Ethanol alters the balance of Sox2, Oct4, and Nanog expression in distinct subpopulations during differentiation of embryonic stem cells

Abstract

The transcription factors Sox2, Oct4, and Nanog regulate within a narrow dose-range embryonic stem (ES) cell pluripotency and cell lineage commitment. Excess of Oct4 relative to Sox2 guides cells to mesoendoderm (ME), while abundance of Sox2 promotes neuroectoderm (NE) formation. Literature does not address whether ethanol interferes with these regulatory interactions during neural development. We hypothesized that ethanol exposure of ES cells in early differentiation causes an imbalance of Oct4 and Sox2 that diverts cells away from NE to ME lineage, consistent with the teratogenesis effects caused by prenatal alcohol exposure. Mouse ES cells were exposed to ethanol (0, 25, 50, and 100 mM) during retinoic acid (10 nM)-directed differentiation to NE for 0-6 days, and the expression of Sox2, Oct4, and Nanog was measured in single live cells by multiparametric flow cytometry, and the cellular phenotype was characterized by immunocytochemistry. Our data showed an ethanol dose- and time-dependent asymmetric modulation of Oct4 and Sox2 expression, as early as after 2 days of exposure. Single-cell analysis of the correlated expression of Sox2, Oct4, and Nanog revealed that ethanol promoted distinct subpopulations with a high Oct4/Sox2 ratio. Ethanol-exposed cells differentiated to fewer β-III tubulin-immunoreactive cells with an immature neuronal phenotype by 4 days. We interpret these data as suggesting that ethanol diverted cells in early differentiation from the NE fate toward the ME lineage. Our results provide a novel insight into the mode of ethanol action and opportunities for discovery of prenatal biomarkers at early stages.

FIG. 1.

(A) Schematic of experimental design. Embryonic stem (ES) cells were differentiated to neuroectoderm (NE) with retinoic acid (RA) (10 nM). Ethanol was added to the culture medium during differentiation at different doses (0, 25, 50, and 100 mM) and for various exposure periods (0, 2, 3, 4, and 6 days). The protein level of the lineage-specific transcription factors (TF) Sox2, Oct4, and Nanog, cell size, and viability (L/D: Live vs. Dead) were assessed by multiparametric flow cytometry in 100,000 cells. The cell size was determined by forward scatter (FSC), and used to normalize the expression of transcription factors. Live cells (green) were identified with a fixable dye that separates these from dead cells (purple) and cell debris (blue), as shown in the viability panel. Scaled data of correlated protein expression of Sox2, Oct4, and Nanog in 50,000 live cells were subjected to multivariate distribution and expression sector analysis. Eight sectors, S1–S8, were defined according to expression of Sox2, Oct4, and Nanog, where S1 and S8 represent, respectively, the cells with negative and positive expression (see Fig. 2A Table inset). Cell proliferation, cell cycle, and apoptosis were evaluated by flow cytometry in separate measurements. Pluripotency was assessed by staining for alkaline phosphatase (ALP), and the phenotype was characterized by immunocytochemistry of Sox2, Oct4, Nanog, SSEA-1, nestin, and β-III tubulin. (B) Correlated expression of Sox2, Oct4, and Nanog in differentiated cells exposed to ethanol (0, 25, 50, and 100 mM) for 3 days. The protein level was expressed as the mean fluorescence intensity (MFI) in samples (filled bars) and isotypes (open bars). *P (Oct4)=0.0003092 (100 vs. 0 mM ethanol), 0.0003463 (100 vs. 25 mM ethanol), and 0.0019532 (100 vs. 50 mM ethanol). (C) The cell size of 3-day differentiated cells exposed as a function of the ethanol dose. *P (FSC)=0.0000580 (100 vs. 0 mM ethanol), 0.0001183 (100 vs. 25 mM ethanol), 0.0061131 (100 vs. 50 mM ethanol), and 0.0065292 (50 vs. 0 mM ethanol). (D) Correlated expression of Sox2, Oct4, and Nanog normalized to the cell size in differentiated cells exposed to ethanol. Values shown are ×10⁴. *P= 0.0003092 (100 vs. 0 mM ethanol), 0.0003463 (100 vs. 0 mM ethanol), and 0.0019532 (100 vs.50 mM ethanol). (E) Oct4/Sox2 geometric mean ratio as a function of ethanol concentration. Transcription factor expression was scaled to 50,000 live cells across samples. Values in (B–E) are mean±SEM, n=3 biological replicates.

