Chronic pancreatitis (CP) affects ∼3 million people worldwide, yet altering the course of the disease is challenging. We developed a patient-derived organoid (PDO) platform to investigate the molecular pathogenesis of this disease and identify therapeutic strategies. We generated 37 PDOs from patients with idiopathic, hereditary, and alcohol-related CP with a high genetic concordance. PDOs retained inflammation-associated transcriptional and proteomic features. Transcriptomic profiling revealed three molecular subtypes of CP independent of etiology. We discovered widespread dysfunction of the cystic fibrosis transmembrane conductance regulator (CFTR) in half of the CP PDOs, including those with wild-type CFTR. Clinically available CFTR modulators stabilized mutant or wild-type CFTR, restored CFTR function, and decreased mitogenic and inflammatory signaling. This work provides a comprehensive PDO platform for modeling CP. We demonstrate the utility of this platform for precision therapeutic investigations. Our findings reveal CFTR modulators as a broadly applicable and effective therapeutic strategy.

Durable therapeutic efficacy remains a major barrier to improving outcomes for patients with pancreatic ductal adenocarcinoma (PDAC). An immunosuppressive tumor microenvironment (TME) is a hallmark of PDAC and has been demonstrated to be a dominant driver of therapeutic resistance. The aberrant glycan CA19-9 is prevalent in PDAC and drives tumor progression, but the paracrine mechanisms by which it contributes to TME remodeling are unknown. To address this, we mapped TME changes and performed functional analyses using a genetically engineered mouse model (GEMM) harboring KrasG12D mutation and inducible CA19-9 expression. Elevation of CA19-9 led to expansion of antigen-presenting cancer associated fibroblasts (apCAFs) and regulatory T cells (Tregs), which can drive immunosuppression. Antibody blockade of CA19-9 resulted in significant restoration of normal histology and decreased apCAF and Treg populations. We dissected the paracrine signaling mechanisms that drive this TME remodeling in vitro using mouse and human organoid mono- and co-culture models as well as in vivo using GEMMs and syngeneic orthotopic transplantation models. CA19-9 induced IL1a and TGFb expression, reprogramming pancreatic mesothelial cells into apCAFs in vitro, which in turn directly ligated naïve Cd4+ T cells resulting in Treg differentiation in co-cultures. Antibody blockade of IL1a and TGFb in mice led to reduced apCAF and Treg differentiation. We previously reported that CA19-9 modification of the secreted Fbln3 protein increased Egfr engagement and now find that the induction of IL1a and TGFb expression by CA19-9 is dependent on Fbln3 hyperactivation of EGFR signaling. Genetic depletion of Fbln3 led to reduced tumor progression and increased Cd8+ T cell infiltration in mice. Together these findings identify a previously unknown signaling axis driving immunosuppressive phenotypes in PDAC, uncovering multiple potential nodes to relieve the immunosuppressive pressures within the PDAC TME.

Pancreatic ductal adenocarcinoma (PDAC) frequently metastasizes to the liver, which drives patient mortality. CA19-9 is elevated in most PDAC tumors and is widely used as a clinical biomarker. Elevated serum levels are associated with poor outcomes. However, whether CA19-9 functionally contributes to metastatic progression has not been fully defined, in part because mice lack endogenous CA19-9 expression. Here, using syngeneic murine PDAC cells engineered to express CA19-9, we investigated its functional role in liver metastasis. In splenic injection models, CA19-9 expression markedly increased liver metastatic burden by promoting both metastatic seeding and subsequent metastatic outgrowth. In vitro, CA19-9 enhanced tumor cell adhesion to endothelial cells through interaction with E-selectin. Metastatic seeding of CA19-9-expressing cells was reduced by genetic deletion of E-selectin or antibody neutralization of either CA19-9 or E-selectin in vivo. Therapeutic targeting of CA19-9 with a neutralizing antibody markedly reduced liver metastatic burden after metastatic seeding. CA19-9 expression increased AKT signaling in PDAC cells and liver metastases, and CA19-9 levels correlated with AKT activation in human PDAC tissues. These findings show that CA19-9 promotes PDAC liver metastasis through E-selectin-dependent metastatic seeding and AKT-associated metastatic outgrowth, highlighting CA19-9 as a functional mediator of PDAC metastasis and a potential therapeutic target.

