Self-renewal of human embryonic stem cells requires insulin-like growth factor-1 receptor and ERBB2 receptor signaling

Abstract

Despite progress in developing defined conditions for human embryonic stem cell (hESC) cultures, little is known about the cell-surface receptors that are activated under conditions supportive of hESC self-renewal. A simultaneous interrogation of 42 receptor tyrosine kinases (RTKs) in hESCs following stimulation with mouse embryonic fibroblast (MEF) conditioned medium (CM) revealed rapid and prominent tyrosine phosphorylation of insulin receptor (IR) and insulin-like growth factor-1 receptor (IGF1R); less prominent tyrosine phosphorylation of epidermal growth factor receptor (EGFR) family members, including ERBB2 and ERBB3; and trace phosphorylation of fibroblast growth factor receptors. Intense IGF1R and IR phosphorylation occurred in the absence of MEF conditioning (NCM) and was attributable to high concentrations of insulin in the proprietary KnockOut Serum Replacer (KSR). Inhibition of IGF1R using a blocking antibody or lentivirus-delivered shRNA reduced hESC self-renewal and promoted differentiation, while disruption of ERBB2 signaling with the selective inhibitor AG825 severely inhibited hESC proliferation and promoted apoptosis. A simple defined medium containing an IGF1 analog, heregulin-1beta (a ligand for ERBB2/ERBB3), fibroblast growth factor-2 (FGF2), and activin A supported long-term growth of multiple hESC lines. These studies identify previously unappreciated RTKs that support hESC proliferation and self-renewal, and provide a rationally designed medium for the growth and maintenance of pluripotent hESCs.

Figure 1

CM triggers IGF1R/IR and ERBB-family tyrosine phosphorylation in hESCs. (A) RTK array analysis of H1 cells stimulated by CM for 5 or 15 minutes. Tyrosine phosphorylation of IGF1R/IR and lower-intensity phosphorylation of ERBB (EGFR) family members was observed consistently. (B) RTK analysis of H1 cells stimulated by NCM and CM for 15 minutes. Increased ERBB3 phosphorylation was observed with CM stimulation. (C) RT-PCR analysis of IGF1, IGF2, IGF1R, and IR expression in H1 cells, and IGF1 and IGF2 expression in MEFs; ERBB1-4 expression in BG03 cells (and BG01 and BG02 cells; Figure S4); and HRG and ADAM19 expression in MEFs. Full-length gels are presented in Figure S4 online. (D) Flow cytometry of IGF1R, IR, ERBB2, and ERBB3 expression in H1 cells.

Figure 2

Disruption of IGF1R and ERBB2 signaling inhibits hESC self-renewal. (A) Treatment of H1 cells with the A12 anti-IGF1R blocking antibody led to decreased IGF1R expression on the cell surface as measured by flow cytometry. (B) Colony counting showed that the A12 antibody inhibited H1 cell proliferation (left). Arrows indicate that cultures were passaged on day 3 and 6. Cumulative colony counts increased in the presence of control antibody but not in the presence of A12. A12 also induced hESC differentiation (right) as measured by colony morphology (also see Figure S2). Error bars here and in panels C-H,J are standard deviation (SD). Flow cytometry for (C) SSEA-3 expression confirmed A12 treatment caused increased differentiation, but (D) not an increase in apoptosis. (E) Significant reduction of IGF1R mRNA in the GFP⁺ population of cultures transfected with an IGF1R-targeted shRNA, compared with either the GFP^- population, or GFP⁺ cells transfected with a control shRNA. (F) Decline in the percentage of GFP⁺ cells in H1 cultures (left) transduced with a lentiviral vector containing a shRNA targeting IGF1R, rather than a control shRNA. Middle panel shows flow cytometry profiles that indicate stable expression of IGF1R and percentages of cells that are GFP⁺ in control shRNA–transduced cultures on days 1, 12, and 22, while cultures transduced with the IGF1R-targeted shRNA vector exhibited a decline in IGF1R expression and a reduction in GFP⁺ and GFP⁺/IGF1R⁺ cells. Right panel shows the average percentage of GFP⁺ cells at days 15, 19, and 22 was significantly lower in the IGF1R-targeted shRNA transduced culture. (G) A total of 50 μM AG825 inhibited proliferation of BG02 hESCs growing in CM. Triplicate cell counts from 2 independent experiments are shown. … and - - - indicate pretreatment cell counts from experiments 1 and 2, respectively. (H,I) Increasing concentrations of AG825 caused a dose-dependent reduction in the proportion of H1 hESCs in the G₁ phase of the cell cycle, and (J) a moderate dose-dependent rise in apoptosis.

