Standardization of the Teratoma Assay for Analysis of Pluripotency of Human ES Cells and Biosafety of Their Differentiated Progeny

Michal Gropp,Benjamin E Reubinoff,Miri Gov,Gilad Vainer,Maria Idelson,Hanita Khaner,Juri Kopolovic,Yaniv Gil,Limor Matzrafi,Naomi B Zak,Vitali Shilo,Dimas Tadeu Covas

doi:10.1371/journal.pone.0045532

Michal Gropp, Benjamin E Reubinoff + Show 10 more

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https://doi.org/10.1371/journal.pone.0045532

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Journal: PloS one	Publication Date: Sep 25, 2012
Citations: 127	License type: CC BY 4.0

Affiliation: Hadassah Medical Center, Emcure Pharmaceuticals (India)

Abstract

Teratoma tumor formation is an essential criterion in determining the pluripotency of human pluripotent stem cells. However, currently there is no consistent protocol for assessment of teratoma forming ability. Here we present detailed characterization of a teratoma assay that is based on subcutaneous co-transplantation of defined numbers of undifferentiated human embryonic stem cells (hESCs) with mitotically inactivated feeder cells and Matrigel into immunodeficient mice. The assay was highly reproducible and 100% efficient when 100,000 hESCs were transplanted. It was sensitive, promoting teratoma formation after transplantation of 100 hESCs, though larger numbers of animals and longer follow-up were required. The assay could detect residual teratoma forming cells within differentiated hESC populations however its sensitivity was decreased in the presence of differentiated cells. Our data lay the foundation, for standardization of a teratoma assay for pluripotency analysis. The assay can also be used for bio-safety analysis of pluripotent stem cell-derived differentiated progeny.

Highlights

Human embryonic stem cells are pluripotent cells derived from preimplantation embryos
Defining uniform standards for the assessment of the pluripotency of new putative pluripotent stem cell lines is of major importance
We show that the assay was highly reproducible and 100% efficient when 16105256105 Human embryonic stem cells (hESCs) were transplanted, with decreasing efficiency when smaller numbers of hESCs were transplanted

Summary

Introduction

Human embryonic stem cells (hESCs) are pluripotent cells derived from preimplantation embryos These cells can self-renew for long periods, and have the potential to differentiate into any cell type [1,2]. In the consensus guidance for the banking, testing and distribution of hESC lines, published by the International Stem Cell Banking Initiative (ISCBI), the teratoma formation assay is defined as the ‘‘gold standard’’ for pluripotency [6]. In accordance with these guidelines many publications on the derivation of new hESC lines include characterization of the line’s pluripotency by a teratoma formation assay. Recently Mueller and colleagues published a call for the standardization of the teratoma formation assay [8]

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