Glycosylation is a complex post-translational modification essential for development, growth, and survival. Altered glycosylation is a hallmark of cancer, yet the roles of many glycoproteins remain unclear, hampering translation of glycoprotein-related vulnerabilities into therapeutic strategies. Pancreatic ductal adenocarcinoma (PDA) is one of the most lethal malignancies, with a five-year survival rate under 13%. For decades, serum levels of CA19-9, a terminal tetra-saccharide glycan, conjugating many secreted and cell surface proteins, has been the single-most effective biomarker to track PDA progression in patients. Recent studies have identified Fibulin-3 (Fbln3) as a secreted, CA19-9–modified matricellular glycoprotein that drives EGFR hyperactivation in the pancreatic epithelium, which is essential for PDA development. However, the functional role of Fbln3 in PDA has remained largely unexplored. Here, we show that Fbln3 promotes PDA progression and tumor microenvironment (TME) remodeling. Fbln3 expression is significantly upregulated in both human and mouse PDA tissues. Overexpression and knockdown of Fbln3 modulate PDA growth rates both in vitro and in vivo. In CA19-9–expressing KRASG12D mutant PDA organoids, Fbln3 enhances activation of key oncogenic pathways, including EGFR, NFκB, and TGFβ signaling. Notably, Fbln3 regulates the expression of IL1A and TGF, cytokines known to impact both autocrine and paracrine signaling. In syngeneic orthotopic tumor models, Fbln3 promotes the expansion of antigen-presenting cancer associated fibroblasts and reduces infiltration of CD8+ T cells, contributing to an immunosuppressive microenvironment. Taken together, these findings demonstrate that CA19-9-modified Fbln3 drives pancreatic tumor progression and TME remodeling by regulating IL1A and TGFb production, highlighting the need to further investigate its potential as a therapeutic target. Hyemin Song, Jasper Hsu, Satoshi Ogawa, Kristina Peck, Kassidy Curtis, Chelsea Bottomely, McKenna Stamp, Dannielle D. Engle. Fibulin-3 drives tumor progression and microenvironment remodeling in CA19-9-induced pancreatic ductal adenocarcinoma [abstract]. In: Proceedings of the AACR Special Conference in Cancer Research: Advances in Pancreatic Cancer Research—Emerging Science Driving Transformative Solutions; Boston, MA; 2025 Sep 28-Oct 1; Boston, MA. Philadelphia (PA): AACR; Cancer Res 2025;85(18_Suppl_3):Abstract nr B009.

Chronic pancreatitis (CP) affects ~3 million people worldwide, yet altering the course of disease is challenging. We developed a patient-derived organoid (PDO) platform to investigate the molecular pathogenesis of this disease and identify therapeutic strategies. We generated 36 PDOs from patients with idiopathic, hereditary, and alcohol-related CP with high genetic concordance. PDOs retained inflammation-associated transcriptional and proteomic features. Transcriptomic profiling revealed three molecular subtypes of CP independent of etiology. We discovered widespread dysfunction of the cystic fibrosis transmembrane conductance regulator (CFTR) in half of the CP PDOs, including those with wildtype CFTR. Clinically available CFTR modulators stabilized mutant or wildtype CFTR, restored CFTR function, and decreased mitogenic and inflammatory signaling. This work provides the first comprehensive PDO platform for modeling CP. We demonstrate the utility of this platform for precision therapeutic investigations. Our findings reveal CFTR modulators as a broadly applicable and effective therapeutic strategy.

Abstract Pancreatic cancer is a deadly malignancy. We have setup a pipeline to interrogate cancer cell intrinsic and extrinsic mechanisms contributing to treatment response. Using a combination of mouse and human organoids models together with in vivo investigation, we have systematically dissected key signaling hubs driving pro-tumorigenic cancer cell intrinsic features as well as remodeling of the tumor microenvironment (TME). We found that the glycan epitope, CA19-9, drives pancreatic inflammation, cancer, and metastasis. CA19-9 drives pro-tumorigenic intrinsic features in part through CA19-9 modification of the secreted glycoprotein Fibulin 3. We have also discovered direct and indirect mechanisms through which CA19-9 causes remodeling of the TME. Specifically, CA19-9 elevation causes expansion of all cancer associated fibroblast (CAF) subtypes, including an increase in antigen presenting CAFs and resulting increase in regulatory T cells. Further, there are dramatic increases in tumor associated macrophages (TAMs) derived from both inflammatory monocytes as well as tissue resident macrophages. To delineate the mechanisms by which these TME changes are mediated, we use novel co-culture model incorporating macrophages, organoids, and fibroblasts (MOrF). Blocking CA19-9 signaling in vitro and in vivo reverses these TME alterations. These changes to the TME are mediated through indirect effectors as well as previously unknown CA19-9 modified proteins. Overall, we elucidate previously unexplored mechanisms driving pancreatic tumorigenesis while also uncovered vulnerabilities for therapeutic exploitation. Citation Format: Jasper Hsu, Hyemin Song, Kristina Peck, Chelsea Bottomley, McKenna Stamp, Shira Okhovat, Dannielle D. Engle. Interception of pro-tumorigenic glycan signaling in pancreatic cancer [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2024; Part 1 (Regular Abstracts); 2024 Apr 5-10; San Diego, CA. Philadelphia (PA): AACR; Cancer Res 2024;84(6_Suppl):Abstract nr 1600.

