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Low-molecular-weight inhibitors of cell differentiation enable efficient growth of mouse iPS cells under feeder-free conditions

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Abstract

Embryonic stem cells and induced pluripotent stem (iPS) cells are usually maintained on feeder cells derived from mouse embryonic fibroblasts (MEFs). In recent years, the cell culture of iPS cells under serum- and feeder-free conditions is gaining attention in overcoming the biosafety issues for clinical applications. In this study, we report on the use of multiple small-molecular inhibitors (i.e., CHIR99021, PD0325901, and Thiazovivin) to efficiently cultivate mouse iPS cells without feeder cells in a chemically-defined and serum-free condition. In this condition, we showed that mouse iPS cells are expressing the Nanog, Oct3/4, and SSEA-1 pluripotent markers, indicating that the culture condition is optimized to maintain the pluripotent status of iPS cells. Without these small-molecular inhibitors, mouse iPS cells required the adaptation period to start the stable cell proliferation. The application of these inhibitors enabled us the shortcut culture method for the cellular adaptation. This study will be useful to efficiently establish mouse iPS cell lines without MEF-derived feeder cells.

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Abbreviations

ES:

Embryonic stem

iPS:

Induced pluripotent stem

MEFs:

Mouse embryonic fibroblasts

SSEA:

Stage specific embryonic antigen

HPV:

Human papilloma virus

STEMCCA:

Stem cell cassette

DMEM:

Dulbecco’s modified Eagle’s medium

KSR:

Knockout serum replacement

LIF:

Leukemia inhibitory factor

2i:

2 Inhibitors (CHIR99021 and PD0325901)

Tzv:

Thiazovivin

GSK-3β:

Glycogen synthase kinase 3β

MAPK:

Mitogen-activated protein kinase

ROCK:

Rho-associated Kinase

PD:

Population doubling

EBs:

Embryoid bodies

AP:

Alkaline phosphatase

DAPI:

4′,6-Diamidino-2-phenylindole

RT:

Reverse transcription

PCR:

Polymerase chain reaction

cDNA:

Complementary DNA

References

  • Amit M, Shariki C, Margulets V, Itskovitz-Eldor J (2004) Feeder layer- and serum-free culture of human embryonic stem cells. Biol Reprod 70:837–845. doi:10.1095/biolreprod.103.021147

    Article  CAS  Google Scholar 

  • Bain J, Plater L, Elliott M, Shpiro N, Hastie CJ, McLauchlan H, Klevernic I, Arthur JS, Alessi DR, Cohen P (2007) The selectivity of protein kinase inhibitors: a further update. Biochem J 408:297–315. doi:10.1042/BJ20070797

    Article  CAS  Google Scholar 

  • Besser D (2004) Expression of nodal, lefty-a, and lefty-B in undifferentiated human embryonic stem cells requires activation of Smad2/3. J Biol Chem 279:45076–45084. doi:10.1074/jbc.M404979200

    Article  CAS  Google Scholar 

  • Donai K, Kuroda K, Guo Y, So KH, Sone H, Kobayashi M, Nishimori K, Fukuda T (2013) Establishment of a reporter system to monitor silencing status in induced pluripotent stem cell lines. Anal Biochem 443:104–112. doi:10.1016/j.ab.2013.08.014

    Article  CAS  Google Scholar 

  • Fukusumi H, Shofuda T, Kanematsu D, Yamamoto A, Suemizu H, Nakamura M, Yamasaki M, Ohgushi M, Sasai Y, Kanemura Y (2013) Feeder-free generation and long-term culture of human induced pluripotent stem cells using pericellular matrix of decidua derived mesenchymal cells. PLoS ONE 8:e55226. doi:10.1371/journal.pone.0055226

    Article  CAS  Google Scholar 

  • Furue M, Okamoto T, Hayashi Y, Okochi H, Fujimoto M, Myoishi Y, Abe T, Ohnuma K, Sato GH, Asashima M, Sato JD (2005) Leukemia inhibitory factor as an anti-apoptotic mitogen for pluripotent mouse embryonic stem cells in a serum-free medium without feeder cells. In Vitro Cell Dev Biol Anim 41:19–28. doi:10.1290/0502010.1

    Article  CAS  Google Scholar 

  • Hayashi Y, Chan T, Warashina M, Fukuda M, Ariizumi T, Okabayashi K, Takayama N, Otsu M, Eto K, Furue MK, Michiue T, Ohnuma K, Nakauchi H, Asashima M (2010) Reduction of N-glycolylneuraminic acid in human induced pluripotent stem cells generated or cultured under feeder- and serum-free defined conditions. PLoS ONE 5:e14099. doi:10.1371/journal.pone.0014099

