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The hypothalamus, composed of several nuclei, is essential for maintaining our body's homeostasis. The arcuate nucleus (ARC), located in the mediobasal hypothalamus, contains neuronal populations with eminent roles in energy and glucose homeostasis as well as reproduction. These neuronal populations are of great interest for translational research. To fulfill this promise, we used a robotic cell culture platform to provide a scalable and chemically defined approach for differentiating human pluripotent stem cells (hPSCs) into pro-opiomelanocortin (POMC), somatostatin (SST), tyrosine hydroxylase (TH) and gonadotropin-releasing hormone (GnRH) neuronal subpopulations with an ARC-like signature. This robust approach is reproducible across several distinct hPSC lines and exhibits a stepwise induction of key ventral diencephalon and ARC markers in transcriptomic profiling experiments. This is further corroborated by direct comparison to human fetal hypothalamus, and the enriched expression of genes implicated in obesity and type 2 diabetes (T2D). Genome-wide chromatin accessibility profiling by ATAC-seq identified accessible regulatory regions that can be utilized to predict candidate enhancers related to metabolic disorders and hypothalamic development. In depth molecular, cellular, and functional experiments unveiled the responsiveness of the hPSC-derived hypothalamic neurons to hormonal stimuli, such as insulin, neuropeptides including kisspeptin, and incretin mimetic drugs such as Exendin-4, highlighting their potential utility as physiologically relevant cellular models for disease studies. In addition, differential glucose and insulin treatments uncovered adaptability within the generated ARC neurons in the dynamic regulation of POMC and insulin receptors. In summary, the establishment of this model represents a novel, chemically defined, and scalable platform for manufacturing large numbers of hypothalamic arcuate neurons and serves as a valuable resource for modeling metabolic and reproductive disorders., Competing Interests: Conflict of Interest Statement The authors declare no competing interests.

Human pluripotent stem cells (hPSCs) represent a powerful model system to study early developmental processes. However, lineage specification into trophectoderm (TE) and trophoblast (TB) differentiation 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 syncytiotrophoblast (STB), followed by the establishment of trophoblast stem cells (TSCs) capable of differentiating into extravillous trophoblast (EVT) and STB after long-term expansion. We confirmed stepwise and controlled induction of lineage- and cell-type-specific genes consistent with developmental biology principles and benchmarked typical features of placental cells using morphological, biochemical, genomics, epigenomics, and single-cell analyses. Charting a well-defined roadmap from hPSCs to distinct placental phenotypes provides invaluable opportunities for studying early human development, infertility, and pregnancy-associated diseases., Competing Interests: J.S., T.D., A.S., and I.S. are co-inventors on a US Department of Health and Human Services patent application covering the trophectoderm/trophoblast differentiation method and its utilization., (© 2024 The Author(s).)

There has been an alarming trend toward earlier puberty in girls, suggesting the influence of an environmental factor(s). As the reactivation of the reproductive axis during puberty is thought to be mediated by the hypothalamic neuropeptides kisspeptin and gonadotropin-releasing hormone (GnRH), we asked whether an environmental compound might activate the kisspeptin (KISS1R) or GnRH receptor (GnRHR). We used GnRHR or KISS1R-expressing HEK293 cells to screen the Tox21 10K compound library, a compendium of pharmaceuticals and environmental compounds, for GnRHR and KISS1R activation. Agonists were identified using Ca2+ flux and phosphorylated extracellularly regulated kinase (p-ERK) detection assays. Follow-up studies included measurement of genes known to be upregulated upon receptor activation using relevant murine or human cell lines and molecular docking simulation. Musk ambrette was identified as a KISS1R agonist, and treatment with musk ambrette led to increased expression of Gnrh1 in murine and human hypothalamic cells and expansion of GnRH neuronal area in developing zebrafish larvae. Molecular docking demonstrated that musk ambrette interacts with the His309, Gln122, and Gln123 residues of the KISS1R. A group of cholinergic agonists with structures similar to methacholine was identified as GnRHR agonists. When applied to murine gonadotrope cells, these agonists upregulated Fos, Jun, and/or Egr1. Molecular docking revealed a potential interaction between GnRHR and 5 agonists, with Asn305 constituting the most conservative GnRHR binding site. In summary, using a Tox21 10K compound library screen combined with cellular, molecular, and structural biology techniques, we have identified novel environmental agents that may activate the human KISS1R or GnRHR., (Published by Oxford University Press on behalf of the Endocrine Society 2024.)

