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Human serum-derived protein removes the need for coating in defined human pluripotent stem cell culture

ABSTRACT: Reliable, scalable and time-efficient culture methods are required to fully realize the clinical and industrial applications of human pluripotent stem (hPS) cells. Here we present a completely defined, xeno-free medium that supports long-term propagation of hPS cells on uncoated tissue-culture plastic. The medium consists of the Essential 8 (E8) formulation supplemented with Inter-α-inhibitor (IαI), a human serum-derived protein, recently demonstrated to activate key pluripotency pathways in mouse PS cells. IαI efficiently induces attachment and long-term growth of both embryonic and induced hPS cell lines when added as a soluble protein to the medium at seeding. IαI-supplementation efficiently supports adaptation of feeder-dependent hPS cells to xeno-free conditions, clonal growth as well as single-cell survival in the absence of Rho-associated kinase inhibitor (ROCKi). This time-efficient and simplified culture method paves the way for large-scale, high-throughput hPS cell culture, and will be valuable for both basic research and commercial applications.

ORGANISM(S): Homo sapiens

PROVIDER: GSE82103 | GEO | 2016/06/02

SECONDARY ACCESSION(S): PRJNA324010

REPOSITORIES: GEO

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Human serum-derived protein removes the need for coating in defined human pluripotent stem cell culture

Project description:Reliable, scalable and time-efficient culture methods are required to fully realize the clinical and industrial applications of human pluripotent stem (hPS) cells. Here we present a completely defined, xeno-free medium that supports long-term propagation of hPS cells on uncoated tissue-culture plastic. The medium consists of the Essential 8 (E8) formulation supplemented with Inter-Î±-inhibitor (IÎ±I), a human serum-derived protein, recently demonstrated to activate key pluripotency pathways in mouse PS cells. IÎ±I efficiently induces attachment and long-term growth of both embryonic and induced hPS cell lines when added as a soluble protein to the medium at seeding. IÎ±I-supplementation efficiently supports adaptation of feeder-dependent hPS cells to xeno-free conditions, clonal growth as well as single-cell survival in the absence of Rho-associated kinase inhibitor (ROCKi). This time-efficient and simplified culture method paves the way for large-scale, high-throughput hPS cell culture, and will be valuable for both basic research and commercial applications. SNP genotyping with Illumina HumanOmniExpressExome-array version 8v1-2_A, encompassing >964.000 single nucleotide polymorphism (SNP)-markers, were performed by the SNP&SEQ technology Platform in Uppsala (www.genotyping.se), according to the manufacturerâ??s instructions. Each of the four hPS cell lines (i.e. H181, H207, HUES1 and K2C) were analyzed in three separate SNP-array experiments for the 12 samples described here. A first analysis was performed after 2-5 passages of growth for initial reference (4 samples), and subsequently, two analyses were performed per cell line after 16-21 passages in E8:VN or E8:IÎ±I culture (8 samples). The CN-calls of the autosomal chromosomes were illustrated using the Nexus-software.

2016-06-02 | E-GEOD-82103 | biostudies-arrayexpress

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

Human corneal endothelial cell cultures obtained from discarded tissues using a xenogeneic free culture medium

Project description:To investigate the use of Plasma Rich in Growth Factors (PRGF) as the unique source of growth factors in primary cultures of human corneal endothelial cells obtained from discarded tissues. We performed gene expression profiling analysis using data obtained from RNA-seq of 8 different cell cultures. 4 of them obtained with a standard culture medium and the other 4 using a xeno free PRGF based culture medium

2023-04-03 | GSE228460 | GEO

Global transcriptional analysis of xeno-free and feeder-cultured human extended pluripotent stem cells by RNA-seq

Project description:Extended pluripotent stem (EPS) cells have shown great applicative potentials in generating synthetic embryos, directed differentiation and disease modeling. However, the lack of a xenofree culture condition has significantly limited their wide applications. We developed a chemically defined and xeno-free culture condition for culturing and deriving human EPS cells, which can be long-term stably propagated in vitro, as well as preserve their embryonic and extraembryonic developmental potentials.

