Pancreatic Progenitor Research Articles

Harnessing Proliferation for the Expansion of Stem Cell-Derived Pancreatic Cells: Advantages and Limitations.

Restoring the number of glucose-responsive β-cells in patients living with diabetes is critical for achieving normoglycemia since functional β-cells are lost during the progression of both type 1 and 2 diabetes. Stem cell-derived β-cell replacement therapies offer an unprecedented opportunity to replace the lost β-cell mass, yet differentiation efficiencies and the final yield of insulin-expressing β-like cells are low when using established protocols. Driving cellular proliferation at targeted points during stem cell-derived pancreatic progenitor to β-like cell differentiation can serve as unique means to expand the final cell therapeutic product needed to restore insulin levels. Numerous studies have examined the effects of β-cell replication upon functionality, using primary islets in vitro and mouse models in vivo, yet studies that focus on proliferation in stem cell-derived pancreatic models are only just emerging in the field. This mini review will discuss the current literature on cell proliferation in pancreatic cells, with a focus on the proliferative state of stem cell-derived pancreatic progenitors and β-like cells during their differentiation and maturation. The benefits of inducing proliferation to increase the final number of β-like cells will be compared against limitations associated with driving replication, such as the blunted capacity of proliferating β-like cells to maintain optimal β-cell function. Potential strategies that may bypass the challenges induced by the up-regulation of cell cycle-associated factors during β-cell differentiation will be proposed.

Pancreatic progenitor epigenome maps prioritize type 2 diabetes risk genes with roles in development.

Genetic variants associated with type 2 diabetes (T2D) risk affect gene regulation in metabolically relevant tissues, such as pancreatic islets. Here, we investigated contributions of regulatory programs active during pancreatic development to T2D risk. Generation of chromatin maps from developmental precursors throughout pancreatic differentiation of human embryonic stem cells (hESCs) identifies enrichment of T2D variants in pancreatic progenitor-specific stretch enhancers that are not active in islets. Genes associated with progenitor-specific stretch enhancers are predicted to regulate developmental processes, most notably tissue morphogenesis. Through gene editing in hESCs, we demonstrate that progenitor-specific enhancers harboring T2D-associated variants regulate cell polarity genes LAMA1 and CRB2. Knockdown of lama1 or crb2 in zebrafish embryos causes a defect in pancreas morphogenesis and impairs islet cell development. Together, our findings reveal that a subset of T2D risk variants specifically affects pancreatic developmental programs, suggesting that dysregulation of developmental processes can predispose to T2D.

Pancreatic β cell regeneration induced by clinical and preclinical agents.

Diabetes, one of the most common chronic diseases in the modern world, has pancreatic β cell deficiency as a major part of its pathophysiological mechanism. Pancreatic regeneration is a potential therapeutic strategy for the recovery of β cell loss. However, endocrine islets have limited regenerative capacity, especially in adult humans. Almost all hypoglycemic drugs can protect β cells by inhibiting β cell apoptosis and dedifferentiation via correction of hyperglycemia and amelioration of the consequent inflammation and oxidative stress. Several agents, including glucagon-like peptide-1 and γ-aminobutyric acid, have been shown to promote β cell proliferation, which is considered the main source of the regenerated β cells in adult rodents, but with less clarity in humans. Pancreatic progenitor cells might exist and be activated under particular circumstances. Artemisinins and γ-aminobutyric acid can induce α-to-β cell conversion, although some disputes exist. Intestinal endocrine progenitors can transdeterminate into insulin-producing cells in the gut after FoxO1 deletion, and pharmacological research into FoxO1 inhibition is ongoing. Other cells, including pancreatic acinar cells, can transdifferentiate into β cells, and clinical and preclinical strategies are currently underway. In this review, we summarize the clinical and preclinical agents used in different approaches for β cell regeneration and make some suggestions regarding future perspectives for clinical application.

Gene Expression in Pancreatic Cancer-Like Cells and Induced Pancreatic Stem Cells Generated by Transient Overexpression of Reprogramming Factors.

