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CD47 is a cell surface receptor with two primary ligands, soluble thrombospondin-1 (TSP1) and cell surface signal regulatory protein alpha (SIRPα). Both interactions provide checkpoints for immune cell activity. We determined that fresh human islets display CD47 and secrete TSP1. However, human islet endocrine cells lack SIRPα. These gene signatures are likely important given the increasing use of CD47 and SIRPα blocking molecules in individuals with cancer.

Pancreatic ducts function to deliver digestive enzymes into the intestines. Upon injury, ducts can become proliferative and contribute to tissue regeneration; however, the identity of the ductal cells that contribute to these processes is unknown. We combined fluorescence-activated cell sorting, a methylcellulose-containing 3-dimensional culture, droplet RNA-sequencing, and a clonal lineage tracing tool to identify and isolate a distinct subpopulation of pancreatic ductal cells that exhibit progenitor cell properties. These ductal cells are unique in that they form tightly-bound clusters (termed FSCmid-high), with an average of 8 cells per cluster. FSCmid-high clusters comprise only about 0.1% of the total pancreas, are tri-potent for duct, acinar and endocrine lineages, and self-renew robustly in vitro. Transcriptomic analysis of FSCmid-high clusters reveals enrichment for genes involved in cell-cell interactions, organ development, and cancer pathways. FSCmid-high clusters express embryonic pancreatic progenitor markers Sox9, Pdx1, and Nkx6-1 at both transcription and protein levels. FSCmid-high clusters are resistant to enzymatic dissociation and survive severe in vivo acinar injury, which induces formation of ductal rosettes that become proliferative within 14 days. Thus, FSCmid-high clusters represent a small subset of ductal cells with progenitor cell properties. These rare progenitor-like duct cell clusters have implications in pancreas regeneration and tumor initiation/progression.

In pancreatic islet transplantation studies, bioluminescence imaging enables quantitative and noninvasive tracking of graft survival. Amid the recent heightened interest in extrahepatic sites for islet and stem cell-derived beta-like cell transplantations, proper understanding the nature of bioluminescence imaging in these sites is important.Islets isolated from Firefly rats ubiquitously expressing luciferase reporter gene in Lewis rats were transplanted into subcutaneous or kidney capsule sites of wild-type Lewis rats or immunodeficient mice. Posttransplant changes of bioluminescence signal curves and absorption of bioluminescence signal in transplantation sites were examined.The bioluminescence signal curve dynamically changed in the early posttransplantation phase; the signal was low within the first 5 days after transplantation. A substantial amount of bioluminescence signal was absorbed by tissues surrounding islet grafts, correlating to the depth of the transplanted site from the skin surface. Grafts in kidney capsules were harder to image than those in the subcutaneous site. Within the kidney capsule, locations that minimized depth from the skin surface improved the graft detectability.Posttransplant phase and graft location/depth critically impact the bioluminescence images captured in islet transplantation studies. Understanding these parameters is critical for reducing experimental biases and proper interpretation of data.

Background Human pancreata contain many types of cells, such as endocrine islets, acinar, ductal, fat, and mesenchymal stromal cells (MSCs). MSCs are important and shown to have a promising therapeutic potential to treat various disease conditions. Methods We investigated intra-pancreatic tissue-derived (IPTD) MSCs isolated from tissue fractions that are routinely discarded during pancreatic islet isolation of human cadaveric donors. Furthermore, whether pro-angiogenic and anti-inflammatory properties of these cells could be enhanced was investigated. Results IPTD-MSCs were expanded in GMP-compatible CMRL-1066 medium supplemented with 5% human platelet lysate (hPL). IPTD-MSCs were found to be highly pure, with > 95% positive for CD90, CD105, and CD73, and negative for CD45, CD34, CD14, and HLA-DR. Immunofluorescence staining of pancreas tissue demonstrated the presence of CD105+ cells in the vicinity of islets. IPTD-MSCs were capable of differentiation into adipocytes, chondrocytes, and osteoblasts in vitro, underscoring their multipotent features. When these cells were cultured in the presence of a low dose of TNF-α, gene expression of tumor necrosis factor alpha-stimulated gene-6 (TSG-6) was significantly increased, compared to control. In contrast, treating cells with dimethyloxallyl glycine (DMOG) (a prolyl 4-hydroxylase inhibitor) enhanced mRNA levels of nuclear factor erythroid 2-related factor 2 (NRF2) and vascular endothelial growth factor (VEGF). Interestingly, a combination of TNF-α and DMOG stimulated the optimal expression of all three genes in IPTD-MSCs. Conditioned medium of IPTD-MSCs treated with a combination of DMOG and TNF-α contained higher levels of pro-angiogenic (VEGF, IL-6, and IL-8) compared to controls, promoting angiogenesis of human endothelial cells in vitro. In contrast, levels of MCP-1, a pro-inflammatory cytokine, were reduced in the conditioned medium of IPTD-MSCs treated with a combination of DMOG and TNF-α. Conclusions The results demonstrate that IPTD-MSCs reside within the pancreas and can be separated as part of a standard islet-isolation protocol. These IPTD-MSCs can be expanded and potentiated ex vivo to enhance their anti-inflammatory and pro-angiogenic profiles. The fact that IPTD-MSCs are generated in a GMP-compatible procedure implicates a direct clinical application.

