Defining multistep cell fate decision pathways during pancreatic development at single-cell resolution.

The generation of terminally differentiated cell lineages during organogenesis requires multiple, coordinated cell fate choice steps. However, this process has not been clearly delineated, especially in complex solid organs such as the pancreas. Here, we performed single-cell RNA-sequencing in pancreatic cells sorted from multiple genetically modified reporter mouse strains at embryonic stages E9.5-E17.5. We deciphered the developmental trajectories and regulatory strategies of the exocrine and endocrine pancreatic lineages as well as intermediate progenitor populations along the developmental pathways. Notably, we discovered previously undefined programs representing the earliest events in islet α- and ß-cell lineage allocation as well as the developmental pathway of the "first wave" of α-cell generation. Furthermore, we demonstrated that repressing ERK pathway activity is essential for inducing both α- and ß-lineage differentiation. This study provides key insights into the regulatory mechanisms underlying cell fate choice and stepwise cell fate commitment and can be used as a resource to guide the induction of functional islet lineage cells from stem cells in vitro.

Understanding generation and regeneration of pancreatic ß cells from a single-cell perspective.

Understanding the mechanisms that underlie the generation and regeneration of ß cells is crucial for developing treatments for diabetes. However, traditional research methods, which are based on populations of cells, have limitations for defining the precise processes of ß-cell differentiation and trans-differentiation, and the associated regulatory mechanisms. The recent development of single-cell technologies has enabled re-examination of these processes at a single-cell resolution to uncover intermediate cell states, cellular heterogeneity and molecular trajectories of cell fate specification. Here, we review recent advances in understanding ß-cell generation and regeneration, in vivo and in vitro, from single-cell technologies, which could provide insights for optimization of diabetes therapy strategies.

The contributions of mesoderm-derived cells in liver development.

The liver is an indispensable organ for metabolism and drug detoxification. The liver consists of endoderm-derived hepatobiliary lineages and various mesoderm-derived cells, and interacts with the surrounding tissues and organs through the ventral mesentery. Liver development, from hepatic specification to liver maturation, requires close interactions with mesoderm-derived cells, such as mesothelial cells, hepatic stellate cells, mesenchymal cells, liver sinusoidal endothelial cells and hematopoietic cells. These cells affect liver development through precise signaling events and even direct physical contact. Through the use of new techniques, emerging studies have recently led to a deeper understanding of liver development and its related mechanisms, especially the roles of mesodermal cells in liver development. Based on these developments, the current protocols for in vitro hepatocyte-like cell induction and liver-like tissue construction have been optimized and are of great importance for the treatment of liver diseases. Here, we review the roles of mesoderm-derived cells in the processes of liver development, hepatocyte-like cell induction and liver-like tissue construction.

CCM2L (Cerebral Cavernous Malformation 2 Like) Deletion Aggravates Cerebral Cavernous Malformation Through Map3k3-KLF Signaling Pathway.

[Figure: see text].

Dynamics of chromatin marks and the role of JMJD3 during pancreatic endocrine cell fate commitment.

Pancreatic endocrine lineages are derived from pancreatic progenitors that undergo a cell fate transition requiring a switch from low to high Ngn3 expression. However, the underlying chromatin regulatory mechanisms are unclear. Here, we performed epigenomic analysis of gene regulatory loci featuring histone marks in cells with low or high level of Ngn3 expression. In combination with transcriptomic analysis, we discovered that in Ngn3-high cells, the removal of H3K27me3 was associated with the activation of key transcription factors and the establishment of primed and active enhancers. Deletion of Jmjd3, a histone demethylase for H3K27me3, at the pancreatic progenitor stage impaired the efficiency of endocrine cell fate transition and thereafter islet formation. Curiously, single-cell RNA-seq revealed that the transcriptome and developmental pathway of Ngn3-high cells were not affected by the deletion of Jmjd3 Our study indicates sequential chromatin events and identifies a crucial role for Jmjd3 in regulating the efficiency of the transition from Ngn3-low to Ngn3-high cells.

