Biology subjects

Wang, K.

Publications and source records attributed to Wang, K..

At least 451 records · Page 25Linked to original sources

bioRxiv2020

Predicting tissue-specific gene expression from whole blood transcriptome

Complex diseases are systemic, largely mediated via transcriptional dysregulation in multiple tissues. Thus, knowledge of tissue-specific transcriptome in an individual can provide important information about an individuals health. Unfortunately, with a few exceptions such as blood, skin, and muscle, an individuals tissue-specific transcriptome is not accessible through non-invasive means. However, due to shared genetics and regulatory programs between tissues, the transcriptome in blood may be predictive of those in other tissues, at least to some extent. Here, based on GTEx data, we address this question in a rigorous, systematic manner, for the first time. We find that an individuals whole blood gene expression and splicing profile can predict tissue-specific expression levels in a significant manner (beyond demographic variables) for many genes. On average, across 32 tissues, the expression of about 60% of the genes is predictable from blood expression in a significant manner, with a maximum of 81% of the genes for the musculoskeletal tissue. Remarkably, the tissue-specific expression inferred from the blood transcriptome is almost as good as the actual measured tissue expression in predicting disease state for six different complex disorders, including Hypertension and Type 2 diabetes, substantially surpassing predictors built directly from the blood transcriptome. The code for our pipeline for tissue-specific gene expression prediction - TEEBoT, is provided, enabling others to study its potential translational value in other indications.

genetics↗

bioRxiv2020

Cryo-EM structure of C9ORF72-SMCR8-WDR41 reveals the role as a GAP for Rab8a and Rab11a

A massive intronic hexanucleotide repeat (GGGGCC) expansion in C9ORF72 is a genetic origin of amyotrophic lateral sclerosis (ALS) and frontotemporal dementia (FTD). Recently, C9ORF72, together with SMCR8 and WDR41, has been shown to regulate autophagy and function as Rab GEF. However, the precise function of C9ORF72 remains unclear. Here, we report the cryo-EM structure of the human C9ORF72-SMCR8-WDR41 complex at a resolution of 3.2 [A]. The structure reveals the dimeric assembly of a heterotrimer of C9ORF72-SMCR8-WDR41. Notably, the C-terminal tail of C9ORF72 and the DENN domain of SMCR8 play critical roles in the dimerization of the two protomers of the C9ORF72-SMCR8-WDR41 complex. In the protomer, C9ORF72 and WDR41 are joined by SMCR8 without direct interaction. WDR41 binds to the DENN domain of SMCR8 by the C-terminal helix. Interestingly, the prominent structural feature of C9ORF72-SMCR8 resembles that of the FLNC-FNIP2 complex, the GTPase activating protein (GAP) of RagC/D. Structural comparison and sequence alignment revealed that Arg147 of SMCR8 is conserved and corresponds to the arginine finger of FLCN, and biochemical analysis indicated that the Arg147 of SMCR8 is critical to the stimulatory effect of the C9ORF72-SMCR8 complex on Rab8a and Rab11a. Our study not only illustrates the basis of C9ORF72-SMCR8-WDR41 complex assembly but also reveals the GAP activity of the C9ORF72-SMCR8 complex. Significance StatementC9ORF72, together with SMCR8 and WDR41, has been shown to form a stable complex that participates in the regulation of membrane trafficking. We report the cryo-EM structure of the C9ORF72-SMCR8-WDR41 complex at atomic resolution. Notably, the stoichiometry of the three subunits in the C9ORF72-SMCR8-WDR41 complex is 2:2:2. Interestingly, the C-termini of C9ORF72 and the DENN domain of SMCR8 mediate the dimerization of the two C9ORF72-SMCR8-WDR41 protomers in the complex. Moreover, WDR41 binds to the DENN domain of SMCR8 by the C-terminal helix without direct contact with C9ORF72. Most importantly, the C9ORF72-SMCR8 complex works as a GAP for Rab8a and Rab11a in vitro, and the Arg147 of SMCR8 is the arginine finger.

biochemistry↗

bioRxiv2020

Cancer SIGVAR: A semi-automated interpretation tool for germline variants of hereditary cancer-related genes

