Cytoplasmic Shotgun Proteomic Points to Key Proteins and Pathways in Temozolomide-Resistant Glioblastoma Multiforme.

Temozolomide (TMZ) is the first line of chemotherapy to treat primary brain tumors of the type glioblastoma multiforme (GBM). TMZ resistance (TMZR) is one of the main barriers to successful treatment and is a principal factor in relapse, resulting in a poor median survival of 15 months. The present paper focuses on proteomic analyses of cytosolic fractions from TMZ-resistant (TMZR) LN-18 cells. The experimental workflow includes an easy, cost-effective, and reproducible method to isolate subcellular fraction of cytosolic (CYTO) proteins, mitochondria, and plasma membrane proteins for proteomic studies. For this study, enriched cytoplasmic fractions were analyzed in replicates by nanoflow liquid chromatography tandem high-resolution mass spectrometry (nLC-MS/MS), and proteins identified were quantified using a label-free approach (LFQ). Statistical analysis of control (CTRL) and temozolomide-resistant (TMZR) proteomes revealed proteins that appear to be differentially controlled in the cytoplasm. The functions of these proteins are discussed as well as their roles in other cancers and TMZ resistance in GBM. Key proteins are also described through biological processes related to gene ontology (GO), molecular functions, and cellular components. For protein-protein interactions (PPI), network and pathway involvement analyses have been performed, highlighting the roles of key proteins in the TMZ resistance phenotypes. This study provides a detailed insight into methods of subcellular fractionation for proteomic analysis of TMZ-resistant GBM cells and the potential to apply this approach to future large-scale studies. Several key proteins, protein-protein interactions (PPI), and pathways have been identified, underlying the TMZ resistance phenotype and highlighting the proteins' biological functions.

The Effect of Choline and Resistance Training on Strength and Lean Mass in Older Adults.

Choline plays many important roles, including the synthesis of acetylcholine, and may affect muscle responses to exercise. We previously observed correlations between low choline intake and reduced gains in strength and lean mass following a 12-week resistance exercise training (RET) program for older adults. To further explore these findings, we conducted a randomized controlled trial. Three groups of 50-to-69-year-old healthy adults underwent a 12-week RET program (3x/week, 3 sets, 8-12 reps, 70% of maximum strength (1RM)) and submitted >48 diet logs (>4x/week for 12 weeks). Participants' diets were supplemented with 0.7 mg/kg lean/d (low, n = 13), 2.8 mg/kg lean/d (med, n = 11), or 7.5 mg/kg lean/d (high, n = 13) of choline from egg yolk and protein powder. The ANCOVA tests showed that low choline intake, compared with med or high choline intakes, resulted in significantly diminished gains in composite strength (leg press + chest press 1RM; low, 19.4 ± 8.2%; med, 46.8 ± 8.9%; high, 47.4 ± 8.1%; p = 0.034) and thigh-muscle quality (leg press 1RM/thigh lean mass; low, 12.3 ± 9.6%; med/high, 46.4 ± 7.0%; p = 0.010) after controlling for lean mass, protein, betaine, and vitamin B12. These data suggest that low choline intake may negatively affect strength gains with RET in older adults.

An overview of glioblastoma multiforme and temozolomide resistance: can LC-MS-based proteomics reveal the fundamental mechanism of temozolomide resistance?

Glioblastoma multiforme (GBM) is a primary type of lethal brain tumor. Over the last two decades, temozolomide (TMZ) has remained the primary chemotherapy for GBM. However, TMZ resistance in GBM constitutes an underlying factor contributing to high rates of mortality. Despite intense efforts to understand the mechanisms of therapeutic resistance, there is currently a poor understanding of the molecular processes of drug resistance. For TMZ, several mechanisms linked to therapeutic resistance have been proposed. In the past decade, significant progress in the field of mass spectrometry-based proteomics has been made. This review article discusses the molecular drivers of GBM, within the context of TMZ resistance with a particular emphasis on the potential benefits and insights of using global proteomic techniques.

Breast cancer cell secretome analysis to decipher miRNA regulating the tumor microenvironment and discover potential biomarkers.

