ABSTRACT
The blue whale, Balaenoptera musculus, is the largest animal known to have ever existed, making it an important case study in longevity and resistance to cancer. To further this and other blue whale-related research, we report a reference-quality, long-read-based genome assembly of this fascinating species. We assembled the genome from PacBio long reads and utilized Illumina/10×, optical maps, and Hi-C data for scaffolding, polishing, and manual curation. We also provided long read RNA-seq data to facilitate the annotation of the assembly by NCBI and Ensembl. Additionally, we annotated both haplotypes using TOGA and measured the genome size by flow cytometry. We then compared the blue whale genome with other cetaceans and artiodactyls, including vaquita (Phocoena sinus), the world's smallest cetacean, to investigate blue whale's unique biological traits. We found a dramatic amplification of several genes in the blue whale genome resulting from a recent burst in segmental duplications, though the possible connection between this amplification and giant body size requires further study. We also discovered sites in the insulin-like growth factor-1 gene correlated with body size in cetaceans. Finally, using our assembly to examine the heterozygosity and historical demography of Pacific and Atlantic blue whale populations, we found that the genomes of both populations are highly heterozygous and that their genetic isolation dates to the last interglacial period. Taken together, these results indicate how a high-quality, annotated blue whale genome will serve as an important resource for biology, evolution, and conservation research.
Subject(s)
Balaenoptera , Neoplasms , Animals , Balaenoptera/genetics , Segmental Duplications, Genomic , Genome , Demography , Neoplasms/geneticsABSTRACT
Considerable effort has been devoted to refining experimental protocols to reduce levels of technical variability and artifacts in single-cell RNA-sequencing data (scRNA-seq). We here present evidence that equalizing the concentration of cDNA libraries prior to pooling, a step not consistently performed in single-cell experiments, improves gene detection rates, enhances biological signals, and reduces technical artifacts in scRNA-seq data. To evaluate the effect of equalization on various protocols, we developed Scaffold, a simulation framework that models each step of an scRNA-seq experiment. Numerical experiments demonstrate that equalization reduces variation in sequencing depth and gene-specific expression variability. We then performed a set of experiments in vitro with and without the equalization step and found that equalization increases the number of genes that are detected in every cell by 17-31%, improves discovery of biologically relevant genes, and reduces nuisance signals associated with cell cycle. Further support is provided in an analysis of publicly available data.
Subject(s)
Gene Library , RNA-Seq/methods , Single-Cell Analysis/methods , Algorithms , Computational Biology/methods , Databases, Genetic , Gene Expression Profiling/methods , Humans , RNA-Seq/standards , Sequence Analysis, RNA/methods , Single-Cell Analysis/standards , SoftwareABSTRACT
Primary hepatocytes are widely used in the pharmaceutical industry to screen drug candidates for hepatotoxicity, but hepatocytes quickly dedifferentiate and lose their mature metabolic function in culture. Attempts have been made to better recapitulate the in vivo liver environment in culture, but the full spectrum of signals required to maintain hepatocyte function ex vivo remains elusive. To elucidate molecular changes that accompany, and may contribute to dedifferentiation of hepatocytes ex vivo, we performed lineage tracing and comprehensive profiling of alterations in their gene expression profiles and chromatin landscape during culture. First, using genetically tagged hepatocytes we demonstrate that expression of the fetal gene alpha-fetoprotein in cultured hepatocytes comes from cells that previously expressed the mature gene albumin, and not from a population of albumin-negative precursor cells, proving mature hepatocytes undergo true dedifferentiation in culture. Next we studied the dedifferentiation process in detail through bulk RNA-sequencing of hepatocytes cultured over an extended period. We identified three distinct phases of dedifferentiation: an early phase, where mature hepatocyte genes are rapidly downregulated in a matter of hours; a middle phase, where fetal genes are activated; and a late phase, where initially rare contaminating non-parenchymal cells proliferate, taking over the culture. Lastly, to better understand the signaling events that result in the rapid downregulation of mature genes in hepatocytes, we examined changes in chromatin accessibility in these cells during the first 24 h of culture using Assay for Transposase-Accessible Chromatin using sequencing (ATAC-seq). We find that drastic and rapid changes in chromatin accessibility occur immediately upon the start of culture. Using binding motif analysis of the areas of open chromatin sharing similar temporal profiles, we identify several candidate transcription factors potentially involved in the dedifferentiation of primary hepatocytes in culture.
