Multipotent Pancreatic Progenitor Cells Research Articles

P53 Inhibition in Pancreatic Progenitors Enhances the Differentiation of Human Pluripotent Stem Cells into Pancreatic β-Cells

The multipotent pancreatic progenitor cells (MPCs) co-expressing the transcription factors, PDX1 and NKX6.1, are the source of functional pancreatic β-cells. The aim of this study was to examine the effect of p53 inhibition in MPCs on the generation of PDX1+/NKX6.1+ MPCs and pancreatic β-cell generation. Human embryonic stem cells (hESCs) were differentiated into MPCs and β-cells. hESC-MPCs (stage 4) were treated with different concentrations of p53 inhibitors, and their effect was evaluated using different approaches. NKX6.1 was overexpressed during MPCs specification. Inhibition of p53 using pifithrin-μ (PFT-μ) at the MPC stage resulted in a significant increase in the number of PDX1+/NKX6.1+ cells and a reduction in the number of CHGA+/NKX6.1− cells. Further differentiation of MPCs treated with PFT-μ into pancreatic β-cells showed that PFT-μ treatment did not significantly change the number of C-Peptide+ cells; however, the number of C-PEP+ cells co-expressing glucagon (polyhormonal) was significantly reduced in the PFT-μ treated cells. Interestingly, overexpression of NKX6.1 in hESC-MPCs enhanced the expression of key MPC genes and dramatically suppressed p53 expression. Our findings demonstrated that the p53 inhibition during stage 4 of differentiation enhanced MPC generation, prevented premature endocrine induction and favored the differentiation into monohormonal β-cells. These findings suggest that adding a p53 inhibitor to the differentiation media can significantly enhance the generation of monohormonal β-cells.Graphical

Jag1-Notch cis-interaction determines cell fate segregation in pancreatic development

The Notch ligands Jag1 and Dll1 guide differentiation of multipotent pancreatic progenitor cells (MPCs) into unipotent pro-acinar cells (PACs) and bipotent duct/endocrine progenitors (BPs). Ligand-mediated trans-activation of Notch receptors induces oscillating expression of the transcription factor Hes1, while ligand-receptor cis-interaction indirectly represses Hes1 activation. Despite Dll1 and Jag1 both displaying cis- and trans-interactions, the two mutants have different phenotypes for reasons not fully understood. Here, we present a mathematical model that recapitulates the spatiotemporal differentiation of MPCs into PACs and BPs. The model correctly captures cell fate changes in Notch pathway knockout mice and small molecule inhibitor studies, and a requirement for oscillatory Hes1 expression to maintain the multipotent state. Crucially, the model entails cell-autonomous attenuation of Notch signaling by Jag1-mediated cis-inhibition in MPC differentiation. The model sheds light on the underlying mechanisms, suggesting that cis-interaction is crucial for exiting the multipotent state, while trans-interaction is required for adopting the bipotent fate.

Abstract PO-067: A multi-omics study in patient-derived organoids reveals MNX1-HNF1B axis to be indispensable for intraductal mucinous papillary neoplasm lineages

