Phosphoproteomic analysis of human embryonic stem cells

Abstract

Protein phosphorylation, while critical to cellular behavior, has been undercharacterized in pluripotent cells. Therefore, we performed phosphoproteomic analyses of human embryonic stem cells (hESCs) and their differentiated derivatives. A total of 2546 phosphorylation sites were identified on 1602 phosphoproteins; 389 proteins contained more phosphorylation site identifications in undifferentiated hESCs, whereas 540 contained more such identifications in differentiated derivatives. Phosphoproteins in receptor tyrosine kinase (RTK) signaling pathways were numerous in undifferentiated hESCs. Cellular assays corroborated this observation by showing that multiple RTKs cooperatively supported undifferentiated hESCs. In addition to bFGF, EGFR, VEGFR, and PDGFR activation was critical to the undifferentiated state of hESCs. PDGF-AA complemented a subthreshold bFGF concentration to maintain undifferentiated hESCs. Also consistent with phosphoproteomics, JNK activity participated in maintenance of undifferentiated hESCs. These results support the utility of phosphoproteomic data, provide guidance for investigating protein function in hESCs, and complement transcriptomics/epigenetics for broadening our understanding of hESC fate determination.

Figure 1. Undifferentiated hESCs Expressed Markers of Pluripotency, whereas the Markers were Down-Regulated upon Differentiation

Cells were cultured to yield undifferentiated hESCs (“hESCs”), or differentiated hESC-derivatives (“derivs”) under feeder-free conditions by withdrawing bFGF and including 5 µM RA in the media for the final 4 days of culture. Nuclei were stained with DAPI (A, B; left column). (A) Cells were stained with antibodies detecting OCT4 (center column), and OCT4 and DAPI images were merged (right column). (B) Cells were stained with antibodies detecting SSEA-4 (center column), and SSEA-4 and DAPI images were merged (right column). All photomicrographs were at the same magnification. The scale bar represents 50 µM.

Figure 2. Number of Protein Phosphorylation Sites and Phosphoproteins Identified in hESCs and their Differentiated Derivatives, Prominence of Tyrosine Phosphorylation, Predicted Sub-Cellular Location of the Phosphoproteins and Phosphoprotein Categories

(A) Total number of non-redundant phosphorylation sites and (B) number of proteins with more phosphorylation site identifications in undifferentiated hESCs (line H1/WA01) (represented in red), RA-differentiated, H1-hESC-derivatives (represented in gold) or with a similar number of phosphorylation site identifications in the two cell populations (represented in gray). The percentage of the phosphorylation sites and phosphoproteins in each of the 3 groups of proteins is shown in parentheses. (C) Percentage of non-redundant Tyr phosphorylation sites, among the sites for which the phosphorylated residue could be defined as Ser, Thr or Tyr (94% of all sites), that were on proteins containing more identified sites in undifferentiated hESCs, differentiated hESC derivatives or that were on proteins with a similar number of identified sites between undifferentiated and differentiated cells. (D–F) The sub-cellular localization of the phosphoproteins is shown; those widely associated with more than one sub-cellular location are designated as variable. (G–I) Phosphoprotein categories, among those whose functions are known, are shown. The percentage of proteins with known functions are 45.8, 55.7 and 57.2% for proteins with more phosphorylation site identifications in undifferentiated hESCs, differentiated hESC-derivatives or a similar number of phosphorylation site identifications between the 2 cell populations, respectively. Each chart progresses from the protein category containing the most to the fewest entries. Abbreviations and definitions: transcript. reg. transcription regulator; enzyme, protein with enzymatic activity outside of the other categories; RNA meta., RNA-binding proteins and proteins participating in metabolic processes involving RNA; prot. degr., protein degradation; transport., transporter; apop. reg., apoptosis regulator; transmem. recep., transmembrane receptor; GEF, GAP, guanine nucleotide exchange factor, GTPase activating protein; cytoskel., proteins that are components of, closely associated with, or regulate cytoskeletal function; cell prolif., proteins participating in regulation of cellular proliferation and/or cell cycle progression; tum. sup., tumor suppressor; translat. reg., translation regulator; phosphoinos. sig., proteins participating in phosphoinositide signaling; gen. assem., genome assembly; GF, growth factor; cell adhes., proteins functioning in cell adhesion; telomere mainten., protein functioning in telomere maintenance; prom. differ., proteins promoting cellular differentiation; GF buffer, proteins regulating the availability of growth factors; comp. casc., complement cascade; nuc. receptor, ligand-dependent nuclear receptor; and hormone biosyn., hormone biosynthesis.

Figure 3. Protein Kinase Inhibitors Resulted in Differentiation of hESCs

(A) Expression of NANOG (Chambers et al., 2003) and OCT4 mRNAs was assessed by RT-PCR, in the presence of protein kinase inhibitors that resulted in differentiation of hESCs. Cells were cultured with 20 ng/ml of bFGF, and inhibitors (10 µM) were included in the cultures for the final 4 days. Inhibitor identities are indicated in the figure. Slower decline of OCT4 than NANOG was typically observed during hESC differentiation. GAPDH was an internal control. (B, C) Undifferentiated, vehicle-only control (B) and differentiated, KDR inhibitor-treated (C) cells are shown under imaging conditions indicated above the columns. All photomicrographs were at the same magnification, and the scale bar (bottom right) represents 50 µM (B, C). Abbreviations: i, inhibitor; uhESCs, undifferentiated hESCs.

Figure 4. PDGF and a Sub-threshold Concentration of bFGF Sustained Long-Term Culture of hESCs

(A). RT-PCR to amplify NANOG and OCT4 transcripts in long-term hESC cultures (>15 passages) in CDM containing 10 ng/ml of PDGF-AA and 4 ng/ml of bFGF (lane PDGF, bFGF4). Lanes bFGF20 or bFGF4 refer to 20 or 4 ng/ml of bFGF in the CDM for 4 days, respectively, in the absence of PDGF, following culture in 10 ng/ml of PDGF-AA and 4 ng/ml of bFGF for >15 passages. (B–D): Colony morphology, OCT4-staining and fluorescence-activated cell sorting (FACS) demonstrated that PDGF/bFGF in CDM maintained undifferentiated hESCs passaged >15 times. Imaging conditions or FACS analyses of SSEA-4 expression, detected via Cy3 conjugated secondary antibodies, is indicated above the columns, and the culture additives that were varied are indicated beside the rows. In FACS plots, dotted lines delineate boundaries of fluorescence intensity approximately indicative of cellular identity as undifferentiated hESCs (“uhESC”), and differentiated hESC-derivatives (“deriv”). Decline of SSEA-4 is incomplete in differentiated hESCs after 4 days (Figure 1). Following maintenance of the hESCs in CDM containing bFGF at 4 ng/ml and PDGF-AA at 10 ng/ml for >15 passages, cells were cultured for 4 d in CDM lacking PDGF and containing bFGF at 4 ng/ml (B) or 20 ng/ml (C), or in the continued presence of bFGF at 4 ng/ml and PDGF-AA at 10 ng/ml (D). All photomicrographs were at the same magnification, and the scale bar (bottom center panel) represents 100 µM (B–D).