Abstract Pancreatic ductal adenocarcinoma (PDA) is an intractable common malignancy with a pro-tumorigenic and immunosuppressive microenvironment (ME) that often limits treatment efficacy. Only 10-15% of patients are eligible for surgical resection, which is the only cure for PDA. The glycan CA19-9 is used to follow treatment response in PDA patients, but its functional role in PDA remained unknown until recently because rodents lack the ability to produce this carbohydrate. CA19-9 elevation in a KRAS-mutant background results in increased tumor proliferation and ME remodeling in mice. The tumor ME is encompassed by hypovascularized stroma consisting of cancer associated fibroblasts (CAFs) and tumor associated macrophages (TAMs) that contribute to desmoplasia and immunosuppression. All prior work investigating the tumor ME in PDA mouse models was performed in CA19-9 negative context, potentially missing a key element of PDA biology. Elevation of CA19-9 in mice caused expansion of both antigen-presenting (ap)CAFs and TAMs. CA19-9 induction in KRAS-mutant organoids increased gene expression of M-CSF, IL1a, and TGFb and led to increased apCAF and TAM differentiation. This project aims to uncover the mediators of CA19-9-mediated ME remodeling. CA19-9-modified macrophage colony-stimulating factor (M-CSF) was identified via immunoprecipitation and mass spectrometry from KRAS-mutant CA19-9-inducible pancreatic organoids. CA19-9-M-CSF induced increased immunosuppressive TAM differentiation in vivo and in vitro using a novel Macrophage, Organoid, and Fibroblast (MOrF) co-culture platform. This project will identify the mechanisms by which CA19-9 directly contributes to PDA ME immune modulation and unveil essential molecular underpinnings of PDA biology that will inform effective therapies in the future. Citation Format: Jasper Hsu, Tae Gyu Oh, Kristina L. Peck, Angelica E. Rock, Garret Bishop, Shira R. Okhovat, Susan M. Kaech, Dannielle D. Engle. Glycan CA19-9 mediated immune modulation in the pancreatic tumor microenvironment [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2024; Part 1 (Regular Abstracts); 2024 Apr 5-10; San Diego, CA. Philadelphia (PA): AACR; Cancer Res 2024;84(6_Suppl):Abstract nr 5553.

Abstract Pancreatic ductal adenocarcinoma (PDA) is a highly lethal malignancy with a five-year survival rate of less than 11%, making it one of the deadliest cancers. Notably, 95% of PDA cases exhibit KRAS mutations, contributing to highly dysregulated cell signaling networks. Despite extensive research into the interplay of various oncogenic pathways in pancreatic tumorigenesis, targeted therapies against these signaling networks have displayed modest efficacy, with most patients rapidly developing therapeutic resistance. Recent studies highlight the significance of the carbohydrate antigen 19-9 (CA19-9) as a functional biomarker linked to disease progression and resistance. However, understanding the detailed functions of CA19-9-modified proteins in PDA has been limited due to the lack of adequate models for synthesizing CA19-9. To address this challenge, we have developed unique mouse and 3D organoid culture models capable of producing CA19-9. CA19-9 elevation in mice with KRAS-mutations results in an aggressive PDA phenotype through increased tumor proliferation and microenvironment (TME) remodeling. Furthermore, our research has identified Fibulin 3 (FBLN3), an extracellular matrix glycoprotein, as a secreted, CA19-9-modified protein that stimulates tumorigenesis. In this study, we found that FBLN3 not only facilitates tumor proliferation but also drives epithelial-mesenchymal transition (EMT) and promotes STAT3 pathways activation in the CA19-9pos, KRAS-mutant PDA organoids. Additionally, in both CA19-9pos, KRAS-mutant PDA mouse models and human PDA, FBLN3 is expressed by cancer-associated fibroblasts (CAFs), suggesting its role in TME remodeling. These findings have significant implications for providing novel insights into PDA biology and paracrine signaling mechanisms, identifying FBLN3 as a potential therapeutic target for the improved treatment of this devastating disease. Citation Format: Hyemin Song, Jasper Hsu, Xiaoxue Lin, Satoshi Ogawa, Vasiliki Pantazopoulou, Kristina Peck, Chelsea Bottomley, Shira Okhovat, McKenna Stamp, Kassidy Curtis, Jeffrey D. Esko, Dannielle D. Engle. Investigating the role of fibulin 3 in pancreatic tumorigenesis [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2024; Part 1 (Regular Abstracts); 2024 Apr 5-10; San Diego, CA. Philadelphia (PA): AACR; Cancer Res 2024;84(6_Suppl):Abstract nr 3937.