We previously reported that transient overexpression of reprogramming factors can be used to generate induced pluripotent stem (iPS) cells, induced tissue-specific stem (iTS) cells, and fibroblast-like (iF) cells from pancreatic tissue. iF cells have tumorigenic ability and behave similarly to pancreatic cancer cells. In this study, we analyzed gene expression in iF cells and iTS-P cells (iTS cells from pancreatic tissue) via microarray analysis and quantitative reverse transcription-polymerase chain reaction (qRT-PCR). The expression levels of the Mybl2 and Lyn genes, which are reported to be oncogenes, were significantly higher in iF cells than in iTS-P cells. The expression level of Nestin, which is expressed in not only pancreatic progenitor cells but also pancreatic ductal adenocarcinomas, was also higher in iF cells than in iTS-P cells. Itgb6 and Fgf13, which are involved in the pathogenesis of diseases such as cancer, exhibited higher expression levels in iF cells than in iTS-P cells. Unexpectedly, the expression levels of genes related to epithelial-mesenchymal transition (EMT), except Bmp4, were lower in iF cells than in iTS-P cells. These data suggest that the Mybl2, Lyn, Nestin, Itgb6, and Fgf13 genes could be important biomarkers to distinguish iTS-P cells from iF cells.

Effect of Sonic hedgehog pathway inhibition on PDX1 expression during pancreatic differentiation of human embryonic stem cells.

Differentiation processes for generating pancreatic progenitors from pluripotent stem cells inhibit Sonic hedgehog signaling through synthetic antagonists. However, the effect of sonic hedgehog inhibition in differentiating human embryonic stem cells remains unclear. The primary aim of this study was to understand the effect of Sonic hedgehog inhibition on key pancreas-specific transcription factors during differentiation of human embryonic stem cells towards a pancreatic lineage. We differentiated human embryonic stem (ES) cells towards the pancreatic progenitor stage. To analyze the effect of Sonic hedgehog inhibition, we differentiated human ES cells in the presence or absence of pathway antagonist, cyclopamine, using the same concentration (0.25µM) as reported earlier. Changes in gene expression between the groups were examined by quantitative reverse-transcription PCR and immunoblot analyses. Surprisingly, we found that expression of key transcription factors, PDX1 and SOX9, was not majorly affected by inhibition of Sonic hedgehog signals. Effects of inhibiting Hedgehog signals on pancreas-specific markers in differentiating human embryonic stem cells were analyzed in the study. We identified that the expression of pancreas-specific PDX1 and SOX9 was not affected by the Sonic hedgehog pathway in pancreatic progenitor populations from human ES cells. Thus, the restrictive nature of Hedgehog signaling during the early stages of pancreas formation could be facilitated through a transcriptional network beyond PDX1 and SOX9.

Response of Pluripotent Stem Cells to Environmental Stress and Its Application for Directed Differentiation.

