Dataset Information

Proteomic analysis of ECM supporting hESC maintenance

ABSTRACT: Comparative analysis of extracellular matrix proteins in substrates supportive and unsupportive for human embryonic stem cell maintenance

INSTRUMENT(S): 4000 Q TRAP, instrument model

ORGANISM(S): Homo Sapiens (human) Mus Musculus (mouse)

SUBMITTER: Adam Byron

PROVIDER: PRD000600 | Pride | 2013-05-30

REPOSITORIES: Pride

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Dataset's files

Source:

			Action	DRS
	PRIDE_Exp_Complete_Ac_19910.pride.mgf.gz	Mgf
	PRIDE_Exp_Complete_Ac_19910.pride.mztab.gz	Mztab
	PRIDE_Exp_Complete_Ac_19910.xml.gz	Xml
	PRIDE_Exp_Complete_Ac_19911.pride.mgf.gz	Mgf
	PRIDE_Exp_Complete_Ac_19911.pride.mztab.gz	Mztab

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Publications

Comparative proteomic analysis of supportive and unsupportive extracellular matrix substrates for human embryonic stem cell maintenance.

Soteriou Despina D Iskender Banu B Byron Adam A Humphries Jonathan D JD Borg-Bartolo Simon S Haddock Marie-Claire MC Baxter Melissa A MA Knight David D Humphries Martin J MJ Kimber Susan J SJ

The Journal of biological chemistry 20130508 26

Human embryonic stem cells (hESCs) are pluripotent cells that have indefinite replicative potential and the ability to differentiate into derivatives of all three germ layers. hESCs are conventionally grown on mitotically inactivated mouse embryonic fibroblasts (MEFs) or feeder cells of human origin. In addition, feeder-free culture systems can be used to support hESCs, in which the adhesive substrate plays a key role in the regulation of stem cell self-renewal or differentiation. Extracellular ...[more]

PMID: 23658023

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Project description:Human ES cells (hESCs) and human induced pluripotent stem cells (hiPSCs) are usually generated and maintained on living feeder cells like mouse embryonic fibroblasts or on a cell-free substrate like Matrigel. For clinical applications, a quality-controlled, xenobiotic-free culture system is required to minimize risks from contaminating animal-derived pathogens and immunogens. We previously reported that the pericellular matrix of decidua-derived mesenchymal cells (PCM-DM) is an ideal human-derived substrate on which to maintain hiPSCs/hESCs. In this study, we examined whether PCM-DM could be used for the generation and long-term stable maintenance of hiPSCs. Decidua-derived mesenchymal cells (DMCs) were reprogrammed by the retroviral transduction of four factors (OCT4, SOX2, KLF4, c-MYC) and cultured on PCM-DM. The established hiPSC clones expressed alkaline phosphatase, hESC-specific genes and cell-surface markers, and differentiated into three germ layers in vitro and in vivo. At over 20 passages, the hiPSCs cultured on PCM-DM held the same cellular properties with genome integrity as those at early passages. Global gene expression analysis showed that the GDF3, FGF4, UTF1, and XIST expression levels varied during culture, and GATA6 was highly expressed under our culture conditions; however, these gene expressions did not affect the cellsM-bM-^@M-^Y pluripotency. PCM-DM can be conveniently prepared from DMCs, which have a high proliferative potential. Our findings indicate that PCM-DM is a versatile and practical human-derived substrate that can be used for the feeder-cell-free generation and long-term stable maintenance of hiPSCs. Keywords: Induced pluripotent stem cells; Extracellular matrix; Decidua; Mesenchymal cells The microarray study was carried out using Affymetrix Human Genome U133 Plus 2.0 gene expression arrays. Total RNA was extracted from cells with RNeasy (Qiagen), and 100ng of total RNA was used to synthesize aRNA using the 3M-bM-^@M-^Y IVT Express Kit, according to the manufacturerM-bM-^@M-^Ys instructions (Affymetrix Inc., Santa Clara, CA). After aRNA purification, 15 M-NM-<g of aRNA was fragmented and hybridized with a pre-equilibrated GeneChip array (Human Genome U133 Plus 2.0 gene expression array, Affymetrix) at 45M-BM-0C for 16 hours. The GeneChip array was then washed, stained, and scanned according to the manufacturerM-bM-^@M-^Ys instructions. The gene expression data were extracted using Affymetrix Expression Console software.