    Article  Google Scholar 

  • Kitajima H, Niwa H (2010) Clonal expansion of human pluripotent stem cells on gelatin-coated surface. Biochem Biophys Res Commun 396:933–938. doi:10.1016/j.bbrc.2010.05.026

    Article  CAS  Google Scholar 

  • Lin T, Ambasudhan R, Yuan X, Li W, Hilcove S, Abujarour R, Lin X, Hahm HS, Hao E, Hayek A, Ding S (2009) A chemical platform for improved induction of human iPSCs. Nat Methods 6:805–808. doi:10.1038/nmeth.1393

    Article  CAS  Google Scholar 

  • Lu J, Hou R, Booth CJ, Yang SH, Snyder M (2006) Defined culture conditions of human embryonic stem cells. Proc Natl Acad Sci USA 103:5688–5693. doi:10.1073/pnas.0601383103

    Article  CAS  Google Scholar 

  • Miyazaki T, Futaki S, Hasegawa K, Kawasaki M, Sanzen N, Hayashi M, Kawase E, Sekiguchi K, Nakatsuji N, Suemori H (2008) Recombinant human laminin isoforms can support the undifferentiated growth of human embryonic stem cells. Biochem Biophys Res Commun 375:27–32. doi:10.1016/j.bbrc.2008.07.111

    Article  CAS  Google Scholar 

  • Navarro-Alvarez N, Soto-Gutierrez A, Yuasa T, Yamatsuji T, Shirakawa Y, Nagasaka T, Sun SD, Javed MS, Tanaka N, Kobayashi N (2008) Long-term culture of Japanese human embryonic stem cells in feeder-free conditions. Cell Transpl 17:27–33

    Article  Google Scholar 

  • Polychronopoulos P, Magiatis P, Skaltsounis AL, Myrianthopoulos V, Mikros E, Tarricone A, Musacchio A, Roe SM, Pearl L, Leost M, Greengard P, Meijer L (2004) Structural basis for the synthesis of indirubins as potent and selective inhibitors of glycogen synthase kinase-3 and cyclin-dependent kinases. J Med Chem 47:935–946. doi:10.1021/jm031016d

    Article  CAS  Google Scholar 

  • Qin XY, Fukuda T, Yang L, Zaha H, Akanuma H, Zeng Q, Yoshinaga J, Sone H (2012) Effects of bisphenol a exposure on the proliferation and senescence of normal human mammary epithelial cells. Cancer Biol Ther 13:296–306. doi:10.4161/cbt.18942

    Article  CAS  Google Scholar 

  • Rodin S, Domogatskaya A, Strom S, Hansson EM, Chien KR, Inzunza J, Hovatta O, Tryggvason K (2010) Long-term self-renewal of human pluripotent stem cells on human recombinant laminin-511. Nat Biotechnol 28:611–615. doi:10.1038/nbt.1620

    Article  CAS  Google Scholar 

  • Rosler ES, Fisk GJ, Ares X, Irving J, Miura T, Rao MS, Carpenter MK (2004) Long-term culture of human embryonic stem cells in feeder-free conditions. Dev Dyn 229:259–274. doi:10.1002/dvdy.10430

    Article  CAS  Google Scholar 

  • Sato N, Meijer L, Skaltsounis L, Greengard P, Brivanlou AH (2004) Maintenance of pluripotency in human and mouse embryonic stem cells through activation of Wnt signaling by a pharmacological GSK-3-specific inhibitor. Nat Med 10:55–63. doi:10.1038/nm979

    Article  CAS  Google Scholar 

  • Sebolt-Leopold JS, Herrera R (2004) Targeting the mitogen-activated protein kinase cascade to treat cancer. Nat Rev Cancer 4:937–947. doi:10.1038/nrc1503

    Article  CAS  Google Scholar 

  • Sommer CA, Stadtfeld M, Murphy GJ, Hochedlinger K, Kotton DN, Mostoslavsky G (2009) Induced pluripotent stem cell generation using a single lentiviral stem cell cassette. Stem Cells 27:543–549. doi:10.1634/stemcells.2008-1075

