HTR8/SVneo cells display trophoblast progenitor cell-like characteristics indicative of self-renewal, repopulation activity, and expression of "stemness-" associated transcription factors

Abstract

JEG3 is a choriocarcinoma--and HTR8/SVneo a transformed extravillous trophoblast--cell line often used to model the physiologically invasive extravillous trophoblast. Past studies suggest that these cell lines possess some stem or progenitor cell characteristics. Aim was to study whether these cells fulfill minimum criteria used to identify stem-like (progenitor) cells. In summary, we found that the expression profile of HTR8/SVneo (CDX2+, NOTCH1+, SOX2+, NANOG+, and OCT-) is distinct from JEG3 (CDX2+ and NOTCH1+) as seen only in human-serum blocked immunocytochemistry. This correlates with HTR8/SVneo's self-renewal capacities, as made visible via spheroid formation and multi-passagability in hanging drops protocols paralleling those used to maintain embryoid bodies. JEG3 displayed only low propensity to form and reform spheroids. HTR8/SVneo spheroids migrated to cover and seemingly repopulate human chorionic villi during confrontation cultures with placental explants in hanging drops. We conclude that HTR8/SVneo spheroid cells possess progenitor cell traits that are probably attained through corruption of "stemness-" associated transcription factor networks. Furthermore, trophoblastic cells are highly prone to unspecific binding, which is resistant to conventional blocking methods, but which can be alleviated through blockage with human serum.

Figure 1

Schematic diagram of spheroid formation.

Figure 2

Spheroid formation of HTR8/SVneo and JEG3 cells via hanging drops. Forty hanging drops per cell line were produced with 20 000 cells per 30 μL drop. The environment of the hanging drop delivers the prerequisite for spheroid formation. After an incubation period of 48 h, 100% of HTR8/SVneo-containing drops formed visible spheroids (a, b), while only 50% of JEG3-containing drops formed spheroids (c). JEG3 spheroids appeared smaller in circumference, less globular (oblong shapes), and rather on the verge of disaggregation (d).

Figure 3

Expression of trophoblast (CDX2), cell fate determining (NOTCH1), and core “stemness-” associated (OCT4, NANOG, and SOX2) stem cell transcription factors in HTR8/SVneo cells. HTR8/SVneo cells express CDX2, NOTCH1, NANOG, and SOX2 ((a), (e), (i), and (q)). OCT4 signaling is lost after blocking the samples with human serum ((m) versus (n)). Human serum is needed to further block exceptional, unspecific binding on trophoblastic cells that cannot be eliminated via conventional blocking measures. Immunofluorescent staining seems less specific with or without blockage with human serum ((c), (d), (g), (h), (k), (l), (o), (p), (s), and (t)).

Figure 4

Expression of trophoblast (CDX2), cell stypo determining (NOTCH1), and core “stemmness-” associated (OCT4, NANOG, and SOX2) stem cell transcription factors in JEG3 cells. JEG3 cells express CDX2 and NOTCH1 ((a) and (i)). OCT4, NANOG and SOX2 signaling are not visible (with human serum: (e), (m), and (q); without human serum (f), (n), and (r)). Immunofluorescent staining seems less specific with or without blockage with human serum (all positive signals: (c), (d), (g), (h), (k), (l), (o), (p), (s), and (t)).

Figure 5

HTR8/SVneo spheroid cells repopulate chorionic villi of placental biopsies. HTR8/SVneo spheroids were confronted with placental biopsies derived from healthy pregnancies after elective, ceasarian section. After 48 h, HTR8/SVneo spheroid cells (green) have covered chorionic villi (endothelium of fetal capillaries within the villi are depicted in red).