MicroRNA (miRNA/miR) 526 b- and miR655-overexpressed tumor cell-free secretions regulate the breast cancer tumor microenvironment (TME) by promoting tumor-associated angiogenesis, oxidative stress, and hypoxic responses. Additionally, premature miRNA (pri-miR526b and pri-miR655) are established breast cancer blood biomarkers. However, the mechanisms of how these miRNAs regulate the TME has yet to be investigated. Mass spectrometry analysis of miRNA-overexpressed cell lines MCF7-miR526b, MCF7-miR655, and miRNA-low MCF7-Mock cell-free secretomes identified 34 differentially expressed proteins coded by eight genes. In both miRNA-high cell secretomes, four markers are upregulated: YWHAB, SFN, TXNDC12, and MYL6B, and four are downregulated: PEA15, PRDX4, PSMB6, and FN1. All upregulated marker transcripts are significantly high in both total cellular RNA pool and cell-free secretions of miRNA-high cell lines, validated with quantitative RT-PCR. Bioinformatics tools were used to investigate these markers' roles in breast cancer. These markers' top gene ontology functions are related to apoptosis, oxidative stress, membrane transport, and motility supporting oncogenic miR526b- and miR655-induced functions. Gene transcription factor analysis tools were used to show how these miRNAs regulate the expression of each secretory marker. Data extracted from the Human Protein Atlas showed that YWHAB, SFN, and TXNDC12 expression could distinguish early and late-stage breast cancer in various breast cancer subtypes and are associated with poor patient survival. Additionally, immunohistochemistry analysis showed the expression of each marker in breast tumors. A stronger correlation between miRNA clusters and upregulated secretory markers gene expression was found in the luminal A tumor subtype. YWHAB, SFN, and MYL6B are upregulated in breast cancer patient's blood, showing biomarker potential. Of these identified novel miRNA secretory markers, SFN and YWHAB successfully passed all validations and are the best candidates to further investigate their roles in miRNA associated TME regulation. Also, these markers show the potential to serve as blood-based breast cancer biomarkers, especially for luminal-A subtypes.

Integrated Network Discovery Using Multi-Proteomic Data.

A fundamental goal of systems biology is to seek a better understanding of the cell's molecular mechanisms. Experimentalists most frequently rely upon reductionist methods to isolate and analyze discrete signaling compartments, including subcellular domains, organelles, and protein-protein interactions. Among the systems-biology community, there is a growing need to integrate multiple datasets to resolve complex cellular networks. In this chapter, we share our procedures for the discovery of integrated signaling networks, across multi-proteomic data. Demonstrating these procedures, we provide an integrated analysis of the cellular proteome and extracellular (secretome) of human glioma LN229.

Ephrin-A3 is required for tonotopic map precision and auditory functions in the mouse auditory brainstem.

Tonotopy is a prominent feature of the vertebrate auditory system and forms the basis for sound discrimination, but the molecular mechanism that underlies its formation remains largely elusive. Ephrin/Eph signaling is known to play important roles in axon guidance during topographic mapping in other sensory systems, so we investigated its possible role in the establishment of tonotopy in the mouse cochlear nucleus. We found that ephrin-A3 molecules are differentially expressed along the tonotopic axis in the cochlear nucleus during innervation. Ephrin-A3 forward signaling is sufficient to repel auditory nerve fibers in a developmental stage-dependent manner. In mice lacking ephrin-A3, the tonotopic map is degraded and isofrequency bands of neuronal activation upon pure tone exposure become imprecise in the anteroventral cochlear nucleus. Ephrin-A3 mutant mice also exhibit a delayed second wave in auditory brainstem responses upon sound stimuli and impaired detection of sound frequency changes. Our findings establish an essential role for ephrin-A3 in forming precise tonotopy in the auditory brainstem to ensure accurate sound discrimination.

Examination of sulfonamide-based inhibitors of MMP3 using the conditioned media of invasive glioma cells.

Glioblastoma multiforme (GBM) is the deadliest and the most common primary malignant brain tumour. The median survival for patients with GBM is around one year due to the nature of glioma cells to diffusely invade that make the complete surgical resection of tumours difficult. Based upon the connexin43 (Cx43) model of glioma migration we have developed a computational framework to evaluate MMP inhibition in materials relevant to GBM. Using the ilomastat Leu-Trp backbone, we have synthesised novel sulphonamides and monitored the performance of these compounds in conditioned media expressing MMP3. From the results discussed herein we demonstrate the performance of sulfonamide based MMPIs included AP-3, AP-6, and AP-7.