Single-cell transcriptomic analyses reveal distinct dorsal/ventral pancreatic programs.

The pancreas of vertebrates is separately derived from both the dorsal and ventral endodermal domains. However, the difference between these two programs has been unclear. Here, using a pancreatic determination gene, Pdx1, driven GFP transgenic mouse strain, we identified Pdx1-GFP highly expressing cells (Pdx1high) and Pdx1-GFP lowly expressing cells (Pdx1low) in both embryonic dorsal Pdx1-expressing region (DPR) and ventral Pdx1-expressing region (VPR). We analyzed the transcriptomes of single Pdx1low and Pdx1high cells from the DPR and VPR. In the VPR, Pdx1low cells have an intermediate progenitor identity and can generate hepatoblasts, extrahepatobiliary cells, and Pdx1high pancreatic progenitor cells. In the DPR, Pdx1high cells are directly specified as pancreatic progenitors, whereas Pdx1low cells are precocious endocrine cells. Therefore, our study defines distinct road maps for dorsal and ventral pancreatic progenitor specification. The findings provide guidance for optimization of current ß-cell induction protocols by following the in vivo dorsal pancreatic specification program.

Dynamics of genomic H3K27me3 domains and role of EZH2 during pancreatic endocrine specification.

Endoderm cells undergo sequential fate choices to generate insulin-secreting beta cells. Ezh2 of the PRC2 complex, which generates H3K27me3, modulates the transition from endoderm to pancreas progenitors, but the role of Ezh2 and H3K27me3 in the next transition to endocrine progenitors is unknown. We isolated endoderm cells, pancreas progenitors, and endocrine progenitors from different staged mouse embryos and analyzed H3K27me3 genome-wide. Unlike the decline in H3K27me3 domains reported during embryonic stem cell differentiation in vitro, we find that H3K27me3 domains increase in number during endocrine progenitor development in vivo. Genes that lose the H3K27me3 mark typically encode transcriptional regulators, including those for pro-endocrine fates, whereas genes that acquire the mark typically are involved in cell biology and morphogenesis. Deletion of Ezh2 at the pancreas progenitor stage enhanced the production of endocrine progenitors and beta cells. Inhibition of EZH2 in embryonic pancreas explants and in human embryonic stem cell cultures increased endocrine progenitors in vitro. Our studies reveal distinct dynamics in H3K27me3 targets in vivo and a means to modulate beta cell development from stem cells.

A single-cell transcriptomic analysis reveals precise pathways and regulatory mechanisms underlying hepatoblast differentiation.

How bipotential hepatoblasts differentiate into hepatocytes and cholangiocytes remains unclear. Here, using single-cell transcriptomic analysis of hepatoblasts, hepatocytes, and cholangiocytes sorted from embryonic day 10.5 (E10.5) to E17.5 mouse embryos, we found that hepatoblast-to-hepatocyte differentiation occurred gradually and followed a linear default pathway. As more cells became fully differentiated hepatocytes, the number of proliferating cells decreased. Surprisingly, proliferating and quiescent hepatoblasts exhibited homogeneous differentiation states at a given developmental stage. This unique feature enabled us to combine single-cell and bulk-cell analyses to define the precise timing of the hepatoblast-to-hepatocyte transition, which occurs between E13.5 and E15.5. In contrast to hepatocyte development at almost all levels, hepatoblast-to-cholangiocyte differentiation underwent a sharp detour from the default pathway. New cholangiocyte generation occurred continuously between E11.5 and E14.5, but their maturation states at a given developmental stage were heterogeneous. Even more surprising, the number of proliferating cells increased as more progenitor cells differentiated into mature cholangiocytes. Based on an observation from the single-cell analysis, we also discovered that the protein kinase C/mitogen-activated protein kinase signaling pathway promoted cholangiocyte maturation. CONCLUSION: Our studies have defined distinct pathways for hepatocyte and cholangiocyte development in vivo, which are critically important for understanding basic liver biology and developing effective strategies to induce stem cells to differentiate toward specific hepatic cell fates in vitro. (Hepatology 2017;66:1387-1401).

