U.S. Department of Health and Human ServicesHHS National Institutes of HealthNIH National Center for Advancing Translational SciencesNCATS

Resources

The iPSC Portal is a unique resource that allows stem cell-related data to be openly shared with the scientific community. Once SCTL publications are available online, the supporting omics data, qHTS, and other data sets will be made available to the public in interactive and/or raw formats. To address the issues of reproducibility that have been a significant obstacle in the stem cell field, SCTL will also provide detailed protocols for researchers seeking to replicate or expand upon our work. If you have any questions, please contact carlos.tristan@nih.gov.

 

Title Citation
SEQUIN: rapid and reproducible analysis of RNA-seq data in R/Shiny

Abstract

Title: SEQUIN: rapid and reproducible analysis of RNA-seq data in R/Shiny
Authors: Weber, et al.
Abstract: SEQUIN is a new web application (app) that allows fast and intuitive RNA-sequencing data analysis for organisms, tissues, and single cells. Integrated app functions enable uploading datasets, quality control, gene set enrichment, data visualization, and differential gene expression analysis. We also present the iPSC Profiler, a practical tool that helps to measure pluripotency and cell differentiation. Freely available to the public, SEQUIN empowers scientists to investigate transcriptome data firsthand with cutting edge statistical methods.

Link:

Weber, et al. bioRxiv.
A Comprehensive Roadmap of Human Placental Development in vitro

Abstract

Title: A Comprehensive Roadmap of Human Placental Development in vitro
Authors: Slamecka, et al.
Abstract: Human pluripotent stem cells (hPSCs) represent a powerful model system to study early developmental processes. However, lineage specification into trophectoderm (TE) remains poorly understood and access to well-characterized placental cells for biomedical research is limited, largely depending on fetal tissues or cancer cell lines. Here, we developed novel strategies enabling highly efficient TE specification that generates cytotrophoblast (CTB) and multinucleated primary syncytiotrophoblast (STB) followed by establishment of trophoblast stem cells (TSCs) capable of differentiating into extravillous trophoblast (EVT) and STB after long-term expansion. We confirmed stepwise induction of lineage- and cell-type-specific genes and substantiated typical features of placental cells using morphological, biochemical, integrated multi-omics, and single-cell analyses. Our data provide conclusive evidence that conventional hPSCs can be directly and exclusively converted into TE, thereby providing an unlimited source of diverse placental cell types suitable for a broad range of biomedical applications.

Slamecka, et al. bioRxiv.
Enhancing the Fitness of Embryoid Bodies and Organoids by Chemical Cytoprotection

Abstract

Title: Enhancing the Fitness of Embryoid Bodies and Organoids by Chemical Cytoprotection
Authors: Ryu, et al.
Abstract: Embryoid bodies (EBs) and self-organizing organoids derived from human pluripotent stem cells (hPSCs) recapitulate tissue development in a dish and hold great promise for disease modeling and drug development. However, current protocols are hampered by cellular stress and apoptosis during cell aggregation, resulting in variability and impaired cell differentiation. Here, we demonstrate that EBs and various organoid models (e.g., brain, gut, and kidney) can be optimized by using the CEPT small molecule cocktail, a polypharmacology approach that ensures cytoprotection and cell survival. Application of CEPT (chroman 1, emricasan, polyamines, trans-ISRIB) for just 24 hours during cell aggregation has long-lasting consequences affecting morphogenesis, gene expression, and cellular differentiation. Various qualification methods confirmed that CEPT treatment consistently improved EB and organoid fitness as compared to the widely used ROCK inhibitor Y-27632. Collectively, we discovered that stress-free cell aggregation and superior cell survival in the presence of CEPT are critical quality control determinants that establish a robust foundation for bioengineering complex tissue and organ models.

