Expression and localization pattern of ABCA1 in diverse human placental primary cells and tissues  L. Nikitina, F. Wenger, M. Baumann, D. Surbek, M. Körner, C. Albrecht  Placenta  Volume 32, Issue 6, Pages 420-430 (June 2011) DOI: 10.1016/j.placenta.2011.03.003 Copyright © 2011 Elsevier Ltd Terms and Conditions

Fig. 1 Characterization of isolated primary placental cells using flow cytometry. FACS analysis was performed on human chorionic mesenchymal cells (HCMC, panel A), villous mesenchymal cells (VMCs, panel B, D–K), term placenta villous mesenchymal cells (TVMCs, panel C), cytotrophoblast cells (L, M) and placental macrophages (N, O). The homogeneous mesenchymal cell populations were positive for the mesenchymal cell markers CD105 (D), CD90 (E), CD73 (F) and the immunological marker HLA-ABC (G), and negative for the hematopoietic stem cell markers CD45 (H) and CD34 (I), the immunological marker HLA-DR (J) and the marker of monocytes CD14 (K) (representative data are shown for VMCs). Primary cytotrophoblast cell populations were also homogeneous (L) and were positive for the trophoblast cell marker CK7 (M). In the lymphocyte cell mixture, approx. 20% of cells were positive for the macrophage/monocyte marker CD14 (N, O). Placenta 2011 32, 420-430DOI: (10.1016/j.placenta.2011.03.003) Copyright © 2011 Elsevier Ltd Terms and Conditions

Fig. 2 Localization of ABCA1 in the villous trophoblast layer of first trimester and term placental tissues. Panels A, C, E, G: immunohistochemistry and light microscopy; ABCA1 (brown), counterstaining with hematoxylin. Panels B, D, F, H: immunofluorescence of placental tissues; ABCA1/Alexa 488 (green), cytokeratin-7/Alexa 594 (red) is used as a marker of trophoblast cells, counterstaining of the nuclei with DAPI (blue). In first trimester placentas (A–D), either intensive staining of the entire cytoplasm of syncytiotrophoblasts (black arrows) and of cytotrophoblast cells (white arrows) was observed (A, B), or in the syncytiotrophoblast the basal cytoplasm was strongly stained while the apical brush border was negative (C, D). In term placentas (E, F), cytotrophoblast cells (white arrows) were strongly ABCA1-positive and syncytiotrophoblasts (black arrows) showed only focal weak staining. Note negative endothelial cells (black arrowheads) surrounding villous capillary lumina (asterisks) in the term peripheral villi in panel E. No positive staining was found in negative (isotype) controls where primary antibodies were substituted with immunoglobulins of the appropriate species (G, H). Placenta 2011 32, 420-430DOI: (10.1016/j.placenta.2011.03.003) Copyright © 2011 Elsevier Ltd Terms and Conditions

Fig. 3 Localization of ABCA1 in primary isolated cytotrophoblast cells from term placenta (38 weeks of gestation). Panels A, C: bright field images of cytotrophoblast cells in culture after 12 and 24 h respectively. Panels B, D: immunocytochemistry with immunofluorescence microscopy; ABCA1/Alexa 488 (green), cytokeratin-7/Alexa 594 (red) is used as a marker of trophoblast cells, counterstaining of the nuclei with DAPI (blue). After 12 h of cultivation cytotrophoblasts were observed as single cells (A). Formation of syncytium islands was found after 24 h of cultivation (C). Intensive staining for ABCA1 was observed in non-syncytialised cytotrophoblast cells after 12 h of cultivation (exposure time 300 ms) (B). The staining was localized in cytoplasmic compartments and at the cell membrane (B). After 24 h of cultivation (exposure time 300 ms), the forming syncytium showed impressively weaker ABCA1 staining localized only focally in the cytoplasm at not in the cell membrane (D). Placenta 2011 32, 420-430DOI: (10.1016/j.placenta.2011.03.003) Copyright © 2011 Elsevier Ltd Terms and Conditions