Subject(s)
Hepatocytes , Liver , Cells, Cultured , Hepatocytes/metabolism , Albumins , Chromatin/geneticsABSTRACT
Human pluripotent stem cells hold significant promise for regenerative medicine. However, long differentiation protocols and immature characteristics of stem cell-derived cell types remain challenges to the development of many therapeutic applications. In contrast to the slow differentiation of human stem cells in vitro that mirrors a nine-month gestation period, mouse stem cells develop according to a much faster three-week gestation timeline. Here, we tested if co-differentiation with mouse pluripotent stem cells could accelerate the differentiation speed of human embryonic stem cells. Following a six-week RNA-sequencing time course of neural differentiation, we identified 929 human genes that were upregulated earlier and 535 genes that exhibited earlier peaked expression profiles in chimeric cell cultures than in human cell cultures alone. Genes with accelerated upregulation were significantly enriched in Gene Ontology terms associated with neurogenesis, neuron differentiation and maturation, and synapse signaling. Moreover, chimeric mixed samples correlated with in utero human embryonic samples earlier than human cells alone, and acceleration was dose-dependent on human-mouse co-culture ratios. The altered gene expression patterns and developmental rates described in this report have implications for accelerating human stem cell differentiation and the use of interspecies chimeric embryos in developing human organs for transplantation.
Subject(s)
Chimerism , Human Embryonic Stem Cells , Neurogenesis , Pluripotent Stem Cells , Animals , Cells, Cultured , Computational Biology , Human Embryonic Stem Cells/cytology , Human Embryonic Stem Cells/physiology , Humans , Mice , Neurogenesis/genetics , Neurogenesis/physiology , Pluripotent Stem Cells/cytology , Pluripotent Stem Cells/physiology , Species Specificity , Transcriptome/geneticsABSTRACT
Pluripotent stem cells retain the developmental timing of their species of origin in vitro, an observation that suggests the existence of a cell-intrinsic developmental clock, yet the nature and machinery of the clock remain a mystery. We hypothesize that one possible component may lie in species-specific differences in the kinetics of transcriptional responses to differentiation signals. Using a liquid-handling robot, mouse and human pluripotent stem cells were exposed to identical neural differentiation conditions and sampled for RNA-sequencing at high frequency, every 4 or 10 minutes, for the first 10 hours of differentiation to test for differences in transcriptomic response rates. The majority of initial transcriptional responses occurred within a rapid window in the first minutes of differentiation for both human and mouse stem cells. Despite similarly early onsets of gene expression changes, we observed shortened and condensed gene expression patterns in mouse pluripotent stem cells compared to protracted trends in human pluripotent stem cells. Moreover, the speed at which individual genes were upregulated, as measured by the slopes of gene expression changes over time, was significantly faster in mouse compared to human cells. These results suggest that downstream transcriptomic response kinetics to signaling cues are faster in mouse versus human cells, and may offer a partial account for the vast differences in developmental rates across species.
Subject(s)
Cell Differentiation/genetics , Pluripotent Stem Cells/cytology , Pluripotent Stem Cells/metabolism , RNA-Seq/statistics & numerical data , Animals , Cell Line , Computational Biology , Gene Expression Regulation, Developmental , Human Embryonic Stem Cells/cytology , Human Embryonic Stem Cells/metabolism , Humans , Kinetics , Mice , Mouse Embryonic Stem Cells/cytology , Mouse Embryonic Stem Cells/metabolism , Regenerative Medicine , Species SpecificityABSTRACT
Here, we report the derivation of arterial endothelial cells from human pluripotent stem cells that exhibit arterial-specific functions in vitro and in vivo. We combine single-cell RNA sequencing of embryonic mouse endothelial cells with an EFNB2-tdTomato/EPHB4-EGFP dual reporter human embryonic stem cell line to identify factors that regulate arterial endothelial cell specification. The resulting xeno-free protocol produces cells with gene expression profiles, oxygen consumption rates, nitric oxide production levels, shear stress responses, and TNFα-induced leukocyte adhesion rates characteristic of arterial endothelial cells. Arterial endothelial cells were robustly generated from multiple human embryonic and induced pluripotent stem cell lines and have potential applications for both disease modeling and regenerative medicine.