Abstract Chromatin architecture governs cell lineages by regulating the specific gene expression; however, its role in the diversity of cancer development remains unknown. Among pancreatic cancers, pancreatic ductal adenocarcinoma (PDAC) and invasive carcinoma with an associated intraductal papillary mucinous neoplasms (invasive IPMNs) arise from two distinct precursors, and their fundamental differences remain obscure. Here, we hypothesized that chromatin profiles may clarify their intrinsic molecular features. We originally established 28 human organoids from distinct subtypes of pancreatic tumors, including IPMN, invasive IPMN, and PDAC as well as from normal ductal cells and performed exome-seq, RNA-seq, ATAC-seq, ChIP-seq, Hi-C, and phenotypic analyses with shRNA or CRISPR interference. Established organoids successfully reproduced the histology of primary tumors. IPMN and invasive IPMN organoids specifically harbored GNAS, RNF43, and KLF4 mutations and showed the distinct lineage related expression profiles compared to PDAC. In addition, chromatin accessibility profiles well stratified the respective tumor groups. Notably, this analysis supported the histological features of tumor subtypes; gastric IPMN gained the stomach-specific accessible regions and the accessible pattern of invasive IPMN linked to diverse gastrointestinal tissues. In contrast, PDAC was characterized by the significant loss of chromatin accessibility seen in normal pancreatic ductal cells. Footprint analysis of transcription factors (TFs) identified specific TFs that are enriched in each tumor subtype. Of note we found the footprint of HNF1B to be active in IPMN lineages but not in PDAC. To address its biological significance, we analyzed the effects of HNF1B by knockdown (KD) experiments and revealed that HNF1B is biologically indispensable for IPMN lineages. We further identified MNX1 as an upstream regulator of HNF1B expression, another TF expressed in multipotent pancreatic progenitor cells. ChIP experiment revealed the enriched binding of MNX1 on the promoter elements of HNF1B in invasive IPMN. Importantly, KD or CRISPR interference of MNX1 impaired the survival of the organoids from invasive IPMN. Moreover, the correlated and high expression patterns of MNX1 and HNF1B in IPMN lineages were validated in a set of human tissues. To identify the common downstream genes of MNX1 and HNF1B, we analyzed RNA-seq and ATAC-seq after KD of the two genes. We found that MNX1-HNF1B axis governed a set of genes including MYC, SOX9, and OLFM4, which are known to regulate stem cell properties in gastrointestinal epithelium. Lastly, to get a broader view of chromatin architecture in these tumors, we performed Hi-C. We found that HNF1B target genes to be three-dimensionally condensed in the genome of invasive IPMN but not in that of PDAC, supporting the functional importance of those genes in invasive IPMN. Collectively, our organoid analyses unraveled the different lineage related chromatin structures correlating to the specific biological behaviors in pancreatic tumor subtypes. Citation Format: Hiroyuki Kato, Keisuke Tateishi, Keisuke Yamamoto, Dousuke Iwadate, Hiroaki Fujiwara, Takuma Nakatsuka, Koji Miyabayashi, Yotaro Kudo, Ijichi Hideaki, Kazuhiko Koike, Mitsuhiro Fujishiro. A multi-omics study in patient-derived organoids reveals MNX1-HNF1B axis to be indispensable for intraductal mucinous papillary neoplasm lineages [abstract]. In: Proceedings of the AACR Virtual Special Conference on Pancreatic Cancer; 2021 Sep 29-30. Philadelphia (PA): AACR; Cancer Res 2021;81(22 Suppl):Abstract nr PO-067.

Modeling HNF1B-associated monogenic diabetes using human iPSCs reveals an early stage impairment of the pancreatic developmental program.

SummaryHeterozygous mutations in HNF1B in humans result in a multisystem disorder, including pancreatic hypoplasia and diabetes mellitus. Here we used a well-controlled human induced pluripotent stem cell pancreatic differentiation model to elucidate the molecular mechanisms underlying HNF1B-associated diabetes. Our results show that lack of HNF1B blocks specification of pancreatic fate from the foregut progenitor (FP) stage, but HNF1B haploinsufficiency allows differentiation of multipotent pancreatic progenitor cells (MPCs) and insulin-secreting β-like cells. We show that HNF1B haploinsufficiency impairs cell proliferation in FPs and MPCs. This could be attributed to impaired induction of key pancreatic developmental genes, including SOX11, ROBO2, and additional TEAD1 target genes whose function is associated with MPC self-renewal. In this work we uncover an exhaustive list of potential HNF1B gene targets during human pancreas organogenesis whose downregulation might underlie HNF1B-associated diabetes onset in humans, thus providing an important resource to understand the pathogenesis of this disease.

Jag1 Modulates an Oscillatory Dll1-Notch-Hes1 Signaling Module to Coordinate Growth and Fate of Pancreatic Progenitors.

Notch signaling controls proliferation of multipotent pancreatic progenitor cells (MPCs) and their segregation into bipotent progenitors (BPs) and unipotent pro-acinar cells (PACs). Here, we showed that fast ultradian oscillations of the ligand Dll1 and the transcriptional effector Hes1 were crucial for MPC expansion, and changes in Hes1 oscillation parameters were associated with selective adoption of BP or PAC fate. Conversely, Jag1, a uniformly expressed ligand, restrained MPC growth. However, when its expression later segregated to PACs, Jag1 became critical for the specification of all but the most proximal BPs, and BPs were entirely lost in Jag1; Dll1 double mutants. Anatomically, ductal morphogenesis and organ architecture are minimally perturbed in Jag1 mutants until later stages, when ductal remodeling fails, and signs of acinar-to-ductal metaplasia appear. Our study thus uncovers that oscillating Notch activity in the developing pancreas, modulated by Jag1, is required to coordinate MPC growth and fate.