The existence and regenerative potential of resident stem and progenitor cells in the adult pancreas are controversial topics. A question that has been only minimally addressed is the capacity of a progenitor cell to self-renew, a key attribute that defines stem cells. Previously, our laboratory has identified putative stem and progenitor cells from the adult murine pancreas. Using an ex vivo colony/organoid culture system, we demonstrated that these stem/progenitor-like cells have self-renewal and multilineage differentiation potential. We have named these cells pancreatic colony-forming units (PCFUs) because they can give rise to three-dimensional colonies. However, the molecular mechanisms by which PCFUs self-renew have remained largely unknown. Here, we tested the hypothesis that PCFU self-renewal requires GLIS family zinc finger 3 (GLIS3), a zinc-finger transcription factor important in pancreas development. Pancreata from 2- to 4-month-old mice were dissociated, sorted for CD133highCD71low ductal cells, known to be enriched for PCFUs, and virally transduced with shRNAs to knock down GLIS3 and other proteins. We then plated these cells into our colony assays and analyzed the resulting colonies for protein and gene expression. Our results revealed a previously unknown GLIS3–to–CD133–to–WNT signaling axis in which GLIS3 and CD133 act as factors necessary for maintaining WNT receptors and signaling molecules in colonies, allowing responses to WNT ligands. Additionally, we found that CD133, but not GLIS3 or WNT, is required for phosphoinositide 3-kinase (PI3K)/AKT Ser/Thr kinase (AKT)-mediated PCFU survival. Collectively, our results uncover a molecular pathway that maintains self-renewal of adult murine PCFUs.

Significant progress has been made in recent years in characterizing human multipotent progenitor cells (hMPCs) of the early pancreas; however, the identity and persistence of these cells during the second trimester, after the initiation of branching morphogenesis, remain elusive. Additionally, studies on hMPCs have been hindered by few isolation methods that allow for the recovery of live cells. Here, we investigated the tip progenitor domain in the branched epithelium of human fetal pancreas between 13.5 and 17.5 gestational weeks by immunohistological staining. We also used a novel RNA‐based technology to isolate live cells followed by gene expression analyses. We identified cells co‐expressing SOX9 and PTF1A, two transcription factors known to be important for pancreatic MPCs, within the tips of the epithelium and observed a decrease in their proportions over time. Pancreatic SOX9+/PTF1A+ cells were enriched for MPC markers, including MYC and GATA6. These cells were proliferative and appeared active in branching morphogenesis and matrix remodeling, as evidenced by gene set enrichment analysis. We identified a hub of genes pertaining to the expanding tip progenitor niche, such as FOXF1, GLI3, TBX3, FGFR1, TGFBR2, ITGAV, ITGA2, and ITGB3. YAP1 of the Hippo pathway emerged as a highly enriched component within the SOX9+/PTF1A+ cells. Single‐cell RNA‐sequencing further corroborated the findings by identifying a cluster of SOX9+/PTF1A+ cells with multipotent characteristics. Based on these results, we propose that the SOX9+/PTF1A+ cells in the human pancreas are uncommitted MPC‐like cells that reside at the tips of the expanding pancreatic epithelium, directing self‐renewal and inducing pancreatic organogenesis. stem cells translational medicine 2019;8:1249&1264, Schematic representation of SOX9+/PTF1A+ cell isolation and characterization.