Subject(s)
Arteries/cytology , Endothelial Cells/transplantation , Neovascularization, Physiologic , Pluripotent Stem Cells/physiology , Tissue Engineering/methods , Animals , CRISPR-Cas Systems , Cell Line , Endothelial Cells/cytology , Humans , Mice , Myocardial Infarction/therapy , Sequence Analysis, RNAABSTRACT
How species-specific developmental timing is controlled is largely unknown. By following human embryonic stem (ES) cell and mouse epiblast stem (EpiS) cell differentiation through detailed RNA-sequencing time courses, here we show that pluripotent stem cells closely retain in vivo species-specific developmental timing in vitro. In identical neural differentiation conditions in vitro, gene expression profiles are accelerated in mouse EpiS cells compared to human ES cells with relative rates of differentiation closely reflecting the rates of progression through the Carnegie stages in utero. Dynamic Time Warping analysis identified 3389 genes that were regulated more quickly in mouse EpiS cells and identified none that were regulated more quickly in human ES cells. Interestingly, we also find that human ES cells differentiated in teratomas maintain the same rate of differentiation observed in vitro in spite of being grown in a mouse host. These results suggest the existence of a cell autonomous, species-specific developmental clock that pluripotent stem cells maintain even out of context of an intact embryo.
Subject(s)
Pluripotent Stem Cells/cytology , Pluripotent Stem Cells/metabolism , Animals , Cell Differentiation/genetics , Cell Line , Embryonic Stem Cells/cytology , Female , Gene Expression Profiling , Gene Expression Regulation , Humans , Mice, SCID , Neurons/cytology , Species Specificity , Teratoma/pathology , Time FactorsABSTRACT
Human pluripotent stem cell-based in vitro models that reflect human physiology have the potential to reduce the number of drug failures in clinical trials and offer a cost-effective approach for assessing chemical safety. Here, human embryonic stem (ES) cell-derived neural progenitor cells, endothelial cells, mesenchymal stem cells, and microglia/macrophage precursors were combined on chemically defined polyethylene glycol hydrogels and cultured in serum-free medium to model cellular interactions within the developing brain. The precursors self-assembled into 3D neural constructs with diverse neuronal and glial populations, interconnected vascular networks, and ramified microglia. Replicate constructs were reproducible by RNA sequencing (RNA-Seq) and expressed neurogenesis, vasculature development, and microglia genes. Linear support vector machines were used to construct a predictive model from RNA-Seq data for 240 neural constructs treated with 34 toxic and 26 nontoxic chemicals. The predictive model was evaluated using two standard hold-out testing methods: a nearly unbiased leave-one-out cross-validation for the 60 training compounds and an unbiased blinded trial using a single hold-out set of 10 additional chemicals. The linear support vector produced an estimate for future data of 0.91 in the cross-validation experiment and correctly classified 9 of 10 chemicals in the blinded trial.
