell derived definitive endoderm enriched cells towards a pancreatic fate. Further studies using MIN-6 condition medium showed that the factor(s) involved was restricted to MIN-6 cells, could be concentrated with ammonium sulphate, and was sensitive to heat treatment, suggesting that it was a protein or peptide. Further analyses showed that insulin or proinsulin failed to mimic the effects of the conditioned media. Collectively, these results suggest that β-cells secrete a factor(s) capable of controlling their own differentiation and maturation. The culture system described here presents unique advantages in the identification and characterisation of these factors.

Synthesized basement membranes direct the differentiation of mouse embryonic stem cells into pancreatic lineages

We previously reported that embryonic stem (ES) cells cultured on M15 cells, a mesoderm-derived supportive cell line, were efficiently differentiated towards an endodermal fate, finally adopting the specific lineages of various digestive organs such as the pancreas and liver. We show here that the endoderm-inducing activity of M15 cells is in part mediated through the extracellular matrices, and that laminin alpha5 is one of the crucial components. In an attempt to establish a feeder-free ES-cell procedure for pancreatic differentiation, we used a synthesized basement membrane (sBM) substratum using an HEK293 cell line stably expressing laminin-511. On the sBM, mouse ES or induced pluripotent stem (iPS) cells sequentially differentiated into the definitive endoderm, pancreatic progenitor cells, and then insulin-expressing pancreatic beta-cells in vitro. Knockdown of ES cells with integrin beta1 (Itgb1) reduces differentiation towards pancreatic cells. Heparan sulfate proteoglycan 2 (HSPG2) knockdown and heparitinase treatment synergistically decreased the number of Pdx1-expressing cells. These findings indicate that components of the basement membrane have an important role in the differentiation of definitive endoderm lineages. This novel procedure will be useful for the study of pancreatic differentiation of ES or iPS cells and the generation of potential sources of surrogate cells for regenerative medicine.