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A stress-reduced passaging technique improves the viability of human pluripotent cells.

ABSTRACT: Xeno-free culture systems have expanded the clinical and industrial application of human pluripotent stem cells (PSCs). However, reproducibility issues, often arising from variability during passaging steps, remain. Here, we describe an improved method for the subculture of human PSCs. The revised method significantly enhances the viability of human PSCs by lowering DNA damage and apoptosis, resulting in more efficient and reproducible downstream applications such as gene editing and directed differentiation. Furthermore, the method does not alter PSC characteristics after long-term culture and attenuates the growth advantage of abnormal subpopulations. This robust passaging method minimizes experimental error and reduces the rate of PSCs failing quality control of human PSC research and application.

SUBMITTER: Takahashi K

PROVIDER: S-EPMC9017214 | biostudies-literature | 2022 Feb

REPOSITORIES: biostudies-literature

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A stress-reduced passaging technique improves the viability of human pluripotent cells.

Takahashi Kazutoshi K Okubo Chikako C Nakamura Michiko M Iwasaki Mio M Kawahara Yuka Y Tabata Tsuyoshi T Miyamoto Yousuke Y Woltjen Knut K Yamanaka Shinya S

Cell reports methods 20220124 2

Xeno-free culture systems have expanded the clinical and industrial application of human pluripotent stem cells (PSCs). However, reproducibility issues, often arising from variability during passaging steps, remain. Here, we describe an improved method for the subculture of human PSCs. The revised method significantly enhances the viability of human PSCs by lowering DNA damage and apoptosis, resulting in more efficient and reproducible downstream applications such as gene editing and directed di ...[more]

PMID: 35474962

Similar Datasets

A stress-reduced passaging technique improves the viability of human pluripotent cells

Project description:Xeno-free culture systems have expanded the application of human pluripotent stem cells (PSCs). Here we describe an improved method for the subculture of human PSCs. The new method significantly facilitated the viability of human PSCs by lowering DNA damage and apoptosis, resulting in more efficient and reproducible downstream applications. Furthermore, the method did not alter the PSC characteristics after long-term culture and attenuated the growth advantage of abnormal subpopulations. This robust passaging method minimizes experimental error and improves the quality control of human PSC research and applications.

2022-01-23 | GSE184071 | GEO

A stress-reduced passaging technique improves the viability of human pluripotent cells

Project description:Xeno-free culture systems have expanded the application of human pluripotent stem cells (PSCs). Here we describe an improved method for the subculture of human PSCs. The revised method significantly facilitated the viability of human PSCs by lowering DNA damage and apoptosis, resulting in more efficient and reproducible downstream applications. Furthermore, the method did not alter the PSC characteristics after long-term culture and attenuated the growth advantage of abnormal subpopulations. This robust passaging method minimizes experimental error and improves the quality control of human PSC research and applications.

2022-01-23 | PXD028731 | JPOST Repository

A stress-reduced passaging technique improves the viability of human pluripotent cells

Project description:A stress-reduced passaging technique improves the viability of human pluripotent cells

| PRJNA763089 | ENA

Physical passaging of embryoid bodies generated from human pluripotent stem cells.

Project description:Spherical three-dimensional cell aggregates called embryoid bodies (EBs), have been widely used in in vitro differentiation protocols for human pluripotent stem cells including human embryonic stem cells (hESCs) and human induced pluripotent stem cells (hiPSCs). Recent studies highlight the new devices and techniques for hEB formation and expansion, but are not involved in the passaging or subculture process. Here, we provide evidence that a simple periodic passaging markedly improved hEB culture condition and thus allowed the size-controlled, mass production of human embryoid bodies (hEBs) derived from both hESCs and hiPSCs. hEBs maintained in prolonged suspension culture without passaging (>2 weeks) showed a progressive decrease in the cell growth and proliferation and increase in the apoptosis compared to 7-day-old hEBs. However, when serially passaged in suspension, hEB cell populations were significantly increased in number while maintaining the normal rates of cell proliferation and apoptosis and the differentiation potential. Uniform-sized hEBs produced by manual passaging using a 1?4 split ratio have been successfully maintained for over 20 continuous passages. The passaging culture method of hEBs, which is simple, readily expandable, and reproducible, could be a powerful tool for improving a robust and scalable in vitro differentiation system of human pluripotent stem cells.

| S-EPMC3086884 | biostudies-literature

Passaging and colony expansion of human pluripotent stem cells by enzyme-free dissociation in chemically defined culture conditions.

Project description:This protocol describes an EDTA-based passaging procedure to be used with chemically defined E8 medium that serves as a tool for basic and translational research into human pluripotent stem cells (PSCs). In this protocol, passaging one six-well or 10-cm plate of cells takes about 6-7 min. This enzyme-free protocol achieves maximum cell survival without enzyme neutralization, centrifugation or drug treatment. It also allows for higher throughput, requires minimal material and limits contamination. Here we describe how to produce a consistent E8 medium for routine maintenance and reprogramming and how to incorporate the EDTA-based passaging procedure into human induced PSC (iPSC) derivation, colony expansion, cryopreservation and teratoma formation. This protocol has been successful in routine cell expansion, and efficient for expanding large-volume cultures or a large number of cells with preferential dissociation of PSCs. Effective for all culture stages, this procedure provides a consistent and universal approach to passaging human PSCs in E8 medium.

| S-EPMC3571618 | biostudies-literature

Extended passaging increases the efficiency of neural differentiation from induced pluripotent stem cells.