Fig. 4 Localization of ABCA1 in primary human amnion epithelial cells (HAEC). Cells were isolated and processed as described in Methods, and analyzed by immunofluorescence confocal microscopy. Panel A: ABCA1/Alexa 488 (green), counterstaining of the nucleus with DAPI (blue), 2 days of cultivation. Panel B: ABCA1/Alexa 488 (green), counterstaining of the nucleus with DAPI (blue), 5 days of cultivation. Panel C: ABCA1/Alexa 488 (green), counterstaining with DAPI (blue). Panel D: ABCA1/Alexa 488 (green), pan-cytokeratin/Alexa 594 (red) is used as a marker for epithelial cells, counterstaining with DAPI (blue). Panel E: ABCA1/Alexa 488 (green), co-staining with calreticulin/Alexa 594 (red), an endoplasmic reticulum marker, counterstaining with DAPI (blue). Panel F: Immunohistochemistry and light microscopy; ABCA1 (brown), counterstaining with hematoxylin. ABCA1 was localized in HAEC of formalin-fixed, paraffin-embedded amnion membranes (arrows; F) and isolated primary HAEC (A–E). In primary cells, staining intensity between 2 (A) and 5 (B) days of cultivation was comparable and showed significant membrane (C, D) and intracellular staining (C, E). Intracellular ABCA1 was observed in cytoplasmic compartments such as the endoplasmic reticulum, as evidenced by co-localization with calreticulin (E; yellow merge, see also magnification in the inset). No positivity was found in negative (isotype) controls where primary antibodies were substituted with immunoglobulins of the appropriate species (data not shown). Placenta 2011 32, 420-430DOI: (10.1016/j.placenta.2011.03.003) Copyright © 2011 Elsevier Ltd Terms and Conditions

Fig. 5 Localization of ABCA1 in mesenchymal cells from chorionic plate, and first trimester and term placental villous stroma. Panels A, C, E: immunohistochemistry and light microscopy; ABCA1 (brown), counterstaining with hematoxylin. Panels B, D, F: immunocytochemistry and immunofluorescence microscopy on isolated cells; ABCA1/Alexa 488 (green), co-localization with calreticulin/Alexa 594 (red), an endoplasmic reticulum marker, counterstaining with DAPI (blue), 40x. ABCA1 was localized in mesenchymal cells (black arrows) from chorionic plate (A), and mesenchymal cells from first trimester (C) and term (E) placental villous tissue. In primary mesenchymal cells isolated from chorion membrane (B), first trimester (D) and term (F) placental villous tissue ABCA1 was localized in the cell membrane and cytoplasmic compartments (co-localization with endoplasmic reticulum (see B, D, F)). No positivity was found in negative (isotype) controls where primary antibodies were substituted with immunoglobulins of the appropriate species (data not shown). Note endothelial cells (white arrow) of stem villous blood vessels staining positive for ABCA1 in E. Placenta 2011 32, 420-430DOI: (10.1016/j.placenta.2011.03.003) Copyright © 2011 Elsevier Ltd Terms and Conditions

Fig. 6 Localization of ABCA1 in placental macrophages (Hofbauer cells). Panel A–B: serial sections of first trimester placental tissue, immunohistochemistry and light microscopy; ABCA1 (left, brown) and CD68 (right, brown), counterstaining with hematoxylin. Panel C–D: confocal microscopy of first trimester placental tissue (C) and primary cells (D1, 2). Human peripheral blood monocytes (D3–4), isolated from venous blood of healthy donors and collected after density gradient centrifugation served as positive control. ABCA1/Alexa 488 (green), co-localization with the monocyte/macrophage markers CD14/Alexa 594 (red, D1, 3) and CD68/Alexa 594 (red, C, D2, 4), counterstaining with DAPI (blue), 100x. ABCA1 was expressed in CD68 and CD14 positive Hofbauer cells from first trimester placental tissue (A, B black arrows, C white arrows), primary isolated macrophages (D1–2) and peripheral blood monocytes (D3–4). In primary isolated macrophages ABCA1 was localized in the cell membrane and cytoplasmic compartments (D1–2). The staining intensity was comparable to the positive control (D3–4). No positivity was found in negative (isotype) controls where primary antibodies were substituted with immunoglobulins of the appropriate species (data not shown). Placenta 2011 32, 420-430DOI: (10.1016/j.placenta.2011.03.003) Copyright © 2011 Elsevier Ltd Terms and Conditions