Differentially Expressed MicroRNA-483 Confers Distinct Functions in Pancreatic ß- and α-Cells.

Insulin secreted from pancreatic ß-cells and glucagon secreted from pancreatic α-cells are the two major hormones working in the pancreas in an opposing manner to regulate and maintain a normal glucose homeostasis. How microRNAs (miRNAs), a population of non-coding RNAs so far demonstrated to be differentially expressed in various types of cells, regulate gene expression in pancreatic ß-cells and its closely associated α-cells is not completely clear. In this study, miRNA profiling was performed and compared between pancreatic ß-cells and their partner α-cells. One novel miRNA, miR-483, was identified for its highly differential expression in pancreatic ß-cells when compared to its expression in α-cells. Overexpression of miR-483 in ß-cells increased insulin transcription and secretion by targeting SOCS3, a member of suppressor of cytokine signaling family. In contrast, overexpression of miR-483 decreased glucagon transcription and secretion in α-cells. Moreover, overexpressed miR-483 protected against proinflammatory cytokine-induced apoptosis in ß-cells. This correlates with a higher expression level of miR-483 and the expanded ß-cell mass observed in the islets of prediabetic db/db mice. Together, our data suggest that miR-483 has opposite effects in α- and ß-cells by targeting SOCS3, and the imbalance of miR-483 and its targets may play a crucial role in diabetes pathogenesis.

The default and directed pathways of hepatoblast differentiation involve distinct epigenomic mechanisms.

The effectiveness of multiomics analyses in defining cell differentiation pathways during development is ambiguous. During liver development, hepatoblasts follow a default or directed pathway to differentiate into hepatocytes or cholangiocytes, respectively, and this provides a practical model to address this issue. Our study discovered that promoter-associated histone modifications and chromatin accessibility dynamics, rather than enhancer-associated histone modifications, effectively delineated the "default vs. directed" process of hepatoblast differentiation. Histone H3K27me3 on bivalent promoters is associated with this asymmetric differentiation strategy in mice and humans. We demonstrated that Ezh2 and Jmjd3 exert opposing regulatory roles in hepatoblast-cholangiocyte differentiation. Additionally, active enhancers, regulated by P300, correlate with the development of both hepatocytes and cholangiocytes. This research proposes a model highlighting the division of labor between promoters and enhancers, with promoter-associated chromatin modifications governing the "default vs. directed" differentiation mode of hepatoblasts, whereas enhancer-associated modifications primarily dictate the progressive development processes of hepatobiliary lineages.

Determination of key events in mouse hepatocyte maturation at the single-cell level.

Hepatocytes, the liver's predominant cells, perform numerous essential biological functions. However, crucial events and regulators during hepatocyte maturation require in-depth investigation. In this study, we performed single-cell RNA sequencing (scRNA-seq) and single-nucleus RNA sequencing (snRNA-seq) to explore the precise hepatocyte development process in mice. We defined three maturation stages of postnatal hepatocytes, each of which establishes specific metabolic functions and exhibits distinct proliferation rates. Hepatic zonation is gradually formed during hepatocyte maturation. Hepatocytes or their nuclei with distinct ploidies exhibit zonation preferences in distribution and asynchrony in maturation. Moreover, by combining gene regulatory network analysis with in vivo genetic manipulation, we identified critical maturation- and zonation-related transcription factors. This study not only delineates the comprehensive transcriptomic profiles of hepatocyte maturation but also presents a paradigm to identify genes that function in the development of hepatocyte maturation and zonation by combining genetic manipulation and measurement of coordinates in a single-cell developmental trajectory.

