Expression of H/358 in Cultured Placental Cell Lines
The HRP/LRP trophoblast stem cells provide an important source of labyrinthine trophoblast cell precursors from which to obtain additional information about placental cell lineages. We therefore determined whether cell lines isolated from the labyrinthine placenta (HRP/LRP) and postulated to contain trophoblast stem cells expressed Hp58. The amount of Hp58 mRNA was similar (Fig. 8A) between the two cell lines in independent RNase protection assays and in cells from different cell passages (data not shown). We determined the spatial distribution of Hp58 in HRP placental cells processed for immunocytochemical detection of the protein (Fig. 8B).
The Hp58 protein was distributed in the nucleus and cytoplasm of positive cells (Fig. 8B, panels A and C). Immunoreactive protein was absent from cells reacted with an equal concentration of preim-mune sera (Fig. 8B, panels B and D). Comparison of phase-contrast images with immunoreactive cells showed that some HRP cells were negative (Fig. 8B, compare A and E). Because negative cells were present in HRP cell lines from different passages and experiments, the results suggest that H358 expression is restricted to particular placental cell types. That interpretation is consistent with our results from the immunolocalization studies in the fetal placenta (Figs. 5 and 6). Similar results of cell-specific expression were obtained from immunocytochemical analysis of LRP placental cell lines (data not shown).
FIG. 8. The Hp58 mRNA and protein are expressed in stable placental cell lines (HRP/LRP) isolated from the labyrinthine placenta. A) Placental cells (passage 12) were cultured to confluence. Total RNA was isolated and the amount of Hp58 mRNA was determined using RNase protection. Data are the mean ± SEM of samples in triplicate analyzed on the same autoradiograph. Results are representative of three independent assays. B) Cell-specific expression of Hp58 in placental cell lines. The HRP placental cells were cultured on collagen-coated coverslips. Cells were treated with Hp58 antibody or preimmune serum as described in the text. Data are representative of results obtained from three independent experiments and cell passages. A) The HRP placental cells treated with Hp58 antibody, X400. B) The HRP placental cells treated with preimmune sera, X400. C) The HRP cells reacted with Hp58 antibody, X1000. D) The HRP placental cells treated with preimmune sera, X1000. E) Corresponding phase-contrast image for A. F) Corresponding phase-contrast image for B. G) Corresponding Nomarski image of C. H) Corresponding Nomarski image of D.