C-Myc downregulation is required for preacinar to acinar maturation and pancreatic homeostasis

Background and aimsc-Myc is highly expressed in pancreatic multipotent progenitor cells (MPC) and in pancreatic cancer. The transition from MPC to unipotent acinar progenitors is associated with c-Myc downregulation; a...

Generation of polyhormonal and multipotent pancreatic progenitor lineages from human pluripotent stem cells

Generation of pancreatic β-cells from human pluripotent stem cells (hPSCs) has enormous importance in type 1 diabetes (T1D), as it is fundamental to a treatment strategy based on cellular therapeutics. Being able to generate β-cells, as well as other mature pancreatic cells, from human embryonic stem cells (hESCs) and human induced pluripotent stem cells (hiPSCs) will also enable the development of platforms that can be used for disease modeling and drug testing for a variety of pancreas-associated diseases, including cystic fibrosis. For this to occur, it is crucial to develop differentiation strategies that are robust and reproducible across cell lines and laboratories. In this article we describe two serum-free differentiation protocols designed to generate specific pancreatic lineages from hPSCs. Our approach employs a variety of cytokines and small molecules to mimic developmental pathways active during pancreatic organogenesis and allows for the in vitro generation of distinct pancreatic populations. The first protocol is designed to give rise to polyhormonal cells that have the potential to differentiate into glucagon-producing cells. The second protocol is geared to generate multipotent pancreatic progenitor cells, which harbor the potential to generate all pancreatic lineages including: monohormonal endocrine cells, acinar, and ductal cells.

Hnf1b controls pancreas morphogenesis and the generation of Ngn3+ endocrine progenitors.

Heterozygous mutations in the human HNF1B gene are associated with maturity-onset diabetes of the young type 5 (MODY5) and pancreas hypoplasia. In mouse, Hnf1b heterozygous mutants do not exhibit any phenotype, whereas the homozygous deletion in the entire epiblast leads to pancreas agenesis associated with abnormal gut regionalization. Here, we examine the specific role of Hnf1b during pancreas development, using constitutive and inducible conditional inactivation approaches at key developmental stages. Hnf1b early deletion leads to a reduced pool of pancreatic multipotent progenitor cells (MPCs) due to decreased proliferation and increased apoptosis. Lack of Hnf1b either during the first or the secondary transitions is associated with cystic ducts. Ductal cells exhibit aberrant polarity and decreased expression of several cystic disease genes, some of which we identified as novel Hnf1b targets. Notably, we show that Glis3, a transcription factor involved in duct morphogenesis and endocrine cell development, is downstream Hnf1b. In addition, a loss and abnormal differentiation of acinar cells are observed. Strikingly, inactivation of Hnf1b at different time points results in the absence of Ngn3(+) endocrine precursors throughout embryogenesis. We further show that Hnf1b occupies novel Ngn3 putative regulatory sequences in vivo. Thus, Hnf1b plays a crucial role in the regulatory networks that control pancreatic MPC expansion, acinar cell identity, duct morphogenesis and generation of endocrine precursors. Our results uncover an unappreciated requirement of Hnf1b in endocrine cell specification and suggest a mechanistic explanation of diabetes onset in individuals with MODY5.