Simple SummaryEnvironmental changes in oxygen concentration, temperature, and mechanical stimulation lead to the activation of specific transcriptional factors and induce the expression of each downstream gene. In general, these responses are protective machinery against such environmental stresses, while these transcriptional factors also regulate cell proliferation, differentiation, and organ development in mammals. In the case of pluripotent stem cells, similar response mechanisms normally work and sometimes stimulate the differentiation cues. Up to now, differentiation protocols utilizing such environmental stresses have been reported to obtain various types of somatic cells from pluripotent stem cells. Basically, environmental stresses as hypoxia (low oxygen), hyperoxia, (high oxygen) and mechanical stress from cell culture plates are relatively safer than chemicals and gene transfers, which affect the genome irreversibly. Therefore, protocols designed with such environments in mind could be useful for the technology development of cell therapy and regenerative medicine. In this manuscript, we summarize recent findings of environmental stress-induced differentiation protocols and discuss their mechanisms.Pluripotent stem cells have unique characteristics compared to somatic cells. In this review, we summarize the response to environmental stresses (hypoxic, oxidative, thermal, and mechanical stresses) in embryonic stem cells (ESCs) and their applications in the differentiation methods directed to specific lineages. Those stresses lead to activation of each specific transcription factor followed by the induction of downstream genes, and one of them regulates lineage specification. In short, hypoxic stress promotes the differentiation of ESCs to mesodermal lineages via HIF-1α activation. Concerning mechanical stress, high stiffness tends to promote mesodermal differentiation, while low stiffness promotes ectodermal differentiation via the modulation of YAP1. Furthermore, each step in the same lineage differentiation favors each appropriate stiffness of culture plate; for example, definitive endoderm favors high stiffness, while pancreatic progenitor favors low stiffness during pancreatic differentiation of human ESCs. Overall, treatments utilizing those stresses have no genotoxic or carcinogenic effects except oxidative stress; therefore, the differentiated cells are safe and could be useful for cell replacement therapy. In particular, the effect of mechanical stress on differentiation is becoming attractive for the field of regenerative medicine. Therefore, the development of a stress-mediated differentiation protocol is an important matter for the future.

Human Pluripotent Stem Cells: A Unique Tool for Toxicity Testing in Pancreatic Progenitor and Endocrine Cells.

Diabetes prevalence is increasing worldwide, and epidemiological studies report an association between diabetes incidence and environmental pollutant exposure. There are >84,000 chemicals in commerce, many of which are released into the environment without a clear understanding of potential adverse health consequences. While in vivo rodent studies remain an important tool for testing chemical toxicity systemically, we urgently need high-throughput screening platforms in biologically relevant models to efficiently prioritize chemicals for in depth toxicity analysis. Given the increasing global burden of obesity and diabetes, identifying chemicals that disrupt metabolism should be a high priority. Pancreatic endocrine cells are key regulators of systemic metabolism, yet often overlooked as a target tissue in toxicology studies. Immortalized β-cell lines and primary human, porcine, and rodent islets are widely used for studying the endocrine pancreas in vitro, but each have important limitations in terms of scalability, lifespan, and/or biological relevance. Human pluripotent stem cell (hPSC) culture is a powerful tool for in vitro toxicity testing that addresses many of the limitations with other β-cell models. Current in vitro differentiation protocols can efficiently generate glucose-responsive insulin-secreting β-like cells that are not fully mature, but still valuable for high-throughput toxicity screening in vitro. Furthermore, hPSCs can be applied as a model of developing pancreatic endocrine cells to screen for chemicals that influence endocrine cell formation during critical windows of differentiation. Given their versatility, we recommend using hPSCs to identify potential β-cell toxins, which can then be prioritized as chemicals of concern for metabolic disruption.

Essential roles of insulin and IGF-1 receptors during embryonic lineage development

The insulin and insulin-like growth factor-1 (IGF-1) receptors are important for the growth and development of embryonic tissues. To directly define their roles in the maintenance of pluripotency and differentiation of stem cells, we knocked out both receptors in induced pluripotent stem cells (iPSCs). iPSCs lacking both insulin and IGF-1 receptors (double knockout, DKO) exhibited preserved pluripotency potential despite decreased expression of transcription factors Lin28a and Tbx3 compared to control iPSCs. While embryoid body and teratoma assays revealed an intact ability of DKO iPSCs to form all three germ layers, the latter were composed of primitive neuroectodermal tumor-like cells in the DKO group. RNA-seq analyses of control vs DKO iPSCs revealed differential regulation of pluripotency, developmental, E2F1, and apoptosis pathways. Signaling analyses pointed to downregulation of the AKT/mTOR pathway and upregulation of the STAT3 pathway in DKO iPSCs in the basal state and following stimulation with insulin/IGF-1. Directed differentiation toward the three lineages was dysregulated in DKO iPSCs, with significant downregulation of key markers (Cebpα, Fas, Pparγ, and Fsp27) in adipocytes and transcription factors (Ngn3, Isl1, Pax6, and Neurod1) in pancreatic endocrine progenitors. Furthermore, differentiated pancreatic endocrine progenitor cells from DKO iPSCs showed increased apoptosis. We conclude that insulin and insulin-like growth factor-1 receptors are indispensable for normal lineage development and perturbations in the function and signaling of these receptors leads to upregulation of alternative compensatory pathways to maintain pluripotency.