High-throughput screening (HTS) is a large-scale hierarchical process in which a large number of chemicals are tested in multiple stages. Conventional statistical analyses of HTS studies often suffer from high testing error rates and soaring costs in large-scale settings. This article develops new methodologies for false discovery rate control and optimal design in HTS studies. We propose a two-stage procedure that determines the optimal numbers of replicates at different screening stages while simultaneously controlling the false discovery rate in the confirmatory stage subject to a constraint on the total budget. The merits of the proposed methods are illustrated using both simulated and real data. We show that, at the expense of a limited budget, the proposed screening procedure effectively controls the error rate and the design leads to improved detection power.

The existence and regenerative potential of resident stem and progenitor cells in the adult pancreas are controversial topics. A question that has been only minimally addressed is the capacity of a progenitor cell to self-renew, a key attribute that defines stem cells. Previously, our laboratory has identified putative stem and progenitor cells from the adult murine pancreas. Using an ex vivo colony/organoid culture system, we demonstrated that these stem/progenitor-like cells have self-renewal and multilineage differentiation potential. We have named these cells pancreatic colony-forming units (PCFUs) because they can give rise to three-dimensional colonies. However, the molecular mechanisms by which PCFUs self-renew have remained largely unknown. Here, we tested the hypothesis that PCFU self-renewal requires GLIS family zinc finger 3 (GLIS3), a zinc-finger transcription factor important in pancreas development. Pancreata from 2- to 4-month-old mice were dissociated, sorted for CD133(high)CD71(low) ductal cells, known to be enriched for PCFUs, and virally transduced with shRNAs to knock down GLIS3 and other proteins. We then plated these cells into our colony assays and analyzed the resulting colonies for protein and gene expression. Our results revealed a previously unknown GLIS3–to–CD133–to–WNT signaling axis in which GLIS3 and CD133 act as factors necessary for maintaining WNT receptors and signaling molecules in colonies, allowing responses to WNT ligands. Additionally, we found that CD133, but not GLIS3 or WNT, is required for phosphoinositide 3-kinase (PI3K)/AKT Ser/Thr kinase (AKT)-mediated PCFU survival. Collectively, our results uncover a molecular pathway that maintains self-renewal of adult murine PCFUs.

Obtaining, growing, and analysis of pancreatic progenitor cells. Adult stem and progenitor cells have been successfully used for cell-based therapies such as transplantation of hematopoietic stem cells for various diseases. Whether stem and progenitor cells in the adult pancreas can be identified and used for replacement therapy has been a highly controversial topic. To address this controversy, our laboratory has developed in vitro colony assays to detect and characterize individual pancreatic stem and progenitor-like cells. We found that a subpopulation of ductal cells in the adult murine pancreas has the abilities to self-renew and differentiate into multiple pancreatic lineages in three-dimensional space in methylcellulose-containing semisolid media. This protocol details the techniques used for culturing and characterizing these pancreatic stem and progenitor-like cells, which we have named pancreatic colony-forming units (PCFUs), as well as their progenies (colonies). The techniques presented here include dissociation of pancreases, sorting antibody-stained cells with a fluorescence-activated cell sorter, viral transduction of dissociated pancreatic cells, growth of PCFUs in semi-solid media, whole-mount immunostaining and Western blot analysis for proteins expressed in colonies, and kidney capsule transplantation of colonies for in vivo functional analysis.

Obtaining, growing, and analysis of pancreatic progenitor cells. Adult stem and progenitor cells have been successfully used for cell-based therapies such as transplantation of hematopoietic stem cells for various diseases. Whether stem and progenitor cells in the adult pancreas can be identified and used for replacement therapy has been a highly controversial topic. To address this controversy, our laboratory has developed in vitro colony assays to detect and characterize individual pancreatic stem and progenitor-like cells. We found that a subpopulation of ductal cells in the adult murine pancreas has the abilities to self-renew and differentiate into multiple pancreatic lineages in three-dimensional space in methylcellulose-containing semisolid media. This protocol details the techniques used for culturing and characterizing these pancreatic stem and progenitor-like cells, which we have named pancreatic colony-forming units (PCFUs), as well as their progenies (colonies). The techniques presented here include dissociation of pancreases, sorting antibody-stained cells with a fluorescence-activated cell sorter, viral transduction of dissociated pancreatic cells, growth of PCFUs in semi-solid media, whole-mount immunostaining and Western blot analysis for proteins expressed in colonies, and kidney capsule transplantation of colonies for in vivo functional analysis.

