Impaired oxysterol-liver X receptor signaling underlies aberrant cortical neurogenesis in a stem cell model of neurodevelopmental disorder.

The mechanisms by which genomic risks contribute to the onset of neuropsychiatric conditions remain a key challenge and a prerequisite for successful development of effective therapies. 15q11.2 copy number variation (CNV) containing the CYFIP1 gene is associated with autism and schizophrenia. Using stem cell models, we show that 15q11.2 deletion (15q11.2del) and CYFIP1 loss of function (CYFIP1-LoF) lead to premature neuronal differentiation, while CYFIP1 gain of function (CYFIP1-GoF) favors neural progenitor maintenance. CYFIP1 dosage changes led to dysregulated cholesterol metabolism and altered levels of 24S,25-epoxycholesterol, which can mimic the 15q11.2del and CYFIP1-LoF phenotypes by promoting cortical neuronal differentiation and can restore the impaired neuronal differentiation of CYFIP1-GoF neural progenitors. Moreover, the neurogenic activity of 24S,25-epoxycholesterol is lost following genetic deletion of liver X receptor (LXRß), while compound deletion of LXRß in CYFIP1-/- background rescued their premature neurogenesis. This work delineates LXR-mediated oxysterol regulation of neurogenesis as a pathological mechanism in neural cells carrying 15q11.2 CNV and provides a potential target for therapeutic strategies for associated disorders.

AI-organoid integrated systems for biomedical studies and applications.

In this review, we explore the growing role of artificial intelligence (AI) in advancing the biomedical applications of human pluripotent stem cell (hPSC)-derived organoids. Stem cell-derived organoids, these miniature organ replicas, have become essential tools for disease modeling, drug discovery, and regenerative medicine. However, analyzing the vast and intricate datasets generated from these organoids can be inefficient and error-prone. AI techniques offer a promising solution to efficiently extract insights and make predictions from diverse data types generated from microscopy images, transcriptomics, metabolomics, and proteomics. This review offers a brief overview of organoid characterization and fundamental concepts in AI while focusing on a comprehensive exploration of AI applications in organoid-based disease modeling and drug evaluation. It provides insights into the future possibilities of AI in enhancing the quality control of organoid fabrication, label-free organoid recognition, and three-dimensional image reconstruction of complex organoid structures. This review presents the challenges and potential solutions in AI-organoid integration, focusing on the establishment of reliable AI model decision-making processes and the standardization of organoid research.

The functional maturity of grafted human pluripotent stem cell derived-islets (hSC-Islets) evaluated by the glycemic set point during blood glucose normalizing process in diabetic mice.

Human pluripotent stem cell (hPSCs) derived-pancreatic islets (hSC-islets) are good candidates for cell replacement therapy for patients with diabetes as substitutes for deceased donor-derived islets, because they are pluripotent and have infinite proliferation potential. Grafted hSC-islets ameliorate hyperglycemia in diabetic mice; however, several weeks are needed to normalize the hyperglycemia. These data suggest hSC-islets require maturation, but their maturation process in vivo is not yet fully understood. In this study, we utilized two kinds of streptozotocin (STZ)-induced diabetes model mice by changing the administration timing in order to examine the time course of maturation of hSC-islets and the effects of hyperglycemia on their maturation. We found no hyperglycemia in immune-compromised mice when hSC-islets had been transplanted under their kidney capsules in advance, and STZ was administered 4 weeks after transplantation. Of note, the blood glucose levels of those mice were stably maintained under 100 mg/dl 10 weeks after transplantation; this is lower than the mouse glycemic set point (120-150 mg/dl), suggesting that hSC-islets control blood glucose levels to the human glycemic set point. We confirmed that gene expression of maturation markers of pancreatic beta cells tended to upregulate during 4 weeks after transplantation. Periodical histological analysis revealed that revascularization was observed as early as 1 week after transplantation, but reinnervation in the grafted hSC-islets was not detected at all, even 15 weeks after transplantation. In conclusion, our hSC-islets need at least 4 weeks to mature, and the human glycemic set point is a good index for evaluating ultimate maturity for hSC-islets in vivo.

