Redefining intestinal stemness: The emergence of a new ISC population.

In tissue homeostasis, intestinal stem cells (ISCs) undergo continuous self-renewal to sustain rapid cellular turnover. In this issue of Cell, Capdevila et al.1 and Malagola, Vasciaveo, et al.2 identify a new ISC population in the upper crypt that can generate Lgr5+ stem cells during homeostasis.

Molecular Regulatory Network of Anthocyanin Accumulation in Black Radish Skin as Revealed by Transcriptome and Metabonome Analysis.

To understand the coloring mechanism in black radish, the integrated metabolome and transcriptome analyses of root skin from a black recombinant inbred line (RIL 1901) and a white RIL (RIL 1911) were carried out. A total of 172 flavonoids were detected, and the analysis results revealed that there were 12 flavonoid metabolites in radish root skin, including flavonols, flavones, and anthocyanins. The relative concentrations of most flavonoids in RIL 1901 were higher than those in RIL 1911. Meanwhile, the radish root skin also contained 16 types of anthocyanins, 12 of which were cyanidin and its derivatives, and the concentration of cyanidin 3-o-glucoside was very high at different development stages of black radish. Therefore, the accumulation of cyanidin and its derivatives resulted in the black root skin of radish. In addition, a module positively related to anthocyanin accumulation and candidate genes that regulate anthocyanin synthesis was identified by the weighted gene co-expression network analysis (WGCNA). Among them, structural genes (RsCHS, RsCHI, RsDFR, and RsUGT75C1) and transcription factors (TFs) (RsTT8, RsWRKY44L, RsMYB114, and RsMYB308L) may be crucial for the anthocyanin synthesis in the root skin of black radish. The anthocyanin biosynthesis pathway in the root skin of black radish was constructed based on the expression of genes related to flavonoid and anthocyanin biosynthesis pathways (Ko00941 and Ko00942) and the relative expressions of metabolites. In conclusion, this study not only casts new light on the synthesis and accumulation of anthocyanins in the root skin of black radish but also provides a molecular basis for accelerating the cultivation of new black radish varieties.

The C-terminal fibrinogen-like domain of angiopoietin-like 4 stimulates adipose tissue lipolysis and promotes energy expenditure.

Angptl4 (Angiopoietin-like 4) is a circulating protein secreted by white and brown adipose tissues and the liver. Structurally, Angptl4 contains an N-terminal coiled-coil domain (CCD) connected to a C-terminal fibrinogen-like domain (FLD) via a cleavable linker, and both full-length Angptl4 and its individual domains circulate in the bloodstream. Angptl4 inhibits extracellular lipoprotein lipase (LPL) activity and stimulates the lipolysis of triacylglycerol stored by adipocytes in the white adipose tissue (WAT). The former activity is furnished by the CCD, but the Angptl4 domain responsible for stimulating adipocyte lipolysis is unknown. We show here that the purified FLD of Angptl4 is sufficient to stimulate lipolysis in mouse primary adipocytes and that increasing circulating FLD levels in mice through adenovirus-mediated overexpression (Ad-FLD) not only induces WAT lipolysis in vivo but also reduces diet-induced obesity without affecting LPL activity. Intriguingly, reduced adiposity in Ad-FLD mice was associated with increased oxygen consumption, fat utilization, and the expression of thermogenic genes (Ucp1 and Ppargc1a) in subcutaneous WAT. Moreover, Ad-FLD mice exhibited increased glucose tolerance. Chronically enhancing WAT lipolysis could produce ectopic steatosis because of an overflow of lipids from the WAT to peripheral tissues; however, this did not occur when Ad-FLD mice were fed a high-fat diet. Rather, these mice had reductions in both circulating triacylglycerol levels and the mRNA levels of lipogenic genes in the liver and skeletal muscle. We conclude that separating the FLD from the CCD-mediated LPL-inhibitory activity of full-length Angptl4 reveals lipolytic and thermogenic properties with therapeutic relevance to obesity and diabetes.

A Coculture System for Modeling Intestinal Epithelial-Fibroblast Crosstalk.

Epithelial organoid monoculture is a powerful tool to model stem cell dynamics in vitro. However, extensive efforts have recently revealed various niche players and their significant roles in regulating epithelial stem cells. Among these niche components, fibroblasts have been heavily recognized in the field as a critical niche signal secretor. Thus, understanding the roles of fibroblasts in epithelial dynamics has become increasingly relevant and crucial. This propels the development of approaches to coculture epithelial 3D organoids with fibroblasts to model epithelial-fibroblast crosstalk in vitro. Here, we describe a stepwise coculture method to isolate and culture primary intestinal fibroblasts and epithelial organoids together. Aligned with the recent literature, our coculture protocol allows for primary intestinal fibroblast support of epithelial organoid growth.

Methods for Differentiating hiPSCs into Vascular Smooth Muscle Cells.