Pluripotent stem cells may serve as an alternative source of beta-like cells for replacement therapy of type 1 diabetes; however, the beta-like cells generated in many differentiation protocols are immature. The maturation of endogenous beta cells involves an increase in insulin expression starting in late gestation and a gradual acquisition of the abilities to sense glucose and secrete insulin by week 2 after birth in mice; however, what molecules regulate these maturation processes are incompletely known. In this study, we aim to identify small molecules that affect immature beta cells. A cell-based assay, using pancreatic beta-like cells derived from murine embryonic stem (ES) cells harboring a transgene containing an insulin 1-promoter driven enhanced green fluorescent protein reporter, was used to screen a compound library (NIH Clinical Collection-003). Cortisone, a glucocorticoid, was among five positive hit compounds. Quantitative reverse transcription–polymerase chain reaction analysis revealed that glucocorticoids enhance the gene expression of not only insulin 1 but also glucose transporter-2 (Glut2; Slc2a2) and glucokinase (Gck), two molecules important for glucose sensing. Mifepristone, a pharmacological inhibitor of glucocorticoid receptor (GR) signaling, reduced the effects of glucocorticoids on Glut2 and Gck expression. The effects of glucocorticoids on ES-derived cells were further validated in immature primary islets. Isolated islets from 1-week-old mice had an increased Glut2 and Gck expression in response to a 4-day treatment of exogenous hydrocortisone in vitro. Gene deletion of GR in beta cells using rat insulin 2 promoter-driven Cre crossed with GR(flox/flox) mice resulted in a reduced gene expression of Glut2, but not Gck, and an abrogation of insulin secretion when islets were incubated in 0.5 mM d-glucose and stimulated by 17 mM d-glucose in vitro. These results demonstrate that glucocorticoids positively regulate glucose sensors in immature murine beta-like cells.

Adult pancreatic stem and progenitor cells may serve as an alternative source of insulin-secreting endocrine cells in cell replacement therapy for type 1 diabetes, but much remained unknown about these cells. We previously identified adult murine pancreatic progenitor-like cells that displayed in vitro self-renewal and tri-lineage differentiation activities in a three-dimensional colony/organoid assay containing 1% methylcellulose and 5% Matrigel. However, the presence of other undefined culture components, such as serum and conditioned medium, has prevented a complete understanding of the signals required for progenitor cell growth. Here, we have established a serum-free, conditioned medium-free colony assay with the inclusion of seven defined factors: epidermal growth factor (EGF), R-Spondin 1 (RSPO1), Noggin, nicotinamide, exendin-4, activin B, and vascular endothelial growth factor (VEGF)-A. The requirements for colony growth were characterized and we found that EGF and nicotinamide were necessary and sufficient for the colony growth and long-term self-renewal of these progenitors. However, the seven factor (7F) culture medium better induced colony size and self-renewal in long-term culture than EGF plus nicotinamide alone. Individual 3-week-old colonies grown in the 7F culture medium expressed ductal, acinar, and endocrine lineage markers, suggesting that tri-lineage differentiation of the tri-potent progenitors was occurring without genetic manipulation. A delayed inhibition of Notch signaling using small molecules in 2-week-old cultures enhanced endocrine gene expression in 3-week-old colonies. This better-defined colony assay system will enable our and other laboratories for in-depth mechanistic studies on the biology of these progenitor cells.