    Article  CAS  Google Scholar 

  • Sun N, Panetta NJ, Gupta DM, Wilson KD, Lee A, Jia F, Hu S, Cherry AM, Robbins RC, Longaker MT, Wu JC (2009) Feeder-free derivation of induced pluripotent stem cells from adult human adipose stem cells. Proc Natl Acad Sci USA 106:15720–15725. doi:10.1073/pnas.0908450106

    Article  CAS  Google Scholar 

  • Takahashi K, Yamanaka S (2006) Induction of pluripotent stem cells from mouse embryonic and adult fibroblast cultures by defined factors. Cell 126:663–676. doi:10.1016/j.cell.2006.07.024

    Article  CAS  Google Scholar 

  • Takahashi K, Tanabe K, Ohnuki M, Narita M, Ichisaka T, Tomoda K, Yamanaka S (2007) Induction of pluripotent stem cells from adult human fibroblasts by defined factors. Cell 131:861–872. doi:10.1016/J.Cell.11.019

    Article  CAS  Google Scholar 

  • Xu C, Inokuma MS, Denham J, Golds K, Kundu P, Gold JD, Carpenter MK (2001) Feeder-free growth of undifferentiated human embryonic stem cells. Nat Biotechnol 19:971–974. doi:10.1038/nbt1001-971

    Article  CAS  Google Scholar 

  • Xu Y, Zhu X, Hahm HS, Wei W, Hao E, Hayek A, Ding S (2010) Revealing a core signaling regulatory mechanism for pluripotent stem cell survival and self-renewal by small molecules. Proc Natl Acad Sci USA 107:8129–8134. doi:10.1073/pnas.1002024107

    Article  CAS  Google Scholar 

  • Yamamoto A, Kumakura S, Uchida M, Barrett JC, Tsutsui T (2003) Immortalization of normal human embryonic fibroblasts by introduction of either the human papillomavirus type 16 E6 or E7 gene alone. Int J Cancer 106:301–309. doi:10.1002/ijc.11219

    Article  CAS  Google Scholar 

  • Ying QL, Wray J, Nichols J, Batlle-Morera L, Doble B, Woodgett J, Cohen P, Smith A (2008) The ground state of embryonic stem cell self-renewal. Nature 453:519–523. doi:10.1038/nature06968

    Article  CAS  Google Scholar 

  • Yu J, Vodyanik MA, Smuga-Otto K, Antosiewicz-Bourget J, Frane JL, Tian S, Nie J, Jonsdottir GA, Ruotti V, Stewart R, Slukvin II, Thomson JA (2007) Induced pluripotent stem cell lines derived from human somatic cells. Science 318:1917–1920. doi:10.1126/science.1151526

    Article  CAS  Google Scholar 

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Acknowledgments

We thank Dr. Gustavo Mostoslavsky (Boston University School of Medicine) for providing the STEMCCA-loxP lentiviral vector. We also thank the technical supports and suggestion from and Dr. Takehiro Ito and Ms. Yukiko Kitamura (Cell Science and Technology Institute). This work was supported by research grants from the Tojuro Iijima Foundation for Food Science and Technology, Asahi Group Foundation and a JSPS grant (KAKENHI, #23650587 and #25640117).

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Authors and Affiliations

  1. Graduate School of Agricultural Science, Tohoku University, 1-1 Tsutsumidori-amamiyamachi, Aoba-ku, Sendai, 981-8555, Japan

    Kenichiro Donai, Akane Inagaki, Kyoung-Ha So, Kengo Kuroda, Katsuhiko Nishimori & Tomokazu Fukuda

  2. Environmental Exposure Research Section, Center for Environmental Risk Research, National Institute for Environmental Studies, 16-2 Onogawa, Tsukuba, Ibaraki, 305-8506, Japan

    Hideko Sone

  3. Graduate School of Bioresource Sciences, Akita Prefectural University, 241-438 Kaidobata-Nishi, Nakano, Shimoshinjo, Akita, 010-0195, Japan

    Masayuki Kobayashi

Authors
  1. Kenichiro Donai
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  2. Akane Inagaki
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  3. Kyoung-Ha So
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  4. Kengo Kuroda
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  5. Hideko Sone
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  6. Masayuki Kobayashi
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  7. Katsuhiko Nishimori
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  8. Tomokazu Fukuda
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Corresponding author

Correspondence to Tomokazu Fukuda.

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Donai, K., Inagaki, A., So, KH. et al. Low-molecular-weight inhibitors of cell differentiation enable efficient growth of mouse iPS cells under feeder-free conditions. Cytotechnology 67, 191–197 (2015). https://doi.org/10.1007/s10616-013-9686-8

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