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First published online October 22, 2003
doi: 10.1242/10.1242/jcs.00767

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The 5T4 oncofoetal antigen is an early differentiation marker of mouse ES cells and its absence is a useful means to assess pluripotency

Christopher M. Ward*, Katie Barrow, Andrew M. Woods and Peter L. Stern

Immunology Group, Paterson Institute for Cancer Research, Christie Hospital NHS Trust, Wilmslow Road, Manchester M20 4BX, UK



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  		Fig. 3. 5T4 is transcriptionally upregulated on ES cells following removal of LIF and the antigen associates with all primary germ layers. (a) 5T4 transcript expression in differentiating ES cells. (i) MESC and (ii) OKO160 ES cell lines were differentiated for 12 days as monolayer cultures by removal of LIF from the growth medium. cDNA was prepared at the specified time points, as described in the legend to Fig. 2, and semi-quantitative RT-PCR analysis (25 cycles) of m5T4 was performed. Samples were run on 2% agarose gels containing ethidium bromide and visualised on a UV transilluminator. ß-tubulin (ß-tub; housekeeping gene) is included for standardisation. D0, undifferentiated cells; D12, 12 days following removal of LIF. (b) 5T4 antigen is expressed on cells derived from all three germ layers following removal of LIF from ES cells. MESC ES cells were differentiated for 3, 6 and 9 days as monolayer cultures by removal of LIF from the growth medium and 5T4-positive cells purified using anti-5T4 monoclonal antibody 9A7 and MidiMACS LS columns. cDNA was prepared as described above followed by RT-PCR analysis of various germ layer-specific transcripts (see Table 1). Samples were run on 2% agarose gels containing ethidium bromide and visualised on a UV transilluminator. ß-tub (housekeeping gene) is included for standardisation. D3, 3 days following removal of LIF; D9, 9 days following removal of LIF.

 


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  		Fig. 1. Cell surface 5T4 oncofoetal antigen is upregulated on ES cells following removal of LIF. (a) Cell surface expression of 5T4 on ES cells: (i) MESC, (ii) D3, (iii) OKO160 and (iv) 129 ES cells were differentiated for 12 days as monolayer cultures by removal of LIF from the growth medium and cell-surface 5T4 was measured using rat anti-m5T4 monoclonal antibody 9A7 (uncoloured population) or control rat IgG (coloured population). Primary antibodies were detected using FITC-conjugated rabbit anti-rat Ig and cell fluorescence measured in a Becton Dickinson FACScan. Viable cells were gated using forward and side scatter and the figure shows the fluorescence of this population. Percentages of each population expressing 5T4 is shown. Day 0, undifferentiated cells; Day 12, 12 days following removal of LIF. (b) Total 5T4 protein expression in ES cells. (i) MESC, (ii) D3, (iii) OKO160 and (iv) 129 ES cells were differentiated for 12 days as monolayer cultures by removal of LIF from the culture medium, lysed (1.2x107 cells/ml) and 20 µl of the lysate separated by unreduced SDS-PAGE. The membrane was probed using rabbit anti-m5T4 polyclonal serum followed by HRP-conjugated sheep anti-rabbit immunoglobulins and developed by enhanced chemiluminescence. Bar charts show the densitometric analysis of the 5T4 bands, with arbitrary density values on the y-axis and days post-removal of LIF on the x-axis. Controls are mouse A9 cells transfected with m5T4 cDNA (positive) or vector control (negative).

 


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  		Fig. 2. Upregulation of 5T4 expression following removal of LIF correlates with differentiation of ES cells. (a) Transcript expression profiles of ES cells following removal of LIF. (i) MESC, (ii) D3, (iii) OKO160 and (iv) 129 ES cells were differentiated for 12 days as monolayer cultures by removal of LIF from the growth medium. RNA was extracted from the cells at the specified time points, treated with DNase, and cDNA synthesised from the mRNA transcripts. RT-PCR was performed for 35 cycles, the samples run on 2% agarose gels containing 400 ng/ml ethidium bromide and visualised on a UV transilluminator. ß-tubulin (B-tub; housekeeping gene) is included for comparison purposes. To ensure the absence of genomic DNA, RT-PCR detection of ß-tub was performed on all samples without prior formation of cDNA (mRNA sample). See Table 1 for description of markers used. D0, undifferentiated cells; D12, 12 days following removal of LIF. (b) Expression of Forssman antigen on ES cells following removal of LIF. ES cells were differentiated for 12 days as monolayer cultures by removal of LIF from the growth medium. Forssman antigen was determined at the specified time points on differentiating (i) MESC, (ii) D3, (iii) OKO160 and (iv) 129 ES cells using rat anti-Forssman antibody (uncoloured population) or control rat IgM (coloured population), detected as described in the legend to Fig. 1. Viable cells were gated using forward and side scatter and the figure shows the fluorescence of this population. Day 0, undifferentiated cells; Day 12, 12 days following removal of LIF.

 


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  		Fig. 4. Expression of 5T4 antigen in differentiating ES cells is associated with the differentiation rate. (a) Expression of 5T4 antigen correlates with cell migration in differentiating ES cells. (i) MESC and (ii) 129 ES cells (105 cells/3 cm dish) were grown for 0, 3 and 6 days in DMEMSR in the absence of LIF and viewed under phase contrast optics on an Olympus inverted microscope. Small arrows indicate undifferentiated ES cells and large arrows differentiated/migrating cells. (b) Expression of 5T4 correlates with the proliferation rate of differentiating ES cells. (i) MESC and (ii) 129 ES cells (105 cells/3 cm dish) were grown in DMEMSR medium in the absence (squares) or presence (diamonds) of LIF (arrow indicates day of LIF removal) for 3 days and the number of viable cells determined by light microscopy of cells excluding Trypan Blue. Scale bar: 10 µm.

 


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  		Fig. 5. Expression of EGFP-h5T4 in undifferentiated ES cells alters colony morphology. 129 ES cells were electroporated with 20 µg plasmid DNA and plated onto gelatin-treated 9 cm dishes. (a) After 24 hours, one third of the cells were assayed for EGFP expression in a Becton Dickinson FACScan. Viable cells were gated using forward and side scatter and the figure shows the fluorescence of this population. EGFP-positive cells were isolated from the remainder of the sample by FACSVantage SE and plated out in fresh gelatin-treated 9 cm tissue culture dishes. (b) Cellular localisation of EGFP proteins was determined after 48 hours using fluorescence microscopy. (c) Cell morphology was determined 48 hours after transfection using inverted light microscopy. Scale bar: 10 µm.

 


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  		Fig. 6. Presence of 5T4 on ES cells is a measure of decreased pluripotency. 129 ES cells were cultured in (a) the presence or (b) absence of LIF for 6 days and (i) SSEA-1-positive cells isolated by FACS (boxed area). (ii) 5T4 expression of the SSEA-1-positive population was determined as described in the legend to Fig. 1a. Pluripotency of the SSEA-1-positive cells was determined by chimera formation following injection of 15 cells into 3.5-day-old BL/6 blastocysts and subsequent implantation into pseudo-pregnant BDF-1 female mice.

 

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© The Company of Biologists Ltd 2003