One-Step Generation of Aqueous-Droplet-Filled Hydrogel Fibers as Organoid Carriers Using an All-in-Water Microfluidic System.

Hydrogel fibers are promising carriers for biological applications due to their flexible mechanical properties, well-defined spatial distribution, and excellent biocompatibility. In particular, the droplet-filled hydrogel fibers with the controllable dimension and location of droplets display great advantages to enhance the loading capacity of multiple components and biofunctions. In this work, we proposed a new all-in-water microfluidic system that allows for one-step fabrication of aqueous-droplet-filled hydrogel fibers (ADHFs) with unique morphology and tunable configurations. In the system, the aqueous droplets with equidistance are successfully arranged within the alginate calcium fibers, relying on the design of the pump valve cycle and the select of two immiscible liquids with a stable aqueous interface. The architecture of the ADHF can be flexibly controlled by adjusting the three phase flow rates and the valve switch cycle. The produced ADHFs exhibit high controllability, uniformity, biocompatibility, and stability. The established system enabled the formation of functional human islet organoids in situ through encapsulating pancreatic endocrine progenitor cells within microfibers. The generated islet organoids within droplets exhibit high cell viability and islet-specific function of insulin secretion. The proposed approach provides a new way to fabricate multifunctional hydrogel fibers for materials sciences, tissue engineering, and regenerative medicine.

A Method for Encapsulation and Transplantation into Diabetic Mice of Human Induced Pluripotent Stem Cells (hiPSC)-Derived Pancreatic Progenitors.

Pancreatic islet endocrine cells generated from patient-derived induced pluripotent stem cells represent a great strategy for both disease modeling and regenerative medicine. Nevertheless, these cells inherently miss the effects of the intricate network of systemic signals characterizing the living organisms. Xenotransplantation of in vitro differentiating cells into murine hosts substantially compensates for this drawback.Here we describe our transplantation strategy of encapsulated differentiating pancreatic progenitors into diabetic immunosuppressed (NSG) overtly diabetic mice generated by the total ablation of insulin-producing cells following diphtheria toxin administration. We will detail the differentiation protocol employed, the alginate encapsulation procedure, and the xenotransplantation steps required for a successful and reproducible experiment.

Aberrant development of pancreatic beta cells derived from human iPSCs with FOXA2 deficiency

FOXA2 has been identified as an essential factor for pancreas development and emerging evidence supports an association between FOXA2 and diabetes. Although the role of FOXA2 during pancreatic development is well-studied in animal models, its role during human islet cell development remains unclear. Here, we generated induced pluripotent stem cells (iPSCs) from a patient with FOXA2 haploinsufficiency (FOXA2+/− iPSCs) followed by beta-cell differentiation to understand the role of FOXA2 during pancreatic beta-cell development. Our results showed that FOXA2 haploinsufficiency resulted in aberrant expression of genes essential for the differentiation and proper functioning of beta cells. At pancreatic progenitor (PP2) and endocrine progenitor (EPs) stages, transcriptome analysis showed downregulation in genes associated with pancreatic development and diabetes and upregulation in genes associated with nervous system development and WNT signaling pathway. Knockout of FOXA2 in control iPSCs (FOXA2−/− iPSCs) led to severe phenotypes in EPs and beta-cell stages. The expression of NGN3 and its downstream targets at EPs as well as INSUILIN and GLUCAGON at the beta-cell stage, were almost absent in the cells derived from FOXA2−/− iPSCs. These findings indicate that FOXA2 is crucial for human pancreatic endocrine development and its defect may lead to diabetes based on FOXA2 dosage.

