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Xeno-free and Chemically Defined Human System for Culturing Human Epidermal Keratinocytes

ABSTRACT: Xeno-free and Chemically Defined Human System for Culturing Human Epidermal Keratinocytes

PROVIDER: PRJNA431596 | ENA |

REPOSITORIES: ENA

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			Action	DRS
	SRR6511800_1.fastq.gz	Fastqsanger.gz
	SRR6511800_2.fastq.gz	Fastqsanger.gz
	SRR6511810_1.fastq.gz	Fastqsanger.gz
	SRR6511810_2.fastq.gz	Fastqsanger.gz
	SRR6511801_1.fastq.gz	Fastqsanger.gz

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Xeno-free and Chemically Defined Human System for Culturing Human Epidermal Keratinocytes

Project description:Developing an effective xeno-free and defined system for human keratinocyte culture has been one of the fundamental measures in current cellular therapy. For keratinocytes, in particular, such system will not only fulfill clinical safety and quality criteria, it will allow for the management of less severe burns and other skin injuries such as chronic wounds. To date, the expansion of autologous keratinocytes in the clinical settings still relies on 3T3 feeder co-culture system. Here we report a completely xeno-free and defined culture system by using human recombinant laminins as feeder replacement. We have thoroughly characterized the cells cultured in this new system both in vitro and in vivo. We found that laminin-511/-521, and the unanticipated laminin-411/-421, but not -332, -111, -121, -211, or LN-221 support adult human skin keratinocytes and could maintain their self-renewal properties comparable to the 3T3 system. We believe our new culture method should facilitate broader use of cultured epithelial cell products for today’s regenerative medicine.

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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.

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

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Single-cell RNA-seq of human cardiovascular progenitor cells generated with a chemically defined, xeno-free laminin based differentiation protocol

Project description:Regeneration of injured human heart muscle is limited and an unmet clinical need. There are no methods for the reproducible generation of clinical quality stem-cell-derived cardiovascular progenitors (CVPs). We identified laminin-221 (LN-221) as the most likely expressed cardiac laminin. We produced it as human recombinant protein, and showed that LN-221 promotes differentiation of pluripotent hESCs towards cardiomyocyte lineage and downregulates pluripotency and teratoma associated genes. We developed a chemically defined, xeno-free laminin-based differentiation protocol to generate CVPs. Single-cell RNA sequencing of CVPs derived from hESC lines supported reproducibility and identified three main progenitor subpopulations. These CVPs were transplanted into myocardial infarction mice, where heart function was measured by echocardiogram and human heart muscle bundle formation was identified histologically. This method may provide clinical quality cells for use in regenerative cardiology.

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