Gastrin is a peptide hormone, which in combination with other factors such as TGFα, EGF or GLP-1, is capable of increasing beta cell mass and lowering blood glucose levels in adult diabetic mice. In humans, administration of a bolus of gastrin alone induces insulin secretion suggesting that gastrin may target islet cells. However, whether gastrin alone is sufficient to exert an effect on isolated human islets has been controversial and the mechanism remained poorly understood. Therefore, in this study we started to examine the effects of gastrin alone on cultured adult human islets. Treatment of isolated human islets with gastrin I for 48 h resulted in increased expression of insulin, glucagon and somatostatin transcripts. These increases were significantly correlated with the levels of donor hemoglobin A1c (HbA1c) but not BMI or age. In addition, gastrin treatment resulted in increased expression of PDX1, NKX6.1, NKX2.2, MNX1 and HHEX in islets from donors with HbA1c greater than 42 mmol/mol. The addition of YM022, an antagonist of the gastrin receptor cholecystokinin B receptor (CCKBR), together with gastrin eliminated these effects, verifying that the effects of gastrin are mediated through CCKBR.CCKBR is expressed in somatostatin-expressing delta cells in islets from all donors. However, in the islets from donors with higher HbA1c (greater than 42 mmol/mol [6.0%]), cells triple-positive for CCKBR, somatostatin and insulin were detected, suggesting a de-differentiation or trans-differentiation of endocrine cells. Our results demonstrate a direct effect of gastrin on human islets from prediabetic or diabetic individuals that is mediated through CCKBR+ cells. Further, our data imply that gastrin may be a potential treatment for diabetic patients.

Ten eleven translocation (TET) enzymes (TET1/TET2/TET3) and thymine DNA glycosylase (TDG) play crucial roles in early embryonic and germ cell development by mediating DNA demethylation. However, the molecular mechanisms that regulate TETs/TDG expression and their role in cellular differentiation, including that of the pancreas, are not known. Here, we report that (i) TET1/2/3 and TDG can be direct targets of the microRNA miR-26a, (ii) murine TETs, especially TET2 and TDG, are down-regulated in islets during postnatal differentiation, whereas miR-26a is up-regulated, (iii) changes in 5-hydroxymethylcytosine accompany changes in TET mRNA levels, (iv) these changes in mRNA and 5-hydroxymethylcytosine are also seen in an in vitro differentiation system initiated with FACS-sorted adult ductal progenitor-like cells, and (v) overexpression of miR-26a in mice increases postnatal islet cell number in vivo and endocrine/acinar colonies in vitro. These results establish a previously unknown link between miRNAs and TET expression levels, and suggest a potential role for miR-26a and TET family proteins in pancreatic cell differentiation.

Past studies of pancreatic progenitor cell biology relied mostly on histological analyses. Recent studies, using genetic labelingandtracingofprogenitors,directsinglecellanalyses, colony assays, and enrichment of the minor population of progenitor cells through the use of cell surface markers, have strongly suggested that pancreatic progenitor cells with various frequency and lineage potentials, including the multipotent progenitors that give rise to endocrine, exocrine, and duct cells, exist in the developing and adult pancreas. In this review, it is therefore proposed that pancreatic progenitor cells may be organized in a hierarchy, in which the most primitive pan-pancreatic multipotent progenitors are at the top and rare, and the monopotent progenitors are at the bottom and abundant. This model may explain why only drastic injuries lead to effective activation of the progenitor cell compartment of the higher hierarchy, whereas under steady state, pregnancy, and milder injuries, recruitment of preexisting mature cells or their immediate monopotent progenitors could be sufficient to restore metabolic homeostasis. It is also proposed that the morphologically defined ductal cells are likely to be functionally heterogeneous and that endocrine progenitor cell activity should be determined based on functional analyses rather than histological locations. (Endocrinology 149: 4312–4316, 2008)

In our previous studies, colony-forming progenitor cells isolated from murine embryonic stem cell-derived cultures were differentiated into morphologically distinct insulin-expressing colonies. These colonies were small and not light-reflective when observed by phase-contrast microscopy (therefore termed “Dark” colonies). A single progenitor cell capable of giving rise to a Dark colony was termed a Dark colony-forming unit (CFU-Dark). The goal of the current study was to test whether endogenous pancreas, and its developmentally related liver, harbored CFU-Dark. Here we show that dissociated single cells from liver and pancreas of one-week-old mice give rise to Dark colonies in methylcellulose-based semisolid culture media containing either Matrigel or laminin hydrogel (an artificial extracellular matrix protein). CFU-Dark comprise approximately 0.1% and 0.03% of the postnatal hepatic and pancreatic cells, respectively. Adult liver also contains CFU-Dark, but at a much lower frequency (~0.003%). Microfluidic qRT-PCR, immunostaining, and electron microscopy analyses of individually handpicked colonies reveal the expression of insulin in many, but not all, Dark colonies. Most pancreatic insulin-positive Dark colonies also express glucagon, whereas liver colonies do not. Liver CFU-Dark require Matrigel, but not laminin hydrogel, to become insulin-positive. In contrast, laminin hydrogel is sufficient to support the development of pancreatic Dark colonies that express insulin. Postnatal liver CFU-Dark display a cell surface marker CD133^(+)CD49f^(low)CD107b^(low) phenotype, while pancreatic CFU-Dark are CD133^-. Together, these results demonstrate that specific progenitor cells in the postnatal liver and pancreas are capable of developing into insulin-expressing colonies, but they differ in frequency, marker expression, and matrix protein requirements for growth.

