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A Comparative Study of Protocols for Mouse Embryonic Stem Cell Culturing
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.
2013 (English)In: PLoS ONE, ISSN 1932-6203, Vol. 8, no 12, e81156- p.Article in journal (Refereed) Published
Abstract [en]

Most stem cell laboratories still rely on old culture methods to support the expansion and maintenance of mouse embryonic stem (ES) cells. These involve growing cells on mouse embryonic fibroblast feeder cells or on gelatin in media supplemented with fetal bovine serum and leukemia inhibitory factor (LIF). However, these techniques have several drawbacks including the need for feeder-cells and/or use of undefined media containing animal derived components. Culture of stem cells under undefined conditions can induce spontaneous differentiation and reduce reproducibility of experiments. In recent years several new ES cell culture protocols, using more well-defined conditions, have been published and we have compared the standard culture protocols with two of the newly described ones: 1) growing cells in semi-adherence in a medium containing two small molecule inhibitors (CHIR99021, PD0325901) and; 2) growing cells in a spheroid suspension culture in a defined medium containing LIF and bFGF. Two feeder-dependent mouse ES (mES) cell lines and two cell lines adapted to feeder-independent growth were used in the study. The overall aim has not only been to compare self-renewal and differentiation capacity, but also ease-of-use and cost efficiency. We show that mES cells when grown adherently proliferate much faster than when grown in suspension as free-floating spheres, independent of media used. Although all the tested culture protocols could maintain sustained pluripotency after prolonged culturing, our data confirm previous reports showing that the media containing two chemical inhibitors generate more pure stem cell cultures with negligible signs of spontaneous differentiation as compared to standard mES media. Furthermore, we show that this medium effectively rescues and cleans up cultures that have started to deteriorate, as well as allow for effective adaption of feeder-dependent mES cell lines to be maintained in feeder-free conditions.

Place, publisher, year, edition, pages
2013. Vol. 8, no 12, e81156- p.
National Category
Engineering and Technology Medical and Health Sciences
URN: urn:nbn:se:uu:diva-217668DOI: 10.1371/journal.pone.0081156ISI: 000328707400012OAI: diva2:693918
Available from: 2014-02-05 Created: 2014-02-04 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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Tamm, ChristofferGalito, Sara PijuanAnneren, Cecilia
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