Distinct requirements for beta-catenin in pancreatic epithelial growth and patterning

Pancreatic exocrine and endocrine lineages arise from multipotent pancreatic progenitor cells (MPCs). Exploiting the mechanisms that govern expansion and differentiation of these cells could enhance efforts to generate β-cells from stem cells. Although our prior work indicates that the canonical Wnt signaling component β-catenin is required qualitatively for exocrine acinar but not endocrine development, precisely how this requirement plays out at the level of MPCs and their lineage-restricted progeny is unknown. In addition, the contribution of β-catenin function to β-cell development remains controversial. To resolve the potential roles of β-catenin in development of MPCs and β-cells, we generated pancreas- and pre-endocrine-specific β-catenin knockout mice. Pancreas-specific loss of β-catenin produced not only a dramatic reduction in acinar cell numbers, but also a significant reduction in β-cell mass. The loss of β-cells is due not to a defect in the differentiation of endocrine precursors, but instead correlates with an early and specific loss of MPCs. In turn, this reflects a novel role for β-catenin in maintaining proximal–distal patterning of the early epithelium, such that distal MPCs resort to a proximal, endocrine-competent “trunk” fate when β-catenin is deleted. Moreover, β-catenin maintains proximal–distal patterning, in part, by inhibiting Notch signaling. Subsequently, β-catenin is required for proliferation of both distal and proximal cells, driving overall organ growth. In distinguishing two distinct roles for β-catenin along the route of β-cell development, we suggest that temporally appropriate positive and negative manipulation of this molecule could enhance expansion and differentiation of stem cell-derived MPCs.

Neurogenin3 Cooperates with Foxa2 to Autoactivate Its Own Expression

The transcription factor Neurogenin3 functions as a master regulator of endocrine pancreas formation, and its deficiency leads to the development of diabetes in humans and mice. In the embryonic pancreas, Neurogenin3 is transiently expressed at high levels for a narrow time window to initiate endocrine differentiation in scattered progenitor cells. The mechanisms controlling these rapid and robust changes in Neurogenin3 expression are poorly understood. In this study, we characterize a Neurogenin3 positive autoregulatory loop whereby this factor may rapidly induce its own levels. We show that Neurogenin3 binds to a conserved upstream fragment of its own gene, inducing deposition of active chromatin marks and the activation of Neurog3 transcription. Additionally, we show that the broadly expressed endodermal forkhead factors Foxa1 and Foxa2 can cooperate synergistically to amplify Neurogenin3 autoregulation in vitro. However, only Foxa2 colocalizes with Neurogenin3 in pancreatic progenitors, thus indicating a primary role for this factor in regulating Neurogenin3 expression in vivo. Furthermore, in addition to decreasing Neurog3 autoregulation, inhibition of Foxa2 by RNA interference attenuates Neurogenin3-dependent activation of the endocrine developmental program in cultured duct mPAC cells. Hence, these data uncover the potential functional cooperation between the endocrine lineage-determining factor Neurogenin3 and the widespread endoderm progenitor factor Foxa2 in the implementation of the endocrine developmental program in the pancreas.

On the role of lateral stabilization during early patterning in the pancreas

The cell fate decision of multi-potent pancreatic progenitor cells between the exocrine and endocrine lineages is regulated by Notch signalling, mediated by cell-cell interactions. However, canonical models of Notch-mediated lateral inhibition cannot explain the scattered spatial distribution of endocrine cells and the cell-type ratio in the developing pancreas. Based on evidence from acinar-to-islet cell transdifferentiation in vitro, we propose that lateral stabilization, i.e. positive feedback between adjacent progenitor cells, acts in parallel with lateral inhibition to regulate pattern formation in the pancreas. A simple mathematical model of transcriptional regulation and cell-cell interaction reveals the existence of multi-stability of spatial patterns whose simultaneous occurrence causes scattering of endocrine cells in the presence of noise. The scattering pattern allows for control of the endocrine-to-exocrine cell-type ratio by modulation of lateral stabilization strength. These theoretical results suggest a previously unrecognized role for lateral stabilization in lineage specification, spatial patterning and cell-type ratio control in organ development.

Spatiotemporal patterns of multipotentiality in Ptf1a-expressing cells during pancreas organogenesis and injury-induced facultative restoration