Ryu, et al. bioRxiv.
Scalable Generation of Pseudo-Unipolar Sensory Neurons from Human Pluripotent Stem Cells

Abstract

Title: Scalable Generation of Pseudo-Unipolar Sensory Neurons from Human Pluripotent Stem Cells
Authors: Deng, et al.
Abstract: Development of new non-addictive analgesics requires advanced strategies to differentiate human pluripotent stem cells (hPSCs) into relevant cell types amenable for translational research. Here, we developed a highly efficient and reproducible method that differentiates hPSCs into peptidergic and non-peptidergic nociceptors. By modulating specific cell signaling pathways, hPSCs were first converted into SOX10+ neural crest cells, followed by differentiation into sensory neurons with an in vivo-like pseudo-unipolar morphology. Detailed characterization confirmed that the hPSC-derived nociceptors displayed molecular and cellular features comparable to native dorsal root ganglion (DRG) neurons, and expressed high-threshold primary sensory neuron markers, transcription factors, neuropeptides, and over 150 ion channels and receptors, including critical pain-relevant drug targets (e.g., TRPV1, TAC1, CALCA, NAV1.7, NAV1.8). Moreover, after confirming robust functional activities and differential response to noxious stimuli and specific drugs, a robotic cell culture system was employed to produce large quantities of human sensory neurons, which can be used to develop nociceptor-selective analgesics.

Deng, et al. bioRxiv.
Directed Differentiation of Human Pluripotent Stem Cells into Radial Glia and Astrocytes Bypasses Neurogenesis

Abstract

Title: Directed Differentiation of Human Pluripotent Stem Cells into Radial Glia and Astrocytes Bypasses Neurogenesis
Authors: Jovanovic, et al.
Abstract: Derivation of astrocytes from human pluripotent stem cells (hPSCs) is inefficient and cumbersome, impeding their use in biomedical research. Here, we developed a highly efficient chemically defined astrocyte differentiation strategy that overcomes current limitations. This approach largely bypasses neurogenesis, which otherwise precedes astrogliogenesis during brain development and in vitro experiments. hPSCs were first differentiated into radial glial cells (RGCs) exhibiting in vivo-like radial glia signatures. Activation of NOTCH and JAK/STAT pathways in bona fide RGCs resulted in direct astrogliogenesis confirmed by expression of various glial markers (NFIA, NFIB, SOX9, CD44, S100B, GFAP). Transcriptomic and genome-wide epigenetic analyses confirmed RGC-to-astrocyte differentiation and absence of neurogenesis. The morphological and functional identity of hPSC-derived astrocytes was confirmed by using an array of methods (e.g. electron microscopy, calcium imaging, co-culture with neurons, grafting into mouse brains). Lastly, the scalable protocol was adapted to a robotic platform and used to model Alexander disease. In conclusion, our findings uncover remarkable plasticity in neural lineage progression that can be exploited to manufacture large numbers of human hPSC-derived astrocytes for drug development and regenerative medicine.

Jovanovic, et al. bioRxiv.
Human Pluripotent Stem Cells for High-Throughput Drug Screening and Characterization of Small Molecules

Abstract

Title: Human Pluripotent Stem Cells for High-Throughput Drug Screening and Characterization of Small Molecules
Authors: Ryu, et al.
Abstract: Human pluripotent stem cells (hPSCs), such as induced pluripotent stem cells (iPSCs), hold great promise for drug discovery, toxicology studies, and regenerative medicine. Here, we describe standardized protocols and experimental procedures that combine automated cell culture for scalable production of hPSCs with quantitative high-throughput screening (qHTS) in miniaturized 384-well plates. As a proof of principle, we established dose-response assessments and determined optimal concentrations of 12 small molecule compounds that are commonly used in the stem cell field. Multi-parametric analysis of readouts from diverse assays including cell viability, mitochondrial membrane potential, plasma membrane integrity, and ATP production was used to distinguish normal biological responses from cellular stress induced by small molecule treatment. Collectively, the establishment of integrated workflows for cell manufacturing, qHTS, high-content imaging, and data analysis provides an end-to-end platform for industrial-scale projects and should leverage the drug discovery process using hPSC-derived cell types.