Myeloid neoplasms are clonal disorders driven by acquisition of somatic mutations in hematopoietic stem cells (HSCs). HSCs acquire mutations at each cell division, most of which are thought to be effectively neutral, while only recurrent driver mutations in a relatively small number of cancer-causative genes are thought to promote clonal expansion of HSCs, or clonal hematopoiesis (CH), and leukemia progression. However, nearly 50% of CH cases are not associated with an annotated driver mutation, suggesting that the true number of drivers might be much larger than catalogued to date. Since CH infrequently progresses to myelodysplastic syndrome (MDS) or acute myeloid leukemia (AML), the dynamics of antecedent preleukemic clonal evolution in individuals who develop myeloid neoplasms remain poorly understood. Here, we use induced pluripotent stem cell (iPSC) reprogramming to track genomic and functional evolution of preleukemic clones in 18 patients with MDS or AML. We generated a panel of 42 iPSC lines spanning all stages of premalignant evolution marked by 1, 2, or 3 annotated driver mutations and isogenic wild-type (WT) clones derived from normal HSPCs in the same patient. To measure genome-wide dynamics of mutation acquisition during clonal evolution, we performed whole exome sequencing of MDS/AML patient samples, preleukemic iPSC clones, and matched WT iPSCs. We defined preleukemic mutations as somatic variants present in the primary MDS/AML patient sample and at least one preleukemic clone but absent in the isogenic WT iPSCs. We found that coding mutation burden in 1-, 2-, and 3-driver clones increased linearly. In a neutral process, the ratio of amino acid-changing to neutral mutations is expected to remain constant. By contrast, we found that 2- and 3-drivers clones harbored a significantly higher proportion of amino acid-changing nonsynonymous and indel mutations compared to 1-driver clones. A regression model incorporating driver and neutral mutation counts showed a significant increase in amino acid-changing mutations during progression relative to neutral counts. These findings show that clonal progression is marked by increasing mutation burden with an excess of amino acid-changing variants, supporting a model of intensified positive selection for unannotated “cryptic” drivers. These genes are likely not annotated as drivers because they are infrequently mutated in myeloid malignancies. To understand how cryptic drivers impact HSC fitness, we first sought to better define the impact of recurrent preleukemic DNMT3A R882H mutations. DNMT3A mutations enhanced hematopoietic output of iPSC-derived HSPCs and reduced expression of immune response pathways and HLA genes encoding the major histocompatibility complex class II (MHC-II). MHC-II expression was concordantly reduced on the surface of DNMT3A-mutant HSPCs and dendritic cells. We next carried out single cell transcriptomics on individuals with DNMT3A-mutant CH. Consistent with the iPSC findings, DNMT3A-mutant HSPCs and dendritic cells had decreased expression of MHC-II genes compared to age-matched controls. These data implicate MHC-II as an axis regulated by common preleukemic DNMT3A mutations. We then extended this analysis to candidate cryptic drivers in our dataset. Of these, NSD1 and IRF1 were infrequently mutated (<1%) in patients with MDS and AML and previously implicated as regulators of HLA expression. To investigate how inactivating NSD1 and IRF1 mutations affect hematopoiesis, we performed knockdown (KD) of these genes in human CD34+ cord blood HSPCs. NSD1-KD expanded CD34+CD133+ HSPCs, while IRF1-KD blocked differentiation into myeloid lineages. Notably, both NSD1- and IRF1-KD displayed significant reduction of MHC-II on CD34+CD133+ HSPCs, suggesting that both common and cryptic drivers converge on MHC-II down-regulation. In conclusion, tracking preleukemic hematopoiesis by iPSC reprogramming, we present evidence that unannotated cryptic driver mutations are under positive selection in preleukemic evolution and nominate MHC-II reduction as convergent pathway by which common and cryptic drivers promote clonal fitness. We propose that downregulation of antigen presentation via MHC class II may be a common mechanism by which cryptic drivers contribute to clonal expansion.