The American College of Medical Genetics and Genomics and the Association for Molecular Pathology published guidelines in 2015 for the clinical interpretation of Mendelian disorder sequence variants based on 28 criteria. ClinGen Sequence Variant Interpretation (SVI) Working Groups have developed many adaptations or refinements of these guidelines to improve the consistency of interpretation. We combined the most recent adaptations to expand the criteria from 28 to 48 and developed a tool called Cancer SIGVAR to help healthcare workers and genetic counselors interpret the clinical significance of cancer germline variants, which is critical for the clinical diagnosis and treatment of hereditary cancer. Our tool can accept VCF files as input and realize fully automated interpretation based on 21 criteria and semi-automated interpretation based on 48 criteria. We validated our tool on the ClinVar and CLINVITAE benchmark databases for the accuracy of fully automated interpretation, achieving an average consistency for pathogenic and benign assessment up to 93.40% and 82.54%, respectively. We compared Cancer SIGVAR with a similar tool, InterVar, and analyzed the main differences in criteria and implementation. In addition, to verify the performance of semi-automated interpretation based on 48 criteria, we selected 911 variants from two benchmark databases and reached an average classification consistency of 98.35%. Our findings highlight the need to optimize automated interpretation tools based on constantly updated guidelines.

genetics↗

bioRxiv2020

A Cyclic Phosphoramidate Prodrug of 2-deoxy-2-fluoro-2-C-methylguanosine for the Treatment of Dengue Infection

Monophosphate prodrug analogs of 2-deoxy-2-fluoro-2-C-methylguanosine have been reported as potent inhibitors of hepatitis C virus (HCV) RNA-dependent RNA polymerase. These prodrugs also display potent anti-dengue activities in cellular assays although their prodrug moieties were designed to produce high levels of triphosphate in the liver. Since peripheral blood mononuclear cells (PBMCs) are one of the major targets of dengue virus, different prodrug moieties were designed to effectively deliver 2-deoxy-2-fluoro-2-C-methylguanosine monophosphate prodrugs and their corresponding triphosphates into PBMCs after oral administration. We identified a cyclic phosphoramidate prodrug 17 demonstrating a well-balanced anti-dengue cellular activity and in vitro stability profiles. In dogs, oral administration of 17 resulted in high PBMC triphosphate level, exceeding TP50 (the intracellular triphosphate concentration at which 50% of virus replication is inhibited) at 10 mg/kg. Compound 17 demonstrated 1.6- and 2.2 log viremia reduction in the dengue mouse model at 100 and 300 mg/kg twice daily, respectively. At 100 mg/kg twice daily, the terminal triphosphate concentration in PBMCs reached above TP50, defining for the first time the minimum efficacious dose for a nucleos(t)ide prodrug. In the two-week dog toxicity studies at 30 to 300 mg/kg/day, no observed adverse effect level (NOAEL) could not be achieved due to pulmonary inflammation and hemorrhage. The preclinical safety results suspended further development of 17. Nevertheless, present work has proven the concept that an efficacious monophosphate nucleoside prodrug could be developed for the potential treatment of dengue infection.

microbiology↗

bioRxiv2020

The spatial and cell-type distribution of SARS-CoV-2 receptor ACE2 in human and mouse brain

By engaging angiotensin-converting enzyme 2 (ACE2 or Ace2), the novel pathogenic SARS-coronavirus 2 (SARS-CoV-2) may invade host cells in many organs, including the brain. However, the distribution of ACE2 in the brain is still obscure. Here we investigated the ACE2 expression in the brain by analyzing data from publicly available brain transcriptome databases. According to our spatial distribution analysis, ACE2 was relatively highly expressed in some brain locations, such as the choroid plexus and paraventricular nuclei of the thalamus. According to cell-type distribution analysis, nuclear expression of ACE2 was found in many neurons (both excitatory and inhibitory neurons) and some non-neuron cells (mainly astrocytes, oligodendrocytes, and endothelial cells) in human middle temporal gyrus and posterior cingulate cortex. A few ACE2-expressing nuclei were found in a hippocampal dataset, and none were detected in the prefrontal cortex. Except for the additional high expression of Ace2 in the olfactory bulb areas for spatial distribution as well as in the pericytes and endothelial cells for cell-type distribution, the distribution of Ace2 in mouse brain was similar to that in the human brain. Thus, our results reveal an outline of ACE2/Ace2 distribution in the human and mouse brain, which indicates the brain infection of SARS-CoV-2 may be capable of inducing central nervous system symptoms in coronavirus disease 2019 (COVID-19) patients. Potential species differences should be considered when using mouse models to study the neurological effects of SARS-CoV-2 infection.

neuroscience↗

bioRxiv2020

Organic manure managements increases soil microbial community structure and diversity in double-cropping paddy field of Southern China