Report of the Assay Guidance Workshop on 3-Dimensional Tissue Models for Antiviral Drug Development.

The National Center for Advancing Translational Sciences (NCATS) Assay Guidance Manual (AGM) Workshop on 3D Tissue Models for Antiviral Drug Development, held virtually on 7-8 June 2022, provided comprehensive coverage of critical concepts intended to help scientists establish robust, reproducible, and scalable 3D tissue models to study viruses with pandemic potential. This workshop was organized by NCATS, the National Institute of Allergy and Infectious Diseases, and the Bill and Melinda Gates Foundation. During the workshop, scientific experts from academia, industry, and government provided an overview of 3D tissue models' utility and limitations, use of existing 3D tissue models for antiviral drug development, practical advice, best practices, and case studies about the application of available 3D tissue models to infectious disease modeling. This report includes a summary of each workshop session as well as a discussion of perspectives and challenges related to the use of 3D tissues in antiviral drug discovery.

Generation and Co-culture of Cortical Glutamatergic and GABAergic-Induced Neuronal Cells.

The study of neurological disorders requires experimentation on human neurons throughout their development. Primary neurons can be difficult to obtain, and animal models may not fully recapitulate phenotypes observed in human neurons. Human neuronal culturing schemes which contain a balanced mixture of excitatory and inhibitory neurons that resemble physiological ratios seen in vivo will be useful to probe the neurological basis of excitation-inhibition (E-I) balance. Here, we describe a method for directly inducing a homogenous population of cortical excitatory neurons and cortical interneurons from human pluripotent stem cells, as well as the generation of mixed cultures using these induced neurons. The obtained cells display robust neuronal synchronous network activity as well as complex morphologies that are amenable to studies probing the molecular and cellular basis of disease mutations or other aspects of neuronal and synaptic development.

Generation of Homogeneous Populations of Cortical Interneurons from Human Pluripotent Stem Cells.

Cortical interneurons (cINs), especially those that are derived from the medial ganglionic eminence (MGE) during early development, are associated with various neuropsychiatric disorders. Human pluripotent stem cell (hPSC)-derived cINs can provide unlimited cell sources for studying disease mechanisms and developing novel therapeutics. Here, we describe an optimized method to generate homogeneous cIN populations based on three-dimensional (3D) cIN sphere generation. This optimized differentiation system could sustain generated cINs relatively long term without compromising their survival or phenotypes.

PEAR1 is a potential regulator of early hematopoiesis of human pluripotent stem cells.

Hemogenic endothelial (HE) cells are specialized endothelial cells to give rise to hematopoietic stem/progenitor cells during hematopoietic development. The underlying mechanisms that regulate endothelial-to-hematopoietic transition (EHT) of human HE cells are not fully understand. Here, we identified platelet endothelial aggregation receptor-1 (PEAR1) as a novel regulator of early hematopoietic development in human pluripotent stem cells (hPSCs). We found that the expression of PEAP1 was elevated during hematopoietic development. A subpopulation of PEAR1+ cells overlapped with CD34+ CD144+ CD184+ CD73- arterial-type HE cells. Transcriptome analysis by RNA sequencing indicated that TAL1/SCL, GATA2, MYB, RUNX1 and other key transcription factors for hematopoietic development were mainly expressed in PEAR1+ cells, whereas the genes encoding for niche-related signals, such as fibronectin, vitronectin, bone morphogenetic proteins and jagged1, were highly expressed in PEAR1- cells. The isolated PEAR1+ cells exhibited significantly greater EHT capacity on endothelial niche, compared with the PEAR1- cells. Colony-forming unit (CFU) assays demonstrated the multilineage hematopoietic potential of PEAR1+ -derived hematopoietic cells. Furthermore, PEAR1 knockout in hPSCs by CRISPR/Cas9 technology revealed that the hematopoietic differentiation was impaired, resulting in decreased EHT capacity, decreased expression of hematopoietic-related transcription factors, and increased expression of niche-related signals. In summary, this study revealed a novel role of PEAR1 in balancing intrinsic and extrinsic signals for early hematopoietic fate decision.

