Stress-free cell aggregation by using the CEPT cocktail enhances embryoid body and organoid fitness.

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.

Giant myxoid retroperitoneal liposarcoma in a 41-year-old patient: A case report.

INTRODUCTION: Retroperitoneal Liposarcomas (RL) are a rare form of malignant tumors, they encompass just 5 % ot these and mainly present in female adults between 50 and 60 years of age. They can be divided into 4 morphological subtypes: 1) well-differentiated, 2) undifferentiated, 3) myxoid, and 4) pleomorphic. CASE PRESENTATION: 41-year-old male who present to the outpatient consult of oncological surgery after being diagnosed via imaging with a giant retroperitoneal liposarcoma. The patient reported unsuccessful weight loss attempts and subsequently noticed an increase in abdominal size as well as the development of ulcers in the lower limbs. He underwent radical resection, which included a right nephrectomy, as the primary treatment. DISCUSSION: Giant retroperitoneal liposarcomas are defined as those 30 cm in diameter or more or 20 kg or more in weight are considered "giants" and are extremely rare. The only treatment involves resection of the tumoration and those adjacent involved organs, and sometimes, uninvolved ones. Due to the high risk of recurrence, adjuvant therapy may be provided as well as imaging follow up. CONCLUSION: RL are a complicated entity to manage. They tend to present high rates of recurrence due to the difficulty of performing a full resection without positive margins. These patients should be thoroughly studied before surgery and appropriate follow-up should be provided.

A defined roadmap of radial glia and astrocyte differentiation from human pluripotent stem cells.

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.

SEQUIN is an R/Shiny framework for rapid and reproducible analysis of RNA-seq data.

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.

Scalable generation of sensory neurons from human pluripotent stem cells.

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, NAV1.7, NAV1.8, 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.

Bioprinted 3D outer retina barrier uncovers RPE-dependent choroidal phenotype in advanced macular degeneration.

Age-related macular degeneration (AMD), a leading cause of blindness, initiates in the outer-blood-retina-barrier (oBRB) formed by the retinal pigment epithelium (RPE), Bruch's membrane, and choriocapillaris. The mechanisms of AMD initiation and progression remain poorly understood owing to the lack of physiologically relevant human oBRB models. To this end, we engineered a native-like three-dimensional (3D) oBRB tissue (3D-oBRB) by bioprinting endothelial cells, pericytes, and fibroblasts on the basal side of a biodegradable scaffold and establishing an RPE monolayer on top. In this 3D-oBRB model, a fully-polarized RPE monolayer provides barrier resistance, induces choriocapillaris fenestration, and supports the formation of Bruch's-membrane-like structure by inducing changes in gene expression in cells of the choroid. Complement activation in the 3D-oBRB triggers dry AMD phenotypes (including subRPE lipid-rich deposits called drusen and choriocapillaris degeneration), and HIF-α stabilization or STAT3 overactivation induce choriocapillaris neovascularization and type-I wet AMD phenotype. The 3D-oBRB provides a physiologically relevant model to studying RPE-choriocapillaris interactions under healthy and diseased conditions.

Efficient and safe single-cell cloning of human pluripotent stem cells using the CEPT cocktail.

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.

Correction to: Human Pluripotent Stem Cells for High-Throughput Drug Screening and Characterization of Small Molecules.

This chapter was inadvertently published with an error, the ninth point under 'Materials' section must read.

Human Pluripotent Stem Cells for High-Throughput Drug Screening and Characterization of Small Molecules.

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.

Robotic high-throughput biomanufacturing and functional differentiation of human pluripotent stem cells.

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.

Foreign modeling agent reaction; managed with two staged surgical reconstruction. Case report and review of literature.

INTRODUCTION: In Mexico, body modeling with injectable biomaterials such as liquid silicone is a common practice in non-certified clinics by non-medical personnel; These materials produces a series of complications described as Foreign modeling agent reaction (FMAR) with variable spectrum of severity. CASE PRESENTATION: 38-year-old female with history of biomaterial injection in a non-certified cosmetic clinic 10 years prior to evaluation. Presents with intermittent symptoms characterized by fever, erythema, induration and pain in the gluteal region. An exhaustive debridement and resection with primary closure was performed. Thereafter, reconstruction was done using a combined technique with gluteal implants and autologous fat graft, evolving without complications. DISCUSSION: The use of biomaterials has been widely documented throughout history; liquid silicone being one of the protagonists. Used for aesthetic purposes and modeling areas such as buttocks and breasts. They have been associated with an assortment of early or late onset complications, sometimes resulting in fatal outcomes. Various treatment modalities have been described depending on the severity of presentation, from conservative to surgical management. CONCLUSION: There is a shortage of treatment guidelines regarding FMAR due to its wide variety of presentation, treatment must be individualized to obtain adequate results. Although conservative treatment has shown good results, the anatomical alterations usually condition dissatisfaction that should be addressed with reconstructive techniques [10].