The soil physicochemical properties were affected by different fertilizer managements, and the soil microbial communities were changed. Fertilizer regimes were closely relative to the soil texture and nutrient status in a double-cropping paddy field of southern China. However, there was limited information about the influence of different long-term fertilizer management practices on the soil microbial communities in a double-cropping rice (Oryza sativa L.) fields. Therefore, the 39-year long-term fertilizer regimes on soil bacterial and fungal diversity in a double-cropping paddy field of southern China were studied by using Illumina sequencing and quantitative PCR technology in the present paper. The filed experiment were including chemical fertilizer alone (MF), rice straw residue and chemical fertilizer (RF), 30% organic manure and 70% chemical fertilizer (OM), and without fertilizer input as a control (CK). The results showed that diversity indices of soil microbial communities with application of organic manure and rice straw residue treatments were higher than that without fertilizer input treatment. Application of organic manure and rice straw residue managements increase soil bacterial abundance of the phylum Proteobacteria, Actinobacteria, and Gammaproteobacteria, and soil fungi abundance of the phylum Basidiomycota, Zygomycota and Tremellales were also increased. Compared with CK treatment, the value of Richness, Shannon and McIntosh indices, and taxonomic diversity were increased with RF and OM treatments. This finding demonstrated that RF and OM treatments modify soil bacterial and fungal diversity. Therefore, the combined application of organic manure or rice straw residue with chemical fertilizer managements could significantly increase the abundance of profitable functional bacteria and fungi species in double-cropping rice fields of southern China.

ecology↗

bioRxiv2020

MiR-203 inhibits the proliferation, invasion, and migration of pancreatic cancer cells by down-regulating fibroblast growth factor 2

BackgroundAberrant fibroblast growth factor 2 (FGF2) expression is a major cause of poor prognosis in pancreatic cancer. MiR-203 is a newly discovered microRNA (miRNA) that can affect the biological behavior of tumors. This study investigated whether miR-203 can regulate FGF2 expression and its role in pancreatic cancer cell proliferation, apoptosis, invasion, and migration. MethodsMiR-203 expression in different cell lines was examined by qRT-PCR, followed by the establishment of knockdown and overexpression cell models. We used the CCK-8 assay to examine cell proliferation and the annexin V-APC/7-AAD double-staining method to detect apoptosis. In addition, we used wound healing and transwell assays to investigate the effects of miR-203 on the migration and invasion of pancreatic cancer cells. The effects of miR-203 knockdown and overexpression on FGF2 mRNA expression were detected by qRT-PCR. We also overexpressed FGF2 and examined the effects of FGF2 overexpression on the proliferation, apoptosis, invasion, and migration of pancreatic cancer cells. The binding of miR-203 to FGF2 was assessed by a luciferase reporter assay. ResultsWe found that the miR-203 expression level was significantly down-regulated in pancreatic cancer cells compared to normal pancreatic cells. Functionally, the knockdown of miR-203 inhibited cell proliferation and increased apoptosis. Equally important, miR-203 reduced the migration and invasion of pancreatic cancer cells. In addition, we found that miR-203 overexpression inhibited FGF2 expression in pancreatic cancer cells by qRT-PCR. FGF2 overexpression significantly affected the proliferation, invasion, and metastasis of pancreatic cancer cells. Mechanistically, miR-203 base-paired with the FGF2 mRNA, resulting in the knockdown of the FGF2 mRNA and the down-regulation of the FGF2 protein. ConclusionsMiR-203 inhibits FGF2 expression, regulates the proliferation of pancreatic cancer cells, and inhibits the invasion and metastasis of pancreatic cancer cells.

cancer biology↗

bioRxiv2020

Multi-organ transcriptomic profiling reveals differential regulation of the immune system among the brain-liver-fats organ network of short-term fasted mice

Different fasting regimens are known to promote health, mitigate chronic immunological disorders, and improve age-related pathophysiological parameters in animals and humans. Indeed, several clinical trials are currently ongoing using fasting as a potential therapy for a wide range of conditions. Fasting alters metabolism by acting as a reset for energy homeostasis. However, the molecular mechanisms underlying the beneficial effects of short-term fasting (STF) are still not well understood, particularly at the systems or multi-organ level. Here, we investigated the dynamic gene expression patterns associated with six periods of STF in nine different mouse organs. We cataloged the transcriptional dynamics within and between organs during STF and discovered differential temporal effects of STF among organs. Using gene ontology enrichment analysis, we identified an organ network sharing 37 common biological pathways perturbed by STF. This network incorporates the brain, liver, interscapular brown adipose tissue, and posterior-subcutaneous white adipose tissue, hence we named it the brain-liver-fats organ network. Using Reactome pathways analysis, we identified the immune system, dominated by T cell regulation processes, as a central and prominent target of systemic modulations during STF in this organ network. The changes we identified in specific immune components point to the priming of adaptive immunity and parallel the fine-tuning of innate immune signaling. Our study provides a comprehensive multi-organ transcriptomic profiling of mice subjected to multiple periods of STF, and adds new insights into the molecular modulators involved in the systemic immuno-transcriptomic changes that occur during short-term energy loss.