Despite numerous efforts to generate vascular tissues that recapitulate the physiological characteristics of native vessels, vascular cell source remains one of the principal challenges in the construction of tissue-engineered vascular grafts (TEVGs). Human pluripotent stem cells, therefore, represent an indispensable source to supply a large production of vascular smooth muscle cells (VSMCs) for cell-based therapy. In particular, human induced pluripotent stem cells (hiPSCs) generated from the same individual have opened up new avenues of achieving patient specificity through the derivation of autologous and immunocompatible VSMCs. This book chapter will detail three representative methods of differentiating hiPSCs into VSMCs that are structurally and functionally mature for TEVG engineering. Luo et al. reported an embryoid body (EB)-based approach to generate a robust, large-scale production of mature, functional hiPSC-derived VSMCs as a cell replacement for vascular tissue engineering. EB formation has an advantage of resembling early embryonic development and allowing cellular interactions in three dimensions. Cheung et al. established a system to produce embryological origin-specific hiPSC-derived VSMCs from the neuroectoderm, lateral plate mesoderm, and paraxial mesoderm lineages in a chemically defined manner. This allows site-specific vascular disease modeling. Moreover, Eoh et al. followed Wanjare et al.'s method to construct hiPSC-derived VSMCs using monolayer cultures of extracellular matrix proteins, with the addition of a pulsatile flow for the secretion of mature, organized elastic fibers. The generation of TEVGs, powered by the unlimited supply of hiPSC-derived VSMCs, has begun a new era in cellular therapy for vascular bypass and defective vessel segment replacement, aimed at addressing millions of cases of cardiovascular diseases across the globe.

Development of a scalable method to isolate subsets of stem cell-derived pancreatic islet cells.

Cell replacement therapy using ß cells derived from stem cells is a promising alternative to conventional diabetes treatment options. Although current differentiation methods produce glucose-responsive ß cells, they can also yield populations of undesired endocrine progenitors and other proliferating cell types that might interfere with long-term islet function and safety of transplanted cells. Here, we describe the generation of an array of monoclonal antibodies against cell surface markers that selectively label stem cell-derived islet cells. A high-throughput screen identified promising candidates, including three clones that mark a high proportion of endocrine cells in differentiated cultures. A scalable magnetic sorting method was developed to enrich for human pluripotent stem cell (hPSC)-derived islet cells using these three antibodies, leading to the formation of islet-like clusters with improved glucose-stimulated insulin secretion and reduced growth upon transplantation. This strategy should facilitate large-scale production of functional islet clusters from stem cells for disease modeling and cell replacement therapy.

LIN28B Impairs the Transition of hESC-Derived ß Cells from the Juvenile to Adult State.

Differentiation of human embryonic stem cells into pancreatic ß cells holds great promise for the treatment of diabetes. Recent advances have led to the production of glucose-responsive insulin-secreting cells in vitro, but resulting cells remain less mature than their adult primary ß cell counterparts. The barrier(s) to in vitro ß cell maturation are unclear. Here, we evaluated a potential role for microRNAs. MicroRNA profiling showed high expression of let-7 family microRNAs in vivo, but not in in vitro differentiated ß cells. Reduced levels of let-7 in vitro were associated with increased levels of the RNA binding protein LIN28B, a negative regulator of let-7 biogenesis. Ablation of LIN28B during human embryonic stem cell (hESC) differentiation toward ß cells led to a more mature glucose-stimulated insulin secretion profile and the suppression of juvenile-specific genes. However, let-7 overexpression had little effect. These results uncover LIN28B as a modulator of ß cell maturation in vitro.

Author Correction: Recapitulating endocrine cell clustering in culture promotes maturation of human stem-cell-derived ß cells.

In the version of this article originally published, the Gene Expression Omnibus (GEO) accession number listed in the data availability section was incorrectly given as GSE10979 instead of GSE109795. The sentence should read "RNA-seq data that support the findings of this study have been deposited in the Gene Expression Omnibus (GEO) under accession code GSE109795," and the code should link to https://www.ncbi.nlm.nih.gov/geo/query/acc.cgi?acc=GSE109795. The error has been corrected in the HTML and PDF versions of the paper.

Recapitulating endocrine cell clustering in culture promotes maturation of human stem-cell-derived ß cells.

Despite advances in the differentiation of insulin-producing cells from human embryonic stem cells, the generation of mature functional ß cells in vitro has remained elusive. To accomplish this goal, we have developed cell culture conditions to closely mimic events occurring during pancreatic islet organogenesis and ß cell maturation. In particular, we have focused on recapitulating endocrine cell clustering by isolating and reaggregating immature ß-like cells to form islet-sized enriched ß-clusters (eBCs). eBCs display physiological properties analogous to primary human ß cells, including robust dynamic insulin secretion, increased calcium signalling in response to secretagogues, and improved mitochondrial energization. Notably, endocrine cell clustering induces metabolic maturation by driving mitochondrial oxidative respiration, a process central to stimulus-secretion coupling in mature ß cells. eBCs display glucose-stimulated insulin secretion as early as three days after transplantation in mice. In summary, replicating aspects of endocrine cell clustering permits the generation of stem-cell-derived ß cells that resemble their endogenous counterparts.