2021-03-24 | GSE147839 | GEO

Chromatin accessibility of xeno-free and feeder-cultured human extended pluripotent stem cells by ATAC-seq

2021-03-24 | GSE157237 | GEO

Global transcriptional analysis of xeno-free and feeder-cultured human extended pluripotent stem cells by single cell RNA-seq

2021-03-24 | GSE156920 | GEO

Modulation of Wnt and Activin/Nodal supports efficient derivation, cloning and suspension expansion of human pluripotent stem cells

Project description:Various culture systems have been used to derive and maintain human pluripotent stem cells (hPSCs), but they are inefficient in sustaining cloning and suspension expansion of hPSCs. Through systematically modulating Wnt and Activin/Nodal signaling, we developed a defined medium (termed AIC), which enables efficient cloning and long-term expansion of hPSCs (AIC-hPSCs) through single-cell passage on feeders, matrix or in suspension (25-fold expansion in 4 days) and maintains genomic stability of hPSCs over extensive expansion. Moreover, the AIC medium supports efficient derivation of hPSCs from blastocysts or somatic cells under feeder-free conditions. Compared to conventional-culture hPSCs, AIC-hPSCs have similar gene expression profiles but down-regulated differentiation genes and display higher metabolic activity. The AIC medium shows a good compatibility for suspension expansion of different hPSC lines. Our study provides a robust culture system for derivation, cloning and suspension expansion of high-quality hPSCs that benefits GMP production and processing of therapeutic hPSC products

2020-04-23 | GSE145163 | GEO

Extracellular matrix-dependent generation of integration- and xeno-free iPS cells using a modified mRNA transfection method

Project description:In this study, we sought to examine whether an extracellular matrix (ECM)-based xeno-free culture system that we recently established could be used together with a microRNA-enhanced mRNA reprogramming method for the generation of clinically safe iPS cells. The notable features of this method are (1) the use of a xeno-free/feeder-free culture system for the generation and expansion of iPS cells rather than the conventional labor-intensive culture systems using human feeder cells or human feeder-conditioned medium and (2) the enhancement of mRNA-mediated reprogramming via the delivery of microRNAs. Strikingly, we observed the early appearance of iPS cell colonies (~11 days), substantial reprogramming efficiency (~0.2-0.3%), and a high percentage of ESC-like colonies among the total colonies (~87.5%), indicating enhanced kinetics and reprogramming efficiency. Therefore, the combined method established in this study provides a valuable platform for the generation and expansion of clinically safe (i.e., integration- and xeno-free) iPS cells, facilitating immune-matched cell therapy in the near future.

2016-04-21 | GSE68035 | GEO

DMSO-free chemically defined cryopreservation of human mesenchymal stem cells and chondrocytes

Project description:Serum- and xeno-free cryopreservation is suitable for cells to be used in clinical applications. However, dimethyl sulfoxide (DMSO), which is often used in conventional cryoprotectant medium, has been shown to be toxic to many cell types and can induce morphological and gene expression changes that can lead to changes in cell function when they are cultured. In order to evaluate this issue in depth, we examined the efficiency of a DMSO-free/serum-free/xeno-free chemically defined cryoprotectant media, STEM-CELLBANKER DMSO-Free, when culturing human mesenchymal stem cells (MSCs) and chondrocytes. Interestingly, the cells cryopreserved in STEM-CELLBANKER DMSO-Free had a similar growth rate, morphology, and global gene expression profile compared to that observed for cells preserved in conventional freezing medium, which contains 10% DMSO and 20% fetal bovine serum. From these data, we therefore propose that STEM-CELLBANKER DMSO-Free may be an effective tool for maintaining the cellular characteristics of MSCs and chondrocytes during cryo-storage prior to clinical application.

2019-03-22 | GSE79496 | GEO

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