systems biology↗

bioRxiv2020

Molecular architecture of the luminal ring of the Xenopus laevis nuclear pore complex

Nuclear pore complex (NPC) mediates the flow of substances between the nucleus and cytoplasm in eukaryotic cells. Here we report the cryo-electron tomography (cryo-ET) structure of the luminal ring (LR) of the NPC from Xenopus laevis oocyte. The observed key structural features of the LR are independently confirmed by single-particle cryo-electron microscopy (cryo-EM) analysis. The LR comprises eight butterfly-shaped subunits, each containing two symmetric wings. Each wing consists of four elongated, tubular protomers. Within the LR subunit, the eight protomers form a Finger domain, which directly contacts the fusion between the inner and outer nuclear membranes, and a Grid domain, which serves as a rigid base for the Finger domain. Two neighbouring LR subunits interact with each other through the lateral edges of their wings to constitute a Bumper domain, which displays two major conformations and appears to cushion neighbouring NPCs. Our study reveals previously unknown features of the LR and potentially explains the elastic property of the NPC.

biophysics↗

bioRxiv2020

The need to connect: Acute social isolation causes neural craving responses similar to hunger.

When people are forced to be isolated from one another, do they crave social interactions? To address this question, we used functional magnetic resonance imaging (fMRI) to measure neural responses evoked by food and social cues after participants (n=40) experienced ten hours of mandated fasting or total social isolation. After isolation, people felt lonely and craved social interaction. Midbrain regions showed selective activation to food cues after fasting and to social cues after isolation; these responses were correlated with self-reported craving. By contrast, striatal and cortical regions differentiated between craving food versus social interaction. Across deprivation sessions, we find that deprivation narrows and focuses the brains motivational responses to the deprived target. Our results support the intuitive idea that acute isolation causes social craving, similar to the way fasting causes hunger.

neuroscience↗

bioRxiv2020

A dynamic 6,000-year genetic history of Eurasia's Eastern Steppe

The Eastern Eurasian Steppe was home to historic empires of nomadic pastoralists, including the Xiongnu and the Mongols. However, little is known about the regions population history. Here we reveal its dynamic genetic history by analyzing new genome-wide data for 214 ancient individuals spanning 6,000 years. We identify a pastoralist expansion into Mongolia ca. 3000 BCE, and by the Late Bronze Age, Mongolian populations were biogeographically structured into three distinct groups, all practicing dairy pastoralism regardless of ancestry. The Xiongnu emerged from the mixing of these populations and those from surrounding regions. By comparison, the Mongols exhibit much higher Eastern Eurasian ancestry, resembling present-day Mongolic-speaking populations. Our results illuminate the complex interplay between genetic, sociopolitical, and cultural changes on the Eastern Steppe.