Pancreatic ductal adenocarcinoma (PDA) is an intractable common malignancy with a pro-tumorigenic and immunosuppressive microenvironment (ME) that often limits treatment efficacy. The glycan CA19-9 is used to follow treatment response in PDA patients, but its functional role in PDA remained unknown until recently because rodents lack this carbohydrate. CA19-9 elevation in a KRAS-mutant background promotes rapid progression to invasive carcinoma and induces ME remodeling in mice. The tumor ME includes cancer associated fibroblasts (CAFs) and tumor associated macrophages (TAMs) that contribute to desmoplasia, immunosuppression, and therapeutic resistance. All prior work investigating the tumor ME in PDA mouse models was performed in a CA19-9 negative context, potentially missing a key element of PDA biology. Elevation of CA19-9 in mice caused expansion of both CAFs and TAMs, but the mechanisms by which CA19-9 contributes to ME remodeling remains largely unknown. We aim to uncover the direct and indirect mechanisms of CA19-9-mediated ME remodeling. We identified CA19-9 modified ligands by immunoprecipitation and mass spectrometry that may contribute to reprogramming of the PDA ME. In addition to exploring changes in abundance, localization, and protein interactions, we will functionally characterize these candidate effectors via CRISPR ablation and antibody blockade using a novel Macrophage, Organoid, and Fibroblast (MOrF) co-culture platform. MOrF co-cultures can be sustained for at least 10 days in defined media conditions and enable compartment specific delineation of paracrine and juxtacrine signals. While CA19-9 modification may play a direct role in stromal remodeling, we also investigate effectors that are altered in response to CA19-9 elevation. CA19-9 induction in KRAS-mutant organoid mono-cultures increased gene expression of IL1α, CSF1,and TGFβ. In addition, CA19-9 expression increased gene set enrichment of inflammatory programs inorganoid mono-culture and led to increased expression of CAF and TAM differentiation programs in MOrF co-culture. These results are being validated as discussed above as well as by evaluation in vivo in CA19-9 positive and negative syngeneic orthotopic transplantation and autochthonous genetically engineered mouse models using scRNA-seq, pharmacologic and genetic perturbation, as well as other approaches. Using these orthogonal approaches, we will identify the mechanisms by which CA19-9 directly and indirectly contributes to PDA ME remodeling and ultimately, contributes to treatment response in the future. Citation Format: Jasper Hsu, Sejin Chung, Angelica E. Rock, Shira R. Okhovat, Dannielle D. Engle. CA19-9-mediated remodeling of the pancreatic tumor microenvironment [abstract]. In: Proceedings of the AACR Special Conference on Pancreatic Cancer; 2022 Sep 13-16; Boston, MA. Philadelphia (PA): AACR; Cancer Res 2022;82(22 Suppl):Abstract nr C045.

Pancreatic ductal adenocarcinoma (PDA) is one of the deadliest cancers and is projected to soon be the second leading cause of cancer death. Median survival of PDA patients is 6–10 mo, with the majority of diagnoses occurring at later, metastatic stages that are refractory to treatment and accompanied by worsening prognoses. Glycosylation is one of the most common types of post-translational modifications. The complex landscape of glycosylation produces an extensive repertoire of glycan moieties, glycoproteins, and glycolipids, thus adding a dynamic and tunable level of intra- and intercellular signaling regulation. Aberrant glycosylation is a feature of cancer progression and influences a broad range of signaling pathways to promote disease onset and progression. However, despite being so common, the functional consequences of altered glycosylation and their potential as therapeutic targets remain poorly understood and vastly understudied in the context of PDA. In this review, the functionality of glycans as they contribute to hallmarks of PDA are highlighted as active regulators of disease onset, tumor progression, metastatic capability, therapeutic resistance, and remodeling of the tumor immune microenvironment. A deeper understanding of the functional consequences of altered glycosylation will facilitate future hypothesis-driven studies and identify novel therapeutic strategies in PDA.