Subject(s)
Cell Differentiation , Embryonic Stem Cells/cytology , Neural Stem Cells/cytology , Pluripotent Stem Cells/cytology , Brain/cytology , Brain/growth & development , Brain/metabolism , Cell Communication/drug effects , Cell Communication/genetics , Cells, Cultured , Culture Media, Serum-Free/pharmacology , Embryonic Stem Cells/drug effects , Embryonic Stem Cells/metabolism , Endothelial Cells/cytology , Endothelial Cells/drug effects , Endothelial Cells/metabolism , Gene Expression Regulation, Developmental , Gene Ontology , Humans , Hydrogels/pharmacology , Macrophages/cytology , Macrophages/drug effects , Macrophages/metabolism , Mesenchymal Stem Cells/cytology , Mesenchymal Stem Cells/drug effects , Mesenchymal Stem Cells/metabolism , Microglia/cytology , Microglia/drug effects , Microglia/metabolism , Models, Biological , Neural Stem Cells/drug effects , Neural Stem Cells/metabolism , Neurogenesis/drug effects , Neurogenesis/genetics , Pluripotent Stem Cells/drug effects , Pluripotent Stem Cells/metabolism , Polyethylene Glycols/pharmacology , Support Vector Machine , Tissue Engineering/methods , Xenobiotics/classification , Xenobiotics/pharmacologyABSTRACT
Combining high-mass-accuracy mass spectrometry, isobaric tagging and software for multiplexed, large-scale protein quantification, we report deep proteomic coverage of four human embryonic stem cell and four induced pluripotent stem cell lines in biological triplicate. This 24-sample comparison resulted in a very large set of identified proteins and phosphorylation sites in pluripotent cells. The statistical analysis afforded by our approach revealed subtle but reproducible differences in protein expression and protein phosphorylation between embryonic stem cells and induced pluripotent cells. Merging these results with RNA-seq analysis data, we found functionally related differences across each tier of regulation. We also introduce the Stem Cell-Omics Repository (SCOR), a resource to collate and display quantitative information across multiple planes of measurement, including mRNA, protein and post-translational modifications.
Subject(s)
Embryonic Stem Cells/metabolism , Induced Pluripotent Stem Cells/metabolism , Proteome/analysis , Proteomics , Humans , Proteome/metabolismABSTRACT
We re-examine the individual components for human embryonic stem cell (ESC) and induced pluripotent stem cell (iPSC) culture and formulate a cell culture system in which all protein reagents for liquid media, attachment surfaces and splitting are chemically defined. A major improvement is the lack of a serum albumin component, as variations in either animal- or human-sourced albumin batches have previously plagued human ESC and iPSC culture with inconsistencies. Using this new medium (E8) and vitronectin-coated surfaces, we demonstrate improved derivation efficiencies of vector-free human iPSCs with an episomal approach. This simplified E8 medium should facilitate both the research use and clinical applications of human ESCs and iPSCs and their derivatives, and should be applicable to other reprogramming methods.
Subject(s)
Cell Culture Techniques/methods , Culture Media/chemistry , Induced Pluripotent Stem Cells/cytology , Animals , Biopsy , Cattle , Cell Proliferation , Cell Survival , Coated Materials, Biocompatible , Culture Media, Serum-Free/chemistry , Embryonic Stem Cells/cytology , Embryonic Stem Cells/metabolism , Fibroblasts/cytology , Gene Expression , Growth Substances , Humans , Induced Pluripotent Stem Cells/metabolism , Karyotyping , Serum Albumin, Bovine , Skin/cytology , VitronectinABSTRACT
The salamander has the remarkable ability to regenerate its limb after amputation. Cells at the site of amputation form a blastema and then proliferate and differentiate to regrow the limb. To better understand this process, we performed deep RNA sequencing of the blastema over a time course in the axolotl, a species whose genome has not been sequenced. Using a novel comparative approach to analyzing RNA-seq data, we characterized the transcriptional dynamics of the regenerating axolotl limb with respect to the human gene set. This approach involved de novo assembly of axolotl transcripts, RNA-seq transcript quantification without a reference genome, and transformation of abundances from axolotl contigs to human genes. We found a prominent burst in oncogene expression during the first day and blastemal/limb bud genes peaking at 7 to 14 days. In addition, we found that limb patterning genes, SALL genes, and genes involved in angiogenesis, wound healing, defense/immunity, and bone development are enriched during blastema formation and development. Finally, we identified a category of genes with no prior literature support for limb regeneration that are candidates for further evaluation based on their expression pattern during the regenerative process.