FIG. 2.

Redistribution of transcription factors Sox2, Oct4, and Nanog in 3-day differentiated cells as a function of ethanol. (A) Ethanol promotes in a dose-dependent manner an increase of S8 cells that express all three transcription factors (upper panel). Note that S8 and S1 are the two major cell subpopulations. Definition of cell subpopulations is presented in Table inset. The distribution of the minor cell subpopulations S2–S7 at different ethanol doses is shown (lower panel). (B) Correlation of expression of Sox2, Oct4, and Nanog in S8 cells with cell counts shows Oct4 changes in comparison to Sox2. Transcription factors expression (×10⁴) was normalized to cell size. (C) The Oct4/Sox2 ratio in S8 cells normalized to control cells increased in an ethanol-dependent manner. (D) The population of Oct4-positive (S3+S4+S7+S8) cells increased at the expense of Oct4-negative (S1+S2+S5+S6) cells with increasing ethanol doses. *P=0.0000000 (Ethanol 0 and 100 mM: Oct4⁺ vs. Oct4⁻); *P=0.0000002 (Ethanol 25 mM: Oct4⁺ vs. Oct4⁻). Total cell numbers were normalized to 50,000 across samples in (A–D); Values shown are mean values±SEM, n=3 biological replicates. (E) Multivariate plots of expression profiles of Sox2, Oct4, and Nanog and their binary and tertiary combinations across 50,000 cells at different ethanol doses. The distribution of Sox2 and Oct4 (solid lines) in comparison to the corresponding isotypes (dotted lines) are illustrated by histograms on the plot walls. Bivariate correlation of Sox2 and Oct4 represented in 10% contours (red) at the bottom of the plot, along with corresponding isotypes contours (black). Nanog expression is depicted in color scale. The subpopulation of high Sox2^H-Oct4^H-Nanog^H cells in trivariate graphs, which grows with increasing ethanol dose, is indicated by arrow. Representative data of n=3 biological replicates per time point and treatment are shown. Color images available online at www.liebertpub.com/scd

FIG. 3.

Cell proliferation, cell cycle, and apoptosis in differentiated cells exposed to ethanol (100 mM) for 4 days. (A) Cell proliferation was measured by incorporation of a fluorescent uridine analog in 30,000 cells. (B) Quantification of cell cycle phases based on measurements is shown in (A). *P=0.0000000 (G0/G1: Control vs. ES, and Ethanol vs. ES; S: Control vs. ES, and Ethanol vs. ES). (C) Cell cycle profile of control (blue) and ethanol-exposed (red) cells, compared to that of ES cells (green). Cells were stained with PI before flow cytometry analysis. Inset Table shows quantification of cell cycle phases. (D) Apoptosis measured by Annexin V-PI flow cytometry assay. The percentage of apoptotic cells (Q3), dead cells (Q2), live cells (Q4), and damaged cells (Q1) is indicated in individual quadrants. Representative plots are shown in A, C, D. Values are mean±SEM, n=3 biological replicates. Color images available online at www.liebertpub.com/scd

FIG. 4.