Focal Suppression of Epileptiform Activity in the Hippocampus by a High-frequency Magnetic Field.

Electric current has been used for epilepsy treatment by targeting specific neural circuitries. Despite its success, direct contact between the electrode and tissue could cause side effects including pain, inflammation, and adverse biological reactions. Magnetic stimulation overcomes these limitations by offering advantages over biocompatibility and operational feasibility. However, the underlying neurological mechanisms of its action are largely unknown. In this work, a magnetic generating system was assembled that included a miniature coil. The coil was positioned above the CA3 area of mouse hippocampal slices. Epileptiform activity (EFA) was induced with low Mg2+/high K+ perfusion or with 100 µM 4-aminopyridine (4-AP). The miniature coil generated a sizable electric field that suppressed the local EFA in the hippocampus in the low-Mg2+/high-K+ model. The inhibition effect was dependent on the frequency and duration of the magnetic stimulus, with high frequency being more effective in suppressing EFA. EFA suppression by the magnetic field was also observed in the 4-AP model, in a frequency and duration - dependent manner. The study provides a platform for further investigation of cellular and molecular mechanisms underlying epilepsy treatment with time varying magnetic fields.

Cx43-Associated Secretome and Interactome Reveal Synergistic Mechanisms for Glioma Migration and MMP3 Activation.

Extracellular matrix (ECM) remodeling, degradation and glioma cell motility are critical aspects of glioblastoma multiforme (GBM). Despite being a rich source of potential biomarkers and targets for therapeutic advance, the dynamic changes occurring within the extracellular environment that are specific to GBM motility have yet to be fully resolved. The gap junction protein connexin43 (Cx43) increases glioma migration and invasion in a variety of in vitro and in vivo models. In this study, the upregulation of Cx43 in C6 glioma cells induced morphological changes and the secretion of proteins associated with cell motility. Demonstrating the selective engagement of ECM remodeling networks, secretome analysis revealed the near-binary increase of osteopontin and matrix metalloproteinase-3 (MMP3), with gelatinase and NFF-3 assays confirming the proteolytic activities. Informatic analysis of interactome and secretome downstream of Cx43 identifies networks of glioma motility that appear to be synergistically engaged. The data presented here implicate ECM remodeling and matrikine signals downstream of Cx43/MMP3/osteopontin and ARK1B10 inhibition as possible avenues to inhibit GBM.

Pannexin 2 Localizes at ER-Mitochondria Contact Sites.

Endomembrane specialization allows functional compartmentalization but imposes physical constraints to information flow within the cell. However, the evolution of an endomembrane system was associated with the emergence of contact sites facilitating communication between membrane-bound organelles. Contact sites between the endoplasmic reticulum (ER) and mitochondria are highly conserved in terms of their morphological features but show surprising molecular diversity within and across eukaryote species. ER-mitochondria contact sites are thought to regulate key processes in oncogenesis but their molecular composition remains poorly characterized in mammalian cells. In this study, we investigate the localization of pannexin 2 (Panx2), a membrane channel protein showing tumor-suppressing properties in cancer cells. Using a combination of subcellular fractionation, particle tracking in live-cell, and immunogold electron microscopy, we show that Panx2 localizes at ER-mitochondria contact sites in mammalian cells and sensitizes cells to apoptotic stimuli.

The effects of hindlimb unloading versus dietary cholesterol and resistance training on rat skeletal muscle responses.

BACKGROUND: The loss of muscle mass and concomitantly strength, poses a serious risk to the elderly and to astronauts. Dietary cholesterol (CL), in conjunction with resistance training (RT), has been strongly associated with improvements in lean mass. The purpose of this study was to examine the effects of two opposing environments on rat skeletal muscle: (1) hindlimb unloading and (2) CL and RT. METHODS: In protocol 1, 13 male Sprague-Dawley rats were unloaded for 28 days (HU; n = 6) or served as cage controls (CC; n = 7). In protocol 2, 42 rats were assigned to 1 of 6 groups: CC (n = 7), CC + CL (n = 4), RT controls (RTC; n = 7), RTC + CL (n = 8), RT (n = 8) and RT + CL (n = 8). RT/RTC consisted of squat-like exercise. RT had weights added progressively from 80 to 410 g over 5 weeks. CL was supplemented in the chow with either 180 ppm (controls) or 1800 ppm (CL). Lower limb muscles were harvested at the end of both protocols and analyzed by Western Blotting for sterol regulatory element-binding protein-2 (SREBP-2) and low-density lipoprotein-receptor (LDL-R) and protein synthesis. RESULTS: Gastrocnemius and plantaris masses and their body mass ratios were significantly lower in the HU rats than control rats. The RT rats gained significantly less body and lean mass than the RTC groups, but the plantar flexor muscles did not show any significant differences among groups. Moreover, RT groups had significantly higher plantaris mixed muscle fractional synthesis rate (FSR) than the RTC and CC animals, with the CL groups showing greater FSR than control rats. No significant differences among groups in SREBP-2 or LDL-R were observed in either protocol. CONCLUSIONS: These studies provide evidence for a relationship between skeletal muscle and cholesterol metabolism, but the exact nature of that association remains unclear.