In a previous study we established colony assays suitable for studying murine adult (2-4 months) pancreatic progenitor cells plated in semisolid media containing methylcellulose and extracellular matrix proteins. Using these assays, we found robust in vitro progenitor cell activities (multilineage differentiation and self-renewal) from pancreatic cells of adult mice in the CD-1 outbred background. However, it was not clear whether progenitor cell activities can be detected from inbred mice, a preferred mouse model for various genetic studies. It was also not clear whether a single cell is sufficient to self-renew. Here, we show that fluorescent activated cell sorting pancreatic CD133(+) but not CD133(-) cells from adult C57Bl/6 inbred mice are enriched for progenitor cells that self-renew and give rise to multilineage colonies in vitro. The number of cells in a colony is in proportion to its diameter. Around 60% of single handpicked 3-week-old colonies express trilineage markers, indicating most progenitors are tripotent for ductal, acinar, and endocrine lineage differentiation. Approximately 80% of primary (freshly sorted) colony-forming progenitor cells are capable of giving rise to secondary progenitors in vitro, indicating that a majority of the primary progenitors self-renew. A single cell is sufficient for self-renewal and a Wnt agonist, R-Spondin1, enhances the number of secondary progenitors from the primary progenitors. Together, our pancreatic colony assays allow quantitative analyses of progenitors at a single-cell level from inbred mice. These assays will be useful for elucidating in vitro mechanisms of pancreatic progenitor cell biology.

The study of hematopoietic colony-forming units using semisolid culture media has greatly advanced the knowledge of hematopoiesis. Here we report that similar methods can be used to study pancreatic colony-forming units. We have developed two pancreatic colony assays that enable quantitative and functional analyses of progenitor-like cells isolated from dissociated adult (2–4 mo old) murine pancreas. We find that a methylcellulose-based semisolid medium containing Matrigel allows growth of duct-like “Ring/Dense” colonies from a rare ( ∼ 1%) population of total pancreatic single cells. With the addition of roof plate-specific spondin 1, a wingless-int agonist, Ring/Dense colony-forming cells can be expanded more than 100,000-fold when serially dissociated and replated in the presence of Matrigel. When cells grown in Matrigel are then transferred to a Matrigel-free semisolid medium with a unique laminin-based hydrogel, some cells grow and differentiate into another type of colony, which we name “Endocrine/Acinar.” These Endocrine/Acinar colonies are comprised mostly of endocrine- and acinar-like cells, as ascertained by RNA expression analysis, immunohistochemistry, and electron microscopy. Most Endocrine/Acinar colonies contain beta-like cells that secrete insulin/C-peptide in response to D -glucose and theophylline. These results demonstrate robust self-renewal and differentiation of adult Ring/Dense colony-forming units in vitro and suggest an approach to producing beta-like cells for cell replacement of type 1 diabetes. The methods described, which include microfluidic expression analysis of single cells and colonies, should also advance study of pancreas development and pancreatic progenitor cells.

