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Growth of Human Pluripotent Stem Cells Using Functional Human Extracellular Matrix

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Human Embryonic Stem Cell Protocols

Part of the book series: Methods in Molecular Biology ((MIMB,volume 1307))

Abstract

The use of animal products in the derivation and maintenance of human pluripotent stem cells (hPSCs) limits their possible applications in research and in clinics. Thus, one of the major goals in regenerative medicine is the establishment of animal-free conditions to support the culture and differentiation of human stem cells. Human fibroblasts produce an extracellular matrix (ECM) which can be extracted without the use of detergents, sterilized, and then used to coat tissue culture plates.

We have shown that human embryonic stem cells (hESCs) grown on this matrix maintain their pluripotency in the presence of medium conditioned by fibroblast cells, and that these cells maintain expression of surface proteins (SSEA4, Tra1-60, Tra1-81), alkaline phosphatase activity, and specific intracellular markers (Nanog, Oct-4, Tert, FoxD3) in hESCs. This growth system reduces exposure of hPSCs to feeder layers and animal ingredients, thereby limiting the risk of pathogenic contamination and additionally, facilitating their manipulation. Herein we present an improved version of our previous protocol for extracting ECM from human foreskin fibroblast using a different buffer. Our new hypotonic shock method is detergent-free, reduces costs, and preserves the integrity of the extracted ECM. This improved protocol has been validated for undifferentiated-state hPSC maintenance (more than 40 passages), stem cell differentiation, and for cell migration assays.

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Acknowledgements

The authors would like to thank Dr. Deborah Burks, Dr. Sonia Prado, Dr. X. Chen Xiong, Mrs. Vanesa Blanca, Dr. Angel Ayuso-Sacido, and Ms. Petra Stojkovic for their support. This work was supported by funds for research in the field of Regenerative Medicine from the Regional Government Health Department (Generalitat Valenciana) and the Instituto Carlos III (Spanish Ministry of Health and Consumer Affairs). Drs. Carmen Escobedo-Lucea and Andres Sanz-Garcia are financed by Academy of Finland projects as follows: No. 273689 (Research Fellow), No. 266486 (FINSKIN), No. 276371 (VATURP), and No. 278153 (mobility grant).

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Authors and Affiliations

  1. Tissue Engineering Group, Division of Pharmaceutical Biosciences, Faculty of Pharmacy, University of Helsinki, Viikinkaari 5 E, 56, 00014, Helsinki, Finland

    Andres Sanz-Garcia & Carmen Escobedo-Lucea

  2. Institute of Advanced Biomedical Engineering and Science, Tokyo Women’s Medical University, TWINS, 8-1 Kawada-cho, Shinjuku-ku, Tokyo, 162-8666, Japan

    Andres Sanz-Garcia & Carmen Escobedo-Lucea

  3. SPEBO Medical, Leskovac, Serbia

    Miodrag Stojkovic

  4. Department of Human Genetics, Faculty of Medicine, University of Kragujevac, Kragujevac, Serbia

    Miodrag Stojkovic

Authors
  1. Andres Sanz-Garcia
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  2. Miodrag Stojkovic
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  3. Carmen Escobedo-Lucea
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Corresponding author

Correspondence to Carmen Escobedo-Lucea .

Editor information

Editors and Affiliations

  1. Ottawa Hospital Research Institute, Ottawa, Ontario, Canada

    Kursad Turksen

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© 2014 Springer Science+Business Media New York

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Sanz-Garcia, A., Stojkovic, M., Escobedo-Lucea, C. (2014). Growth of Human Pluripotent Stem Cells Using Functional Human Extracellular Matrix. In: Turksen, K. (eds) Human Embryonic Stem Cell Protocols. Methods in Molecular Biology, vol 1307. Humana Press, New York, NY. https://doi.org/10.1007/7651_2014_154

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  • DOI: https://doi.org/10.1007/7651_2014_154

  • Published:

  • Publisher Name: Humana Press, New York, NY

  • Print ISBN: 978-1-4939-2667-1

  • Online ISBN: 978-1-4939-2668-8

  • eBook Packages: Springer Protocols

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