Myeloid neoplasms, including myelodysplastic syndromes (MDS), are genetically heterogeneous disorders driven by clonal acquisition of somatic mutations in hematopoietic stem and progenitor cells (HPCs). The order of premalignant mutations and their impact on HPC self-renewal and differentiation remain poorly understood. We show that episomal reprogramming of MDS patient samples generates induced pluripotent stem cells from single premalignant cells with a partial complement of mutations, directly informing the temporal order of mutations in the individual patient. Reprogramming preferentially captured early subclones with fewer mutations, which were rare among single patient cells. To evaluate the functional impact of clonal evolution in individual patients, we differentiated isogenic MDS induced pluripotent stem cells harboring up to 4 successive clonal abnormalities recapitulating a progressive decrease in hematopoietic differentiation potential. SF3B1, in concert with epigenetic mutations, perturbed mitochondrial function leading to accumulation of damaged mitochondria during disease progression, resulting in apoptosis and ineffective erythropoiesis. Reprogramming also informed the order of premalignant mutations in patients with complex karyotype and identified 5q deletion as an early cytogenetic anomaly. The loss of chromosome 5q cooperated with TP53 mutations to perturb genome stability, promoting acquisition of structural and karyotypic abnormalities. Reprogramming thus enables molecular and functional interrogation of preleukemic clonal evolution, identifying mitochondrial function and chromosome stability as key pathways affected by acquisition of somatic mutations in MDS.

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 GRflox/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.

Progenitor cells in the adult pancreas are potential sources of endocrine beta cells for treating type 1 diabetes. Previously, we identified tri-potent progenitor cells in the adult (2–4 month-old) murine pancreas that were capable of self-renewal and differentiation into duct, acinar, and endocrine cells in vitro. These progenitor cells were named pancreatic colony-forming units (PCFUs). However, because PCFUs are a minor population in the pancreas (~ 1%) they are difficult to study. To enrich PCFUs, strategies using cell-surface marker analyses and fluorescence-activated cell sorting were developed. We found that CD133highCD71low cells, but not other cell populations, enriched PCFUs by up to 30 fold compared to the unsorted cells. CD133highCD71low cells generated primary, secondary, and subsequent colonies when serially re-plated in Matrigel-containing cultures, suggesting self-renewal abilities. In the presence of a laminin hydrogel, CD133highCD71low cells gave rise to colonies that contained duct, acinar, and Insulin+ Glucagon+ double-hormonal endocrine cells. Colonies from the laminin hydrogel culture were implanted into diabetic mice, and five weeks later duct, acinar, and Insulin+ Glucagon− cells were detected in the grafts, demonstrating tri-lineage differentiation potential of CD133highCD71low cells. These CD133highCD71low cells will enable future studies of putative adult pancreas stem cells in vivo.

Postnatal pancreas is a potential source for progenitor cells to generate endocrine β-cells for treating type 1 diabetes. However, it remains unclear whether young (1-week-old) pancreas harbors multipotent progenitors capable of differentiating into duct, acinar, and endocrine cells. Laminin is an extracellular matrix (ECM) protein important for β-cells' survival and function. We established an artificial extracellular matrix (aECM) protein that contains the functional IKVAV (Ile-Lys-Val-Ala-Val) sequence derived from laminin (designated aECM-lam). Whether IKVAV is necessary for endocrine differentiation in vitro is unknown. To answer these questions, we cultured single cells from 1-week-old pancreas in semi-solid media supplemented with aECM-lam, aECM-scr (which contains a scrambled sequence instead of IKVAV), or Matrigel. We found that colonies were generated in all materials. Individual colonies were examined by microfluidic reverse transcription-polymerase chain reaction, immunostaining, and electron microscopy analyses. The majority of the colonies expressed markers for endocrine, acinar, and ductal lineages, demonstrating tri-lineage potential of individual colony-forming progenitors. Colonies grown in aECM-lam expressed higher levels of endocrine markers Insulin1, Insulin2, and Glucagon compared with those grown in aECM-scr and Matrigel, indicating that the IKVAV sequence enhances endocrine differentiation. In contrast, Matrigel was inhibitory for endocrine gene expression. Colonies grown in aECM-lam displayed the hallmarks of functional β-cells: mature insulin granules and glucose-stimulated insulin secretion. Colony-forming progenitors were enriched in the CD133(high) fraction and among 230 micro-manipulated single CD133(high) cells, four gave rise to colonies that expressed tri-lineage markers. We conclude that young postnatal pancreas contains multipotent progenitor cells and that aECM-lam promotes differentiation of β-like cells in vitro.

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.

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.