Subject(s)
Ambystoma mexicanum/physiology , Gene Expression Profiling/methods , Gene Expression Regulation , Oncogenes , Sequence Analysis, RNA/methods , Ambystoma mexicanum/genetics , Amputation, Surgical , Animals , Cluster Analysis , Extremities/injuries , Extremities/physiology , Regeneration/genetics , Regeneration/physiology , Up-Regulation , Wound Healing/genetics , Wound Healing/physiologyABSTRACT
Macrophages armed with chimeric antigen receptors (CARs) provide a potent new option for treating solid tumors. However, genetic engineering and scalable production of somatic macrophages remains significant challenges. Here, we used CRISPR-Cas9 gene editing methods to integrate an anti-GD2 CAR into the AAVS1 locus of human pluripotent stem cells (hPSCs). We then established a serum- and feeder-free differentiation protocol for generating CAR macrophages (CAR-Ms) through arterial endothelial-to-hematopoietic transition (EHT). CAR-M produced by this method displayed a potent cytotoxic activity against GD2-expressing neuroblastoma and melanoma in vitro and neuroblastoma in vivo. This study provides a new platform for the efficient generation of off-the-shelf CAR-Ms for antitumor immunotherapy.
Subject(s)
Melanoma , Neuroblastoma , Pluripotent Stem Cells , Receptors, Chimeric Antigen , Humans , Receptors, Chimeric Antigen/genetics , Receptors, Antigen, T-Cell/genetics , Immunotherapy/methods , Pluripotent Stem Cells/pathology , Melanoma/therapy , Neuroblastoma/therapy , Neuroblastoma/pathology , Macrophages/pathologyABSTRACT
In a single round: By combining the high-efficiency enrichment through the continuous-flow magnetic separation (CFMS) technique with the analytical power of next-generation sequencing, the generation of antibody mimetics with a single round of mRNA display is made possible. This approach eliminates iterative selection cycles and provides a path to fully automated ligand generation (see picture).
Subject(s)
Biomimetic Materials/metabolism , RNA, Messenger/chemistry , Amino Acid Sequence , Antibodies/chemistry , Antibodies/metabolism , Biomimetic Materials/chemistry , Enzyme-Linked Immunosorbent Assay , Fibronectins/chemistry , Fibronectins/metabolism , Gene Library , Humans , Immunoglobulin Fc Fragments/chemistry , Immunoglobulin Fc Fragments/metabolism , Immunomagnetic Separation , Ligands , Maltose-Binding Proteins/chemistry , Maltose-Binding Proteins/metabolism , RNA, Messenger/isolation & purificationABSTRACT
Skeletal muscle has a robust, inherent ability to regenerate in response to injury from acute to chronic. In severe trauma, however, complete regeneration is not possible, resulting in a permanent loss of skeletal muscle tissue referred to as volumetric muscle loss (VML). There are few consistently reliable therapeutic or surgical options to address VML. A major limitation in investigation of possible therapies is the absence of a well-characterized large animal model. In this study, we present results of a comprehensive transcriptomic, proteomic, and morphologic characterization of wound healing following VML in a novel canine model of VML which we compare to a nine-patient cohort of combat-associated VML. The canine model is translationally relevant as it provides both a regional (spatial) and temporal map of the wound healing processes that occur in human VML. Collectively, these data show the spatiotemporal transcriptomic, proteomic, and morphologic properties of canine VML healing as a framework and model system applicable to future studies investigating novel therapies for human VML. Impact Statement The spatiotemporal transcriptomic, proteomic, and morphologic properties of canine volumetric muscle loss (VML) healing is a translational framework and model system applicable to future studies investigating novel therapies for human VML.
Subject(s)
Muscular Diseases , Transcriptome , Dogs , Animals , Humans , Transcriptome/genetics , Proteomics , Regeneration/physiology , Wound Healing/genetics , Muscle, Skeletal/injuries , Muscular Diseases/therapyABSTRACT
As newer single-cell protocols generate increasingly more cells at reduced sequencing depths, the value of a higher read depth may be overlooked. Using data from three different single-cell RNA-seq protocols that lend themselves to having either higher read depth (Smart-seq) or many cells (MARS-seq and 10X), we evaluate their ability to recapitulate biological signals in the context of spatial reconstruction. Overall, we find gene expression profiles after spatial reconstruction analysis are highly reproducible between datasets despite being generated by different protocols and using different computational algorithms. While UMI-based protocols such as 10X and MARS-seq allow for capturing more cells, Smart-seq's higher sensitivity and read-depth allow for analysis of lower expressed genes and isoforms. Additionally, we evaluate trade-offs for each protocol by performing subsampling analyses and find that optimizing the balance between sequencing depth and number of cells within a protocol is necessary for efficient use of resources. Our analysis emphasizes the importance of selecting a protocol based on the biological questions and features of interest.