Project description:BackgroundThe use of induced pluripotent stem cells (iPSCs) for the functional replacement of damaged neurons and in vitro disease modeling is of great clinical relevance. Unfortunately, the capacity of iPSC lines to differentiate into neurons is highly variable, prompting the need for a reliable means of assessing the differentiation capacity of newly derived iPSC cell lines. Extended passaging is emerging as a method of ensuring faithful reprogramming. We adapted an established and efficient embryonic stem cell (ESC) neural induction protocol to test whether iPSCs (1) have the competence to give rise to functional neurons with similar efficiency as ESCs and (2) whether the extent of neural differentiation could be altered or enhanced by increased passaging.ResultsOur gene expression and morphological analyses revealed that neural conversion was temporally delayed in iPSC lines and some iPSC lines did not properly form embryoid bodies during the first stage of differentiation. Notably, these deficits were corrected by continual passaging in an iPSC clone. iPSCs with greater than 20 passages (late-passage iPSCs) expressed higher expression levels of pluripotency markers and formed larger embryoid bodies than iPSCs with fewer than 10 passages (early-passage iPSCs). Moreover, late-passage iPSCs started to express neural marker genes sooner than early-passage iPSCs after the initiation of neural induction. Furthermore, late-passage iPSC-derived neurons exhibited notably greater excitability and larger voltage-gated currents than early-passage iPSC-derived neurons, although these cells were morphologically indistinguishable.ConclusionsThese findings strongly suggest that the efficiency neuronal conversion depends on the complete reprogramming of iPSCs via extensive passaging.

| S-EPMC3167757 | biostudies-literature

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

Project description: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.

| S-EPMC8314867 | biostudies-literature

Testosterone improves the differentiation efficiency of insulin-producing cells from human induced pluripotent stem cells.

Project description:Human induced pluripotent stem cells (hiPSCs) may provide potential resource for regenerative medicine research, including generation of insulin-producing cells for diabetes research and insulin production. Testosterone (T) is an androgen hormone which promotes protein synthesis and improves the management of type 2 diabetes in clinical studies. Concurrently, co-existed hyperandrogenism and hyperinsulinism is frequently observed in polycystic ovary syndrome, congenital adrenal hyperplasia and some of Wermer's syndrome. However, the relationship among androgens, insulin and the differentiation of pancreatic β cells is still not fully clear. Here we find that T improves the differentiation efficiency of insulin-producing cells from hiPSCs. The addition of T into routine differentiation formula for pancreatic β cells increases the differentiation efficiency from 12% to 35%. The administration of T promotes the expression of key genes associated with β cells differentiation including NGN3, NEUROD1 and INS. This finding benefits the ongoing process to optimize the differentiation protocol of pancreatic β cells from hiPSCs, and provides some degree of understanding the clinical management of T for type 2 diabetes.

| S-EPMC5464652 | biostudies-literature

Simulated microgravity improves maturation of cardiomyocytes derived from human induced pluripotent stem cells.

Project description:Cardiomyocytes derived from human induced pluripotent stem cells (hiPSC-CMs) possess tremendous potential for basic research and translational application. However, these cells structurally and functionally resemble fetal cardiomyocytes, which is a major limitation of these cells. Microgravity can significantly alter cell behavior and function. Here we investigated the effect of simulated microgravity on hiPSC-CM maturation. Following culture under simulated microgravity in a random positioning machine for 7 days, 3D hiPSC-CMs had increased mitochondrial content as detected by a mitochondrial protein and mitochondrial DNA to nuclear DNA ratio. The cells also had increased mitochondrial membrane potential. Consistently, simulated microgravity increased mitochondrial respiration in 3D hiPSC-CMs, as indicated by higher levels of maximal respiration and ATP content, suggesting improved metabolic maturation in simulated microgravity cultures compared with cultures under normal gravity. Cells from simulated microgravity cultures also had improved Ca2+ transient parameters, a functional characteristic of more mature cardiomyocytes. In addition, these cells had improved structural properties associated with more mature cardiomyocytes, including increased sarcomere length, z-disc length, nuclear diameter, and nuclear eccentricity. These findings indicate that microgravity enhances the maturation of hiPSC-CMs at the structural, metabolic, and functional levels.

| S-EPMC10817987 | biostudies-literature

Actin-myosin contractility is responsible for the reduced viability of dissociated human embryonic stem cells.

Project description:Human ESCs are the pluripotent precursor of the three embryonic germ layers. Human ESCs exhibit basal-apical polarity, junctional complexes, integrin-dependent matrix adhesion, and E-cadherin-dependent cell-cell adhesion, all characteristics shared by the epiblast epithelium of the intact mammalian embryo. After disruption of epithelial structures, programmed cell death is commonly observed. If individualized human ESCs are prevented from reattaching and forming colonies, their viability is significantly reduced. Here, we show that actin-myosin contraction is a critical effector of the cell death response to human ESC dissociation. Inhibition of myosin heavy chain ATPase, downregulation of myosin heavy chain, and downregulation of myosin light chain all increase survival and cloning efficiency of individualized human ESCs. ROCK inhibition decreases phosphorylation of myosin light chain, suggesting that inhibition of actin-myosin contraction is also the mechanism through which ROCK inhibitors increase cloning efficiency of human ESCs.

| S-EPMC2916864 | biostudies-literature

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