Establishing the waist as the better location for attaching a single accelerometer to estimate center of pressure trajectories.

BACKGROUND: In this study, we seek to replace conventional force platforms with a single accelerometer for measuring Center of Pressure trajectories, in order to achieve portability and convenience without sacrificing accuracy. METHODS: We measure the actual Anterior/Posterior and Medial/Lateral Center of Pressure trajectories of ten healthy young subjects using a force platform, and compare them with estimated measurements derived from accelerometer signals collected from three body locations (upper trunk, waist, and lower thigh) using three machine learning algorithms (Neural Network, Genetic Algorithm, and Adaptive Network-based Fuzzy Inference System). Error ratios and correlation coefficients corresponding to body locations were compared via one-way repeated-measures ANOVA. The ratios and coefficients corresponding to the three algorithms were also compared using the same approach. FINDINGS: Estimated Anterior/Posterior trajectories indicated that measurements collected from the waist provided the lowest margins of error (8.1-8.4% v. 12.1-13.4%, P ≤ .001) and the highest correlation (.95 v. .82-.86, P ≤ .032). Estimated Medial/Lateral trajectories indicated that measurements collected from both the waist and thigh, as compared to the upper trunk, provided lower margins of error (7.0-7.3% v. 8.5-10.8%). In general, the waist is the better accelerometer attachment location. INTERPRETATION: The results of our study corroborate our deduction that the high correlation between Center of Pressure and body's Center of Mass provides the rationale to place the single accelerometer close to the waist for Center of Pressure estimations. This study also supports the feasibility of using one single accelerometer programmed with algorithms for similar clinical applications.

Hormone replacement therapy decreases the risk of tinnitus in menopausal women: a nationwide study.

The incidence and risk of tinnitus associated with hormone replacement therapy (HRT) in menopausal women have not yet been fully examined. We examined the medical records of menopausal women aged between 45 and 79 years from Taiwan's National Health Insurance Research Database of records between 1 January 2000 and 31 December 2010 to establish matched cohorts (13,920 HRT users and 41,760 nonusers). The incidence of tinnitus in HRT users and nonusers were matched 1:3 based on propensity-score matching over this ten year period. The Cox regression hazard model was used to identify risk factors of tinnitus, and results indicate that a significantly lower percentage of HRT users (P = 0.017) developed tinnitus in comparison with nonusers (0.43%, 60/13, 920 vs. 0.59%, 246/41, 760). Using Cox regressions analysis after adjustments for age and other variables (adjusted hazard ratio: 0.505 (95% confidence interval, 0.342-0.756)), we were also able to show that HRT users appeared to have a reduced risk of developing tinnitus in comparison with nonusers. Based on our observation of the lower incidence of tinnitus among HRT users in this cohort, we speculate that HRT may have provided potential benefits on the management and prevention of tinnitus among menopausal women.

Passive Stretch Induces Structural and Functional Maturation of Engineered Heart Muscle as Predicted by Computational Modeling.

