Introduction: Circulating fetal cells in maternal blood provide a tool for risk-free, non-invasive prenatal diagnosis. However, fetal cells in the maternal circulation are scarce, and to effectively isolate enough of them for reliable diagnostics, it is crucial to know which fetal cell type(s) should be targeted. Materials and Methods: Fetal cells were enriched from maternal blood by magnetic-activated cell sorting using the endothelial cell marker CD105 and identified by XY fluorescence in situ hybridization. Expression pattern was compared between fetal cells and maternal blood cells using stem cell microarray analysis. Results: 39 genes were identified as candidates for unique fetal cell markers. More than half of these are genes known to be expressed in the placenta, especially in extravillous trophoblasts (EVTs). Immunohistochemical staining of placental tissue confirmed CD105 staining in EVTs and 76% of fetal cells enriched by CD105 were found to be cytokeratin-positive. Discussion: The unique combination of mesodermal (CD105) and ectodermal (cytokeratin) markers in EVTs could be a potential marker set for cell enrichment of this cell type in maternal blood and could be the basis for future cell-based non-invasive prenatal diagnosis.

Keywords:
Fetal cells in maternal blood, Extravillous trophoblast, Microarray, Gene expression
1.
Tabor A, Vestergaard CH, Lidegaard O: Fetal loss rate after chorionic villus sampling and amniocentesis: an 11-year national registry study. Ultrasound Obstet Gynecol 2009;34:19-24.
2.
Calabrese G, Baldi M, Fantasia D, Sessa MT, Kalantar M, Holzhauer C, Alunni-Fabbroni M, Palka G, Sitar G: Detection of chromosomal aneuploidies in fetal cells isolated from maternal blood using single-chromosome dual-probe FISH analysis. Clin Genet 2012;82:131-139.
3.
Kolvraa S, Christensen B, Lykke-Hansen L, Philip J: The fetal erythroblast is not the optimal target for non-invasive prenatal diagnosis: preliminary results. J Histochem Cytochem 2005;53:331-336.
4.
Krabchi K, Gros-Louis F, Yan J, Bronsard M, Masse J, Forest JC, Drouin R: Quantification of all fetal nucleated cells in maternal blood between the 18th and 22nd weeks of pregnancy using molecular cytogenetic techniques. Clin Genet 2001;60:145-150.
5.
Bianchi DW, Simpson JL, Jackson LG, Elias S, Holzgreve W, Evans MI, Dukes KA, Sullivan LM, Klinger KW, Bischoff FZ, Hahn S, Johnson KL, Lewis D, Wapner RJ, de la Cruz F: Fetal gender and aneuploidy detection using fetal cells in maternal blood: analysis of NIFTY I data. National Institute of Child Health and Development Fetal Cell Isolation Study. Prenat Diagn 2002;22:609-615.
6.
Christensen B, Kolvraa S, Lykke-Hansen L, Lorch T, Gohel D, Smidt-Jensen S, Bang J, Philip J: Studies on the isolation and identification of fetal nucleated red blood cells in the circulation of pregnant women before and after chorion villus sampling. Fetal Diagn Ther 2003;18:376-384.
7.
Oudejans CB, Tjoa ML, Westerman BA, Mulders MA, Van Wijk IJ, Van Vugt JM: Circulating trophoblast in maternal blood. Prenat Diagn 2003;23:111-116.
8.
Krabchi K, Gadji M, Forest JC, Drouin R: Quantification of all fetal nucleated cells in maternal blood in different cases of aneuploidies. Clin Genet 2006;69:145-154.
9.
Guetta E, Gordon D, Simchen MJ, Goldman B, Barkai G: Hematopoietic progenitor cells as targets for non-invasive prenatal diagnosis: detection of fetal CD34+ cells and assessment of post-delivery persistence in the maternal circulation. Blood Cells Mol Dis 2003;30:13-21.
10.
Brinch M, Hatt L, Singh R, Moller K, Sommer S, Uldbjerg N, Christensen B, Kolvraa S: Identification of circulating fetal cell markers by microarray analysis. Prenat Diagn 2012;32:742-751.
11.
Christensen B, Kolvraa S, Lykke-Hansen L, Lorch T, Gohel D, Smidt-Jensen S, Bang J, Philip J: Studies on the isolation and identification of fetal nucleated red blood cells in the circulation of pregnant women before and after chorion villus sampling. Fetal Diagn Ther 2003;18:376-384.
