Search

Summary: Contractile to synthetic phenotypic switching of smooth muscle cells (SMCs) contributes to stenosis in vascular disease and vascular transplants. To generate more contractile SMCs, we performed a high-throughput differentiation screen using a MYH11-NLuc-tdTomato human embryonic stem cell reporter cell line. We identified RepSox as a factor that promotes differentiation of MYH11-positive cells by promoting NOTCH signaling. RepSox induces SMCs to exhibit a more contractile phenotype than SMCs generated using PDGF-BB and TGF-β1, two factors previously used for SMC differentiation but which also cause intimal hyperplasia. In addition, RepSox inhibited intimal hyperplasia caused by contractile to synthetic phenotypic switching of SMCs in a rat balloon injury model. Thus, in addition to providing more contractile SMCs that could prove useful for constructing artificial blood vessels, this study suggests a strategy for identifying drugs for inhibiting intimal hyperplasia that act by driving contractile differentiation rather than inhibiting proliferation non-specifically. : Thomson, Zhang, and colleagues report a high-throughput screen that can be used for optimization of the fully defined differentiation of contractile smooth muscle cells and identification of intimal hyperplasia inhibitors. Both in vitro and in vivo evidence revealed that RepSox is better than PDGF-BB and TGF-β1 in inducing contractile phenotype of smooth muscle cells and reducing the risk of intimal hyperplasia. Keywords: pluripotent stem cells, contractile smooth muscle cells, differentiation, maturation, RepSox, NOTCH, intima hyperplasia, MYH11-NLuc-tdTomato reporter cell line, high-throughput screen, restenosis

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.

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., Author summary For regenerative medicine, a slow developmental clock operating in human pluripotent stem cells imposes severe obstacles to many pluripotent stem cell therapies. Producing the desired cell types with the necessary level of physiological maturity in clinically-relevant time frames will be essential if the therapy is aimed at being patient-specific. Thus, understanding what controls species-specific rates of differentiation and identifying ways to accelerate it would have a broad impact on improving the therapeutic value of human pluripotent stem cells. In this paper, we measured the immediate-early gene expression responses to neural differentiation signals in mouse and human pluripotent stem cells. We quantitatively summarized the gene expression patterns using a flexible model that allowed us to identify shared trends and dynamic changes across species. We found that responses occurred equally early across species, but expression patterns were prolonged in human cells and the rate of upregulation in gene expression was significantly faster in mouse cells. Our results represent a significant contribution in furthering the knowledge of early developmental transcriptional programs.

Neuroepithelial stem cells (NSC) from different anatomical regions of the embryonic neural tube’s rostrocaudal axis can differentiate into diverse central nervous system tissues, but the transcriptional regulatory networks governing these processes are incompletely understood. Here, we measure region-specific NSC gene expression along the rostrocaudal axis in a human pluripotent stem cell model of early central nervous system development over a 72-hour timecourse spanning the hindbrain to cervical spinal cord. We introduce Escarole, a probabilistic clustering algorithm for non-stationary time series, and combine it with prior-based regulatory network inference to identify genes that are regulated dynamically and predict their upstream regulators. We identify known regulators of patterning and neural development, including the HOX genes, and predict a direct regulatory connection between the transcription factor POU3F2 and target gene STMN2. We demonstrate that POU3F2 is required for expression of STMN2, suggesting that this regulatory connection is important for region-specificity of NSCs.

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.

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.

Shewanella oneidensis is of particular interest for research because of its unique ability to use a variety of metals as final respiratory electron acceptors and reduce them into insoluble oxides. A collection of monoclonal antibodies (mAbs) that were prepared towards Escherichia coli RNA polymerase (RNAP) was tested for reactivity with proteins extracted from S. oneidensis. Two polyol-responsive monoclonal antibodies (PR-mAbs) were used to purify RNA polymerase from S. oneidensis using immunoaffinity purification techniques. A collection of mAbs towards E. coli sigma subunits was also examined for cross-reactivity with S. oneidensis proteins. Reactions were identified with mAbs to E. coli sigma(70) and sigma(54). These mAbs will be useful tools for immunoaffinity purifying and studying the transcriptional machinery of S. oneidensis.

Hemogenic endothelium (HE) has been recognized as a source of hematopoietic stem cells (HSCs) in the embryo. Access to human HE progenitors (HEPs) is essential for enabling the investigation of the molecular determinants of HSC specification. Here, we show that HEPs capable of generating definitive hematopoietic cells can be obtained from human pluripotent stem cells (hPSCs) and identified precisely by a VE-cadherin(+)CD73(-)CD235a/CD43(-) phenotype. This phenotype discriminates true HEPs from VE-cadherin(+)CD73(+) non-HEPs and VE-cadherin(+)CD235a(+)CD41a(-) early hematopoietic cells with endothelial and FGF2-dependent hematopoietic colony-forming potential. We found that HEPs arise at the post-primitive-streak stage of differentiation directly from VE-cadherin-negative KDR(bright)APLNR(+)PDGFRα(low/-) hematovascular mesodermal precursors (HVMPs). In contrast, hemangioblasts, which are capable of forming endothelium and primitive blood cells, originate from more immature APLNR(+)PDGFRα(+) mesoderm. The demarcation of HEPs and HVMPs provides a platform for modeling blood development from endothelium with a goal of facilitating the generation of HSCs from hPSCs.

Nat. Methods 3, 637–646 (2006); published online 21 July 2006; corrected after print 24 August 2006. In the version of this Protocol initially published, the references were numbered incorrectly. This error has been corrected in the HTML and PDF versions of the article.