Surface antigens of human embryonic stem cells: changes upon differentiation in culture

Abstract

We have analysed the surface antigen phenotype of a human embryonic stem (hES) cell line (H7) and the changes that occur upon differentiation induced by retinoic acid, hexamethylene bisacetamide and dimethylsulphoxide. The undifferentiated stem cells expressed Stage Specific Embryonic Antigen-3 (SSEA3), SSEA4, TRA-1-60, and TRA-1-8 but not SSEA1. In these characteristics they closely resemble human embryonal carcinoma (EC) cells derived from testicular teratocarcinomas, and are distinct from murine EC and ES cells. The undifferentiated cells also expressed the liver/bone/kidney isozyme of alkaline phosphatase detected by antibody TRA-2-54, the class 1 major histocompatability antigens, HLA-ABC, and the human Thy1 antigen. Differentiation of hES cells was induced by retinoic acid, HMBA and DMSO with the appearance of various cell types including neurons and muscle cells. The surface antigens characteristically expressed by hES cells were down-regulated following induction of differentiation and other antigens appeared, notably several ganglioside glycolipids detected by antibodies VIN-IS-56 (GD3 and GD2), VIN-2PB-22 (GD2), A2B5 (GT3) and ME311 (9-O-acetyl-GD3). Whereas the expression of HLA was slightly down-regulated upon differentiation, its expression was strongly induced by interferon-y in both the undifferentiated and the differentiated cells, although the induction in the differentiated cultures was considerably stronger than in the stem cells. In all of these features the human ES cells, and their pattern of differentiation, resembled the pluripotent human EC cell line NTERA-2 although clearly the range of cells generated by the hES cells was considerably greater.

Fig. 1

Phase contrast photomicrograph of an undifferentiated human ES cell colony. The cells resembled human EC cells morphologically, forming tight colonies composed of cells with high nucleus/cytoplasm ratios and containing few prominent nucleoli. Scale bar = 50 µm.

Fig. 2

Neurons, glial and muscle cells detected by immunofluorescence with antibodies to neurofilaments (A), GFAP (B) and desmin (C) were found in the hES H7 cultures grown in the presence of 10⁻⁵ m retinoic acid.

Fig. 3

Changes in surface antigen expression by hES H7 cells when cultured on MEF feeders or Matrigel, in the presence or absence of 10⁻⁵ m retinoic acid. For comparison typical antigen expression by NTERA2 EC cells and NTERA2 cells induced to differentiate with retinoic acid for 7 days are also shown.

Fig. 4

The effect of IFNγ on the expression of HLA (HLA-A,B,C and β₂ microglobulin) and the antigens (alkaline phosphatase and SSEA4) by hES H7 cells. The histograms show the mean fluorescence intensity of cells after staining with antibodies W6/32 (anti HLA-A,B,C), BBM1 (anti-β₂ microglobulin), TRA-2-54 (anti-liver/bone/kidney alkaline phosphatase) and MCX813-70 (anti-SSEA4); the percentage of cells fluorescing is indicated above each bar.

Fig. 5

Surface antigen expression by hES. H7 cells were cultured on Matrigel in the presence, or absence, of 3 mm HMBA or 1% (v/v) DMSO.