Time course of Sox2, Oct4, and Nanog expression during ES cell differentiation (0–4 days) exposed to ethanol (100 mM). (A) Higher expression of Sox2, Oct4, and Nanog during differentiation in ethanol-exposed cells normalized to that in control cells. (B) Increased Oct4/Sox2 ratio of differentiated ethanol-exposed cells scaled to control cells. (C) Multivariate plots of single-cell expression of transcription factors in ethanol-exposed (upper panel) and control cells (lower panel) as a function of differentiation time (other conditions described in Fig. 2E). Arrows point out the subpopulation of Sox2^H-Oct4^H-Nanog^H cells in trivariate graphs (yellow peak). Data shown are representative of n=3 biological replicates per time point and treatment. (D) Relative increase with ethanol (red) compared to control (blue) of Oct4-positive cells (closed markers, solid lines) as a function of differentiation time, at the expense of Oct4-negative cell populations (open markers, dotted lines), is reflected in (E) the changes of the S8 and S1 cells, which account for the bulk of Oct4-positive and Oct4-negative cell subpopulations, respectively. Total cell numbers were normalized to 50,000 across samples in A–E. Values in A, B, D, E are mean±SEM, n=3 biological replicates. Color images available online at www.liebertpub.com/scd

FIG. 5.

Cell count distribution and expression profiles of Sox2 and Oct4 in multivariate sectors during ES cell differentiation (2 and 4 days) in the presence or absence of ethanol (0 and 100 mM). (A) Cell count distribution in sectors S1–S8, which are defined in Fig. 2A Table Inset. The sectors are organized as two groups, Oct4-negative and Oct4-positive, separated by a vertical line. The dotted lines connect the separatrix over differentiation time and ethanol exposure indicating relative changes in cell counts between Oct4-negative vs. Oct4-positive groups. Trajectories of (B) Oct4 and (C) Sox2 relate expression and cell counts in each sector. Data values are mean±SEM for both axes, n=3 biological replicates. Data on all sectors are presented, and sectors S6 and S8 contain the majority of cells with MFI values above isotype (Oct4=414±13; Sox2=259±120) and are highlighted in the corresponding boxes. (D) Time course of the Oct4/Sox2 ratio in S8 and S1 cells with ethanol exposure values normalized to control. The ratios were log-transformed and the Ethanol versus Control differences were computed in each group. Data were subsequently normalized to average of log-ratios in the ES group. We estimated 95% confidence intervals and P-values based on Student's t-tests. *P=0.0022 (day 2 vs. day 0) and 0.0282 (day 4 vs. day 0). Color images available online at www.liebertpub.com/scd

FIG. 6.

Immunocytochemical detection of transcription factors and pluripotency and neural lineage markers during differentiation in the presence of ethanol (0 and 100 mM). (A) Pluripotency staining with ALP during RA-directed differentiation (0 and 3 days) in the presence of ethanol (0 and 100 mM), and quantitative analysis of residual colonies. The y-axis represents the percentage of ALP-stained colonies per field. Data were obtained from an average of 305 fields. *P=0.0142 (Ethanol vs. Control) was based on Student's t-test. (B) Expression of Sox2, Oct4, and Nanog and pluripotency marker SSEA-1 in ES cells, with DAPI counterstaining to visualize the nuclei. (C) Expression of nestin in neural progenitors and β-III tubulin (Tuj1) in neurons during differentiation (4 and 6 days) in the presence or absence of ethanol (0 and 100 mM). Color images available online at www.liebertpub.com/scd

FIG. 7.

Model of the reprogrammed regulatory network of Oct4, Sox2, and Nanog that may lead to aberrant neuroectodermal fate with ethanol. The inherently heterogeneous ES cell population is able to interconvert between a fully pluripotent (high Nanog) and a metastable lineage-primed state (low Nanog). Developmental and environmental signals, such as ethanol, control Oct4, and Sox2 levels and cell fate decisions. In the presence of NE-inductive RA signal, the high Sox2 level relative to Oct4 induces cells to the NE fate. Ethanol favors a higher Oct4/Sox2 ratio that diverts cells to the ME fate. The wavy line indicates lineage valleys and in-between barriers that do not permit spontaneous interconversion from one lineage to another.