Subject(s)
Hepatocytes/metabolism , RNA-Seq/methods , Single-Cell Analysis/methods , Spatial Analysis , Transcriptome , Algorithms , Animals , Computer Simulation , Immunohistochemistry , Kinetics , Liver/metabolism , Male , Mice , Mice, Inbred C57BL , Reproducibility of Results , Sensitivity and SpecificityABSTRACT
Lead (Pb) is a well-known toxicant, especially for the developing nervous system, albeit the mechanism is largely unknown. In this study, we use time series RNA-seq to conduct a genome-wide survey of the transcriptome response of human embryonic stem cell-derived neural progenitor cells to lead treatment. Using a dynamic time warping algorithm coupled with statistical tests, we find that lead can either accelerate or decelerate the expression of specific genes during the time series. We further show that lead disrupts a neuron- and brain-specific splicing factor NOVA1 regulated splicing network. Using lead induced transcriptome change signatures, we predict several known and novel disease risks under lead exposure. The findings in this study will allow a better understanding of the mechanism of lead toxicity, facilitate the development of diagnostic biomarkers and treatment for lead exposure, and comprise a highly valuable resource for environmental toxicology. Our study also demonstrates that a human (embryonic stem) cell-derived system can be used for studying the mechanism of toxicants, which can be useful for drug or compound toxicity screens and safety assessment.
Subject(s)
Alternative Splicing , Lead/toxicity , Neural Stem Cells/drug effects , Sequence Analysis, RNA/methods , Transcriptome , Algorithms , Gene Expression Profiling , Humans , Neural Stem Cells/metabolism , Real-Time Polymerase Chain Reaction , Risk Factors , Support Vector MachineABSTRACT
A defined protocol for efficiently deriving endothelial cells from human pluripotent stem cells was established and vascular morphogenesis was used as a model system to understand how synthetic hydrogels influence global biological function compared with common 2D and 3D culture platforms. RNA sequencing demonstrated that gene expression profiles were similar for endothelial cells and pericytes cocultured in polyethylene glycol (PEG) hydrogels or Matrigel, while monoculture comparisons identified distinct vascular signatures for each cell type. Endothelial cells cultured on tissue-culture polystyrene adopted a proliferative phenotype compared with cells cultured on or encapsulated in PEG hydrogels. The proliferative phenotype correlated to increased FAK-ERK activity, and knockdown or inhibition of ERK signaling reduced proliferation and expression for cell-cycle genes while increasing expression for "3D-like" vasculature development genes. Our results provide insight into the influence of 2D and 3D culture formats on global biological processes that regulate cell function.
Subject(s)
Endothelial Cells/cytology , Pericytes/cytology , Pluripotent Stem Cells/cytology , Tissue Engineering/methods , Transcriptome , Cell Culture Techniques/methods , Cell Cycle , Cell Differentiation , Cell Line , Cell Proliferation , Cells, Cultured , Collagen/chemistry , Drug Combinations , Endothelial Cells/metabolism , Humans , Hydrogels/chemistry , Laminin/chemistry , MAP Kinase Signaling System , Neovascularization, Physiologic , Pericytes/metabolism , Pluripotent Stem Cells/metabolism , Polyethylene Glycols/chemistry , Polystyrenes/chemistry , Proteoglycans/chemistry , Tissue Scaffolds/chemistryABSTRACT
The aim of the present study was to test sample reproducibility for model neural tissues formed on synthetic hydrogels. Human embryonic stem (ES) cell-derived precursor cells were cultured on synthetic poly(ethylene glycol) (PEG) hydrogels to promote differentiation and self-organization into model neural tissue constructs. Neural progenitor, vascular, and microglial precursor cells were combined on PEG hydrogels to mimic developmental timing, which produced multicomponent neural constructs with 3D neuronal and glial organization, organized vascular networks, and microglia with ramified morphologies. Spearman's rank correlation analysis of global gene expression profiles and a comparison of coefficient of variation for expressed genes demonstrated that replicate neural constructs were highly uniform to at least day 21 for samples from independent experiments. We also demonstrate that model neural tissues formed on PEG hydrogels using a simplified neural differentiation protocol correlated more strongly to in vivo brain development than samples cultured on tissue culture polystyrene surfaces alone. These results provide a proof-of-concept demonstration that 3D cellular models that mimic aspects of human brain development can be produced from human pluripotent stem cells with high sample uniformity between experiments by using standard culture techniques, cryopreserved cell stocks, and a synthetic extracellular matrix. Impact statement Pluripotent stem (PS) cells have been characterized by an inherent ability to self-organize into 3D "organoids" resembling stomach, intestine, liver, kidney, and brain tissues, offering a potentially powerful tool for modeling human development and disease. However, organoid formation must be quantitatively reproducible for applications such as drug and toxicity screening. Here, we report a strategy to produce uniform neural tissue constructs with reproducible global gene expression profiles for replicate samples from multiple experiments.
