Transcriptional changes and the role of ONECUT1 in hPSC pancreatic differentiation

Cell type specification during pancreatic development is tightly controlled by a transcriptional and epigenetic network. The precise role of most transcription factors, however, has been only described in mice. To convey such concepts to human pancreatic development, alternative model systems such as pancreatic in vitro differentiation of human pluripotent stem cells can be employed. Here, we analyzed stage-specific RNA-, ChIP-, and ATAC-sequencing data to dissect transcriptional and regulatory mechanisms during pancreatic development. Transcriptome and open chromatin maps of pancreatic differentiation from human pluripotent stem cells provide a stage-specific pattern of known pancreatic transcription factors and indicate ONECUT1 as a crucial fate regulator in pancreas progenitors. Moreover, our data suggest that ONECUT1 is also involved in preparing pancreatic progenitors for later endocrine specification. The dissection of the transcriptional and regulatory circuitry revealed an important role for ONECUT1 within such network and will serve as resource to study human development and disease.

Primate-specific ZNF808 is essential for pancreatic development in humans

Identifying genes linked to extreme phenotypes in humans has the potential to highlight new biological processes fundamental for human development. Here we report the identification of homozygous loss of function variants in the primate-specific gene ZNF808 as a cause of pancreatic agenesis. ZNF808 is a member of the KRAB zinc finger protein (KZFPs) family, a large and rapidly evolving group of epigenetic silencers that target transposable elements. Loss of ZNF808 in vitro results in aberrant activation of many transposable elements it normally represses during early pancreas development. We show that this results in inappropriate specification of cell fate with induction of genes associated with liver endoderm and a loss of pancreatic identity. This suggests that ZNF808 and its transposable element targets play a critical role in cell fate specification during human pancreatic development. This is the first report of loss of a primate-specific gene causing a congenital developmental disease and highlights the essential role of ZNF808 for pancreatic development in humans.

Activation of Pancreatic Stellate Cells Is Beneficial for Exocrine but Not Endocrine Cell Differentiation in the Developing Human Pancreas

Pancreatic stellate cells (PaSCs) are non-endocrine, mesenchymal-like cells that reside within the peri-pancreatic tissue of the rodent and human pancreas. PaSCs regulate extracellular matrix (ECM) turnover in maintaining the integrity of pancreatic tissue architecture. Although there is evidence indicating that PaSCs are involved in islet cell survival and function, its role in islet cell differentiation during human pancreatic development remains unclear. The present study examines the expression pattern and functional role of PaSCs in islet cell differentiation of the developing human pancreas from late 1st to 2nd trimester of pregnancy. The presence of PaSCs in human pancreata (8–22 weeks of fetal age) was characterized by ultrastructural, immunohistological, quantitative RT-PCR and western blotting approaches. Using human fetal PaSCs derived from pancreata at 14–16 weeks, freshly isolated human fetal islet-epithelial cell clusters (hIECCs) were co-cultured with active or inactive PaSCs in vitro. Ultrastructural and immunofluorescence analysis demonstrated a population of PaSCs near ducts and newly formed islets that appeared to make complex cell-cell dendritic-like contacts. A small subset of PaSCs co-localized with pancreatic progenitor-associated transcription factors (PDX1, SOX9, and NKX6-1). PaSCs were highly proliferative, with significantly higher mRNA and protein levels of PaSC markers (desmin, αSMA) during the 1st trimester of pregnancy compared to the 2nd trimester. Isolated human fetal PaSCs were identified by expression of stellate cell markers and ECM. Suppression of PaSC activation, using all-trans retinoic acid (ATRA), resulted in reduced PaSC proliferation and ECM proteins. Co-culture of hIECCs, directly on PaSCs or indirectly using Millicell® Inserts or using PaSC-conditioned medium, resulted in a reduction the number of insulin+ cells but a significant increase in the number of amylase+ cells. Suppression of PaSC activation or Notch activity during the co-culture resulted in an increase in beta-cell differentiation. This study determined that PaSCs, abundant during the 1st trimester of pancreatic development but decreased in the 2nd trimester, are located near ductal and islet structures. Direct and indirect co-cultures of hIECCs with PaSCs suggest that activation of PaSCs has opposing effects on beta-cell and exocrine cell differentiation during human fetal pancreas development, and that these effects may be dependent on Notch signaling.