The field of pancreatic stem and progenitor cell biology has been hampered by a lack of in vitro functional and quantitative assays that allow for the analysis of the single cell. Analyses of single progenitors are of critical importance because they provide definitive ways to unequivocally demonstrate the lineage potential of individual progenitors. Although methods have been devised to generate "pancreatospheres" in suspension culture from single cells, several limitations exist. First, it is time-consuming to perform single cell deposition for a large number of cells, which in turn commands large volumes of culture media and space. Second, numeration of the resulting pancreatospheres is labor-intensive, especially when the frequency of the pancreatosphere-initiating progenitors is low. Third, the pancreatosphere assay is not an efficient method to allow both the proliferation and differentiation of pancreatic progenitors in the same culture well, restricting the usefulness of the assay. To overcome these limitations, a semi-solid media based colony assay for pancreatic progenitors has been developed and is presented in this report. This method takes advantage of an existing concept from the hematopoietic colony assay, in which methylcellulose is used to provide viscosity to the media, allowing the progenitor cells to stay in three-dimensional space as they undergo proliferation as well as differentiation. To enrich insulin-expressing colony-forming progenitors from a heterogeneous population, we utilized cells that express neurogenin (Ngn) 3, a pancreatic endocrine progenitor cell marker. Murine embryonic stem (ES) cell-derived Ngn3 expressing cells tagged with the enhanced green fluorescent protein reporter were sorted and as many as 25,000 cells per well were plated into low-attachment 24-well culture dishes. Each well contained 500 μL of semi-solid media with the following major components: methylcellulose, Matrigel, nicotinamide, exendin-4, activin βB, and conditioned media collected from murine ES cell-derived pancreatic-like cells. After 8 to 12 days of culture, insulin-expressing colonies with distinctive morphology were formed and could be further analyzed for pancreatic gene expression using quantitative RT-PCR and immunoflourescent staining to determine the lineage composition of each colony. In summary, our colony assay allows easy detection and quantification of functional progenitors within a heterogeneous population of cells. In addition, the semi-solid media format allows uniform presentation of extracellular matrix components and growth factors to cells, enabling progenitors to proliferate and differentiate in vitro. This colony assay provides unique opportunities for mechanistic studies of pancreatic progenitor cells at the single cell level.

Embryonic stem (ES) cell technology may serve as a platform for the discovery of drugs to treat diseases such as diabetes. However, because of difficulties in establishing reliable ES cell differentiation methods and in creating cost-effective plating conditions for the high-throughput format, screening for molecules that regulate pancreatic beta cells and their immediate progenitors has been limited. A relatively simple and inexpensive differentiation protocol that allows efficient generation of insulin-expressing cells from murine ES cells was previously established in our laboratories. In this report, this system is characterized in greater detail to map developmental cell stages for future screening experiments. Our results show that sequential activation of multiple gene markers for undifferentiated ES cells, epiblast, definitive endoderm, foregut, and pancreatic lineages was found to follow the sequence of events that mimics pancreatic ontogeny. Cells that expressed enhanced green fluorescent protein, driven by pancreatic and duodenal homeobox 1 or insulin 1 promoter, correctly expressed known beta cell lineage markers. Overexpression of Sox17, an endoderm fate-determining transcription factor, at a very early stage of differentiation (days 2-3) enhanced pancreatic gene expression. Overexpression of neurogenin3, an endocrine progenitor cell marker, induced glucagon expression at stages when pancreatic and duodenal homeobox 1 message was present (days 10-16). Forced expression (between days 16 and 25) of MafA, a pancreatic maturation factor, resulted in enhanced expression of insulin genes, glucose transporter 2 and glucokinase, and glucose-responsive insulin secretion. Day 20 cells implanted in vivo resulted in pancreatic-like cells. Together, our differentiation assay recapitulates the proceedings and behaviors of pancreatic development and will be valuable for future screening of beta cell effectors.

The following information was missing from the Funding section: Additional funding was provided by NIH grant 5R01DK081587-01.

Background Insulin is a critical component of metabolic control, and as such, insulin gene expression has been the focus of extensive study. DNA sequences that regulate transcription of the insulin gene and the majority of regulatory factors have already been identified. However, only recently have other components of insulin gene expression been investigated, and in this study we examine the role of DNA methylation in the regulation of mouse and human insulin gene expression. Methodology/Principal Findings Genomic DNA samples from several tissues were bisulfite-treated and sequenced which revealed that cytosine-guanosine dinucleotide (CpG) sites in both the mouse Ins2 and human INS promoters are uniquely demethylated in insulin-producing pancreatic beta cells. Methylation of these CpG sites suppressed insulin promoter-driven reporter gene activity by almost 90% and specific methylation of the CpG site in the cAMP responsive element (CRE) in the promoter alone suppressed insulin promoter activity by 50%. Methylation did not directly inhibit factor binding to the CRE in vitro, but inhibited ATF2 and CREB binding in vivo and conversely increased the binding of methyl CpG binding protein 2 (MeCP2). Examination of the Ins2 gene in mouse embryonic stem cell cultures revealed that it is fully methylated and becomes demethylated as the cells differentiate into insulin-expressing cells in vitro. Conclusions/Significance Our findings suggest that insulin promoter CpG demethylation may play a crucial role in beta cell maturation and tissue-specific insulin gene expression.

