Yue J. Wang

Single-Cell Transcriptomics of the Human Endocrine Pancreas.

Human pancreatic islets consist of multiple endocrine cell types. To facilitate the detection of rare cellular states and uncover population heterogeneity, we performed single-cell RNA sequencing (RNA-seq) on islets from multiple deceased organ donors, including children, healthy adults, and individuals with type 1 or type 2 diabetes. We developed a robust computational biology framework for cell type annotation. Using this framework, we show that α- and β-cells from children exhibit less well-defined gene signatures than those in adults. Remarkably, α- and β-cells from donors with type 2 diabetes have expression profiles with features seen in children, indicating a partial dedifferentiation process. We also examined a naturally proliferating α-cell from a healthy adult, for which pathway analysis indicated activation of the cell cycle and repression of checkpoint control pathways. Importantly, this replicating α-cell exhibited activated Sonic hedgehog signaling, a pathway not previously known to contribute to human α-cell proliferation. Our study highlights the power of single-cell RNA-seq and provides a stepping stone for future explorations of cellular heterogeneity in pancreatic endocrine cells.

Genetic lineage tracing analysis of the cell of origin of hepatotoxin‐induced liver tumors in mice

The expression of biliary/progenitor markers by hepatocellular carcinoma (HCC) is often associated with poor prognosis and stem cell‐like behaviors of tumor cells. Hepatocellular adenomas (HCAs) also often express biliary/progenitor markers and frequently act as precursor lesions for HCC. However, the cell of origin of HCA and HCC that expresses these markers remains unclear. Therefore, to evaluate if mature hepatocytes give rise to HCA and HCC tumors and to understand the molecular pathways involved in tumorigenesis, we lineage‐labeled hepatocytes by injecting adeno‐associated virus containing thyroxine‐binding globulin promoter‐driven causes recombination (AAV‐TBG‐Cre) into RosaYFP mice. Yellow fluorescent protein (YFP) was present in >96% of hepatocytes before exposure to carcinogens. We treated AAV‐TBG‐Cre; RosaYFP mice with diethylnitrosamine (DEN), followed by multiple injections of carbon tetrachloride to induce carcinogenesis and fibrosis and found that HCA and HCC nodules were YFP+ lineage‐labeled; positive for osteopontin, SRY (sex determining region Y)‐box 9, and epithelial cell adhesion molecule; and enriched for transcripts of biliary/progenitor markers such as prominin 1, Cd44, and delta‐like 1 homolog. Next, we performed the converse experiment and lineage‐labeled forkhead box protein L1(Foxl1)‐positive hepatic progenitor cells simultaneously with exposure to carcinogens. None of the tumor nodules expressed YFP, indicating that Foxl1‐expressing cells are not the origin for hepatotoxin‐induced liver tumors. We confirmed that HCA and HCC cells are derived from mature hepatocytes and not from Foxl1‐Cre‐marked cells in a second model of toxin‐induced hepatic neoplasia, using DEN and 3,3′,5,5′‐tetrachloro‐1,4‐bis(pyridyloxy)benzene (TCPOBOP). Conclusion: Hepatocytes are the cell of origin of HCA and HCC in DEN/carbon tetrachloride and DEN/TCPOBOP induced liver tumors. (Hepatology 2016;64:1163‐1177)

Subepithelial telocytes are an important source of Wnts that supports intestinal crypts

