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Mouse Pluripotent Stem Cell Differentiation Under Physiological Oxygen Reduces Residual Teratomas

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Abstract

Introduction

Residual pluripotent stem cells (PSC) within differentiated populations are problematic because of their potential to form tumors. Simple methods to reduce their occurrence are needed.

Methods

Here, we demonstrate that control of the oxygen partial pressure (pO2) to physiological levels typical of the developing embryo, enabled by culture on a highly oxygen permeable substrate, reduces the fraction of PSC within and the tumorigenic potential of differentiated populations.

Results

Differentiation and/or extended culture at low pO2 reduced measured pluripotency markers by up to four orders of magnitude for mouse PSCs (mPSCs). Combination with cell sorting increased the reduction to as much as six orders of magnitude. Upon implantation into immunocompromised mice, mPSCs differentiated at low pO2 either did not form tumors or formed tumors at a slower rate than at high pO2.

Conclusions

Low pO2 culture alone or in combination with other methods is a potentially straightforward method that could be applied to future cell therapy protocols to minimize the possibility of tumor formation.

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Acknowledgments

Oct4-GFP mESC and miPSC lines were generously donated by Professor Douglas Melton (Harvard University). HIF-1α−/− mESC were generously donated by Professor Peter Carmeliet (Katholieke Universiteit Leuven). Helpful discussion and technical assistance with aspects of this work were provided by Professor Susan Bonner-Weir (Joslin Diabetes Center). Helpful discussion and technical assistance with aspects of the cardiomyocyte aspects of this work were provided by Dr. Daryl Powers (MIT). Histopathological assessment was provided by Dr. Nicola M. A. Parry (MIT). The FACS was operated by Michael Jennings and Michele Griffin (MIT). Rose E. Yu, Giulia M. Dula, Julie Paul, and Bhargavi Chevva (MIT) provided technical assistance.

Author Contributions

JRM and CKC conceived the experimental design. JRM and JT conducted the in vitro experiments. JRM conducted the in vivo experiments. JRM and CKC wrote the manuscript. All authors edited and reviewed the manuscript.

Funding

This study was supported by a grant from the Juvenile Diabetes Research Foundation (41-2009-803).

Data Availability

All data associated with this study is presented in the paper. All cell lines used were acquired from third parties.

Conflict of interest

J.R.M. and C.K.C. are inventors on issued patent number 10,179,901 entitled “Methods and compositions for increased safety of stem cell-derived populations”. J.R.M. is a consultant for Sana Biotechnology. J.R.M. is co-founder of Salentra Biosciences. J.T. has no declarations.

Ethical Approval

All animal work was approved by the MIT Committee on Animal Care.

Informed Consent

There were no research subject participants. All authors consent to publication.

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Authors and Affiliations

  1. Division of Endocrinology, Metabolism and Lipid Research, Washington University School of Medicine, St. Louis, MO, 63110, USA

    Jeffrey R. Millman

  2. Department of Biomedical Engineering, Washington University in St. Louis, St. Louis, MO, 63130, USA

    Jeffrey R. Millman

  3. Department of Chemical Engineering, Massachusetts Institute of Technology, 77 Massachusetts Ave, Cambridge, MA, 02139, USA

    Jit Hin Tan & Clark K. Colton

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  1. Jeffrey R. Millman
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Correspondence to Jeffrey R. Millman or Clark K. Colton.

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Associate Editor Michael R. King oversaw the review of this article.

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Millman, J.R., Tan, J.H. & Colton, C.K. Mouse Pluripotent Stem Cell Differentiation Under Physiological Oxygen Reduces Residual Teratomas. Cel. Mol. Bioeng. 14, 555–567 (2021). https://doi.org/10.1007/s12195-021-00687-8

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