EZH2 controls epicardial cell migration during heart development.

Enhancer of zeste homolog 2 (EZH2) is an important transcriptional regulator in development that catalyzes H3K27me3. The role of EZH2 in epicardial development is still unknown. In this study, we show that EZH2 is expressed in epicardial cells during both human and mouse heart development. Ezh2 epicardial deletion resulted in impaired epicardial cell migration, myocardial hypoplasia, and defective coronary plexus development, leading to embryonic lethality. By using RNA sequencing, we identified that EZH2 controls the transcription of tissue inhibitor of metalloproteinase 3 (TIMP3) in epicardial cells during heart development. Loss-of-function studies revealed that EZH2 promotes epicardial cell migration by suppressing TIMP3 expression. We also found that epicardial Ezh2 deficiency-induced TIMP3 up-regulation leads to extracellular matrix reconstruction in the embryonic myocardium by mass spectrometry. In conclusion, our results demonstrate that EZH2 is required for epicardial cell migration because it blocks Timp3 transcription, which is vital for heart development. Our study provides new insight into the function of EZH2 in cell migration and epicardial development.

Identification of HSC/MPP expansion units in fetal liver by single-cell spatiotemporal transcriptomics.

Limited knowledge of cellular and molecular mechanisms underlying hematopoietic stem cell and multipotent progenitor (HSC/MPP) expansion within their native niche has impeded the application of stem cell-based therapies for hematological malignancies. Here, we constructed a spatiotemporal transcriptome map of mouse fetal liver (FL) as a platform for hypothesis generation and subsequent experimental validation of novel regulatory mechanisms. Single-cell transcriptomics revealed three transcriptionally heterogeneous HSC/MPP subsets, among which a CD93-enriched subset exhibited enhanced stem cell properties. Moreover, by employing integrative analysis of single-cell and spatial transcriptomics, we identified novel HSC/MPP 'pocket-like' units (HSC PLUS), composed of niche cells (hepatoblasts, stromal cells, endothelial cells, and macrophages) and enriched with growth factors. Unexpectedly, macrophages showed an 11-fold enrichment in the HSC PLUS. Functionally, macrophage-HSC/MPP co-culture assay and candidate molecule testing, respectively, validated the supportive role of macrophages and growth factors (MDK, PTN, and IGFBP5) in HSC/MPP expansion. Finally, cross-species analysis and functional validation showed conserved cell-cell interactions and expansion mechanisms but divergent transcriptome signatures between mouse and human FL HSCs/MPPs. Taken together, these results provide an essential resource for understanding HSC/MPP development in FL, and novel insight into functional HSC/MPP expansion ex vivo.

The epigenetic profile of Ig genes is dynamically regulated during B cell differentiation and is modulated by pre-B cell receptor signaling.

Ag receptor loci poised for V(D)J rearrangement undergo germline transcription (GT) of unrearranged genes, and the accessible gene segments are associated with posttranslational modifications (PTM) on histones. In this study, we performed a comprehensive analysis of the dynamic changes of four PTM throughout B and T cell differentiation in freshly isolated ex vivo cells. Methylation of lysines 4 and 79 of histone H3, and acetylation of H3, demonstrated stage and lineage specificity, and were most pronounced at the J segments of loci poised for, or undergoing, rearrangement, except for dimethylation of H3K4, which was more equally distributed on V, D, and J genes. Focusing on the IgL loci, we demonstrated there are no active PTM in the absence of pre-BCR signaling. The kappa locus GT and PTM on Jkappa genes are rapidly induced following pre-BCR signaling in large pre-B cells. In contrast, the lambda locus shows greatly delayed onset of GT and PTM, which do not reach high levels until the immature B cell compartment, the stage at which receptor editing is initiated. Analysis of MiEkappa(-/-) mice shows that this enhancer plays a key role in inducing not only GT, but PTM. Using an inducible pre-B cell line, we demonstrate that active PTM on Jkappa genes occur after GT is initiated, indicating that histone PTM do not make the Jkappa region accessible, but conversely, GT may play a role in adding PTM. Our data indicate that the epigenetic profile of IgL genes is dramatically modulated by pre-BCR signaling and B cell differentiation status.

