title: Identification and characterization of epithelial cells derived from human ovarian follicular fluid.
abstract: BACKGROUND
  Follicular fluid is important for follicular development and oocyte maturation. Evidence suggests that follicular fluid is not only rich in proteins but cells. Besides oocytes, the follicular fluid contains granulosa, thecal, and ovarian surface epithelial cells, and both granulosa and thecal cells are well-characterized. However, epithelial cells in follicular fluid are poorly studied. This study aims to isolate and characterize in vitro epithelial cells that originate from human ovarian follicular fluid retrieved in the assisted fertilization program.
  
  
  METHODS
  Follicular fluid samples were collected from 20 women in the assisted reproduction program. Epithelial cells were characterized by flow cytometry assay, immunofluorescence staining, real-time PCR, and time lapse photography.
  
  
  RESULTS
  Epithelial cell cultures were established from 18 samples. A small population of epithelial cells expresses germ-line stem cell markers, such as octamer-binding transcription factor 4 (OCT4), NANOG, and DEAD box polypeptide 4 (DDX4). In the epithelial cell culture system, oocyte-like cells formed spontaneously in vitro and expressed the following transcription markers: deleted in azoospermia-like (DAZL), developmental pluripotency associated protein 3 stella-related protein (STELLA), zona pellucida gene family C (ZPC), Syntaptonemal complex protein (SCP), and growth and differentiation factor 9 (GDF9). Some of the oocyte-like cells developed a zona pellucida-like structure. Both the symmetric and asymmetric division split of epithelial cells and early developing oocytes were observed using time lapse photography. Cell colonies were formed during epithelial culturing, which maintained and proliferated in an undifferentiated way on the feeder layer and expressed some pluripotency markers. These colonies differentiated in vitro into various somatic cell types in all three germ layers, but did not form teratoma when injected into immunodeficient mice. Furthermore, these epithelial cells could be differentiated directly to functional hepatocyte-like cells, which do not exist in ovarian tissues.
  
  
  CONCLUSIONS
  The epithelial cells derived from follicular fluid are a potential stem cell source with a pluripotent/multipotent character for safe application in oogenesis and regenerative medicine.
authors.affiliation: The Center of Research Laboratory, The International Peace Maternity and Child Health Hospital, School of Medicine, Shanghai Jiaotong University, Shanghai, 200030, China. laidongmei@hotmail.com.
authors.forename: Dongmei
authors.initials: D
authors.lastname: Lai
authors.affiliation: The Center of Research Laboratory, The International Peace Maternity and Child Health Hospital, School of Medicine, Shanghai Jiaotong University, Shanghai, 200030, China. xuminhua900505@gmail.com.
authors.forename: Minhua
authors.initials: M
authors.lastname: Xu
authors.affiliation: The Center of Research Laboratory, The International Peace Maternity and Child Health Hospital, School of Medicine, Shanghai Jiaotong University, Shanghai, 200030, China. 10111010022@fudan.edu.cn.
authors.forename: Qiuwan
authors.initials: Q
authors.lastname: Zhang
authors.affiliation: The Center of Research Laboratory, The International Peace Maternity and Child Health Hospital, School of Medicine, Shanghai Jiaotong University, Shanghai, 200030, China. cfychenyifei@yahoo.com.
authors.forename: Yifei
authors.initials: Y
authors.lastname: Chen
authors.affiliation: The Center of Research Laboratory, The International Peace Maternity and Child Health Hospital, School of Medicine, Shanghai Jiaotong University, Shanghai, 200030, China. liting-0002@163.com.
authors.forename: Ting
authors.initials: T
authors.lastname: Li
authors.affiliation: The Center of Research Laboratory, The International Peace Maternity and Child Health Hospital, School of Medicine, Shanghai Jiaotong University, Shanghai, 200030, China. paulial@163.com.
authors.forename: Qian
authors.initials: Q
authors.lastname: Wang
authors.affiliation: State Key Laboratory of Cell Biology, Institute of Biochemistry and Cell Biology, Shanghai Institutes for Biological Sciences, Chinese Academy of Sciences, Shanghai, 200031, China. gaoyimeng@sibcb.ac.cn.
authors.forename: Yimeng
authors.initials: Y
authors.lastname: Gao
authors.affiliation: Eye and ENT Hospital, Fudan University, Shanghai, 200031, China. weics2003@hotmail.com.
authors.forename: Chunsheng
authors.initials: C
authors.lastname: Wei
mesh_terms: D000818:Animals; D002454:Cell Differentiation; D002478:Cells, Cultured; D002868:Chromosomal Proteins, Non-Histone; D003346:Corticotropin-Releasing Hormone / Q000378:metabolism; D053487:DEAD-box RNA Helicases / Q000378:metabolism; D004527:Egg Proteins / Q000378:metabolism; D004847:Epithelial Cells / Q000166:cytology*; D005260:Female; D015571:Follicular Fluid / Q000166:cytology*; D055429:Growth Differentiation Factor 9 / Q000378:metabolism; D022781:Hepatocytes / Q000166:cytology; D018398:Homeodomain Proteins / Q000378:metabolism; D006801:Humans; D008562:Membrane Glycoproteins / Q000378:metabolism; D051379:Mice; D016513:Mice, SCID; D000071317:Nanog Homeobox Protein; D050814:Octamer Transcription Factor-3 / Q000378:metabolism; D009866:Oogenesis / Q000502:physiology*; D011506:Proteins / Q000378:metabolism; D016601:RNA-Binding Proteins / Q000378:metabolism; D011956:Receptors, Cell Surface / Q000378:metabolism; D013724:Teratoma / Q000378:metabolism; D054832:Urocortins / Q000378:metabolism; D015044:Zona Pellucida / Q000378:metabolism; D000072216:Zona Pellucida Glycoproteins
chemical_list: D002868:Chromosomal Proteins, Non-Histone; C101358:DAZL protein, human; C466105:DPPA3 protein, human; D004527:Egg Proteins; C525897:GDF9 protein, human; D055429:Growth Differentiation Factor 9; D018398:Homeodomain Proteins; D008562:Membrane Glycoproteins; C486780:NANOG protein, human; D000071317:Nanog Homeobox Protein; D050814:Octamer Transcription Factor-3; C494173:POU5F1 protein, human; D011506:Proteins; D016601:RNA-Binding Proteins; D011956:Receptors, Cell Surface; C426704:UCN3 protein, human; D054832:Urocortins; D000072216:Zona Pellucida Glycoproteins; D003346:Corticotropin-Releasing Hormone; C498288:DDX4 protein, human; D053487:DEAD-box RNA Helicases