12.
Kaufmann P, Black S, Huppertz B: Endovascular trophoblast invasion: implications for the pathogenesis of intrauterine growth retardation and preeclampsia. Biol Reprod 2003;69:1-7.
13.
Harris LK: Trophoblast-vascular cell interactions in early pregnancy: how to remodel a vessel. Placenta 2010;31(suppl):S93-S98.
14.
Zhou Y, Fisher SJ, Janatpour M, Genbacev O, Dejana E, Wheelock M, Damsky CH: Human cytotrophoblasts adopt a vascular phenotype as they differentiate. A strategy for successful endovascular invasion? J Clin Invest 1997;99:2139-2151.
15.
Huppertz B, Kadyrov M, Kingdom JC: Apoptosis and its role in the trophoblast. Am J Obstet Gynecol 2006;195:29-39.
16.
Attwood HD, Park WW: Embolism to the lungs by trophoblast. J Obstet Gynaecol Br Commonw 1961;68:611-617.
17.
Dubernard G, Oster M, Chareyre F, Antoine M, Rouzier R, Uzan S, Aractingi S, Khosrotehrani K: Increased fetal cell microchimerism in high grade breast carcinomas occurring during pregnancy. Int J Cancer 2009;124:1054-1059.
18.
Khosrotehrani K, Johnson KL, Cha DH, Salomon RN, Bianchi DW: Transfer of fetal cells with multilineage potential to maternal tissue. JAMA 2004;292:75-80.
19.
Nguyen HS, Oster M, Uzan S, Chareyre F, Aractingi S, Khosrotehrani K: Maternal neoangiogenesis during pregnancy partly derives from fetal endothelial progenitor cells. Proc Natl Acad Sci USA 2007;104:1871-1876.
20.
Naik MU, Vuppalanchi D, Naik UP: Essential role of junctional adhesion molecule-1 in basic fibroblast growth factor-induced endothelial cell migration. Arterioscler Thromb Vasc Biol 2003;23:2165-2171.
21.
Weber C, Fraemohs L, Dejana E: The role of junctional adhesion molecules in vascular inflammation. Nat Rev Immunol 2007;7:467-477.
22.
Anfosso F, Bardin N, Vivier E, Sabatier F, Sampol J, Dignat-George F: Outside-in signaling pathway linked to CD146 engagement in human endothelial cells. J Biol Chem 2001;276:1564-1569.
23.
Krause G, Winkler L, Mueller SL, Haseloff RF, Piontek J, Blasig IE: Structure and function of claudins. Biochim Biophys Acta 2008;1778:631-645.
24.
Lampugnani MG, Corada M, Caveda L, Breviario F, Ayalon O, Geiger B, Dejana E: The molecular organization of endothelial cell to cell junctions: differential association of plakoglobin, β-catenin, and α-catenin with vascular endothelial cadherin (VE-cadherin). J Cell Biol 1995;129:203-217.
25.
Dus D, Krawczenko A, Zalecki P, Paprocka M, Wiedlocha A, Goupille C, Kieda C: IL-7 receptor is present on human microvascular endothelial cells. Immunol Lett 2003;86:163-168.
26.
Genersch E, Hayess K, Neuenfeld Y, Haller H: Sustained ERK phosphorylation is necessary but not sufficient for MMP-9 regulation in endothelial cells: involvement of Ras-dependent and -independent pathways. J Cell Sci 2000;113:4319-4330.
27.
Ghosh R, Lipson KL, Sargent KE, Mercurio AM, Hunt JS, Ron D, Urano F: Transcriptional regulation of VEGF-A by the unfolded protein response pathway. PLoS One 2010;5:e9575.
28.
Leppanen VM, Jeltsch M, Anisimov A, Tvorogov D, Aho K, Kalkkinen N, Toivanen P, Yla-Herttuala S, Ballmer-Hofer K, Alitalo K: Structural determinants of vascular endothelial growth factor-D receptor binding and specificity. Blood 2011;117:1507-1515.
29.
Midwood KS, Hussenet T, Langlois B, Orend G: Advances in tenascin-C biology. Cell Mol Life Sci 2011;68:3175-3199.
30.
