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. 2017 Jun 1;20(6):874–890.e7. doi: 10.1016/j.stem.2017.02.014

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© 2017 The Authors

This is an open access article under the CC BY license (http://creativecommons.org/licenses/by/4.0/).

PMC Copyright notice

An Antibody Panel to Distinguish between Naive-State and Primed-State Human PSCs

(A) A list of antibodies that are combined to form a multiplexed panel. The information in brackets shows the fluorophore conjugation of each antibody. See Table S4 for antibody details and Table S5 for flow cytometer parameters.

(B) Flow cytometry contour plots of pairwise antibody combinations. The primed-specific marker CD57 is on the y axes, and different naive-specific (top) and primed-specific (bottom) markers are on the x axes. Primed (red) and t2i/L+PKCi-cultured naive (blue) H9 PSCs are shown for each antibody combination. See Figure S4A for flow cytometry plots that exemplify a typical complete gating scheme for H9 naive PSCs. Note that CD77 shows a greater degree of heterogeneity in naive PSCs compared with the other markers but is still useful when used in combination.

(C) FlowSOM visualization of flow cytometry data for all antibodies in the panel. An unsupervised self-organizing map arranges the cells into clusters (represented by circles) according to similarities in their cell surface protein expression profiles (right). Overlaying the identity of the cell type within each cluster reveals a clear separation of naive (blue) and primed (red) populations. The heatmap panels (left) show the expression level of each cell surface protein in the cell clusters. Clusters are arranged in the same position as for the minimal spanning tree of the self-organizing map. See Figures S4B and S4C for analyses of additional ESC and iPSC lines.

(D) Flow cytometry contour plots show that the identified panel of state-specific markers can discriminate between primed and naive PSCs when the cells are mixed together. Left: the expression levels of two naive-specific proteins (CD130 and CD75) in primed (top) and naive (bottom) H9 PSCs. Top right: the expression levels of the same proteins in a sample of 90% primed + 10% naive PSCs. Bottom right: CD75⁺/CD130⁺ cells do not express the primed-specific markers CD57 and CD24. Gates were drawn based on unstained, live, human PSCs.