Tissues that undergo rapid cellular turnover, such as the mammalian haematopoietic system or the intestinal epithelium, are dependent on stem and progenitor cells that proliferate to provide differentiated cells to maintain organismal health. Stem and progenitor cells, in turn, are thought to rely on signals and growth factors provided by local niche cells to support their function and self-renewal. Several cell types have been hypothesized to provide the signals required for the proliferation and differentiation of the intestinal stem cells in intestinal crypts1–6. Here we identify subepithelial telocytes as an important source of Wnt proteins, without which intestinal stem cells cannot proliferate and support epithelial renewal. Telocytes are large but rare mesenchymal cells that are marked by expression of FOXL1 and form a subepithelial plexus that extends from the stomach to the colon. While supporting the entire epithelium, FOXL1+ telocytes compartmentalize the production of Wnt ligands and inhibitors to enable localized pathway activation. Conditional genetic ablation of porcupine (Porcn), which is required for functional maturation of all Wnt proteins, in mouse FOXL1+ telocytes causes rapid cessation of Wnt signalling to intestinal crypts, followed by loss of proliferation of stem and transit amplifying cells and impaired epithelial renewal. Thus, FOXL1+ telocytes are an important source of niche signals to intestinal stem cells.Subepithelial telocytes are identified as a source of Wnt signals that enable proliferation and differentiation of intestinal stem cells, an essential function for maintenance of the intestinal epithelium.

β-Cells are not uniform after all-Novel insights into molecular heterogeneity of insulin-secreting cells: AVRAHAMI et al.

While the β‐cells of the endocrine pancreas are defined as cells with high levels of insulin production and tight stimulus‐secretion coupling, the existence of functional heterogeneity among them has been known for decades. Recent advances in molecular technologies, in particular single‐cell profiling on both the protein and messenger RNA level, have uncovered that β‐cells exist in several antigenically and molecularly definable states. Using antibodies to cell surface markers or multidimensional clustering of β‐cells using more than 20 protein markers by mass cytometry, 4 distinct groups of β‐cells could be differentiated. However, whether these states represent permanent cell lineages or are readily interconvertible from one group to another remains to be determined. Nevertheless, future analysis of the pathogenesis of type 1 and type 2 diabetes will certainly benefit from a growing appreciation of β‐cell heterogeneity. Here, we aim to summarize concisely the recent advances in the field and their possible impact on our understanding of β‐cell physiology and pathophysiology.

Combinatorial genetics in liver repopulation and carcinogenesis with a novel in vivo CRISPR activation platform

Clustered regularly interspaced short palindromic repeats (CRISPR)/CRISPR‐associated 9 activation (CRISPRa) systems have enabled genetic screens in cultured cell lines to discover and characterize drivers and inhibitors of cancer cell growth. We adapted this system for use in vivo to assess whether modulating endogenous gene expression levels can result in functional outcomes in the native environment of the liver. We engineered the catalytically dead CRISPR‐associated 9 (dCas9)–positive mouse, cyclization recombination–inducible (Cre) CRISPRa system for cell type–specific gene activation in vivo. We tested the capacity for genetic screening in live animals by applying CRISPRa in a clinically relevant model of liver injury and repopulation. We targeted promoters of interest in regenerating hepatocytes using multiple single guide RNAs (gRNAs), and employed high‐throughput sequencing to assess enrichment of gRNA sequences during liver repopulation and to link specific gRNAs to the initiation of carcinogenesis. All components of the CRISPRa system were expressed in a cell type–specific manner and activated endogenous gene expression in vivo. Multiple gRNA cassettes targeting a proto‐oncogene were significantly enriched following liver repopulation, indicative of enhanced division of cells expressing the proto‐oncogene. Furthermore, hepatocellular carcinomas developed containing gRNAs that activated this oncogene, indicative of cancer initiation events. Also, we employed our system for combinatorial cancer genetics in vivo as we found that while clonal hepatocellular carcinomas were dependent on the presence of the oncogene‐inducing gRNAs, they were depleted for multiple gRNAs activating tumor suppressors. Conclusion: The in vivo CRISPRa platform developed here allows for parallel and combinatorial genetic screens in live animals; this approach enables screening for drivers and suppressors of cell replication and tumor initiation. (Hepatology 2017).

Author Correction: Subepithelial telocytes are an important source of Wnts that supports intestinal crypts

Change history: In this Letter, the surname of author Efi E. Massasa was misspelled ‘Massassa’. This error has been corrected online.