genomics↗

bioRxiv2020

The Genomic Formation of Human Populations in East Asia

The deep population history of East Asia remains poorly understood due to a lack of ancient DNA data and sparse sampling of present-day people. We report genome-wide data from 191 individuals from Mongolia, northern China, Taiwan, the Amur River Basin and Japan dating to 6000 BCE - 1000 CE, many from contexts never previously analyzed with ancient DNA. We also report 383 present-day individuals from 46 groups mostly from the Tibetan Plateau and southern China. We document how 6000-3600 BCE people of Mongolia and the Amur River Basin were from populations that expanded over Northeast Asia, likely dispersing the ancestors of Mongolic and Tungusic languages. In a time transect of 89 Mongolians, we reveal how Yamnaya steppe pastoralist spread from the west by 3300-2900 BCE in association with the Afanasievo culture, although we also document a boy buried in an Afanasievo barrow with ancestry entirely from local Mongolian hunter-gatherers, representing a unique case of someone of entirely non-Yamnaya ancestry interred in this way. The second spread of Yamnaya-derived ancestry came via groups that harbored about a third of their ancestry from European farmers, which nearly completely displaced unmixed Yamnaya-related lineages in Mongolia in the second millennium BCE, but did not replace Afanasievo lineages in western China where Afanasievo ancestry persisted, plausibly acting as the source of the early-splitting Tocharian branch of Indo-European languages. Analyzing 20 Yellow River Basin farmers dating to [~]3000 BCE, we document a population that was a plausible vector for the spread of Sino-Tibetan languages both to the Tibetan Plateau and to the central plain where they mixed with southern agriculturalists to form the ancestors of Han Chinese. We show that the individuals in a time transect of 52 ancient Taiwan individuals spanning at least 1400 BCE to 600 CE were consistent with being nearly direct descendants of Yangtze Valley first farmers who likely spread Austronesian, Tai-Kadai and Austroasiatic languages across Southeast and South Asia and mixing with the people they encountered, contributing to a four-fold reduction of genetic differentiation during the emergence of complex societies. We finally report data from Jomon hunter-gatherers from Japan who harbored one of the earliest splitting branches of East Eurasian variation, and show an affinity among Jomon, Amur River Basin, ancient Taiwan, and Austronesian-speakers, as expected for ancestry if they all had contributions from a Late Pleistocene coastal route migration to East Asia.

genomics↗

bioRxiv2020

SARS-CoV-2 invades host cells via a novel route: CD147-spike protein

Currently, COVID-19 caused by severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) has been widely spread around the world; nevertheless, so far there exist no specific antiviral drugs for treatment of the disease, which poses great challenge to control and contain the virus. Here, we reported a research finding that SARS-CoV-2 invaded host cells via a novel route of CD147-spike protein (SP). SP bound to CD147, a receptor on the host cells, thereby mediating the viral invasion. Our further research confirmed this finding. First, in vitro antiviral tests indicated Meplazumab, an anti-CD147 humanized antibody, significantly inhibited the viruses from invading host cells, with an EC50 of 24.86 g/mL and IC50 of 15.16 g/mL. Second, we validated the interaction between CD147 and SP, with an affinity constant of 1.85x10-7M. Co-Immunoprecipitation and ELISA also confirmed the binding of the two proteins. Finally, the localization of CD147 and SP was observed in SARS-CoV-2 infected Vero E6 cells by immuno-electron microscope. Therefore, the discovery of the new route CD147-SP for SARS-CoV-2 invading host cells provides a critical target for development of specific antiviral drugs.

microbiology↗

bioRxiv2020

O-GlcNAcylation of SAMHD1 Indicating a Link between Metabolic Reprogramming and Anti-HBV Immunity

Viruses hijack the host cell machinery to promote viral replication; however, the mechanism by which metabolic reprogramming regulates innate antiviral immunity in the host remains elusive. Herein, we found that Hepatitis B virus (HBV) infection upregulates glucose transporter 1expression, promotes hexosamine biosynthesis pathway (HBP) activity, and enhances O-linked {beta}-N-acetylglucosamine (O-GlcNAc) modification of downstream proteins. HBP-mediated O-GlcNAcylation positively regulates host antiviral response against HBV in vitro and in vivo. Mechanistically, O-GlcNAc transferase (OGT)-mediated O-GlcNAcylation of sterile alpha motif and histidine/aspartic acid domain-containing protein 1 (SAMHD1) on Ser93 stabilizes SAMHD1 and enhances its antiviral activity. In addition, O-GlcNAcylation of SAMHD1 promoted its antiviral activity against human immunodeficiency virus-1 in vitro. In conclusion, the results of our study reveal a link between HBP, O-GlcNAc modification, and innate antiviral immunity by targeting SAMHD1. Therefore, the results of this study demonstrate a strategy for the potential treatment of HBV infection by modulating HBP activity.

microbiology↗

bioRxiv2020

The CDK inhibitor SIAMESE targets both CDKA;1 and CDKB1 complexes to establish endoreplication in trichomes