The ability to differentiate human pluripotent stem cells (hPSCs) into cardiomyocytes (CMs) makes them an attractive source for repairing injured myocardium, disease modeling, and drug testing. Although current differentiation protocols yield hPSC-CMs to >90% efficiency, hPSC-CMs exhibit immature characteristics. With the goal of overcoming this limitation, we tested the effects of varying passive stretch on engineered heart muscle (EHM) structural and functional maturation, guided by computational modeling. Human embryonic stem cells (hESCs, H7 line) or human induced pluripotent stem cells (IMR-90 line) were differentiated to hPSC-derived cardiomyocytes (hPSC-CMs) in vitro using a small molecule based protocol. hPSC-CMs were characterized by troponin+ flow cytometry as well as electrophysiological measurements. Afterwards, 1.2 × 106 hPSC-CMs were mixed with 0.4 × 106 human fibroblasts (IMR-90 line) (3:1 ratio) and type-I collagen. The blend was cast into custom-made 12-mm long polydimethylsiloxane reservoirs to vary nominal passive stretch of EHMs to 5, 7, or 9 mm. EHM characteristics were monitored for up to 50 days, with EHMs having a passive stretch of 7 mm giving the most consistent formation. Based on our initial macroscopic observations of EHM formation, we created a computational model that predicts the stress distribution throughout EHMs, which is a function of cellular composition, cellular ratio, and geometry. Based on this predictive modeling, we show cell alignment by immunohistochemistry and coordinated calcium waves by calcium imaging. Furthermore, coordinated calcium waves and mechanical contractions were apparent throughout entire EHMs. The stiffness and active forces of hPSC-derived EHMs are comparable with rat neonatal cardiomyocyte-derived EHMs. Three-dimensional EHMs display increased expression of mature cardiomyocyte genes including sarcomeric protein troponin-T, calcium and potassium ion channels, ß-adrenergic receptors, and t-tubule protein caveolin-3. Passive stretch affects the structural and functional maturation of EHMs. Based on our predictive computational modeling, we show how to optimize cell alignment and calcium dynamics within EHMs. These findings provide a basis for the rational design of EHMs, which enables future scale-up productions for clinical use in cardiovascular tissue engineering. Stem Cells 2018;36:265-277.

Effects of depression and antidepressant medications on hip fracture: A population-based cohort study in Taiwan.

This study was conducted to investigate the effects of depression and antidepressant medications on hip fracture. The database of the Taiwan National Health Insurance with medical records of more than 1,000,000 individuals was searched for patients who had hip fracture with or without depression from 1998 to 2009. Patients with the following conditions were excluded: hip fracture due to cancer or traffic accidents, hip fracture that occurred before the diagnosis of depression, and use of antidepressants before the diagnosis of depression. A matched cohort of 139,110 patients was investigated, including 27,822 (17,309 females; 10,513 males) with depression and 111,288 (69,236 females; 42,052 males) without depression (1:4 randomly matched with age, sex, and index date). Among these patients, 232 (158 females and 74 males) had both hip fracture and depression, and 690 (473 females and 217 males) had hip fracture only. The Cox proportional-hazards regression method was used to determine the effect of depression on hip fracture. The hazard ratio (HR) for each clinical parameter was calculated after adjusting for confounders including sex, age, Charlson comorbidity index, urbanization, osteoporosis, and antidepressants. Results showed that patients with major depressive disorder had a 61% higher incidence of hip fracture than those without depression (HR 1.61, 95% confidence interval [CI] 1.19-2.18, P = 0.002). The risk of hip fracture for patients with less severe depressive disorder (dysthymia or depressive disorder, not otherwise specified) was not statistically higher than that of patients with no depression (HR 1.10, 95% CI = 0.91-1.34, P = 0.327). Among the patients with depression, females had a 49% higher incidence for hip fracture than males (HR 1.49, 95% CI 1.30-1.72, P < 0.001). The incidence of hip fracture also increased with age and Charlson comorbidity index scores. Analyses of both all (139,110) patients and only patients (27,822) with depression revealed that antidepressants had no negative impact on the incidence of hip fracture. In conclusion, major depression was found to be a risk factor for hip fracture and that use of antidepressants had no adverse effect on hip fracture in the Taiwanese population.

Development of a scalable suspension culture for cardiac differentiation from human pluripotent stem cells.