Recent Advances in the Generation of β-Cells from Induced Pluripotent Stem Cells as a Potential Cure for Diabetes Mellitus.

Diabetes mellitus (DM) is a group of metabolic disorders characterized by high blood glucose levels due to insufficient insulin secretion, insulin action, or both. The present-day solution to diabetes mellitus includes regular administration of insulin, which brings about many medical complications in diabetic patients. Although islet transplantation from cadaveric subjects was proposed to be a permanent cure, the increased risk of infections, the need for immunosuppressive drugs, and their unavailability had restricted its use. To overcome this, the generation of renewable and transplantable β-cells derived from autologous induced pluripotent stem cells (iPSCs) has gained enormous interest as a potential therapeutic strategy to treat diabetes mellitus permanently. To date, extensive research has been undertaken to derive transplantable insulin-producing β-cells (iβ-cells) from iPSCs in vitro by recapitulating the in vivo developmental process of the pancreas. This in vivo developmental process relies on transcription factors, signaling molecules, growth factors, and culture microenvironment. This review highlights the various factors facilitating the generation of mature β-cells from iPSCs. Moreover, this review also describes the generation of pancreatic progenitors and β-cells from diabetic patient-specific iPSCs, exploring the potential of the diabetes disease model and drug discovery. In addition, the applications of genome editing strategies have also been discussed to achieve patient-specific diabetes cell therapy. Last, we have discussed the current challenges and prospects of iPSC-derived β-cells to improve the relative efficacy of the available treatment of diabetes mellitus.

Neuron-Glia Interaction in the Developing and Adult Enteric Nervous System.

The enteric nervous system (ENS) constitutes the largest part of the peripheral nervous system. In recent years, ENS development and its neurogenetic capacity in homeostasis and allostasishave gained increasing attention. Developmentally, the neural precursors of the ENS are mainly derived from vagal and sacral neural crest cell portions. Furthermore, Schwann cell precursors, as well as endodermal pancreatic progenitors, participate in ENS formation. Neural precursors enherite three subpopulations: a bipotent neuron-glia, a neuronal-fated and a glial-fated subpopulation. Typically, enteric neural precursors migrate along the entire bowel to the anal end, chemoattracted by glial cell-derived neurotrophic factor (GDNF) and endothelin 3 (EDN3) molecules. During migration, a fraction undergoes differentiation into neurons and glial cells. Differentiation is regulated by bone morphogenetic proteins (BMP), Hedgehog and Notch signalling. The fully formed adult ENS may react to injury and damage with neurogenesis and gliogenesis. Nevertheless, the origin of differentiating cells is currently under debate. Putative candidates are an embryonic-like enteric neural progenitor population, Schwann cell precursors and transdifferentiating glial cells. These cells can be isolated and propagated in culture as adult ENS progenitors and may be used for cell transplantation therapies for treating enteric aganglionosis in Chagas and Hirschsprung’s diseases.

I. How can you choose the fate of iPSCs and stem cells, Regeneration or Carcinogenesis? A hypothetical insight.: II. Modelling human beta cell development with pluripotent stem cells.