Reciprocal patterns of methylation of H3K36 and H3K27 on proximal vs. distal IgVH genes are modulated by IL-7 and Pax5.

The usage of >100 functional murine Ig heavy chain V(H) genes, when rearranged to D(H)J(H) genes, generates a diverse antibody repertoire. The V(H) locus encompasses 2.5 Mb, and rearrangement of V(H) genes in the D(H)-distal half of the locus are controlled very differently from the V(H) genes in the proximal end of the locus. The rearrangement of distal but not proximal V(H) genes is impaired in mice deficient in the cytokine IL-7 or its receptor, in the transcription factor Pax5, or in Ezh2, a histone methyltransferase for Lys-27 of histone H3 (H3K27). The relative role of IL-7, Pax5, and Ezh2 in regulating distal vs. proximal V(H) rearrangement is not clear. Here, we show by ChIP and ChIP-on-chip that the active histone modification H3K36me2 is most highly associated with distal V(H) segments and the repressive histone modification H3K27me3 is exclusively present on proximal V(H) segments. We observed an absence of H3K27me3 in fetal pro-B cells, which predominantly rearrange proximal V(H) genes. Absence of IL-7 signaling reduces H3K36me2, and overexpression of IL-7 increases H3K36me2. In contrast, the major effect of the absence of Pax5 is the reduction in H3K27me3. Our data indicate that the cytokine IL-7 and the transcription factor Pax5 influence the rearrangement of the two regions of the V(H) locus by differentially modulating two reciprocal histone modifications during B lymphocyte development.

Sequential progenitor states mark the generation of pancreatic endocrine lineages in mice and humans.

The pancreatic islet contains multiple hormone+ endocrine lineages (α, ß, δ, PP and ε cells), but the developmental processes that underlie endocrinogenesis are poorly understood. Here, we generated novel mouse lines and combined them with various genetic tools to enrich all types of hormone+ cells for well-based deep single-cell RNA sequencing (scRNA-seq), and gene coexpression networks were extracted from the generated data for the optimization of high-throughput droplet-based scRNA-seq analyses. These analyses defined an entire endocrinogenesis pathway in which different states of endocrine progenitor (EP) cells sequentially differentiate into specific endocrine lineages in mice. Subpopulations of the EP cells at the final stage (EP4early and EP4late) show different potentials for distinct endocrine lineages. ε cells and an intermediate cell population were identified as distinct progenitors that independently generate both α and PP cells. Single-cell analyses were also performed to delineate the human pancreatic endocrinogenesis process. Although the developmental trajectory of pancreatic lineages is generally conserved between humans and mice, clear interspecies differences, including differences in the proportions of cell types and the regulatory networks associated with the differentiation of specific lineages, have been detected. Our findings support a model in which sequential transient progenitor cell states determine the differentiation of multiple cell lineages and provide a blueprint for directing the generation of pancreatic islets in vitro.

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.

Comparative analysis of cell lineage differentiation during hepatogenesis in humans and mice at the single-cell transcriptome level.