A versatile polypharmacology platform promotes cytoprotection and viability of human pluripotent and differentiated cells.

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.

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

Efficient translation of human induced pluripotent stem cells (hiPSCs) depends on implementing scalable cell manufacturing strategies that ensure optimal self-renewal and functional differentiation. Currently, manual culture of hiPSCs is highly variable and labor-intensive posing significant 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 streamlined 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 to generate primary embryonic germ layers and more mature phenotypes such as neurons, cardiomyocytes, and hepatocytes. To validate our approach, we carefully compared robotic and manual cell culture and performed molecular and functional cell characterizations (e.g. bulk culture and single-cell transcriptomics, mass cytometry, metabolism, electrophysiology, Zika virus experiments) in order to benchmark industrial-scale cell culture operations towards building an integrated platform for efficient cell manufacturing for disease modeling, drug screening, and cell therapy. Combining stem cell-based models and non-stop robotic cell culture may become a powerful strategy to increase scientific rigor and productivity, which are particularly important during public health emergencies (e.g. opioid crisis, COVID-19 pandemic).

Chemically Defined Neural Conversion of Human Pluripotent Stem Cells.

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.

Disabled cell density sensing leads to dysregulated cholesterol synthesis in glioblastoma.

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.

Role of apoptosis signal-regulating kinase 1 (ASK1) as an activator of the GAPDH-Siah1 stress-signaling cascade.

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.

The diverse functions of GAPDH: views from different subcellular compartments.

Multiple roles for glyceraldehyde-3-phosphate dehydrogenase (GAPDH) have been recently appreciated. In addition to the cytoplasm where the majority of GAPDH is located under the basal condition, GAPDH is also found in the particulate fractions, such as the nucleus, the mitochondria, and the small vesicular fractions. When cells are exposed to various stressors, dynamic subcellular re-distribution of GAPDH occurs. Here we review these multifunctional properties of GAPDH, especially linking them to its oligomerization, posttranslational modification, and subcellular localization. This includes mechanistic descriptions of how S-nitrosylation of GAPDH under oxidative stress may lead to cell death/dysfunction via nuclear translocation of GAPDH, which is counteracted by a cytosolic GOSPEL. GAPDH is also involved in various diseases, especially neurodegenerative disorders and cancers. Therapeutic strategies to these conditions based on molecular understanding of GAPDH are discussed.

Treating metastatic solid tumors with bortezomib and a tumor necrosis factor-related apoptosis-inducing ligand receptor agonist antibody.

BACKGROUND: Resistance of tumors to cell death signals poses a complex clinical problem. We explored the therapeutic potential and in vivo toxicity of a combination of bortezomib, a proteasome inhibitor, and MD5-1, a tumor necrosis factor-related apoptosis-inducing ligand (TRAIL) receptor (DR5) agonist monoclonal antibody, in mouse carcinomas. METHODS; Mice bearing Renca-FLAG (renal) or 4T1 (mammary) tumors were treated with bortezomib and/or MD5-1 and examined for lung metastases (Renca-FLAG: n = 93; 4T1: n = 40) or monitored for survival (Renca-FLAG: n = 143). Toxicity was assessed by histopathology and hematology. Viability and apoptotic signaling in Renca-FLAG and 4T1 cells treated with bortezomib alone or in combination with TRAIL were analyzed using 3-[4,5-dimethyiazol-2-yl-5]-[3-carboxymethyloxyphenyl]-2-[4-sulfophenyl]-2H tetrazolium assay and by measuring mitochondrial membrane depolarization and caspase-8 and caspase-3 activation. All statistical tests were two-sided. RESULTS: Bortezomib (20 nM) sensitized Renca-FLAG and 4T1 cells to TRAIL-mediated apoptosis (mean percent decrease in numbers of viable cells, bortezomib + TRAIL vs TRAIL: Renca-FLAG, 95% vs 34%, difference = 61%, 95% confidence interval [CI] = 52% to 69%, P < .001; 4T1, 85% vs 20%, difference = 65%, 95% CI = 62% to 69%, P < .001). Sensitization involved activation of caspase-8 and caspase-3 but not mitochondrial membrane depolarization, suggesting an amplified signaling of the extrinsic cell death pathway. Treatment with bortezomib and MD5-1 reduced lung metastases in mice carrying Renca and 4T1 tumors (mean number of metastases, bortezomib + MD5-1 vs MD5-1: Renca-FLAG, 1 vs 8, difference = 7, 95% CI = 5 to 9, P < .001; 4T1, 1 vs 12, difference = 11, 95% CI = 9 to 12, P < .001) and increased median survival of mice bearing Renca-FLAG tumors (bortezomib + MD5-1 vs bortezomib + control isotype antibody: 22 of 30 [73%] were still alive at day 180 vs median survival of 42 days [95% CI = 41 to 44 days, P < .001]) in the absence of obvious toxicity. CONCLUSION: Bortezomib combined with DR5 agonist monoclonal antibody may be a useful treatment for metastatic solid tumors.