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Wu, X.* ; Schneider, N.* ; Platen, A.* ; Mitra, I.* ; Blazek, M.* ; Zengerle, R.* ; Schüle, R.* ; Meier, M.

In situ characterization of the mTORC1 during adipogenesis of human adult stem cells on chip.

Proc. Natl. Acad. Sci. U.S.A. 113, E4143-E4150 (2016)
Verlagsversion DOI PMC
Open Access Gold
Creative Commons Lizenzvertrag
Mammalian target of rapamycin (mTOR) is a central kinase integrating nutrient, energy, and metabolite signals. The kinase forms two distinct complexes: mTORC1 and mTORC2. mTORC1 plays an essential but undefined regulatory function for regeneration of adipose tissue. Analysis of mTOR in general is hampered by the complexity of regulatory mechanisms, including protein interactions and/or phosphorylation, in an ever-changing cellular microenvironment. Here, we developed a microfluidic large-scale integration chip platform for culturing and differentiating human adipose-derived stem cells (hASCs) in 128 separated microchambers under standardized nutrient conditions over 3 wk. The progression of the stem cell differentiation was measured by determining the lipid accumulation rates in hASC cultures. For in situ protein analytics, we developed a multiplex in situ proximity ligation assay (mPLA) that can detect mTOR in its two complexes selectively in single cells and implemented it on the same chip. With this combined technology, it was possible to reveal that the mTORC1 is regulated in its abundance, phosphorylation state, and localization in coordination with lysosomes during adipogenesis. High-content image analysis and parameterization of the in situ PLA signals in over 1 million cells cultured on four individual chips showed that mTORC1 and lysosomes are temporally and spatially coordinated but not in its composition during adipogenesis.
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Publikationstyp Artikel: Journalartikel
Dokumenttyp Wissenschaftlicher Artikel
Schlagwörter Adipogenesis ; Mtorc1 Regulation ; Microfluidics ; Multiplexed Pla ; Stem Cell Differentiation
Sprache englisch
Veröffentlichungsjahr 2016
HGF-Berichtsjahr 2016
ISSN (print) / ISBN 0027-8424
e-ISSN 1091-6490
Quellenangaben Band: 113, Heft: 29, Seiten: E4143-E4150 Artikelnummer: , Supplement: ,
Verlag National Academy of Sciences
Begutachtungsstatus Peer reviewed
Institut(e) Helmholtz Pioneer Campus (HPC)
PubMed ID 27382182
Erfassungsdatum 2019-01-18