It is nowadays taken granted that induced pluripotent stem cells (iPSCs) are available for the regeneration therapy since iPSCs differentiate into any kind of phenotypes. If iPSCs can choose their fate in every way of differentiation why they do not choose cancer phenotype. As a body develops for one fertilized egg, embryonic stem cell must choose every phenotype of tissues such as blood, neuron, lung, liver, pancreas and so on depending on the stages. And sometimes the cells get cancer. So do iPSCs because iPSCs are almost equivalent to embryonic cells. Then how can the safety of the regeneration therapy be maintained with iPSCs? When inducing the differentiation of iPSCs it is considered important to choose the proper conditions of culture such as 3D-platform for embryoid, supplement of cytokines and growth factors, inhibition of signaling and so on. On the other hand, several conditions have been reported to induce cancer stem cells. The cancer inducing conditions are possibly summarized as the factors chronically exposed to iPSCs. It is further worthwhile noticing that the conditions do not appear to induce mutations but affecting the epigenetics. Collectively, to secure the safety of regeneration therapy, it appears the best way to avoid the conditions to induce cancer stem cells. Further insights in details will be discussed in the lecture. Type 1 Diabetes (T1D) is an autoimmune disease characterized by destruction of the pancreatic beta cells and loss of insulin. Using the Edmonton protocol, donor-derived islets seeded into the liver successfully restore glycemia in 58% of T1D patients. However, donor scarcity, risks associated with immunosuppressants and poor engraftment limit this therapeutic application to a small number of patients. To overcome these challenges, the developmental potential of human embryonic stem cells and human induced pluripotent stem cells is being harnessed to produce surrogate islets in vitro. We and others have been able to mimic human embryonic development and generate pancreatic progenitors (PP) that have the ability to mature into insulin-producing beta-like cells both in vitro and in vivo. Transplantation of pancreatic progenitors in the kidney capsule of immunodeficient mice leads to formation of islet-like structures that secrete human insulin. However, there are some limitations to the use of pancreatic progenitors for the treatment of T1D. First and foremost, their safety as the PP population can be heterogenous and highly proliferative, which might lead to formation of cellular outgrowth or teratoma after transplantation. Second, while insulin-producing cells develop in vivo 6 weeks after transplantation, restoration of normoglycemia occurs ~5 months later, suggesting that these "early" insulin-producing cells are immature, or poorly connected to the host vasculature. We have been addressing these two limitations and developed approaches to 1) improve safety by identifying markers to purify the PP populations and 2) accelerate functionality by improving vascularization at the time of transplantation.

Single-cell lineage analysis reveals extensive multimodal transcriptional control during directed beta-cell differentiation.

The in vitro differentiation of insulin-producing β-like cells can model aspects of human pancreatic development. Here we generate 95,308 single cell transcriptomes and reconstruct a lineage tree of the entire differentiation process from hESCs to β-like cells to study temporally regulated genes during differentiation. We identify so-called ‘switch genes’ at the branch point of endocrine/non-endocrine cell fate choice, revealing insights into the mechanisms of differentiation promoting reagents, such as NOTCH and ROCKII inhibitors, and providing improved differentiation protocols. Over 20% of all detectable genes are activated multiple times during differentiation, even though their enhancer activation is usually unimodal, indicating extensive gene reuse driven by different enhancers. We also identify a stage-specific enhancer in the TCF7L2 diabetes GWAS locus that drives a transient wave of gene expression in pancreatic progenitors. Finally, we develop a web app to visualize gene expression on the lineage tree, providing a comprehensive single cell data resource for researchers studying islet biology and diabetes.

Mesenchymal stem cells promote pancreatic \u03b2-cell regeneration through downregulation of FoxO1 pathway

