Four steps for validating stem cells

The key to utilizing stem cells for regenerative medicine and tissue engineering lies in a property of theirs called pluripotency. This refers to the cells’ ability to differentiate into different types of cells. This means that we need to be able to reliably obtain, culture and maintain fully pluripotent stem cells. It has been difficult to generate human embryonic stem cells at the earliest stage of pluripotency, in what is named “ground” or “naïve” state, whereas this is readily done with mouse cells. The labs of Rudolf Jaenisch at MIT, Joe Ecker at the Salk Institute, and Didier Trono at EPFL have now developed a four-step process for determining accurate signatures of human embryonic stem cells and relating them to precise developmental stages. The work, a first for human embryonic stem cells, is published in Cell Stem Cell.

The first criterion involves a rigorous assay to see how much the naïve stem cells contribute to a mouse-human embryo. If the resulting organism (a so-called “chimera”) contains any human DNA, it signals successful engraftment of the stem cells.

The second criterion looks at the expression profile of 4.5 million RNA biomarkers called “transposable elements”, which are genetic units that can move around the genome – in fact, they make up half of the human genome. Because they can cause dangerous mutations by inserting themselves inside genes, transposable elements are actually suppressed in the early developmental stages of the embryo. However, transposable elements also regulate gene expression, and are essential in maintaining the organism’s homeostasis. The researchers demonstrated that profiling which transposable elements are active in the stem cells is an extremely sensitive and highly reproducible indicator of their pluripotency stage.

The third criterion focuses on DNA methylation state of the cells, which is lower in the naïve compared to the primed state. Finally, the fourth criterion is the epigenetic state of the X chromosome in female naïve cells, which resembles that found in the human pre-implantation embryo.

The study provides a roadmap for broadly evaluating stage, state and quality of human pluripotent cells, and can overcome current limitations with using such cells in research and clinical applications. Based on this work, the researchers have developed a startup project named Cellphmed. The company’s mission is to streamline the experimental work of the second criterion, which involves the transcriptional profiling of transposable elements to generate human cell markers for broad research and clinical applications.

This work involved a collaboration between EPFL’s School of Life Sciences, MIT’s Whitehead Institute for Biomedical Research (Cambridge, MA), and the Salk Institute for Biological Studies (La Jolla, CA). It was funded by the Simons Foundation, the National Institutes of Health (NIH), the Swiss National Science Foundation (SNSF), the European Research Council (ERC), the Howard Hughes Medical Institute, the Gordon and Betty Moore Foundation, the Mary K. Chapman Foundation, the Wellcome Trust, a Foundation Bettencourt Award, the Association pour la Recherche sur le Cancer (ARC), and the Fonds de la Recherche en Santé du Québec.

Reference

Theunissen TW, Friedli M, He Y, Planet E, Oneil R, Markoulaki S, Pontis J, Wang H, Iouranova A, Imbeault M, Duc J, Cohen MA, Wert KJ, Castanon RG, Zhang Z, Huang Y, Nery JR, Drotar J, Lungjangwa T, Trono D, Ecker JR, Jaenisch R. Molecular Criteria for Defining the Naive Human Pluripotent State. Cell Stem Cell 14 July 2016. DOI: 10.1016/j.stem.2016.06.011