Pancreatic multipotent progenitor cells (MPCs) produce acinar, endocrine and duct cells during organogenesis, but their existence and location in the mature organ remain contentious. We used inducible lineage-tracing from the MPC-instructive gene Ptf1a to define systematically in mice the switch of Ptf1a(+) MPCs to unipotent proacinar competence during the secondary transition, their rapid decline during organogenesis, and absence from the mature organ. Between E11.5 and E15.5, we describe tip epithelium heterogeneity, suggesting that putative Ptf1a(+)Sox9(+)Hnf1β(+) MPCs are intermingled with Ptf1a(HI)Sox9(LO) proacinar progenitors. In the adult, pancreatic duct ligation (PDL) caused facultative reactivation of multipotency factors (Sox9 and Hnf1β) in Ptf1a(+) acini, which undergo rapid reprogramming to duct cells and longer-term reprogramming to endocrine cells, including insulin(+) β-cells that are mature by the criteria of producing Pdx1(HI), Nkx6.1(+) and MafA(+). These Ptf1a lineage-derived endocrine/β-cells are likely formed via Ck19(+)/Hnf1β(+)/Sox9(+) ductal and Ngn3(+) endocrine progenitor intermediates. Acinar to endocrine/β-cell transdifferentiation was enhanced by combining PDL with pharmacological elimination of pre-existing β-cells. Thus, we show that acinar cells, without exogenously introduced factors, can regain aspects of embryonic multipotentiality under injury, and convert into mature β-cells.

PS21 - 103. Multipotent pancreatic progenitor cells in 3D culture of human pancreatic ductal tissue: potential relevance for beta cell therapy in Type 1 diabetes

An attractive regenerative medicine approach towards β-cell replacement therapy is the derivation of insulinsecreting cells from stem/progenitor cells. We applied a novel 3-dimensional (3D) matrigel-based culture system to generate human pancreatic tissue (‘pancreatic organoids’) in vitro as a potential source for human β-cells.

Notch-mediated patterning and cell fate allocation of pancreatic progenitor cells

Early pancreatic morphogenesis is characterized by the transformation of an uncommitted pool of pancreatic progenitor cells into a branched pancreatic epithelium that consists of 'tip' and 'trunk' domains. These domains have distinct molecular signatures and differentiate into distinct pancreatic cell lineages. Cells at the branched tips of the epithelium develop into acinar cells, whereas cells in the trunk subcompartment differentiate into endocrine and duct cells. Recent genetic analyses have highlighted the role of key transcriptional regulators in the specification of these subcompartments. Here, we analyzed in mice the role of Notch signaling in the patterning of multipotent pancreatic progenitor cells through mosaic overexpression of a Notch signaling antagonist, dominant-negative mastermind-like 1, resulting in a mixture of wild-type and Notch-suppressed pancreatic progenitor cells. We find that attenuation of Notch signaling has pronounced patterning effects on multipotent pancreatic progenitor cells prior to terminal differentiation. Relative to the wild-type cells, the Notch-suppressed cells lose trunk marker genes and gain expression of tip marker genes. The Notch-suppressed cells subsequently differentiate into acinar cells, whereas duct and endocrine populations are formed predominantly from the wild-type cells. Mechanistically, these observations could be explained by a requirement of Notch for the expression of the trunk determination gene Nkx6.1. This was supported by the finding of direct binding of RBP-jκ to the Nkx6.1 proximal promoter.

Ptf1a-mediated control of Dll1 reveals an alternative to the lateral inhibition mechanism

Neurog3-induced Dll1 expression in pancreatic endocrine progenitors ostensibly activates Hes1 expression via Notch and thereby represses Neurog3 and endocrine differentiation in neighboring cells by lateral inhibition. Here we show in mouse that Dll1 and Hes1 expression deviate during regionalization of early endoderm, and later during early pancreas morphogenesis. At that time, Ptf1a activates Dll1 in multipotent pancreatic progenitor cells (MPCs), and Hes1 expression becomes Dll1 dependent over a brief time window. Moreover, Dll1, Hes1 and Dll1/Hes1 mutant phenotypes diverge during organ regionalization, become congruent at early bud stages, and then diverge again at late bud stages. Persistent pancreatic hypoplasia in Dll1 mutants after eliminating Neurog3 expression and endocrine development, together with reduced proliferation of MPCs in both Dll1 and Hes1 mutants, reveals that the hypoplasia is caused by a growth defect rather than by progenitor depletion. Unexpectedly, we find that Hes1 is required to sustain Ptf1a expression, and in turn Dll1 expression in early MPCs. Our results show that Ptf1a-induced Dll1 expression stimulates MPC proliferation and pancreatic growth by maintaining Hes1 expression and Ptf1a protein levels.

