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. 2009 Nov 5;4(11):e7708.
doi: 10.1371/journal.pone.0007708.

A continuum of cell states spans pluripotency and lineage commitment in human embryonic stem cells

Shelley R Hough  1 , Andrew L Laslett, Sean B Grimmond, Gabriel Kolle, Martin F Pera
Affiliations

A continuum of cell states spans pluripotency and lineage commitment in human embryonic stem cells

Shelley R Hough et al. PLoS One. .

Abstract

Background: Commitment in embryonic stem cells is often depicted as a binary choice between alternate cell states, pluripotency and specification to a particular germ layer or extraembryonic lineage. However, close examination of human ES cell cultures has revealed significant heterogeneity in the stem cell compartment.

Methodology/principal findings: We isolated subpopulations of embryonic stem cells using surface markers, then examined their expression of pluripotency genes and lineage specific transcription factors at the single cell level, and tested their ability to regenerate colonies of stem cells. Transcript analysis of single embryonic stem cells showed that there is a gradient and a hierarchy of expression of pluripotency genes in the population. Even cells at the top of the hierarchy generally express only a subset of the stem cell genes studied. Many cells co-express pluripotency and lineage specific genes. Cells along the continuum show a progressively decreasing likelihood of self renewal as their expression of stem cell surface markers and pluripotency genes wanes. Most cells that are positive for stem cell surface markers express Oct-4, but only those towards the top of the hierarchy express the nodal receptor TDGF-1 and the growth factor GDF3.

Significance: These findings on gene expression in single embryonic stem cells are in concert with recent studies of early mammalian development, which reveal molecular heterogeneity and a stochasticity of gene expression in blastomeres. Our work indicates that only a small fraction of the population resides at the top of the hierarchy, that lineage priming (co-expression of stem cell and lineage specific genes) characterizes pluripotent stem cell populations, and that extrinsic signaling pathways are upstream of transcription factor networks that control pluripotency.

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Conflict of interest statement

Competing Interests: The authors have declared that no competing interests exist.

Figures

Figure 1
Figure 1. Gene expression in immunologically defined subsets of human embryonic stem cells.
A. Fractionation of HES 3 or H9 cells by flow cytometry according to the levels of expression of cell surface markers (GCTM-2, pericellular matrix proteoglycan, and CD9). Cells were separated into High, Mid, Low, and Negative subpopulations as shown. B. Heat map showing gene expression in the four subpopulations isolated as shown in A above. Data are for cell line HES2 at passages 48, 49 and 50 (1,2 and 3) respectively. Subpopulations labeled as follows: P7, High; P6, Mid; P5, Low; P4, Negative. Results for 3752 genes showing a B-statistic greater than zero between P4 and P7 in all experiments are depicted. C. Patterns of expression of selected pluripotency genes in subpopulations isolated as shown in A above. Results are shown for HES-2 at passage 48 (top), passage 49 (middle) and passage 50 (bottom).
Figure 2
Figure 2. Patterns of gene expression in single cells vary according to position within a colony.
Single cell Q-RTPCR for the genes indicated was carried out on 87 cells isolated from different regions of ES colonies. (A) Expression of 5 stem marker genes in single cells isolated from the edge (E), mid (M) or adjacent center (C) region of HES2 colonies. Data are normalized to cyclophilin and expressed as ΔCt (marker gene–cyclophilin). Marker genes expressed at very low or undetectable levels (Ct 35–40) fall above the upper dotted line. Each point represents a Q-RTPCR measurement on a single cell; eighty seven cells were analyzed. Cripto is TDGF-1. (B) Percent of isolated single cells expressing stem or lineage markers according to location with the colony. Endoderm markers were GATA-4, GATA-6, and SOX-17; Mesoderm markers were GSC, MIXL1, and T; Neural markers were PAX-6 and LHX-2. (C) Incidence of co-expression of 0–5 stem marker genes (identified in panel A) in single cells isolated from three colony regions.
Figure 3
Figure 3. Heterogeneity in gene and protein expression in HES cells.
Single cell Q-RTPCR for the genes indicated was carried out on cells separated by flow cytometry on the basis of cell surface marker expression. (A) Expression of 5 stem marker genes in single HES3 cells isolated by FACS into four populations according to levels of expression of GCTM2 and CD9 surface antigens (H = HIGH, M = MID, L = LOW, N = NEG). Data are normalized to cyclophilin and expressed as ΔCt (marker gene–cyclophilin). Marker genes expressed at very low or undetectable levels (Ct 35–40) fall above the upper dotted line. Each point represents a Q-RTPCR measurement on a single cell; one hundred cells were analyzed. Cripto is TDGF-1. (B) Percent of sorted single HES3 cells in (A) expressing stem or lineage markers according to level of surface marker expression. Endoderm markers were GATA-4, GATA-6, and SOX-17; Mesoderm markers were GSC, MIXL1, and T; Neural markers were PAX-6 and LHX-2. (C) Incidence of co-expression of 0–5 stem marker genes (identified in panel A) in sorted single HES3 cells. (D) Detection of lineage marker expression in single sorted HES3 cells. Lineage markers were the same as those in 3B. (E) Immunolocalization of OCT and DNMT3b, (F) OCT4 and Nanog, and (G) OCT4 and GATA6 in HES colonies in culture. Scale bars represent 100 µM.
Figure 4
Figure 4. Biological fate of HES3 subpopulations isolated by FACS.
(A) Representative images of cell types emerging following culture of sorted subpopulations. After 17 days, cells were fixed and stained for expression of GCTM2 and counterstained with hematoxylin (a) stem colony, (b) mixed or abortive colony, (c) cystic colony, (d) GCTM2-negative pebble-like cells. Scale bars equal 100 µM. (B) Image of cell culture wells following staining for GCTM2 at day 17. (C) Incidence of stem colony formation generated from sorted subpopulations in culture. Colony phenotypes were tabulated across three replicate experiments. (D) Neuronal-like outgrowths emerge within 72 hr following transfer of clusters of small GCTM-2 negative pebble-like cells (panel A–d above), to conditions supporting neural development. Scale bar equals 100 µM. (E) Immunocytochemical localization of β-III tubulin on neuronal outgrowths. Scale bars equal 50 µM.
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References

    1. Murry CE, Keller G. Differentiation of embryonic stem cells to clinically relevant populations: lessons from embryonic development. Cell. 2008;132:661–680. - PubMed
    1. Boyer LA, Lee TI, Cole MF, Johnstone SE, Levine SS, et al. Core transcriptional regulatory circuitry in human embryonic stem cells. Cell. 2005;122:947–956. - PMC - PubMed
    1. Meshorer E, Yellajoshula D, George E, Scambler PJ, Brown DT, et al. Hyperdynamic plasticity of chromatin proteins in pluripotent embryonic stem cells. Dev Cell. 2006;10:105–116. - PMC - PubMed
    1. Bernstein BE, Mikkelsen TS, Xie X, Kamal M, Huebert DJ, et al. A bivalent chromatin structure marks key developmental genes in embryonic stem cells. Cell. 2006;125:315–326. - PubMed
    1. Enver T, Pera M, Peterson C, Andrews PW. Stem cell states, fates, and the rules of attraction. Cell Stem Cell. 2009;4:387–397. - PubMed

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