Protein kinase C mediated extraembryonic endoderm differentiation of human embryonic stem cells

Abstract

Unlike mouse embryonic stem cells (ESCs), which are closely related to the inner cell mass, human ESCs appear to be more closely related to the later primitive ectoderm. For example, human ESCs and primitive ectoderm share a common epithelial morphology, growth factor requirements, and the potential to differentiate to all three embryonic germ layers. However, it has previously been shown that human ESCs can also differentiate to cells expressing markers of trophoblast, an extraembryonic lineage formed before the formation of primitive ectoderm. Here, we show that phorbol ester 12-O-tetradecanoylphorbol 13-acetate causes human ESCs to undergo an epithelial mesenchymal transition and to differentiate into cells expressing markers of parietal endoderm, another extraembryonic lineage. We further confirmed that this differentiation is through the activation of protein kinase C (PKC) pathway and demonstrated that a particular PKC subtype, PKC-δ, is most responsible for this transition.

Figure 1

TPA induces epithelial-mesenchymal transition of human embryonic stem cells (ESCs) with the concomitant downregulation of OCT4. (A): (I) Morphological changes induced by TPA. Human ESCs were treated with or without TPA for 48 hours. Scale bar = 100 µm. (II) Loss of E-cadherin during TPA-induced cell-cell dissociation. Cells stably expressing EGFP-E-cadherin fusion protein were treated with TPA. Before treatment, EGFP-E-cadherin was localized at the plasma membrane. After exposure to TPA, EGFP-E-cadherin disappeared from the membrane. Scale bar = 25 µm. (III) Disruption of tight junction during TPA-induced cell-cell dissociation. Scale bar = 100 µm. (IV) Expression of vimentin in TPA stimulated cells. Scale bar = 100 µm. (B): The expression of OCT4 mRNA in 0, 24, 48, and 96-hour TPA-treated cells examined by quantitative PCR. (C): Fluorescence-activated cell sorting analysis of OCT4 expression in 96-hour TPA-treated cells. Gray line: isotype control; black line: antibody staining. Abbreviations: EGFP, enhanced green fluorescent protein; % of MAX, % of maximum; TPA, 12-O-tetradecanoylphorbol 13-acetate; and PCR, polymerase chain reaction.

Figure 2

TPA differentiated human embryonic stem cells express markers of parietal endoderm. (A): Marker gene expression along TPA treatment time course. Heatmap produced in MeV program was used to display the changes of selected lineage markers in TPA-treated cells over untreated cells. The color of the tiles represented the fold change of each gene in a log2 scale. Upper panel: The expression of different embryonic and extraembryonic tissue markers. Lower panel: The expression of two extraembryonic endoderm sublineage markers. (B): Fluorescence-activated cell sorting (FACS) analysis of GATA6 expression in 0-hour and 48-hour TPA-treated cells. Gray line: isotype control; black line: antibody staining. (C): FACS analysis of THBD expression in 0-day and 5-day TPA-treated cells. (D): Identification of genes that exhibited earliest and strongest upregulation during TPA treatment time course. Hierarchical cluster analysis of the 685 genes that belonged to selected functional classes was conducted based on the Euclidean Distance. The fold changes were displayed in a log2 ratio. Abbreviations:% ofMAX,% of maximum; TPA, 12-O-tetradecanoylphorbol 13-acetate.

Figure 3

Protein kinase C (PKC)-specific inhibitor blocks TPA-induced differentiation. (A): Western blot analysis of phosphorylated MARCKS protein in 0-minute and 15-minute TPA or TPA plus GF 109203X-treated cells. (B): TPA-induced morphological changes were inhibited by PKC inhibitor. Scale bar = 200 µm. (C): TPA-induced downregulation of OCT4 was inhibited by PKC inhibitor. Abbreviations: MARCKS, myristoylated alanine-rich C-kinase substrate; TPA, 12-O-tetradecanoylphorbol 13-acetate.

Figure 4

PKC-δ plays dominant role in TPA-induced differentiation. (A): Morphologies of cells transfected with nontargeting or PKC isoform-specific siRNA followed by TPA treatment. (B): (I and II) Quantitative PCR analysis of OCT4 and SNAI2 in cells transfected with nontargeting or PKC isoform-specific siRNA followed by TPA treatment. (III) Quantitative PCR analysis of extraembryonic endoderm markers in PKC-δ knockdown cells. The data are presented as mean ± SD (n = 3). Cells were treated with 50 nM TPA at 48 hours after transfection for another 48 hours. The fold changes are normalized to non-TPA-treated cells. (C): (I) Overexpression of PKC-δ generated the same morphology as TPA treatment. Cells overexpressing EGFP was included as control. Scale bar = 100 µM. (II) Quantitative PCR analysis of lineage-specific markers in PKC-δ overexpressed cells. The data are normalized to EGFP overexpressed cells and presented as mean ± SD (n = 3). Abbreviations: EGFP, enhanced green fluorescent protein; PKC, protein kinase C; TPA, 12-O-tetradecanoylphorbol 13-acetate; and PCR, polymerase chain reaction.