Engineering Skeletal Muscle Grafts with PAX7::GFP-Sorted Human Pluripotent Stem Cell-Derived Myogenic Progenitors on Fibrin Microfiber Bundles for Tissue Regeneration.

Tissue engineering strategies that combine human pluripotent stem cell-derived myogenic progenitors (hPDMs) with advanced biomaterials provide promising tools for engineering 3D skeletal muscle grafts to model tissue development in vitro and promote muscle regeneration in vivo. We recently demonstrated (i) the potential for obtaining large numbers of hPDMs using a combination of two small molecules without the overexpression of transgenes and (ii) the application of electrospun fibrin microfiber bundles for functional skeletal muscle restoration following volumetric muscle loss. In this study, we aimed to demonstrate that the biophysical cues provided by the fibrin microfiber bundles induce hPDMs to form engineered human skeletal muscle grafts containing multinucleated myotubes that express desmin and myosin heavy chains and that these grafts could promote regeneration following skeletal muscle injuries. We tested a genetic PAX7 reporter line (PAX7::GFP) to sort for more homogenous populations of hPDMs. RNA sequencing and gene set enrichment analyses confirmed that PAX7::GFP-sorted hPDMs exhibited high expression of myogenic genes. We tested engineered human skeletal muscle grafts derived from PAX7::GFP-sorted hPDMs within in vivo skeletal muscle defects by assessing myogenesis, engraftment and immunogenicity using immunohistochemical staining. The PAX7::GFP-sorted groups had moderately high vascular infiltration and more implanted cell association with embryonic myosin heavy chain (eMHC) regions, suggesting they induced pro-regenerative microenvironments. These findings demonstrated the promise for the use of PAX7::GFP-sorted hPDMs on fibrin microfiber bundles and provided some insights for improving the cell-biomaterial system to stimulate more robust in vivo skeletal muscle regeneration.

Single Nucleotide Polymorphism (SNP) Arrays and Their Sensitivity for Detection of Genetic Changes in Human Pluripotent Stem Cell Cultures.

Human pluripotent stem cells (hPSCs) can be grown in culture indefinitely, making them a valuable tool for use in basic biology, disease modeling, and regenerative medicine. However, over prolonged periods in culture, hPSCs tend to acquire genomic aberrations that confer growth advantages, similar to those seen in some cancers. Monitoring the genomic stability of cultured hPSCs is critical to ensuring their efficacy and safety as a therapeutic tool. Most commonly employed methods for monitoring of hPSC genomes are cytogenetic methods, such as G-banding. Nonetheless, such methods have limited resolution and sensitivity for detecting mosaicism. Single nucleotide polymorphism (SNP) array platforms are a potential alternative that could improve detection of abnormalities. Here, we outline protocols for SNP array whole-genome screening of hPSCs. Moreover, we detail the procedure for assessing the SNP array's sensitivity in detecting low-level mosaic copy-number changes. We show that mosaicism can be confidently identified in samples only once they contain 20% variants, although samples containing 10% variants typically display enough variation to warrant further investigation and confirmation, for example by using a more sensitive targeted method. Finally, we highlight the advantages and limitations of SNP arrays, including a cost comparison of SNP arrays versus other commonly employed methods for detection of genetic changes in hPSC cultures. © 2022 The Authors. Current Protocols published by Wiley Periodicals LLC. Basic Protocol 1: DNA sample preparation for SNP arrays Basic Protocol 2: SNP array hybridization, washing, and scanning Basic Protocol 3: SNP array data analysis Support Protocol: Assessment of SNP array sensitivity for detection of mosaicism.

Highly Efficient CRISPR/Cas9-Mediated Genome Editing in Human Pluripotent Stem Cells.