Schiessl B, Innes BA, Bulmer JN, Otun HA, Chadwick TJ, Robson SC, Lash GE: Localization of angiogenic growth factors and their receptors in the human placental bed throughout normal human pregnancy. Placenta 2009;30:79-87.
31.
Bamberger AM, Makrigiannakis A, Roser K, Radde J, Carstens T, Flohr AM, Bamberger CM, Bullerdiek J, Loning T: Expression of the high-mobility group protein HMGI(Y) in human trophoblast: potential role in trophoblast invasion of maternal tissue. Virchows Arch 2003;443:649-654.
32.
Bamberger AM, Bamberger CM, Aupers S, Milde-Langosch K, Loning T, Makrigiannakis A: Expression pattern of the activating protein-1 family of transcription factors in the human placenta. Mol Hum Reprod 2004;10:223-228.
33.
Schorpp-Kistner M, Wang ZQ, Angel P, Wagner EF: JunB is essential for mammalian placentation. EMBO J 1999;18:934-948.
34.
Hirano T, Higuchi T, Ueda M, Inoue T, Kataoka N, Maeda M, Fujiwara H, Fujii S: CD9 is expressed in extravillous trophoblasts in association with integrin alpha3 and integrin alpha5. Mol Hum Reprod 1999;5:162-167.
35.
Hirano T, Higuchi T, Katsuragawa H, Inoue T, Kataoka N, Park KR, Ueda M, Maeda M, Fujiwara H, Fujii S: CD9 is involved in invasion of human trophoblast-like choriocarcinoma cell line, BeWo cells. Mol Hum Reprod 1999;5:168-174.
36.
Hurtado CW, Golden-Mason L, Brocato M, Krull M, Narkewicz MR, Rosen HR: Innate immune function in placenta and cord blood of hepatitis C-seropositive mother-infant dyads. PLoS One 2010;5:e12232.
37.
James JL, Stone PR, Chamley LW: The isolation and characterization of a population of extravillous trophoblast progenitors from first trimester human placenta. Hum Reprod 2007;22:2111-2119.
38.
Li HW, Cheung AN, Tsao SW, Cheung AL, O WS: Expression of e-cadherin and β-catenin in trophoblastic tissue in normal and pathological pregnancies. Int J Gynecol Pathol 2003;22:63-70.
39.
Liu Q, Yan X, Li Y, Zhang Y, Zhao X, Shen Y: Pre-eclampsia is associated with the failure of melanoma cell adhesion molecule (MCAM/CD146) expression by intermediate trophoblast. Lab Invest 2004;84:221-228.
40.
Luo J, Qiao F, Yin X: Hypoxia induces FGF2 production by vascular endothelial cells and alters MMP9 and TIMP1 expression in extravillous trophoblasts and their invasiveness in a cocultured model. J Reprod Dev 2011;57:84-91.
41.
Luo J, Qiao F, Yin X: Impact of silencing MMP9 gene on the biological behaviors of trophoblasts. J Huazhong Univ Sci Technolog Med Sci 2011;31:241-245.
42.
Pollheimer J, Loregger T, Sonderegger S, Saleh L, Bauer S, Bilban M, Czerwenka K, Husslein P, Knofler M: Activation of the canonical wingless/T-cell factor signaling pathway promotes invasive differentiation of human trophoblast. Am J Pathol 2006;168:1134-1147.
43.
Proll J, Blaschitz A, Hartmann M, Thalhamer J, Dohr G: Human first-trimester placenta intra-arterial trophoblast cells express the neural cell adhesion molecule. Early Pregnancy 1996;2:271-275.
44.
Shih IM, Kurman RJ: Expression of melanoma cell adhesion molecule in intermediate trophoblast. Lab Invest 1996;75:377-388.
45.
Uszynski M, Uszynski W, Zekanowska E: P-selectin in placenta and gestational myometrium: its measurements and hypothetical role in hemostasis of placental bed after labor. J Perinat Med 2008;36:213-216.
46.
Guetta E, Gutstein-Abo L, Barkai G: Trophoblasts isolated from the maternal circulation: in vitro expansion and potential application in non-invasive prenatal diagnosis. J Histochem Cytochem 2005;53:337-339.
47.
Oudejans CB, Tjoa ML, Westerman BA, Mulders MA, van Wijk IJ, van Vugt JM: Circulating trophoblast in maternal blood. Prenat Diagn 2003;23:111-116.
48.