The glucocorticoid-Angptl4-ceramide axis induces insulin resistance through PP2A and PKCζ.

Chronic glucocorticoid exposure is associated with the development of insulin resistance. We showed that glucocorticoid-induced insulin resistance was attenuated upon ablation of Angptl4, a glucocorticoid target gene encoding the secreted protein angiopoietin-like 4, which mediates glucocorticoid-induced lipolysis in white adipose tissue. Through metabolomic profiling, we revealed that glucocorticoid treatment increased hepatic ceramide concentrations by inducing enzymes in the ceramide synthetic pathway in an Angptl4-dependent manner. Angptl4 was also required for glucocorticoids to stimulate the activities of the downstream effectors of ceramide, protein phosphatase 2A (PP2A) and protein kinase Cζ (PKCζ). We further showed that knockdown of PP2A or inhibition of PKCζ or ceramide synthesis prevented glucocorticoid-induced glucose intolerance in wild-type mice. Moreover, the inhibition of PKCζ or ceramide synthesis did not further improve glucose tolerance in Angptl4-/- mice, suggesting that these molecules were major downstream effectors of Angptl4. Overall, our study demonstrates the key role of Angptl4 in glucocorticoid-augmented hepatic ceramide production that induces whole-body insulin resistance.

Relationships between circulating matrix metalloproteinase-2 and -9 and renal function in patients with chronic kidney disease.

BACKGROUND: It has been proven that extracellular matrix turnover is involved in the pathogenesis of various renal fibrosis diseases. Matrix metalloproteinase-2 and -9 (MMP-2 and -9) are the extracellular matrix degrading enzymes that are believed to play important roles in renal diseases. However, the relationship of circulating levels of MMP-2, -9 and serum creatinine in the patients of chronic kidney disease (CKD) has not yet been investigated. METHODS: Gelatin zymography and ELISA were employed to measure MMP-2 and MMP-9 activities in the plasma samples of 60 CKD patients and 40 control subjects. RESULTS: Serum creatinine concentrations and MMP-2 activities were significantly higher (p<0.001) while MMP-9 activity and creatinine clearance (CCr) were significantly lower (p<0.05 and p<0.001, respectively) in CKD patients, as compared with those of control subjects. In addition, serum creatinine concentrations correlated with MMP-2 activity (R=0.288, p<0.05) and inversely correlated with that of MMP-9 (R=0.344, p<0.01). CONCLUSIONS: This study demonstrated a correlation between MMP-2, -9 and serum creatinine in CKD patients to suggest that MMP-2 and MMP-9 might contribute in the pathogenesis of CKD.

[An early warning method of cucumber downy mildew in solar greenhouse based on canopy temperature and humidity modeling].

The greenhouse environmental parameters can be used to establish greenhouse nirco-climate model, which can combine with disease model for early warning, with aim of ecological controlling diseases to reduce pesticide usage, and protecting greenhouse ecological environment to ensure the agricultural product quality safety. Greenhouse canopy leaf temperature and air relative humidity, models were established using energy balance and moisture balance principle inside the greenhouse. The leaf temperature model considered radiation heat transfer between the greenhouse crops, wall, soil and cover, plus the heat exchange caused by indoor net radiation and crop transpiration. Furthermore, the water dynamic balance in the greenhouse including leaf transpiration, soil evaporation, cover and leaf water vapor condensation, was considered to develop a relative humidity model. The primary infection and latent period warning models for cucumber downy mildew (Pseudoperonospora cubensis) were validated using the results of the leaf temperature and relative humidity model, and then the estimated disease occurrence date of cucumber downy mildew was compared with actual disease occurrence date of field observation. Finally, the results were verified by the measured temperature and humidity data of September and October, 2014. The results showed that the root mean square deviations (RMSDs) of the measured and estimated leaf temperature were 0.016 and 0.024 °C, and the RMSDs of the measured and estimated air relative humidity were 0.15% and 0.13%, respectively. Combining the result of estimated temperature and humidity models, a cucumber disease early warning system was established to forecast the date of disease occurrence, which met with the real date. Thus, this work could provide the micro-environment data for the early warning system of cucumber diseases in solar greenhouses.

A Method of the Simultaneous Purification of Cardiac Troponin T and I.

A method of the simultaneous purification of cardiac troponin T (cTnT) and troponin I (cTnI) from human cardiac left ventricular muscle have been developed. Five mg cTnT and 10.2 mg of cTnI were obtained from 100 mg of cardiac muscle. The purity of cTnT and cTnI could reach to 97.6% and 97.2% respectively. Their immunoactivity and specificity have been identified by ELISA method.