Human pluripotent stem cells hold tremendous potential for both basic biology and cell-based therapies for a wide variety of diseases. The ability to manipulate the genome of these cells using the CRISPR/Cas9 technology has expanded this potential by providing a valuable tool to engineer or correct disease-associated mutations. Because of the high efficiency with which CRISPR/Cas9 creates targeted double-strand breaks, a major challenge has been the introduction of precise genetic modifications on one allele without indel formation on the non-targeted allele. To overcome this obstacle, we describe use of two oligonucleotide repair templates: one expressing the sequence change and the other maintaining the normal sequence. In addition, we have streamlined both the transfection and screening methodologies to make the protocols efficient, with small numbers of cells and a limited amount of labor-intensive clone passaging. This article provides a technically simple approach for generating valuable tools to model human disease in stem cells. © 2022 Wiley Periodicals LLC. Basic Protocol 1: Application and optimization of CRISPR-based genome editing in human pluripotent stem cells Basic Protocol 2: Genetic modification of human pluripotent stem cells using a double-oligonucleotide CRISPR/Cas9 recombination system.

Assessing Cell Competition in Human Pluripotent Stem Cell (hPSC) Cultures.

Cell-cell interactions are required for development and homeostasis in multicellular organisms from insects to mammals. A critical process governed by these interactions is cell competition, which functions throughout development to control tissue composition by eliminating cells that possess a lower fitness status than their neighbors. Human pluripotent stem cells (hPSCs) are a key biological tool in modeling human development and offer further potential as a source of clinically relevant cell populations for regenerative medicine applications. Recently, cell competition has been demonstrated in hPSC cultures and during induced pluripotent stem cell reprogramming. In turn, these findings suggest that hPSCs can be used as a tool to study and model cell-cell interactions during different stages of development and disease. Here, we provide a panel of protocols optimized for hPSCs to investigate the potential role that cell competition may have in determining the fate and composition of cell populations during culture. The protocols entail assessment of the competitive phenotype and the mode through which cell competition may lead to elimination of less-fit cells from mosaic cultures with fitter counterparts. © 2022 The Authors. Current Protocols published by Wiley Periodicals LLC. Basic Protocol 1: Electroporation of hPSCs to establish a fluorescent reference cell line Support Protocol 1: Single-cell dissociation of hPSCs Support Protocol 2: Single-cell cloning of fluorescently labeled hPSCs Basic Protocol 2: Separate culture and co-culture proliferation assays Basic Protocol 3: Assessing levels of apoptosis in hPSC cultures using flow cytometry Basic Protocol 4: Transwell assay Support Protocol 3: Immunohistochemistry and image quantification of cleaved caspase-3 Basic Protocol 5: Cell confrontation assay Basic Protocol 6: Cell compression assay Basic Protocol 7: Time-lapse imaging to assess mechanical extrusion.

Human Pluripotent Stem Cell-Derived Alveolar Epithelial Cells as a Tool to Assess Cytotoxicity of Particulate Matter and Cigarette Smoke Extract.

Human pluripotent stem cells (hPSCs) can give rise to a vast array of differentiated derivatives, which have gained great attention in the field of in vitro toxicity evaluation. We have previously demonstrated that hPSC-derived alveolar epithelial cells (AECs) are phenotypically and functionally similar to primary AECs and could be more biologically relevant alternatives for assessing the potential toxic materials including in fine dust and cigarette smoking. Therefore, in this study, we employed hPSC-AECs to evaluate their responses to exposure of various concentrations of diesel particulate matter (dPM), cigarette smoke extract (CSE) and nicotine for 48 hrs in terms of cell death, inflammation, and oxidative stress. We found that all of these toxic materials significantly upregulated the transcription of pro-inflammatory cytokines such as IL-1α, IL-ß, IL-6, and TNF-α. Furthermore, the exposure of dPM (100 µg/mL) strongly induced upregulation of genes related with cell death, inflammation, and oxidative stress compared with other concentrations of CSE and nicotine. These results suggest that hPSC-AECs could be a robust in vitro platform to evaluate pulmotoxicity of various air pollutants and harmful chemicals.

Characterizing the Genetic Stability of Human Naïve and Primed Pluripotent Stem Cells.