During embryogenesis, the liver is the site of hepatogenesis and hematopoiesis and contains many cell lineages derived from the endoderm and mesoderm. However, the characteristics and developmental programs of many of these cell lineages remain unclear, especially in humans. Here, we performed single-cell RNA sequencing of whole human and mouse fetal livers throughout development. We identified four cell lineage families of endoderm-derived, erythroid, non-erythroid hematopoietic, and mesoderm-derived non-hematopoietic cells, and defined the developmental pathways of the major cell lineage families. In both humans and mice, we identified novel markers of hepatic lineages and an ID3+ subpopulation of hepatoblasts as well as verified that hepatoblast differentiation follows the "default-directed" model. Additionally, we found that human but not mouse fetal hepatocytes display heterogeneity associated with expression of metabolism-related genes. We described the developmental process of erythroid progenitor cells during human and mouse hematopoiesis. Moreover, despite the general conservation of cell differentiation programs between species, we observed different cell lineage compositions during hematopoiesis in the human and mouse fetal livers. Taken together, these results reveal the dynamic cell landscape of fetal liver development and illustrate the similarities and differences in liver development between species, providing an extensive resource for inducing various liver cell lineages in vitro.

Characterizing pancreatic ß-cell heterogeneity in the streptozotocin model by single-cell transcriptomic analysis.

OBJECTIVES: The streptozotocin (STZ) model is widely used in diabetes research. However, the cellular and molecular states of pancreatic endocrine cells in this model remain unclear. This study explored the molecular characteristics of islet cells treated with STZ and re-evaluated ß-cell dysfunction and regeneration in the STZ model. METHODS: We performed single-cell RNA sequencing of pancreatic endocrine cells from STZ-treated mice. High-quality sequencing data from 2,999 cells were used to identify clusters via Louvain clustering analysis. Principal component analysis (PCA), t-distributed stochastic neighbor embedding (t-SNE), uniform manifold approximation and projection (UMAP), force-directed layout (FDL), and differential expression analysis were used to define the heterogeneity and transcriptomic changes in islet cells. In addition, qPCR and immunofluorescence staining were used to confirm findings from the sequencing data. RESULTS: Untreated ß-cells were divided into two populations at the transcriptomic level, a large high-Glut2 expression (Glut2high) population and a small low-Glut2 expression (Glut2low) population. At the transcriptomic level, Glut2low ß-cells in adult mice did not represent a developmentally immature state, although a fraction of genes associated with ß-cell maturation and function were downregulated in Glut2low cells. After a single high-dose STZ treatment, most Glut2high cells were killed, but Glut2low cells survived and over time changed to a distinct cell state. We did not observe conversion of Glut2low to Glut2high ß-cells up to 9 months after STZ treatment. In addition, we did not detect transcriptomic changes in the non-ß endocrine cells or a direct trans-differentiation pathway from the α-cell lineage to the ß-cell lineage in the STZ model. CONCLUSIONS: We identified the heterogeneity of ß-cells in both physiological and pathological conditions. However, we did not observe conversion of Glut2low to Glut2high ß-cells, transcriptomic changes in the non-ß endocrine cells, or direct trans-differentiation from the α-cell lineage to the ß-cell lineage in the STZ model. Our results clearly define the states of islet cells treated with STZ and allow us to re-evaluate the STZ model widely used in diabetes studies.

Large-scale Generation of Functional and Transplantable Hepatocytes and Cholangiocytes from Human Endoderm Stem Cells.

The ever-increasing therapeutic and pharmaceutical demand for liver cells calls for systems that enable mass production of hepatic cells. Here we describe a large-scale suspension system that uses human endoderm stem cells (hEnSCs) as precursors to generate functional and transplantable hepatocytes (E-heps) or cholangiocytes (E-chos). hEnSC-derived hepatic populations are characterized by single-cell transcriptomic analyses and compared with hESC-derived counterparts, in-vitro-maintained or -expanded primary hepatocytes and adult cells, which reveals that hepatic differentiation of hEnSCs recapitulates in vivo development and that the heterogeneities of the resultant populations can be manipulated by regulating the EGF and MAPK signaling pathways. Functional assessments demonstrate that E-heps and E-chos possess properties comparable with adult counterparts and that, when transplanted intraperitoneally, encapsulated E-heps were able to rescue rats with acute liver failure. Our study lays the foundation for cell-based therapeutic agents and in vitro applications for liver diseases.