SETD4-expressing cells contribute to pancreatic development and response to cerulein induced pancreatitis injury

In the adult pancreas, the presence of progenitor or stem cells and their potential involvement in homeostasis and regeneration remains unclear. Here, we identify that SET domain-containing protein 4 (SETD4), a histone lysine methyltransferase, is expressed in a small cell population in the adult mouse pancreas. Genetic lineage tracing shows that during pancreatic development, descendants of SETD4+ cells make up over 70% of pancreatic cells and then contribute to each pancreatic lineage during pancreatic homeostasis. SETD4+ cells generate newborn acinar cells in response to cerulein-induced pancreatitis in acinar compartments. Ablation of SETD4+ cells compromises regeneration of acinar cells, in contrast to controls. Our findings provide a new cellular narrative for pancreatic development, homeostasis and response to injury via a small SETD4+ cell population. Potential applications may act to preserve pancreatic function in case of pancreatic disease and/or damage.

Hippo Signaling Pathway in Pancreas Development

The Hippo signaling pathway is a vital regulator of pancreatic development and homeostasis, directing cell fate decisions, morphogenesis, and adult pancreatic cellular plasticity. Through loss-of-function research, Hippo signaling has been found to play key roles in maintaining the proper balance between progenitor cell renewal, proliferation, and differentiation in pancreatic organogenesis. Other studies suggest that overactivation of YAP, a downstream effector of the pathway, promotes ductal cell development and suppresses endocrine cell fate specification via repression of Ngn3. After birth, disruptions in Hippo signaling have been found to lead to de-differentiation of acinar cells and pancreatitis-like phenotype. Further, Hippo signaling directs pancreatic morphogenesis by ensuring proper cell polarization and branching. Despite these findings, the mechanisms through which Hippo governs cell differentiation and pancreatic architecture are yet to be fully understood. Here, we review recent studies of Hippo functions in pancreatic development, including its crosstalk with NOTCH, WNT/β-catenin, and PI3K/Akt/mTOR signaling pathways.

Human Pluripotent Stem Cells Go Diabetic: A Glimpse on Monogenic Variants

Diabetes, as one of the major diseases in industrial countries, affects over 350 million people worldwide. Type 1 (T1D) and type 2 diabetes (T2D) are the most common forms with both types having invariable genetic influence. It is accepted that a subset of all diabetes patients, generally estimated to account for 1–2% of all diabetic cases, is attributed to mutations in single genes. As only a subset of these genes has been identified and fully characterized, there is a dramatic need to understand the pathophysiological impact of genetic determinants on β-cell function and pancreatic development but also on cell replacement therapies. Pluripotent stem cells differentiated along the pancreatic lineage provide a valuable research platform to study such genes. This review summarizes current perspectives in applying this platform to study monogenic diabetes variants.

Overdosage of HNF1B Gene Associated With Annular Pancreas Detected in Neonate Patients With 17q12 Duplication

The annular pancreas (AP) is a congenital anomaly of the pancreas that can cause acute abdominal pain and vomiting after birth. However, the genetic cause of AP is still unknown, and no study has reported AP in patients with 17q12 duplication. This study retrospectively analyzed the next-generation sequencing (NGS) data of individuals from January 2016 to June 2020 for 17q12 duplication. To identify the function of the key gene of HNF1B in the 17q12 duplication region, human HNF1B mRNA was microinjected into LiPan zebrafish transgenic embryos. A total of 19 cases of 17q12 duplication were confirmed. AP was diagnosed during exploratory laparotomy in four patients (21.1%). The other common features of 17q12 duplication included intellectual disability (50%), gross motor delay (50%), and seizures/epilepsy (31.58%). The ratio of the abnormal pancreas in zebrafish was significantly higher in the HNF1B overexpression models. In conclusion, we first reported AP in patients with duplication of the 17q12 region, resulting in the phenotype of 17q12 duplication syndrome. Furthermore, our zebrafish studies verified the role of the HNF1B gene in pancreatic development.

miR-375 Promotes Pancreatic Differentiation In Vitro by Affecting Different Target Genes at Different Stages

Human embryonic stem cells (hESCs) possess the ability to differentiate into insulin-producing cells (IPCs), which can be used to treat type I diabetes. miR-375 is an essential transcriptional regulator in the development and maturation of the pancreas. In this study, we optimized a protocol to differentiate hESCs into IPCs and successfully obtained IPCs. Then, we performed overexpression and inhibition experiments of miR-375 on cells at different stages of differentiation and performed RNA-seq. The results showed that the expression of miR-375 fluctuated during hESC differentiation and was affected by miR-375 mimics and inhibitors. miR-375 influences global gene expression and the target genes of miR-375. The overexpression of miR-375 can cause changes in multiple signaling pathways during pancreatic development. miR-375 is a major participant in the differentiation of pancreatic β-cells through different target genes at different stages. This study provides new ideas for further investigation of how microRNAs affect cell fate and gene transcription.