title: Isolation, purification and in vitro differentiation of cytotrophoblast cells from human term placenta.
abstract: BACKGROUND
  The syncytialization of cytotrophoblast cells to syncytiotrophoblast is central to human placental transport and hormone production. Many techniques for in vitro study of this process have been proposed and new investigators to the field may find the literature in the field daunting. Here, we present a straightforward and reliable method to establish this important model using modern but readily available tools and reagents.
  
  
  METHODS
  Villous cytotrophoblast cells are obtained from term placenta using mild enzymatic degradation, Percoll gradient centrifugation, negative magnetic cell sorting using an antibody against classical major histocompatibility complex molecules and in vitro culture on a matrix-coated growth surface.
  
  
  RESULTS
  The purity of isolated cytotrophoblast cells exceeds 98 % as assessed by cytokeratin-7 expression using flow cytometry. Contamination by mesenchymal cells, extravillous trophoblast cells, leukocytes, Hofbauer and endothelial cells is minimized (less than 2 % when analyzed for vimentin, HLA-G, CD45, CD163 and CD31 using flow cytometry). Isolated cytotrophoblast cells began to aggregate into monolayers of mononucleated cells within about 12 h of plating. By 72 h in culture, most cytotrophoblast cells have differentiated into syncytiotrophoblast as demonstrated by a loss of intercellular E-cadherin expression upon fusion into multinucleated syncytia. After 72 h in culture, nearly every cultured cell expresses syncytiotrophoblast markers, including cytokeratin-7, human chorionic gonadotropin-β (β-hCG) and the fusion-related proteins glial cell missing-1 (GCM-1) and syncytin.
  
  
  CONCLUSIONS
  We present an efficient and reliable method for isolating of cytotrophoblast cells with high purity and complete differentiation into syncytiotrophoblast in vitro.
authors.affiliation: Department of Obstetrics, Gynecology and Women's Health, University of Missouri School of Medicine, 500 N. Keene Street, Columbia, MO, USA. doctorlipingli@foxmail.com.|Department of Obstetrics and Gynecology, Guangzhou Medical University Affiliated Guangzhou First People's Hospital, 1 Panfu Road, Guangzhou, China. doctorlipingli@foxmail.com.
authors.forename: Liping
authors.initials: L
authors.lastname: Li
authors.affiliation: Department of Obstetrics, Gynecology and Women's Health, University of Missouri School of Medicine, 500 N. Keene Street, Columbia, MO, USA. schustd@health.missouri.edu.
authors.forename: Danny J
authors.initials: DJ
authors.lastname: Schust
mesh_terms: D015820:Cadherins / Q000378:metabolism; D002454:Cell Differentiation / Q000502:physiology*; D002469:Cell Separation; D002478:Cells, Cultured; D006063:Chorionic Gonadotropin / Q000378:metabolism; D004268:DNA-Binding Proteins; D005260:Female; D005434:Flow Cytometry; D015726:Giant Cells / Q000166:cytology* / Q000378:metabolism; D006801:Humans; D066298:In Vitro Techniques; D053552:Keratin-7 / Q000378:metabolism; D009687:Nuclear Proteins / Q000378:metabolism; D010920:Placenta / Q000166:cytology* / Q000378:metabolism; D011247:Pregnancy; D014157:Transcription Factors / Q000378:metabolism; D014327:Trophoblasts / Q000166:cytology* / Q000378:metabolism
chemical_list: D015820:Cadherins; D006063:Chorionic Gonadotropin; D004268:DNA-Binding Proteins; C491089:GCM1 protein, human; D053552:Keratin-7; D009687:Nuclear Proteins; D014157:Transcription Factors