To meet the need of a large quantity of hPSC-derived cardiomyocytes (CM) for pre-clinical and clinical studies, a robust and scalable differentiation system for CM production is essential. With a human pluripotent stem cells (hPSC) aggregate suspension culture system we established previously, we developed a matrix-free, scalable, and GMP-compliant process for directing hPSC differentiation to CM in suspension culture by modulating Wnt pathways with small molecules. By optimizing critical process parameters including: cell aggregate size, small molecule concentrations, induction timing, and agitation rate, we were able to consistently differentiate hPSCs to >90% CM purity with an average yield of 1.5 to 2×10(9) CM/L at scales up to 1L spinner flasks. CM generated from the suspension culture displayed typical genetic, morphological, and electrophysiological cardiac cell characteristics. This suspension culture system allows seamless transition from hPSC expansion to CM differentiation in a continuous suspension culture. It not only provides a cost and labor effective scalable process for large scale CM production, but also provides a bioreactor prototype for automation of cell manufacturing, which will accelerate the advance of hPSC research towards therapeutic applications.

Human Engineered Heart Muscles Engraft and Survive Long Term in a Rodent Myocardial Infarction Model.

RATIONALE: Tissue engineering approaches may improve survival and functional benefits from human embryonic stem cell-derived cardiomyocyte transplantation, thereby potentially preventing dilative remodeling and progression to heart failure. OBJECTIVE: Assessment of transport stability, long-term survival, structural organization, functional benefits, and teratoma risk of engineered heart muscle (EHM) in a chronic myocardial infarction model. METHODS AND RESULTS: We constructed EHMs from human embryonic stem cell-derived cardiomyocytes and released them for transatlantic shipping following predefined quality control criteria. Two days of shipment did not lead to adverse effects on cell viability or contractile performance of EHMs (n=3, P=0.83, P=0.87). One month after ischemia/reperfusion injury, EHMs were implanted onto immunocompromised rat hearts to simulate chronic ischemia. Bioluminescence imaging showed stable engraftment with no significant cell loss between week 2 and 12 (n=6, P=0.67), preserving ≤25% of the transplanted cells. Despite high engraftment rates and attenuated disease progression (change in ejection fraction for EHMs, -6.7±1.4% versus control, -10.9±1.5%; n>12; P=0.05), we observed no difference between EHMs containing viable and nonviable human cardiomyocytes in this chronic xenotransplantation model (n>12; P=0.41). Grafted cardiomyocytes showed enhanced sarcomere alignment and increased connexin 43 expression at 220 days after transplantation. No teratomas or tumors were found in any of the animals (n=14) used for long-term monitoring. CONCLUSIONS: EHM transplantation led to high engraftment rates, long-term survival, and progressive maturation of human cardiomyocytes. However, cell engraftment was not correlated with functional improvements in this chronic myocardial infarction model. Most importantly, the safety of this approach was demonstrated by the lack of tumor or teratoma formation.

The DIONESUS algorithm provides scalable and accurate reconstruction of dynamic phosphoproteomic networks to reveal new drug targets.

Many drug candidates fail in clinical trials due to an incomplete understanding of how small-molecule perturbations affect cell phenotype. Cellular responses can be non-intuitive due to systems-level properties such as redundant pathways caused by co-activation of multiple receptor tyrosine kinases. We therefore created a scalable algorithm, DIONESUS, based on partial least squares regression with variable selection to reconstruct a cellular signaling network in a human carcinoma cell line driven by EGFR overexpression. We perturbed the cells with 26 diverse growth factors and/or small molecules chosen to activate or inhibit specific subsets of receptor tyrosine kinases. We then quantified the abundance of 60 phosphosites at four time points using a modified microwestern array, a high-confidence assay of protein abundance and modification. DIONESUS, after being validated using three in silico networks, was applied to connect perturbations, phosphorylation, and cell phenotype from the high-confidence, microwestern dataset. We identified enhancement of STAT1 activity as a potential strategy to treat EGFR-hyperactive cancers and PTEN as a target of the antioxidant, N-acetylcysteine. Quantification of the relationship between drug dosage and cell viability in a panel of triple-negative breast cancer cell lines validated proposed therapeutic strategies.

The suspension culture of undifferentiated human pluripotent stem cells using spinner flasks.

Suspension cell culture systems with superior scalability, controllability, and monitoring options are an attractive alternative to static adherent culture methods for expansion and production of human pluripotent stem cells (hPSCs), including embryonic stem cells (ESCs) and induced pluripotent cells (iPSCs). In this chapter, we describe a scalable suspension culture system using serum-free, feeder-free, matrix-free, and defined culture conditions with spinner flasks for hPSC maintenance and expansion. This suspension culture system provides an efficient and GMP-compatible process for large-scale manufacture of hPSCs.