Gussin HA, Sharma AK, Elias S: Culture of endothelial cells isolated from maternal blood using anti-CD105 and CD133. Prenat Diagn 2004;24:189-193.
49.
Gussin HA, Sharma AK, Elias S: Culture of cells from maternal circulation, in conditions favoring fetal endothelial cell expansion, does not facilitate the preferential expansion of circulating fetal cells. Fetal Diagn Ther 2005;20:64-69.
50.
Toutain J, Labeau-Gauzere C, Barnetche T, Horovitz J, Saura R: Confined placental mosaicism and pregnancy outcome: a distinction needs to be made between types 2 and 3. Prenat Diagn 2010;30:1155-1164.
51.
Chan KC, Ding C, Gerovassili A, Yeung SW, Chiu RW, Leung TN, Lau TK, Chim SS, Chung GT, Nicolaides KH, Lo YM: Hypermethylated RASSF1A in maternal plasma: a universal fetal DNA marker that improves the reliability of noninvasive prenatal diagnosis. Clin Chem 2006;52:2211-2218.
52.
Tsaliki E, Papageorgiou EA, Spyrou C, Koumbaris G, Kypri E, Kyriakou S, Sotiriou C, Touvana E, Keravnou A, Karagrigoriou A, Lamnissou K, Velissariou V, Patsalis PC: MeDIP real-time qPCR of maternal peripheral blood reliably identifies trisomy 21. Prenat Diagn 2012;32:996-1001.
53.
Palomaki GE, Deciu C, Kloza EM, Lambert-Messerlian GM, Haddow JE, Neveux LM, Ehrich M, van den Boom D, Bombard AT, Grody WW, Nelson SF, Canick JA: DNA sequencing of maternal plasma reliably identifies trisomy 18 and trisomy 13 as well as Down syndrome: an international collaborative study. Genet Med 2012;14:296-305.
54.
Dan S, Wang W, Ren J, Li Y, Hu H, Xu Z, Lau TK, Xie J, Zhao W, Huang H, Xie J, Sun L, Zhang X, Wang W, Liao S, Qiang R, Cao J, Zhang Q, Zhou Y, Zhu H, Zhong M, Guo Y, Lin L, Gao Z, Yao H, Zhang H, Zhao L, Jiang F, Chen F, Jiang H, Li S, Li Y, Wang J, Wang J, Duan T, Su Y, Zhang X: Clinical application of massively parallel sequencing-based prenatal noninvasive fetal trisomy test for trisomies 21 and 18 in 11,105 pregnancies with mixed risk factors. Prenat Diagn 2012;32:1225-1232.
55.
Mersy E, Smits LJ, van Winden LA, de Die-Smulders CE, Paulussen AD, Macville MV, Coumans AB, Frints SG: Noninvasive detection of fetal trisomy 21: systematic review and report of quality and outcomes of diagnostic accuracy studies performed between 1997 and 2012. Hum Reprod Update 2013;19:318-329.
56.
Hahn S, Lapaire O, Tercanli S, Kolla V, Hosli I: Determination of fetal chromosome aberrations from fetal DNA in maternal blood: has the challenge finally been met? Expert Rev Mol Med 2011;13:e16.
© 2013 S. Karger AG, Basel
2013
Copyright / Drug Dosage / Disclaimer
Copyright: All rights reserved. No part of this publication may be translated into other languages, reproduced or utilized in any form or by any means, electronic or mechanical, including photocopying, recording, microcopying, or by any information storage and retrieval system, without permission in writing from the publisher.
Drug Dosage: The authors and the publisher have exerted every effort to ensure that drug selection and dosage set forth in this text are in accord with current recommendations and practice at the time of publication. However, in view of ongoing research, changes in government regulations, and the constant flow of information relating to drug therapy and drug reactions, the reader is urged to check the package insert for each drug for any changes in indications and dosage and for added warnings and precautions. This is particularly important when the recommended agent is a new and/or infrequently employed drug.
Disclaimer: The statements, opinions and data contained in this publication are solely those of the individual authors and contributors and not of the publishers and the editor(s). The appearance of advertisements or/and product references in the publication is not a warranty, endorsement, or approval of the products or services advertised or of their effectiveness, quality or safety. The publisher and the editor(s) disclaim responsibility for any injury to persons or property resulting from any ideas, methods, instructions or products referred to in the content or advertisements.
You do not currently have access to this content.