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Conditioned media of naive human pluripotent stem cells cultured in microfluidics and RSet medium

ABSTRACT: These are 3 TMT10 samples. The indicated "standard" sub-samples all refer to the same biological sample (obtained as a mixture of all other samples), split and tagged as indicated, to play as a reference for normalization. TMT10 sample1 contains the following sub-samples: -TAG 126: Standard. -TAG 127N: Day 3 conditioned medium of replication-incompetent MEFs cultured on VTN coating in RSeT medium. -TAG 127C: Day 3 conditioned medium of naive human pluripotent stem cells cultured on a MEF feeder-layer in RSeT medium. -TAG 128N: Day 3 conditioned medium of naive human pluripotent stem cells cultured on a VTN coating in RSeT medium. -TAG 128C: Not relevant. -TAG 129N: Not relevant. -TAG 129C: Not relevant. -TAG 130N: Not relevant. -TAG 130C: RSet medium. -TAG 131: Standard. TMT10 sample2 contains the following sub-samples: -TAG 126: Standard -TAG 127N: Day 3 conditioned medium of replication-incompetent MEFs cultured on VTN coating in RSeT medium. -TAG 127C: Day 3 conditioned medium of naive human pluripotent stem cells cultured on a MEF feeder-layer in RSeT medium. -TAG 128N: Day 3 conditioned medium of naive human pluripotent stem cells cultured on a VTN coating in RSeT medium. -TAG 128C: Not relevant. -TAG 129N: Day 2 conditioned medium of naive human pluripotent stem cells cultured on a MEF feeder-layer in RSeT medium. -TAG 129C: Day 2 conditioned medium of naive human pluripotent stem cells cultured on a VTN coating in RSeT medium. -TAG 130N: Not relevant. -TAG 130C: RSet medium. -TAG 131: Standard. TMT10 sample3 contains the following sub-samples: -TAG 126: Standard. -TAG 127N: Day 3 conditioned medium of replication-incompetent MEFs cultured on VTN coating in RSeT medium. -TAG 127C: Day 3 conditioned medium of naive human pluripotent stem cells cultured on a MEF feeder-layer in RSeT medium. -TAG 128N: Day 2 conditioned medium of replication-incompetent MEFs cultured on VTN coating in RSeT medium. -TAG 128C: Day 2 conditioned medium of replication-incompetent MEFs cultured on VTN coating in RSeT medium. -TAG 129N: Day 2 conditioned medium of naive human pluripotent stem cells cultured on a MEF feeder-layer in RSeT medium. -TAG 129C: Day 2 conditioned medium of naive human pluripotent stem cells cultured on a VTN coating in RSeT medium. -TAG 130N: Not relevant. -TAG 130C: RSet medium. -TAG 131: Standard. Detailed culture and processing methods are described in Cesare et al, under submission.

INSTRUMENT(S): Orbitrap Fusion ETD

ORGANISM(S): Homo Sapiens (ncbitaxon:9606) Mus Musculus (ncbitaxon:10090)

SUBMITTER: Nicola Elvassore

PROVIDER: MSV000086068 | MassIVE |

SECONDARY ACCESSION(S): PXD021288

REPOSITORIES: MassIVE

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Project description:Leptin receptors (Lepr) are expressed by various types of stem cells including mesenchymal stem cells, hematopoietic stem cells, embryonic stem cells, and induced pluripotent stem cells. Leptin/lepr signaling is also a central regulator of metabolism. However, the role of Lepr in pluripotency, metabolic disease progression and growth development is still controversial and poorly understood. In the present study, we explored the Lepr function in disease progression, pluripotency and metabolism using day 14.5 mouse embryonic fibroblasts (MEFs) and their reprogrammed induced pluripotent stem cells (iPSCs) as model system. We successfully reprogrammed mouse embryonic fibroblasts into iPSCs from control and db/db (Lepr deficient) mice. Using a global quantitative proteomic approach, we identified key pathways regulating pluripotency, metabolic homeostasis and protein synthesis during fetal growth and development. The Lepr MEFs show abnormal metabolic abnormalities and mitochondrial dysfunction as compared to control MEFs, while Lepr iPSCs show upregulated elongated factor 4 e (eIF4e) protein synthesis pathway and altered Oct4 and Stat3 pathways which are involved in normal fetal growth development. Furthermore, chip analysis revealed that higher Stat3 binding on the promoter of eIF4e in Lepr iPSCs leads to higher protein synthesis in these cell types as compared to control iPSCs. Finally, point mutation corrected Lepr iPSCs using CRISPR/Cas9 gene editing method showed recovered pluripotency, metabolic and protein synthesis pathways. In conclusion, we have shown that Lepr signaling is involved in the regulation of the metabolic properties and key developmental pathways in MEFs and stemness of pluripotent stem cells. Disruption of Lepr signaling has been shown to involve in the pathophysiology of various diseases including obesity and diabetes. The generated MEFs and iPSCs in this present study provide valuable tools to explore the role of Lepr in the progression of obesity, diabetes and metabolic abnormalities, and to find the putative targets of Lepr signaling during the development of these diseases.

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Conditioned media of naive human pluripotent stem cells cultured in microfluidics and RSet medium

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