BackgroundMesenchymal stem cells (MSC) are non-haematopoietic, fibroblast-like multipotent stromal cells. In the injured pancreas, these cells are assumed to secrete growth factors and immunomodulatory molecules, which facilitate the regeneration of pre-existing β-cells. However, when MSC are delivered intravenously, their majority is entrapped in the lungs and does not reach the pancreas. Therefore, the aim of this investigation was to compare the regenerative support of hTERT-MSC (human telomerase reverse transcriptase mesenchymal stem cells) via intrapancreatic (IPR) and intravenous route (IVR).MethodshTERT-MSC were administered by IPR and IVR to 50% pancreatectomized NMRI nude mice. After eight days, blood glucose level, body weight, and residual pancreatic weight were measured. Proliferating pancreatic β-cells were labelled and identified with bromodeoxyuridine (BrdU) in vivo. The number of residual islets and the frequency of proliferating β-cells were compared in different groups with sequential pancreatic sections. The pancreatic insulin content was evaluated by enzyme-linked immunosorbent assay (ELISA) and the presence of hTERT-MSC with human Alu sequence. Murine gene expression of growth factors, β-cell specific molecules and proinflammatory cytokines were inspected by real-time polymerase chain reaction (RT-PCR) and Western blot.ResultsThis study evaluated the regenerative potential of the murine pancreas post-hTERT-MSC administration through the intrapancreatic (IPR) and intravenous route (IVR). Both routes of hTERT-MSC transplantation (IVR and IPR) increased the incorporation of BrdU by pancreatic β-cells compared to control. MSC induced epidermal growth factor (EGF) expression and inhibited proinflammatory cytokines (IFN-γ and TNF-α). FOXA2 and PDX-1 characteristics for pancreatic progenitor cells were activated via AKT/ PDX-1/ FoxO1 signalling pathway.ConclusionThe infusion of hTERT-MSC after partial pancreatectomy (Px) through the IVR and IPR facilitated the proliferation of autochthonous pancreatic β-cells and provided evidence for a regenerative influence of MSC on the endocrine pancreas. Moderate benefit of IPR over IVR was observed which could be a new treatment option for preventing diabetes mellitus after pancreas surgery.

Abstract PO-058: Transcriptional subtype-specific microenvironmental crosstalk and tumor cell plasticity in metastatic pancreatic cancer

Abstract Metastatic pancreatic ductal adenocarcinoma (PDAC) is an aggressive and lethal malignancy with few therapeutic options. Tumor transcriptional state is a strong predictor of clinical outcome in PDAC, with two primary cell states, basal-like and classical, identified by bulk transcriptional profiling. Basal-like tumors carry a worse prognosis, but the mechanisms underlying this survival difference, the degree of cellular heterogeneity within a given tumor, and the subtype-specific contributions from the local immune microenvironment are not well understood. In addition, there are ongoing efforts to use patient-derived organoid models as functional surrogates for an individual patient’s disease, but the degree to which patient transcriptional phenotypes are preserved in their matched organoid models remains unclear. Here, we describe a pipeline that enables both direct characterization of the liver metastatic niche via single-cell RNA-sequencing and functional assessment of PDAC tumor biology in patient-matched organoid models. Starting from core needle biopsies of metastatic PDAC lesions, we applied this approach to profile 22 patient samples and their matched organoid models using single-cell RNA-sequencing with Seq-Well. We demonstrate significant heterogeneity at the single-cell level across the basal-like to classical transcriptional spectrum. Basal-like cells expressed more mesenchymal and stem-like features, while classical cells expressed features of epithelial and pancreatic progenitor transcriptional programs. A population of “hybrid” malignant cells co-expressed markers of both basal-like and classical states, suggesting that these phenotypes lie on a continuum rather than as discrete entities. Microenvironmental composition also differed by subtype across T/NK and macrophage populations. Specifically, basal-like tumors exhibited tumor cell crosstalk with specific macrophage subsets, while classical tumors harbored greater immune infiltration and a relatively pro-angiogenic microenvironment, raising important considerations for subtype-specific microenvironmental directed therapy. Finally, we found that matched organoids exhibited transcriptional drift along the basal-like to classical axis relative to their parent tumors, with evidence for selection against basal-like phenotypes in vitro. However, tumor cells in organoid culture exhibited remarkable plasticity and could recover in vivo basal-like phenotypes in response to changes in their growth conditions. Taken together, our work provides a framework for the analysis of human cancers and their matched models using single-cell methods to dissect tumor-intrinsic and extrinsic contributions, and reveals novel insights into the transcriptional heterogeneity and plasticity of PDAC. Citation Format: Srivatsan Raghavan, Peter S. Winter, Andrew W. Navia, Hannah L. Williams, Alan DenAdel, Radha L. Kalekar, Jennyfer Galvez-Reyes, Kristen E. Lowder, Nolawit Mulugeta, Manisha S. Raghavan, Ashir A. Borah, Sara A. Vayrynen, Andressa Dias Costa, Junning Wang, Emma Reilly, Dorisanne Y. Ragon, Lauren K. Brais, Alex M. Jaeger, James M. Cleary, Lorin Crawford, Jonathan A. Nowak, Brian M. Wolpin, William C. Hahn, Andrew J. Aguirre, Alex K. Shalek. Transcriptional subtype-specific microenvironmental crosstalk and tumor cell plasticity in metastatic pancreatic cancer [abstract]. In: Proceedings of the AACR Virtual Special Conference on Pancreatic Cancer; 2020 Sep 29-30. Philadelphia (PA): AACR; Cancer Res 2020;80(22 Suppl):Abstract nr PO-058.

