Culture adaptation alters transcriptional hierarchies among single human embryonic stem cells reflecting altered patterns of differentiation.

Paul J Gokhale,David N Keys,Peter W Andrews,Jon K Sherlock,Srividya Dadi,Shamit Soneji,Neil J Harrison,Tariq Enver,Janice K Au-Young,Mark Jones,Kai Wang

doi:10.1371/journal.pone.0123467

Paul J Gokhale, David N Keys + Show 9 more

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https://doi.org/10.1371/journal.pone.0123467

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Journal: PloS one	Publication Date: Apr 14, 2015
Citations: 19	License type: CC BY 4.0

Affiliation: University of Sheffield, University of Oxford

Abstract

We have used single cell transcriptome analysis to re-examine the substates of early passage, karyotypically Normal, and late passage, karyotypically Abnormal (‘Culture Adapted’) human embryonic stem cells characterized by differential expression of the cell surface marker antigen, SSEA3. The results confirmed that culture adaptation is associated with alterations to the dynamics of the SSEA3(+) and SSEA3(-) substates of these cells, with SSEA3(-) Adapted cells remaining within the stem cell compartment whereas the SSEA3(-) Normal cells appear to have differentiated. However, the single cell data reveal that these substates are characterized by further heterogeneity that changes on culture adaptation. Notably the Adapted population includes cells with a transcriptome substate suggestive of a shift to a more naïve-like phenotype in contrast to the cells of the Normal population. Further, a subset of the Normal SSEA3(+) cells expresses genes typical of endoderm differentiation, despite also expressing the undifferentiated stem cell genes, POU5F1 (OCT4) and NANOG, whereas such apparently lineage-primed cells are absent from the Adapted population. These results suggest that the selective growth advantage gained by genetically variant, culture adapted human embryonic stem cells may derive in part from a changed substate structure that influences their propensity for differentiation.

Highlights

Populations of stem cells are subject to selection pressure for variants that favor self-renewal over the alternative fates of differentiation or cell death [1] [2]
One possibility is that the growth advantages of culture adapted ES cells can arise from alterations to the dynamics of the various lineage primed substates in which they may exist, in turn affecting their patterns of differentiation. To address this we have re-examined the SSEA3(+) and (-) subsets that we previously identified in the early passage, normal, and late passage, culture adapted, sublines of H7 human ES cells [4], to determine whether at a single cell level there is evidence of lineage priming, and whether this is influenced by culture adaptation in a way that might contribute to a growth advantage for the variant cells
The results of our present single cell transcriptome study are consistent with our earlier population based study of early passage, Normal, and late passage, Adapted, human ES cells, and confirm that the surface antigen SSEA3 defines substates, the dynamics of which are altered during culture adaptation [4]

Summary

Introduction

Populations of stem cells are subject to selection pressure for variants that favor self-renewal over the alternative fates of differentiation or cell death [1] [2]. Single Cell PCR in Culture Adapted Human Embryonic Stem Cells was a collaboration with Thermo Fisher (JKA-Y SVD DNK, JS) who provided reagents, helped design the study, performed experiments, collected and analysed data and helped prepare the manuscript

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Results

Conclusion