Subject(s)
Brain/cytology , Models, Biological , Neural Stem Cells/cytology , Pluripotent Stem Cells/cytology , Tissue Engineering/methods , Cell Culture Techniques/methods , Cell Differentiation/physiology , Cells, Cultured , Humans , Hydrogels , Polyethylene GlycolsABSTRACT
Here, we describe an in vitro strategy to model vascular morphogenesis where human induced pluripotent stem cell-derived endothelial cells (iPSC-ECs) are encapsulated in peptide-functionalized poly(ethylene glycol) (PEG) hydrogels, either on standard well plates or within a passive pumping polydimethylsiloxane (PDMS) tri-channel microfluidic device. PEG hydrogels permissive towards cellular remodeling were fabricated using thiol-ene photopolymerization to incorporate matrix metalloproteinase (MMP)-degradable crosslinks and CRGDS cell adhesion peptide. Time lapse microscopy, immunofluorescence imaging, and RNA sequencing (RNA-Seq) demonstrated that iPSC-ECs formed vascular networks through mechanisms that were consistent with in vivo vasculogenesis and angiogenesis when cultured in PEG hydrogels. Migrating iPSC-ECs condensed into clusters, elongated into tubules, and formed polygonal networks through sprouting. Genes upregulated for iPSC-ECs cultured in PEG hydrogels relative to control cells on tissue culture polystyrene (TCP) surfaces included adhesion, matrix remodeling, and Notch signaling pathway genes relevant to in vivo vascular development. Vascular networks with lumens were stable for at least 14days when iPSC-ECs were encapsulated in PEG hydrogels that were polymerized within the central channel of the microfluidic device. Therefore, iPSC-ECs cultured in peptide-functionalized PEG hydrogels offer a defined platform for investigating vascular morphogenesis in vitro using both standard and microfluidic formats. STATEMENT OF SIGNIFICANCE: Human induced pluripotent stem cell-derived endothelial cells (iPSC-ECs) cultured in synthetic hydrogels self-assemble into capillary networks through mechanisms consistent with in vivo vascular morphogenesis.
Subject(s)
Blood Vessels/physiology , Endothelial Cells/cytology , Hydrogels/pharmacology , Induced Pluripotent Stem Cells/cytology , Tissue Engineering/methods , Blood Vessels/drug effects , Capillaries/drug effects , Capillaries/physiology , Cell Adhesion/drug effects , Cells, Cultured , Endothelial Cells/drug effects , Endothelial Cells/metabolism , Extracellular Matrix/drug effects , Extracellular Matrix/metabolism , Gene Expression Profiling , Gene Expression Regulation/drug effects , Humans , Induced Pluripotent Stem Cells/drug effects , Induced Pluripotent Stem Cells/metabolismABSTRACT
RNA sequencing has increasingly become an indispensable tool for biological research. While sequencing costs have fallen dramatically in recent years, the current cost of RNA sequencing, nonetheless, remains a barrier to even more widespread adoption. Here, we present a simple RNA sequencing protocol with substantially reduced costs. This protocol uses as little as 10â ng of total RNA, allows multiplex sequencing of up to 96 samples per lane, and is strand specific. Extensive validation using human embryonic stem cells showed high consistency between technical replicates at various multiplexing levels.