Astrocytes, the most abundant glial cell type in the brain, play crucial roles in maintaining homeostasis within the central nervous system (CNS). Impairment or abnormalities of typical astrocyte functions in the CNS serve as a causative or contributing factor in numerous neurodevelopmental, neurodegenerative, and neuropsychiatric disorders. Currently, disease-modeling and drug-screening approaches, primarily focused on human astrocytes, rely on human pluripotent stem cell (hPSC)-derived astrocytes. However, it is important to acknowledge that these hPSC-derived astrocytes exhibit notable differences across studies and when compared to their in vivo counterparts. These differences may potentially compromise translational outcomes if not carefully accounted for. This review aims to explore state-of-the-art in vitro models of human astrocyte development, focusing on the developmental processes, functional maturity, and technical aspects of various hPSC-derived astrocyte differentiation protocols. Additionally, it summarizes their successful application in modeling neurological disorders. The discussion extends to recent advancements in the large-scale production of human astrocytes and their application in developing high-throughput assays conducive to therapeutic drug discovery.

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, kidney) can be optimized by using the small molecule cocktail named CEPT (chroman 1, emricasan, polyamines, trans-ISRIB), a polypharmacological approach that ensures cytoprotection and cell survival. Application of CEPT for just 24 h during cell aggregation has long-lasting consequences affecting morphogenesis, gene expression, cellular differentiation, and organoid function. Various qualification methods confirmed that CEPT treatment enhanced experimental reproducibility and 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., (Creative Commons Attribution license.)

Human gliogenesis remains poorly understood, and derivation of astrocytes from human pluripotent stem cells (hPSCs) is inefficient and cumbersome. Here, we report controlled glial differentiation from hPSCs that bypasses neurogenesis, which otherwise precedes astrogliogenesis during brain development and in vitro differentiation. hPSCs were first differentiated into radial glial cells (RGCs) resembling resident RGCs of the fetal telencephalon, and modulation of specific cell signaling pathways resulted in direct and stepwise induction of key astroglial markers (NFIA, NFIB, SOX9, CD44, S100B, glial fibrillary acidic protein [GFAP]). Transcriptomic and genome-wide epigenetic mapping and single-cell analysis confirmed RGC-to-astrocyte differentiation, obviating neurogenesis and the gliogenic switch. Detailed molecular and cellular characterization experiments uncovered new mechanisms and markers for human RGCs and astrocytes. In summary, establishment of a glia-exclusive neural lineage progression model serves as a unique serum-free platform of manufacturing large numbers of RGCs and astrocytes for neuroscience, disease modeling (e.g., Alexander disease), and regenerative medicine., Competing Interests: Conflict of interests V.M.J., A.S., and I.S. are coinventors on a US Department of Health and Human Services patent covering the described radial glia and astrocyte differentiation method and its use., (Published by Elsevier Inc.)

Development of new non-addictive analgesics requires advanced strategies to differentiate human pluripotent stem cells (hPSCs) into relevant cell types. Following principles of developmental biology and translational applicability, here we developed an efficient stepwise differentiation method for peptidergic and non-peptidergic nociceptors. By modulating specific cell signaling pathways, hPSCs were first converted into SOX10 + neural crest, followed by differentiation into sensory neurons. Detailed characterization, including ultrastructural analysis, confirmed that the hPSC-derived nociceptors displayed cellular and molecular 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 relevant for pain research and axonal growth/regeneration studies (e.g., TRPV1, NA V 1.7, NA V 1.8, TAC1, CALCA, GAP43, DPYSL2, NMNAT2). 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., Competing Interests: Conflict of interests T.D., A.S., and I.S. are co-inventors on a US Department of Health and Human Services patent covering the nociceptor differentiation method and its utilization., (Published by Elsevier Inc.)

SEQUIN is a web-based application (app) that allows fast and intuitive analysis of RNA sequencing data derived for model 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 developed the iPSC Profiler, a practical gene module scoring tool that helps measure and compare pluripotent and differentiated cell types. Benchmarking to other commercial and non-commercial products underscored several advantages of SEQUIN. Freely available to the public, SEQUIN empowers scientists using interdisciplinary methods to investigate and present transcriptome data firsthand with state-of-the-art statistical methods. Hence, SEQUIN helps democratize and increase the throughput of interrogating biological questions using next-generation sequencing data with single-cell resolution., Competing Interests: The authors declare no conflicts of interest.

