Int. J. Dev. Biol. 57: 715 - 724 (2013)

https://doi.org/10.1387/ijdb.130173to

Vol 57, Issue 9-10

Long-term serial cultivation of mouse induced pluripotent stem cells in serum-free and feeder-free defined medium

Original Article | Published: 15 November 2013

Sachiko Yamasaki¹, Kou Nabeshima¹, Yusuke Sotomaru², Yuki Taguchi³, Hanae Mukasa³, Miho K. Furue⁴, J. Denry Sato⁵ and Tetsuji Okamoto*^,1

¹Department of Molecular Oral Medicine and Maxillofacial Surgery, Applied Life Sciences, Institute of Biomedical & Health Sciences, Hiroshima University, ²Natural Science Center for Basic Research and Development, Hiroshima University ³Department of Molecular Oral Medicine and Maxillofacial Surgery, Division of Frontier Medical Sciences, Graduate School of Biomedical & Health Sciences, Hiroshima University, ⁴Laboratory of Stem Cell Cultures, Department of Disease Bioresources Research, National Institute of Biomedical Innovation, Manzoni Project, Hiroshima, Japan and ⁵Manzanar Project Foundation, Wenham, MA, USA

Abstract

Mouse embryonic stem (mES) cells and mouse induced pluripotent stem (miPS) cells are commonly maintained on inactivated mouse embryonic fibroblast feeder cells in medium supplemented with fetal bovine serum or proprietary replacements. An undefined medium containing unknown quantities of reagents has limited the development of applications for pluripotent cells because of the relative lack of knowledge regarding cell responses to differentiating growth factors. Therefore we developed a serum-free medium, designated ESF7, in which mES cells can be maintained in an undifferentiated state without feeder cells. The medium was tested for culturing miPS cells. The miPS cells have been maintained in ESF7 medium for more than 3 years with an undifferentiated phenotype manifested by the expression of pluripotency marker genes and alkaline phosphatase, and these cells exhibited largely normal karyotypes. Furthermore, we found that fibroblast growth factor-2 (FGF-2) with heparin induced miPS cell differentiation into neuronal cells, both in an adherent monolayer and in embryoid body suspension culture. Moreover, we found that FGF-2 with bone morphogenetic protein 2 induced miPS cell differentiation into cardiomyocytes in embryoid body suspension culture. Furthermore, we transplanted subcutaneously miPS cells maintained in ESF7 into the dorsal flanks of SCID mice; all of the transplants produced tumors with tissues derived from all three embryonic germ layers. As this simple serum-free adherent monoculture system supports the long-term propagation of pluripotent iPS cells in vitro, it will allow us to elucidate cell responses to growth factors under defined conditions, and it should provide useful information for differentiation protocols for human iPS cells.

Keywords

iPS cell, serum-free, LIF, neural differentiation, cardiomyocyte