Endoreplication, also known as endoreduplication, is a modified cell cycle in which DNA is replicated without subsequent cell division. Endoreplication plays important roles in both normal plant development and in stress responses. The SIAMESE (SIM) gene of Arabidopsis (Arabidopsis thaliana) encodes a cyclin-dependent kinase inhibitor that plays an central role in establishing endoreplication, and is the founding member of the SIAMESE-RELATED (SMR) family of plant-specific cyclin-dependent kinase inhibitors genes. However, there has been conflicting evidence regarding which specific cyclin/CDK complexes are inhibited by SIM in vivo. In this work, we use genetic evidence to show that SIM likely inhibits both CDKA;1- and CDKB1-containing CDK complexes in vivo to promote endoreplication in developing Arabidopsis trichomes. We also show that SIM interacts with CYCA2;3, a binding partner of CDKB1;1, via SIM Motif A, which we previously identified as a CDK-binding motif. In contrast, SIM Motif C, which has been indicated as a cyclin binding motif in other contexts, appears to be relatively unimportant for interaction between SIM and CYCA2;3. Together with earlier results, our work suggests that SIM and other SMRs likely have a multivalent interaction with CYC/CDK complexes. One sentence summaryThe cyclin-dependent kinase inhibitor SIAMESE (SIM) targets both CDKA;1 and CDKB1 complexes to establish endoreplication, and that SIM interacts with the cyclin CYCA2;3 via SIM Motif A.

plant biology↗

bioRxiv2020

Implications of error-prone long-read whole-genome shotgun sequencing on characterizing reference microbiomes

Long-read sequencing techniques, such as the Oxford Nanopore Technology, can generate reads that are tens of kilobases in length, and are therefore particularly relevant for microbiome studies. However, due to the higher per-base error rates than typical short-read sequencing, the application of long-read sequencing on microbiomes remains largely unexplored. Here we deeply sequenced two human microbiota mock community samples (HM-276D and HM-277D) from the Human Microbiome Project. We showed that assembly programs consistently achieved high accuracy (~99%) and completeness (~99%) for bacterial strains with adequate coverage. We also found that long-read sequencing provides accurate estimates of species-level abundance (R=0.94 for 20 bacteria with abundance ranging from 0.005% to 64%). Our results demonstrate the feasibility to characterize complete microbial genomes and populations from error-prone Nanopore sequencing data, but also highlight necessary bioinformatics improvements for future metagenomics tool development.

genomics↗

bioRxiv2020

A transcription-centric model of SNP-Age interaction

Complex age-associated phenotypes are caused, in part, by an interaction between an individuals genotype and age. The mechanisms governing such interactions are however not entirely understood. Here, we provide a novel transcriptional mechanism-based framework - SNiPage, to investigate such interactions, whereby a transcription factor (TF) whose expression changes with age (age-associated TF), binds to a polymorphic regulatory element in an allele-dependent fashion, rendering the target genes expression dependent on both, the age and the genotype. Applying SNiPage to GTEx, we detected ~637 interacting TF-SNP-Gene triplets on average across 25 tissues, where the TF binds to a regulatory SNP in the genes promoter or putative enhancer and potentially regulates its expression in an age- and allele-dependent fashion. The detected SNPs are enriched for epigenomic marks indicative of regulatory activity, exhibit allele-specific chromatin accessibility, and spatial proximity to their putative gene targets. Furthermore, the TF-SNP interaction-dependent target genes have established links to aging and to age-associated diseases. In six hypertension-implicated tissues, detected interactions significantly inform hypertension state of an individual. Lastly, the age-interacting SNPs exhibit a greater proximity to the reported phenotype/diseases-associated SNPs than eSNPs identified in an interaction-independent fashion. Overall, we present a novel mechanism-based model, and a novel framework SNiPage, to identify functionally relevant SNP-age interactions in transcriptional control and illustrate their potential utility in understanding complex age-associated phenotypes.

genetics↗

bioRxiv2020

Robust differentiation of human pluripotent stem cells into endothelial cells via temporal modulation of ETV2 with modified mRNA

Human induced pluripotent stem cell (h-iPSC)-derived endothelial cells (h-iECs) have become a valuable tool in regenerative medicine. However, current differentiation protocols remain inefficient and lack reliability. Here, we describe a method for rapid, consistent, and highly efficient generation of h-iECs. The protocol entails the delivery of modified mRNA encoding the transcription factor ETV2 at the intermediate mesodermal stage of differentiation. This approach reproducibly differentiated thirteen diverse h-iPSC lines into h-iECs with exceedingly high efficiency. In contrast, standard differentiation methods that relied on endogenous ETV2 were inefficient and notably inconsistent. Our h-iECs were functionally competent in many respects, including the ability to form perfused vascular networks in vivo. Importantly, timely activation of ETV2 was critical, and bypassing the mesodermal stage produced putative h-iECs with reduced expansion potential and inability to form functional vessels. Our protocol has broad applications and could reliably provide an unlimited number of h-iECs for vascular therapies.

cell biology↗