Human pluripotent stem cells (hPSCs) are inherently sensitive cells. Single-cell dissociation and the establishment of clonal cell lines have been long-standing challenges. This inefficiency of cell cloning represents a major obstacle for the standardization and streamlining of gene editing in induced pluripotent stem cells for basic and translational research. Here we describe a chemically defined protocol for robust single-cell cloning using microfluidics-based cell sorting in combination with the CEPT small-molecule cocktail. This advanced strategy promotes the viability and cell fitness of self-renewing stem cells. The use of low-pressure microfluidic cell dispensing ensures gentle and rapid dispensing of single cells into 96- and 384-well plates, while the fast-acting CEPT cocktail minimizes cellular stress and maintains cell structure and function immediately after cell dissociation. The protocol also facilitates clone picking and produces genetically stable clonal cell lines from hPSCs in a safe and cost-efficient fashion. Depending on the proliferation rate of the clone derived from a single cell, this protocol can be completed in 7-14 d and requires experience with aseptic cell culture techniques. Altogether, the relative ease, scalability and robustness of this workflow should boost gene editing in hPSCs and leverage a wide range of applications, including cell line development (e.g., reporter and isogenic cell lines), disease modeling and applications in regenerative medicine., (© 2022. This is a U.S. Government work and not under copyright protection in the US; foreign copyright protection may apply.)

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., (© 2021. Springer Science+Business Media, LLC.)

This chapter was inadvertently published with an error, the ninth point under 'Materials' section must read., (© 2021. Springer Science+Business Media, LLC.)

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., (Copyright © 2021 The Authors. Published by Elsevier Inc. All rights reserved.)

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.

Human pluripotent stem cells (hPSCs) are characterized by their ability to self-renew and differentiate into any cell type of the human body. To fully utilize the potential of hPSCs for translational research and clinical applications, it is critical to develop rigorous cell differentiation protocols under feeder-free conditions that are efficient, reproducible, and scalable for high-throughput projects. Focusing on neural conversion of hPSCs, here we describe robust small molecule-based procedures that generate neural stem cells (NSCs) in less than a week under chemically defined conditions. These protocols can be used to dissect the mechanisms of neural lineage entry and to further develop systematic protocols that produce the cellular diversity of the central nervous system at industrial scale.

A hallmark of cellular transformation is the evasion of contact-dependent inhibition of growth. To find new therapeutic targets for glioblastoma, we looked for pathways that are inhibited by high cell density in astrocytes but not in glioma cells. Here we report that glioma cells have disabled the normal controls on cholesterol synthesis. At high cell density, astrocytes turn off cholesterol synthesis genes and have low cholesterol levels, but glioma cells keep this pathway on and maintain high cholesterol. Correspondingly, cholesterol pathway upregulation is associated with poor prognosis in glioblastoma patients. Densely-plated glioma cells increase oxygen consumption, aerobic glycolysis, and the pentose phosphate pathway to synthesize cholesterol, resulting in a decrease in reactive oxygen species, TCA cycle intermediates, and ATP. This constitutive cholesterol synthesis is controlled by the cell cycle, as it can be turned off by cyclin-dependent kinase inhibitors and it correlates with disabled cell cycle control though loss of p53 and RB. Finally, glioma cells, but not astrocytes, are sensitive to cholesterol synthesis inhibition downstream of the mevalonate pathway, suggesting that specifically targeting cholesterol synthesis might be an effective treatment for glioblastoma.

Glyceraldehyde-3-phosphate dehydrogenase (GAPDH) plays roles in both energy maintenance, and stress signaling by forming a protein complex with seven in absentia homolog 1 (Siah1). Mechanisms to coordinate its glycolytic and stress cascades are likely to be very important for survival and homeostatic control of any living organism. Here we report that apoptosis signal-regulating kinase 1 (ASK1), a representative stress kinase, interacts with both GAPDH and Siah1 and is likely able to phosphorylate Siah1 at specific amino acid residues (Thr-70/Thr-74 and Thr-235/Thr-239). Phosphorylation of Siah1 by ASK1 triggers GAPDH-Siah1 stress signaling and activates a key downstream target, p300 acetyltransferase in the nucleus. This novel mechanism, together with the established S-nitrosylation/oxidation of GAPDH at Cys-150, provides evidence of how the stress signaling involving GAPDH is finely regulated. In addition, the present results imply crosstalk between the ASK1 and GAPDH-Siah1 stress cascades., (© 2015 by The American Society for Biochemistry and Molecular Biology, Inc.)