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Serum Inter-α-inhibitor Activates the Yes Tyrosine Kinase and YAP/TEAD Transcriptional Complex in Mouse Embryonic Stem Cells
Uppsala University, Disciplinary Domain of Medicine and Pharmacy, Faculty of Medicine, Department of Medical Biochemistry and Microbiology.
Uppsala University, Disciplinary Domain of Medicine and Pharmacy, Faculty of Medicine, Department of Medical Biochemistry and Microbiology.
Uppsala University, Disciplinary Domain of Medicine and Pharmacy, Faculty of Medicine, Department of Medical Biochemistry and Microbiology.
2014 (English)In: Journal of Biological Chemistry, ISSN 0021-9258, E-ISSN 1083-351X, Vol. 289, no 48, 33492-33502 p.Article in journal (Refereed) Published
Abstract [en]

We have previously demonstrated that the Src family kinase Yes, the Yes-associated protein (YAP) and TEA domain TEAD2 transcription factor pathway are activated by leukemia inhibitory factor (LIF) and contribute to mouse embryonic stem (mES) cell maintenance of pluripotency and self-renewal. In addition, we have shown that fetal bovine serum (FBS) induces Yes auto-phosphorylation and activation. In the present study we confirm that serum also activates TEAD-dependent transcription in a time- and dose-dependent manner and we identify Inter-α-inhibitor (IαI) as a component in serum capable of activating the Yes/YAP/TEAD pathway by inducing Yes auto-phosphorylation, YAP nuclear localization and TEAD-dependent transcription. The cleaved heavy chain 2 (HC2) sub-component of IαI, is demonstrated to be responsible for this effect. Moreover, IαI is also shown to efficiently increase expression of TEAD-downstream target genes including well-known stem cell factors Nanog and Oct 3/4. IαI is not produced by the ES cells per se but is added to the cells via the cell culture medium containing serum or serum-derived components such as bovine serum albumin (BSA). In conclusion, we describe a novel function of IαI in activating key pluripotency pathways associated with ES cell maintenance and self-renewal.

Place, publisher, year, edition, pages
2014. Vol. 289, no 48, 33492-33502 p.
National Category
Basic Medicine Cell and Molecular Biology
URN: urn:nbn:se:uu:diva-237578DOI: 10.1074/jbc.M114.580076ISI: 000345636600037PubMedID: 25301940OAI: oai:DiVA.org:uu-237578DiVA: diva2:768283
Available from: 2014-12-03 Created: 2014-12-03 Last updated: 2015-07-07Bibliographically approved
In thesis
1. Novel Culture Strategies and Signal Transduction Pathways of Pluripotent Stem Cells
Open this publication in new window or tab >>Novel Culture Strategies and Signal Transduction Pathways of Pluripotent Stem Cells
2015 (English)Doctoral thesis, comprehensive summary (Other academic)
Abstract [en]

Pluripotent stem cells (PSCs) can self-renew indefinitely in culture while maintaining their capacity to differentiate into any cell type of an organism, thus offering novel sources for drug screening, in vitro disease modelling, and cell replacement therapies. However, due to their sensitive nature, many PSC lines are still cultured using undefined components such as serum or serum-derived components, on either feeder cells or complex protein mixes such as Matrigel or gelatine. In order to fully realize the potential of these cells we need controlled, completely defined and xeno-free culturing conditions that maintain growth and survival of homogenous, non-differentiated colonies. This thesis focuses on the in vitro maintenance of both mouse and human PSCs, analysing the media and substrate requirements of these cells and linking them to the intracellular signalling pathways involved in the maintenance of pluripotency and self-renewal. Benchmarking of commercially available culture methods for PSCs has been performed, evaluating their capacity to maintain pluripotency and growth of undifferentiated PSCs over several passages and reporting new characteristics, like the tendency of mouse PSCs to grow as floating spheres in 2i medium, a novel media formulation that uses two inhibitors to hinder differentiation capacity and subsequently induce pure, undifferentiated cultures. The major finding in this thesis is the identification of Inter-α-Inhibitor (IαI) as a protein able to activate the previously described signal-transduction pathway Yes/YAP/TEAD in mouse PSCs and to induce transcription of the well-known stem cell transcription factors Nanog and Oct3/4. IαI is a serum protein found in high concentration in human serum that had been traditionally described as an extracellular matrix remodelling protein. For the first time, we describe IαI to have signalling capacity on PSCs. Moreover, IαI is demonstrated to induce attachment, growth and long-term survival of undifferentiated mouse and human PSCs when added to serum-free, chemically defined media. IαI is the first molecule described to date to induce attachment of human PSCs on uncoated, standard tissue-culture treated plastic, just by supplementation as a soluble molecule at the seeding step. Following this discovery, we evaluate a novel culture method using the completely defined, serum-free E8 medium supplemented with IαI (E8:IαI) for long-term propagation of four different human PSC lines and discover that IαI can indeed support long-term culture with maintained pluripotency, differentiation capacity, growth rate and genetic stability. Moreover, in contrast to the control culture method using a commercially available surface coating, IαI supplementation can support single cell passaging of human PSCs, and adapt feeder-dependent cultured human PSCs to E8:IαI with high efficiency. A mouse PSC line is also grown for over 20 passages in IαI with retained pluripotency, differentiation capacity and genetic stability.

IαI is inexpensive to produce and derived from human plasma, and could therefore be produced in compliance with Good Manufacturing Practices. Ultimately, our group aims to develop and test large-scale, completely defined, xeno-free culturing methods for PSCs, suitable for pharmacological and medical applications. 

Place, publisher, year, edition, pages
Uppsala: Acta Universitatis Upsaliensis, 2015. 54 p.
Digital Comprehensive Summaries of Uppsala Dissertations from the Faculty of Medicine, ISSN 1651-6206 ; 1090
Pluripotent stem cells, Inter-alpha inhibitor, culture method
National Category
Medical and Health Sciences
Research subject
Biology with specialization in Molecular Cell Biology; Biology with specialization in Molecular Biotechnology
urn:nbn:se:uu:diva-248124 (URN)978-91-554-9218-2 (ISBN)
Public defence
2015-05-15, A/B1:111a, Husargatan 3, Uppsala, 09:15 (English)
Available from: 2015-04-24 Created: 2015-03-27 Last updated: 2015-07-07

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Pijuan Galitó, SaraAnnerén, Cecilia
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