Ryu, et al. 2021.
A Versatile Polypharmacology Platform Promotes Cytoprotection and Viability of Human Pluripotent and Differentiated Cells

Abstract and links

Title: A Versatile Polypharmacology Platform Promotes Cytoprotection and Viability of Human Pluripotent and Differentiated Cells
Authors: Chen, Tristan, et al.
Abstract: Human pluripotent stem cells (hPSCs) are capable of extensive self-renewal yet remain highly sensitive to environmental perturbations in vitro, posing challenges to their therapeutic use. There is an urgent need to advance strategies that ensure safe and robust long-term growth and functional differentiation of these cells. Here, we deployed high-throughput screening strategies to identify a small-molecule cocktail that improves viability of hPSCs and their differentiated progeny. The combination of chroman 1, emricasan, polyamines, and trans-ISRIB (CEPT) enhanced cell survival of genetically stable hPSCs by simultaneously blocking several stress mechanisms that otherwise compromise cell structure and function. CEPT provided strong improvements for several key applications in stem-cell research, including routine cell passaging, cryopreservation of pluripotent and differentiated cells, embryoid body (EB) and organoid formation, single-cell cloning, and genome editing. Thus, CEPT represents a unique poly-pharmacological strategy for comprehensive cytoprotection, providing a rationale for efficient and safe utilization of hPSCs.

Links:

Chen, et al. 2021.

Robotic High-Throughput Biomanufacturing and Functional Differentiation of Human Pluripotent Stem Cells

Abstract and links

Title: Robotic High-Throughput Biomanufacturing and Functional Differentiation of Human Pluripotent Stem Cells
Authors:
Tristan, et al.
Abstract:
Efficient translation of human induced pluripotent stem cells (hiPSCs) requires scalable cell manufacturing strategies for optimal self-renewal and functional differentiation. Traditional manual cell culture is variable and labor intensive, posing challenges for high-throughput applications. Here, we established a robotic platform and automated all essential steps of hiPSC culture and differentiation under chemically defined conditions. This approach allowed rapid and standardized manufacturing of billions of hiPSCs that can be produced in parallel from up to 90 different patient- and disease-specific cell lines. Moreover, we established automated multi-lineage differentiation and generated functional neurons, cardiomyocytes, and hepatocytes. To validate our approach, we compared robotic and manual cell culture operations and performed comprehensive molecular and cellular characterizations (e.g., single-cell transcriptomics, mass cytometry, metabolism, electrophysiology) to benchmark industrial-scale cell culture operations toward building an integrated platform for efficient cell manufacturing for disease modeling, drug screening, and cell therapy.

Tristan, et al. 2021.

Quantitative Analysis of Human Pluripotency and Neural Specification by In-Depth (Phospho)Proteomic Profiling

Abstract and links

 Title: Quantitative Analysis of Human Pluripotency and Neural Specification by In-Depth (Phospho)Proteomic Profiling
Authors:
Singeç, et al.
Abstract:
Controlled differentiation of human embryonic stem cells (hESCs) can be utilized for precise analysis of cell type identities during early development. We established a highly efficient neural induction strategy and an improved analytical platform, and determined proteomic and phosphoproteomic profiles of hESCs and their specified multipotent neural stem cell derivatives (hNSCs). This quantitative dataset (nearly 13,000 proteins and 60,000 phosphorylation sites) provides unique molecular insights into pluripotency and neural lineage entry. Systems-level comparative analysis of proteins (e.g., transcription factors, epigenetic regulators, kinase families), phosphorylation sites, and numerous biological pathways allowed the identification of distinct signatures in pluripotent and multipotent cells. Furthermore, as predicted by the dataset, we functionally validated an autocrine/paracrine mechanism by demonstrating that the secreted protein midkine is a regulator of neural specification. This resource is freely available to the scientific community, including a searchable website, PluriProt.

Singeç, et al. 2016.
NCATS web toolbox

The NCATS Toolbox is a collection of publicly available web apps developed in NCATS Informatics.