Past studies of pancreatic progenitor cell biology relied mostly on histological analyses. Recent studies, using genetic labeling and tracing of progenitors, direct single cell analyses, colony assays, and enrichment of the minor population of progenitor cells through the use of cell surface markers, have strongly suggested that pancreatic progenitor cells with various frequency and lineage potentials, including the multipotent progenitors that give rise to endocrine, exocrine, and duct cells, exist in the developing and adult pancreas. In this review, it is therefore proposed that pancreatic progenitor cells may be organized in a hierarchy, in which the most primitive pan-pancreatic multipotent progenitors are at the top and rare, and the monopotent progenitors are at the bottom and abundant. This model may explain why only drastic injuries lead to effective activation of the progenitor cell compartment of the higher hierarchy, whereas under steady state, pregnancy, and milder injuries, recruitment of preexisting mature cells or their immediate monopotent progenitors could be sufficient to restore metabolic homeostasis. It is also proposed that the morphologically defined ductal cells are likely to be functionally heterogeneous and that endocrine progenitor cell activity should be determined based on functional analyses rather than histological locations.

Previous studies describe a unique culture method for the commitment of murine embryonic stem cells to early endocrine pancreata. In this report, early pancreatic-like β-cell progenitors were enriched and a colony assay devised to allow these progenitors to differentiate into insulin-expressing colonies in vitro. An embryonic stem cell line with enhanced green fluorescent protein (EGFP) inserted into one allele of neurogenin 3 (Ngn3), a marker for pancreatic endocrine progenitors, was differentiated. During the late stage of culture, 20–30% of cells were Ngn3-EGFP+. Gene expression profiling using the PancChip microarray platform demonstrated that Ngn3-EGFP+ cells differentially express endocrine-related genes. A novel semisolid culture method was developed to support the formation of individual insulin/C-peptide–expressing colonies from dissociated single cells. Approximately 0.1–0.6% of Ngn3-EGFP+ cells gave rise to insulin-expressing colonies, a three- to fivefold enrichment of β-cell–like progenitors, or insulin-expressing colony-forming units (ICFUs), compared with nonsorted cells. All of the single colonies expressed insulin II, while 69% coexpressed insulin I and 44% coexpressed glucagon. Some single colonies expressed insulin I, insulin II, and Pdx-1 (pancreatic duodenal homeobox–1), but not glucagon. In other colonies, glucagon expression overlapped with C-peptide II in double immunostaining analysis, suggesting heterogeneity among the ICFUs and their resulting colonies. Together, these results demonstrate that progenitors that have the potential to give rise to insulin-expressing cells can be derived from murine embryonic stem cells.

A panel of genetic markers was used to assess the in vitro commitment of murine embryonic stem (ES) cells toward the endoderm-derived pancreas and to distinguish insulin-expressing cells of this lineage from other lineages such as neuron, liver, and yolk sac. There are two nonallelic insulin genes in mice. Neuronal cells express only insulin II, whereas the pancreas expresses both insulin I and II. Yolk sac and fetal liver express predominately insulin II, small amounts of insulin I, and no glucagon. We found that ES-derived embryoid bodies cultured in the presence of stage-specific concentrations of monothio-glycerol and 15% fetal calf serum, followed by serum-free conditions, give rise to a population that expresses insulin I, insulin II, pdx-1 (a pancreas marker), and Sox17 (an endoderm marker). Immunohistochemical staining shows intracellular insulin particles, and its de novo production was confirmed by staining for C-peptide. Most, but not all, of the insulin+ or C-peptide+ cells coexpress glucagon, demonstrating a differentiation pathway to pancreas rather than yolk sac or fetal liver. Addition of beta-cell specification and differentiation factors activin beta B, nicotinamide, and exendin-4 to later-stage culture increased insulin-positive cells to 2.73% of the total population, compared with the control culture, which gave rise to less than 1% insulin-staining cells. These findings suggest that stepwise culture manipulations can direct ES cells to become early endocrine pancreas.