TRAP-seq identifies cystine/glutamate antiporter as a driver of recovery from liver injury

Understanding the molecular basis of the regenerative response following hepatic injury holds promise for improved treatment of liver diseases. Here, we report an innovative method to profile gene expression specifically in the hepatocytes that regenerate the liver following toxic injury. We used the Fah–/– mouse, a model of hereditary tyrosinemia, which conditionally undergoes severe liver injury unless fumarylacetoacetate hydrolase (FAH) expression is reconstituted ectopically. We used translating ribosome affinity purification followed by high-throughput RNA sequencing (TRAP-seq) to isolate mRNAs specific to repopulating hepatocytes. We uncovered upstream regulators and important signaling pathways that are highly enriched in genes changed in regenerating hepatocytes. Specifically, we found that glutathione metabolism, particularly the gene Slc7a11 encoding the cystine/glutamate antiporter (xCT), is massively upregulated during liver regeneration. Furthermore, we show that Slc7a11 overexpression in hepatocytes enhances, and its suppression inhibits, repopulation following toxic injury. TRAP-seq allows cell type–specific expression profiling in repopulating hepatocytes and identified xCT, a factor that supports antioxidant responses during liver regeneration. xCT has potential as a therapeutic target for enhancing liver regeneration in response to liver injury.

Targeted demethylation at the CDKN1C/p57 induces human β cell replication

The loss of insulin-secreting &bgr; cells is characteristic among type I and type II diabetes. Stimulating proliferation to expand sources of &bgr; cells for transplantation remains a challenge because adult &bgr; cells do not proliferate readily. The cell cycle inhibitor p57 has been shown to control cell division in human &bgr; cells. Expression of p57 is regulated by the DNA methylation status of the imprinting control region 2 (ICR2), which is commonly hypomethylated in Beckwith-Wiedemann syndrome patients who exhibit massive &bgr; cell proliferation. We hypothesized that targeted demethylation of the ICR2 using a transcription activator–like effector protein fused to the catalytic domain of TET1 (ICR2-TET1) would repress p57 expression and promote cell proliferation. We report here that overexpression of ICR2-TET1 in human fibroblasts reduces p57 expression levels and increases proliferation. Furthermore, human islets overexpressing ICR2-TET1 exhibit repression of p57 with concomitant upregulation of Ki-67 while maintaining glucose-sensing functionality. When transplanted into diabetic, immunodeficient mice, the epigenetically edited islets show increased &bgr; cell replication compared with control islets. These findings demonstrate that epigenetic editing is a promising tool for inducing &bgr; cell proliferation, which may one day alleviate the scarcity of transplantable &bgr; cells for the treatment of diabetes.

The Dysregulation of the DLK1-MEG3 Locus in Islets From Type 2 Diabetics is Mimicked by Targeted Epimutation of its Promoter with TALE-DNMT Constructs

Type 2 diabetes mellitus (T2DM) is characterized by the inability of the insulin-producing β-cells to overcome insulin resistance. We previously identified an imprinted region on chromosome 14, the DLK1-MEG3 locus, as being downregulated in islets from humans with T2DM. In this study, using targeted epigenetic modifiers, we prove that increased methylation at the promoter of Meg3 in mouse βTC6 β-cells results in decreased transcription of the maternal transcripts associated with this locus. As a result, the sensitivity of β-cells to cytokine-mediated oxidative stress was increased. Additionally, we demonstrate that an evolutionarily conserved intronic region at the MEG3 locus can function as an enhancer in βTC6 β-cells. Using circular chromosome conformation capture followed by high-throughput sequencing, we demonstrate that the promoter of MEG3 physically interacts with this novel enhancer and other putative regulatory elements in this imprinted region in human islets. Remarkably, this enhancer is bound in an allele-specific manner by the transcription factors FOXA2, PDX1, and NKX2.2. Overall, these data suggest that the intronic MEG3 enhancer plays an important role in the regulation of allele-specific expression at the imprinted DLK1-MEG3 locus in human β-cells, which in turn impacts the sensitivity of β-cells to cytokine-mediated oxidative stress.