The presence of genetic changes in human pluripotent stem cells (hPSCs) can affect their behavior and impact on the utility of hPSC-based applications in research and clinic. The spectrum of spontaneously arising genetic abnormalities in hPSCs is wide and ranges from numerical and structural chromosomal anomalies down to point mutations. The detection of genetic changes in hPSCs is confounded by the fact that no single method detects all types of abnormalities with the same accuracy and sensitivity, therefore necessitating the use of a combination of different methods. Here, we provide detailed protocols for two methods commonly utilized for the detection of genetic changes in naïve and primed hPSCs: karyotyping by G-banding and fluorescent in situ hybridization (FISH).

Differentiation of Human Pluripotent Stem Cells Into Specific Neural Lineages.

Human pluripotent stem cells (hPSCs) are sources of several somatic cell types for human developmental studies, in vitro disease modeling, and cell transplantation therapy. Improving strategies of derivation of high-purity specific neural and glial lineages from hPSCs is critical for application to the study and therapy of the nervous system. Here, we will focus on the principles behind establishment of neuron and glia differentiation methods according to developmental studies. We will also highlight the limitations and challenges associated with the differentiation of several "difficult" neural lineages and delay in neuronal maturation and functional integration. To overcome these challenges, we will introduce strategies and novel technologies aimed at improving the differentiation of various neural lineages to expand the application potential of hPSCs to the study of the nervous system.

Three-dimensional culture models to study glioblastoma - current trends and future perspectives.

Glioblastoma (GBM) is the most prevalent form of primary malignant brain tumor in adults and remains almost invariably lethal owing to its aggressive and invasive nature. There have only been marginal improvements in its bleak survival rate of 12-15 months over the last four decades. The lack of preclinical models that efficiently recapitulate tumor biology and the tumor microenvironment is also in part responsible for the slow phase of translational GBM research. Emerging three-dimensional (3D) organoids and cell culture systems offer new and innovative possibilities for GBM modelling. These 3D models find their application to engineer the disease, screen drugs, establishing live biobank, and explore personalized therapy. Furthermore, these models can also be genetically modified by using the clustered regularly interspaced short palindromic repeats (CRISPR)/Cas9 technology, which would allow one to study the specific role of key genes associated with gliomagenesis. Establishment of a coculture system with GBM cells to understand its invasive behavior is yet another major application of this model. Despite these merits, the organoid models also have certain limitations, including the absence of immune responses and vascular systems. In recent years, major progress has been made in the development and refinement of 3D models of GBM. In this review, we intend to highlight these recent advances and the potential future implications of this rapidly evolving field, which should facilitate a better understanding of GBM biology.

In Vitro Generation of Posterior Motor Neurons from Human Pluripotent Stem Cells.

The ability to generate spinal cord motor neurons from human pluripotent stem cells (hPSCs) is of great use for modelling motor neuron-based diseases and cell-replacement therapies. A key step in the design of hPSC differentiation strategies aiming to produce motor neurons involves induction of the appropriate anteroposterior (A-P) axial identity, an important factor influencing motor neuron subtype specification, functionality, and disease vulnerability. Most current protocols for induction of motor neurons from hPSCs produce predominantly cells of a mixed hindbrain/cervical axial identity marked by expression of Hox paralogous group (PG) members 1-5, but are inefficient in generating high numbers of more posterior thoracic/lumbosacral Hox PG(8-13)+ spinal cord motor neurons. Here, we describe a protocol for efficient generation of thoracic spinal cord cells and motor neurons from hPSCs. This step-wise protocol relies on the initial generation of a neuromesodermal-potent axial progenitor population, which is differentiated first to produce posterior ventral spinal cord progenitors and subsequently to produce posterior motor neurons exhibiting a predominantly thoracic axial identity. © 2021 The Authors. Current Protocols published by Wiley Periodicals LLC. Basic Protocol 1: Differentiation of neuromesodermal progenitors Basic Protocol 2: Posterior ventral spinal cord progenitor differentiation Basic Protocol 3: Posterior motor neuron differentiation.

Differentiation and Application of Human Pluripotent Stem Cells Derived Cardiovascular Cells for Treatment of Heart Diseases: Promises and Challenges.