Generation of Pancreatic Progenitors from Human Pluripotent Stem Cells by Small Molecules

Human pluripotent stem cells (hPSCs)-derived pancreatic progenitors (PPs) provide promising cell therapies for Type I Diabetes (T1D). Current PP differentiation requires high amount of Activin A during definitive endoderm (DE) stage, making it economically difficult for commercial ventures. Here we identify a dose-dependent role of Wnt signaling in controlling DE differentiation without Activin A. While high-level Wnt activation induces mesodermal formation, low-level Wnt activation by a small-molecule inhibitor of glycogen synthase kinase (GSK3) is sufficient for DE differentiation, yielding SOX17+FOXA2+ DE cells. BMP inhibition further enhances Wnt activation induced DE differentiation, generating over 87% DE cells, which could be further differentiated into PPs and functional β cells. RNA sequencing analysis of PP differentiation from hPSCs revealed expected transcriptome dynamics and new gene regulators during small molecule PP differentiation protocol. Overall, we establish a robust growth factor-free protocol for generating DE and PP cells, facilitating scalable production of pancreatic cells for regenerative applications.

Combined Omics Approaches Reveal the Roles of Non-canonical WNT7B Signaling and YY1 in the Proliferation of Human Pancreatic Progenitor Cells

The proliferation of human pancreatic progenitor cells (PPCs) is critical for developing cell therapies for diabetes. Here, using transcriptome analysis combined with small interfering RNA (siRNA) screening, we revealed that WNT7B is a downstream growth factor of AT7867, a compound known to promote the proliferation of PPCs generated from human pluripotent stem cells. Feeder cell lines stably expressing mouse Wnt7a or Wnt7b, but not other Wnts, enhanced PPC proliferation in the absence of AT7867. Importantly, Wnt7a/b ligands did not activate the canonical Wnt pathway, and PPC proliferation depended on the non-canonical Wnt/PKC pathway. A comparison of the phosphoproteome in response to AT7867 or a newly synthesized AT7867 derivative uncovered the function of YY1 as a transcriptional regulator of WNT7B. Overall, our data highlight unknown roles of non-canonical WNT7B/PKC signaling and YY1 in human PPC proliferation and will contribute to the stable supply of a cell source for pancreatic disease modeling and therapeutic applications.

Single-cell patterning and axis characterization in the murine and human definitive endoderm.

Defining the precise regionalization of specified definitive endoderm progenitors is critical for understanding the mechanisms underlying the generation and regeneration of respiratory and digestive organs, yet the patterning of endoderm progenitors remains unresolved, particularly in humans. We performed single-cell RNA sequencing on endoderm cells during the early somitogenesis stages in mice and humans. We developed molecular criteria to define four major endoderm regions (foregut, lip of anterior intestinal portal, midgut, and hindgut) and their developmental pathways. We identified the cell subpopulations in each region and their spatial distributions and characterized key molecular features along the body axes. Dorsal and ventral pancreatic progenitors appear to originate from the midgut population and follow distinct pathways to develop into an identical cell type. Finally, we described the generally conserved endoderm patterning in humans and clear differences in dorsal cell distribution between species. Our study comprehensively defines single-cell endoderm patterning and provides novel insights into the spatiotemporal process that drives establishment of early endoderm domains.