Evaluation of Cytokeratin 7 as an accurate intracellular marker with which to assess the purity of human placental villous trophoblast cells by flow cytometry.
Villous trophoblast cells (TC) obtained from first trimester and term human placentae after trypsin/Percoll gradient isolation were immunodepleted of contaminant cells. The level of purity was assessed by the intracellular expression of the pan trophoblast marker cytokeratin-7 (CK7) and comparisons were made with the GB25 trophoblast-specific (cytotrophoblast+syncytiotrophoblast) cell surface marker. The presence of contaminating cells was traced with intracellular vimentin, or cell surface CD2, CD36, and CD163 markers and evaluated by flow cytometric analysis. The pattern of CK7 expression by trophoblast cells was also analyzed by immunofluorescence microscopy. Most batches of TC from first trimester or term placentae (92+/-3% and 96+/-2%, respectively) showed a high percentage of CK7 expressing cells, with less than 2% contaminating vimentin positive cells. In some batches of TC with a lower percentage (65+/-4%) of CK7-expressing cells, no vimentin was found, but a low percentage of CD36-expressing cells was evidenced, with no presence of CD2, and/or CD163-expressing cells. The intracellular CK7 signal correlated significantly with that of GB25 (p<0.05) cell surface expression in TC of term placentae. The choriocarcinoma BeWo and Jar cell lines also showed high levels (>92%) of CK7-expressing cells. Conversely, the control U87 astrocytoma cell line showed a high percentage (>90%) of vimentin but no CK7-expressing cells. These results provide evidence that the mutually exclusive pattern of intracellular CK7/vimentin expression of human TC can be used for evaluation by flow cytometry of the purity of primary human trophoblast cells.
Maldonado-Estrada
Juan
J
INSERM U131, Equipe Cytokines et Régulation Materno Foetale, Maternité, Hôpital Antoine Béclère, Clamart, France.
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Elisabeth
E
Roques
Pierre
P
Barré-Sinoussi
Françoise
F
Chaouat
Gérard
G
D015703:Antigens, CD / Q000276:immunology; D015415:Biomarkers; D002824:Chorionic Villi / Q000276:immunology*; D005260:Female; D005434:Flow Cytometry; D006801:Humans; D053552:Keratin-7; D007633:Keratins / Q000096:biosynthesis / Q000276:immunology*; D008856:Microscopy, Fluorescence; D011247:Pregnancy; D011261:Pregnancy Trimester, First; D014327:Trophoblasts / Q000166:cytology / Q000276:immunology*
D015703:Antigens, CD; D015415:Biomarkers; C507497:KRT7 protein, human; D053552:Keratin-7; D007633:Keratins