Human pluripotent stem cells (hPSCs) are derived from human embryos (human embryonic stem cells) or reprogrammed from human somatic cells (human induced pluripotent stem cells). They can differentiate into cardiovascular cells, which have great potential as exogenous cell resources for restoring cardiac structure and function in patients with heart disease or heart failure. A variety of protocols have been developed to generate and expand cardiovascular cells derived from hPSCs in vitro. Precisely and spatiotemporally activating or inhibiting various pathways in hPSCs is required to obtain cardiovascular lineages with high differentiation efficiency. In this concise review, we summarize the protocols of differentiating hPSCs into cardiovascular cells, highlight their therapeutic application for treatment of cardiac diseases in large animal models, and discuss the challenges and limitations in the use of cardiac cells generated from hPSCs for a better clinical application of hPSC-based cardiac cell therapy.

The Developmental & Molecular Requirements for Ensuring that Human Pluripotent Stem Cell-Derived Hair Follicle Bulge Stem Cells Have Acquired Competence for Hair Follicle Generation Following Transplantation.

When using human induced pluripotent stem cells (hiPSCs) to achieve hair follicle (HF) replacement, we found it best to emulate the earliest fundamental developmental processes of gastrulation, ectodermal lineage commitment, and dermogenesis. Viewing hiPSCs as a model of the epiblast, we exploited insights from mapping the dynamic up- and down-regulation of the developmental molecules that determine HF lineage in order to ascertain the precise differentiation stage and molecular requirements for grafting HF-generating progenitors. To yield an integrin-dependent lineage like the HF in vivo, we show that hiPSC derivatives should co-express, just prior to transplantation, the following combination of markers: integrins α6 and ß1 and the glycoprotein CD200 on their surface; and, intracellularly, the epithelial marker keratin 18 and the hair follicle bulge stem cell (HFBSC)-defining molecules transcription factor P63 and the keratins 15 and 19. If the degree of trichogenic responsiveness indicated by the presence of these molecules is not achieved (they peak on Days 11-18 of the protocol), HF generation is not possible. Conversely, if differentiation of the cells is allowed to proceed beyond the transient intermediate progenitor state represented by the HFBSC, and instead cascades to their becoming keratin 14+ keratin 5+ CD200- keratinocytes (Day 25), HF generation is equally impossible. We make the developmental case for transplanting at Day 16-18 of differentiation-the point at which the hiPSCs have lost pluripotency, have attained optimal expression of HFBSC markers, have not yet experienced downregulation of key integrins and surface glycoproteins, have not yet started expressing keratinocyte-associated molecules, and have sufficient proliferative capacity to allow a well-populated graft. This panel of markers may be used for isolating (by cytometry) HF-generating derivatives away from cell types unsuited for this therapy as well as for identifying trichogenic drugs.

Towards physiologically relevant human pluripotent stem cell (hPSC) models of Parkinson's disease.

The derivation of human embryonic stem cells followed by the discovery of induced pluripotent stem cells and leaps in genome editing approaches have continuously fueled enthusiasm for the development of new models of neurodegenerative diseases such as Parkinson's disease (PD). PD is characterized by the relative selective loss of dopaminergic neurons (DNs) in specific areas of substantia nigra pars compacta (SNpc). While degeneration in late stages can be widespread, there is stereotypic early degeneration of these uniquely vulnerable neurons. Various causes of selective vulnerability have been investigated but much remains unclear. Most studies have sought to identify cell autonomous properties of the most vulnerable neurons. However, recent findings from genetic studies and model systems have added to our understanding of non-cell autonomous contributions including regional-specific neuro-immune interactions with astrocytes, resident or damage-activated microglia, neuro-glia cell metabolic interactions, involvement of endothelial cells, and damage to the vascular system. All of these contribute to specific vulnerability and, along with aging and environmental factors, might be integrated in a complex stressor-threshold model of neurodegeneration. In this forward-looking review, we synthesize recent advances in the field of PD modeling using human pluripotent stem cells, with an emphasis on organoid and complex co-culture models of the nigrostriatal niche, with emerging CRISPR applications to edit or perturb expression of causal PD genes and associated risk factors, such as GBA, to understand the impact of these genes on relevant phenotypes.