Figure 3

Culture of hESCs in a defined medium designed to stimulate IGF1R/IR and ERBB2/3 signaling. (A) Low (4× objective) and high (40× objective) magnification phase contrast images of morphologically undifferentiated BG02 hESCs growing in DC-HAIF. Overlapping fields (dashed black borders) were aligned to image a representative large colony. Scale bar equals 100 and 25 μm, respectively. (B) Colony counting for serial passaging of CyT49 hESCs in different combinations of growth factors. Starter cultures were growing on MEFs in NCM and the proportion of undifferentiated (■) and differentiated (□) colonies at each stage are indicated. A combination of all 4 factors was necessary to enable long-term maintenance of hESCs. Similar results were obtained with repeated experiments and other hESC lines. Panel B and C abbreviations: H indicates 10 ng/mL HRG1β; A, 10 ng/mL ActA; I, 200 ng/mL LR³-IGF1; and F, 8 ng/mL FGF2. (C) Cell-counting analysis of the role of IGF1 and HRG in hESC proliferation using BG02 cells (left panel). The mean cell number/well before the different growth factor combinations were applied on day 1 is indicated (…). Cultures were disaggregated and counted on day 7, and the mean and standard deviation were plotted. Right panel shows OCT4/DAPI immunostaining of a duplicate repeated experiment (4× objective), which demonstrated that IGF1 and HRG significantly increased the proportion of OCT4⁺ cells compared with ActA/FGF2 conditions. Scale bar equals 50 μm. (D) Direct comparison of CM and DC-HAIF growth conditions with serial passaging. BG03 cells growing on MEFs were passaged to CM and DC-HAIF conditions in parallel (p0 plates). Triplicate cell counts were performed at p1 to p3 and the split ratio–corrected total cell number was plotted (mean ± SD). Split ratios were (CM, DC-HAIF): p0 (1:3, 1:3); p1 (1:2, 1:3); and p2 (1:2, 1:2). Similar results were obtained with repeat experiments. (E) Maintenance of markers of undifferentiated cells in BG02 DC-HAIF p5 cells compared with BG02 in CM by RT-PCR. (F) Positive immunofluorescence of hESC markers in BG02 DC-HAIF p5 cells (10× objective). Scale bar equals 50 μm. (G) Representative G-banding of BG02 DC-HAIF p26 cells. (H) RTK blotting analysis of BG01 DC-HAIF hESCs starved of growth factors overnight; starved, then pulsed with DC-HAIF for 15 minutes; or steady-state cultures are shown (left panel). The mean and range of normalized relative intensity is plotted (right panel).

Figure 4

Comparison of the transcriptome of hESCs maintained in DC-HAIF or CM. (A) Venn diagram of the distribution of transcripts detected using high-density Illumina Sentrix Human-6 Expression Beadchips containing 47 296 transcript probes in BG02 cells maintained in CM (64 passages) or DC-HAIF (10 or 32 passages in defined medium). A large proportion of the expressed transcripts were detected in all samples. (B) Scatterplot analysis demonstrated that the transcriptional profile of BG02 DC-HAIF p32 cells was highly similar to that of BG02 cells maintained in CM (top panel), and was not substantially altered in early- and late-passage cultures in DC-HAIF (bottom panel). Correlation coefficients (R²) were generated using all detected transcripts with an expression level of more than 0 (black and blue dots), or with transcripts exhibiting a detection confidence level of more than 0.99 (R² select, blue dots). Red lines delineate the mean and limits of a 2-fold difference. (C) Hierarchic clustering dendrogram of relative gene expression in different populations generated using the Beadstudio software (Illumina, San Diego, CA). Early- and late-passage BG02 cells maintained in DC-HAIF clustered tightly (approximately 0.0075) and retained a close similarity to BG02 and BG03 cells maintained in CM (approximately 0.037). Previous analyses showed that different undifferentiated hESC lines clustered tightly (approximately 0.032) compared with differentiated embryoid body populations (approximately 0.0875) or fibroblasts (approximately 0.160).

Figure 5

In vivo and in vitro differentiation of hESCs maintained in DC-HAIF. (A) Analysis of teratomas from BG02 DC-HAIF p25 cells demonstrated pluripotent differentiation potential to ectoderm, mesoderm, and endoderm. NSE indicates neuron-specific enolase; SMA, smooth-muscle actin; and pCK, pan-cytokeratin. Scale bars equal 20 μm (NSE, p63, 100× oil objective), 50 μm (SMA, Ducts, pCK, 20× objective), and 200 μm (Bone, 10× objective). (B) Differentiation of BG02 DC-HAIF p5 cells to ectoderm (βIII tubulin⁺), mesoderm (SMA⁺), and endoderm (alphafetoprotein⁺ [αFP]) lineages in embryoid bodies (20× objective, same scale as panel D). (C,D) Directed differentiation of BG02 DC-HAIF p48 cells (HAIF) to definitive endoderm (d3) and foregut endoderm (d6) using defined medium differentiation conditions (Document S1). (C) qPCR analyses showed down-regulation of OCT4 expression and minimal PAX6 or CDX2 expression in differentiated samples, suggesting differentiation of pluripotent cells and lack of substantial neuroepithelial or trophectodermal differentiation. The generation of definitive endoderm at day 3 was confirmed by up-regulation of SOX17, CXCR4, and CER expression, and foregut endoderm at day 6 by up-regulation of FOXA2, HNF1β, and HNF4α. Similar results were observed with CyT49 cells. Error bars are plus or minus SD. (D) Immunostaining analyses confirmed homogenous expression of OCT4 and lack of SOX17 expression in undifferentiated cultures (HAIF). After 3 days, most cells express SOX17 with only pockets of OCT4⁺ cells remaining (d3). After 6 days, the differentiation to foregut endoderm was confirmed by expression of HNF1β and HNF4α (d6). Only rare OCT4⁺ cells were present at d6 (not shown). Scale bar equals 50 μm (20× objective).