Rfx6 is an Ngn3-dependent winged helix transcription factor required for pancreatic islet cell development

The transcription factor neurogenin 3 (Neurog3 or Ngn3) controls islet cell fate specification in multipotent pancreatic progenitor cells in the mouse embryo. However, our knowledge of the genetic programs implemented by Ngn3, which control generic and islet subtype-specific properties, is still fragmentary. Gene expression profiling in isolated Ngn3-positive progenitor cells resulted in the identification of the uncharacterized winged helix transcription factor Rfx6. Rfx6 is initially expressed broadly in the gut endoderm, notably in Pdx1-positive cells in the developing pancreatic buds, and then becomes progressively restricted to the endocrine lineage, suggesting a dual function in both endoderm development and islet cell differentiation. Rfx6 is found in postmitotic islet progenitor cells in the embryo and is maintained in all developing and adult islet cell types. Rfx6 is dependent on Ngn3 and acts upstream of or in parallel with NeuroD, Pax4 and Arx transcription factors during islet cell differentiation. In zebrafish, the Rfx6 ortholog is similarly found in progenitors and hormone expressing cells of the islet lineage. Loss-of-function studies in zebrafish revealed that rfx6 is required for the differentiation of glucagon-, ghrelin- and somatostatin-expressing cells, which, in the absence of rfx6, are blocked at the progenitor stage. By contrast, beta cells, whose number is only slightly reduced, were no longer clustered in a compact islet. These data unveil Rfx6 as a novel regulator of islet cell development.

Pdx-1 and Ptf1a concurrently determine fate specification of pancreatic multipotent progenitor cells

The pancreas is derived from a pool of multipotent progenitor cells (MPCs) that co-express Pdx-1 and Ptf1a. To more precisely define how the individual and combined loss of Pdx-1 and Ptf1a affects pancreatic MPC specification and differentiation we derived and studied mice bearing a novel Ptf1aYFP allele. While the expression of Pdx-1 and Ptf1a in pancreatic MPCs coincides between E9.5 and 12.5 the developmental phenotypes of Pdx-1 null and Pdx-1; Ptf1a double null mice are indistinguishable, and an early pancreatic bud is formed in both cases. This finding indicates that Pdx-1 is required in the foregut endoderm prior to Ptf1a for pancreatic MPC specification. We also found that Ptf1a is neither required for specification of Ngn3-positive endocrine progenitors nor differentiation of mature β-cells. In the absence of Pdx-1 Ngn3-positive cells were not observed after E9.5. Thus, in contrast to the deletion of Ptf1a, the loss of Pdx-1 precludes the sustained Ngn3-based derivation of endocrine progenitors from pancreatic MPCs. Taken together, these studies indicate that Pdx-1 and Ptf1a have distinct but interdependent functions during pancreatic MPC specification.

Multipotent Progenitor Cells Isolated From Adult Human Pancreatic Tissue

The supply of islet cells is a limiting factor for the widespread application of islet transplantation of type-1 diabetes. Islets constitute 1% to 2% of pancreatic tissue, leaving approximately 98% as discard after islet isolation and purification. In this report we present our data on the isolation of multipotent progenitor cells from discarded adult human pancreatic tissue. The collected cells from discarded nonislet fractions, after enzymatic digestion and gradient purification of islets, were dissociated for suspension culture in a serum-free medium. The cell clusters grown to a size of 100 to 150 μm contained cells staining for stage-specific embryonic antigens, but not insulin or C-peptide. To direct cell differentiation toward islets, clusters were recultured in a pancreatic differentiation medium. Insulin and C-peptide–positive cells by immunocytochemistry appeared within a week, reaching over 10% of the cell population. Glucagon and somatostatin-positive cells were also detected. The cell clusters were found to secrete insulin in response to glucose stimulation. Cells from the same clusters also had the capacity for differentiation into neural cells, as documented by staining for neural and glial cell markers when cultured as monolayers in media containing neurotrophic factors. These data suggest that multipotent pancreatic progenitor cells exist within the human pancreatic tissue that is typically discarded during islet isolation procedures. These adult progenitor cells can be successfully differentiated into insulin-producing cells, and thus they have the potential for treatment of type-1 diabetes mellitus.