title: Integrin α4-positive human trophoblast progenitors: functional characterization and transcriptional regulation.
abstract: What are the functional characteristics and transcriptional regulators of human trophoblast progenitor cells (TBPCs)?
  
  
  
  TBPC lines established from the human smooth chorion by cell sorting for integrin α4 expressed markers of stemness and trophoblast (TB) stage-specific antigens, invaded Matrigel substrates and contributed to the cytotrophoblasts (CTBs) layer of smooth chorion explants with high-mobility group protein HMGI-C (HMGA2) and transcription factor GATA-4 (GATA4) controlling their progenitor state and TB identity.
  
  
  
  Previously, we reported the derivation of TBPC lines by trypsinization of colonies that formed in cultures of chorionic mesenchyme cells that were treated with an activin nodal inhibitor. Microarray analyses showed that, among integrins, α4 was most highly expressed, and identified HMGA2 and GATA4 as potential transcriptional regulators.
  
  
  
  The aim of this study was to streamline TBPC derivation across gestation. High-cell surface expression of integrin α4 enabled the use of a fluorescence-activated cell sorter (FACS) approach for TBPC isolation from the human smooth chorion (n = 6 lines). To confirm their TBPC identity, we profiled their expression of stemness and TB markers, and growth factor receptors. At a functional level, we assayed their invasive capacity (n = 3) and tropism for the CTB layer of the smooth chorion (n = 3). At a molecular level, we studied the roles of HMGA2 and GATA4.
  
  
  
  Cells were enzymatically disassociated from the human smooth chorion across gestation. FACS was used to isolate the integrin α4-positive population. In total, we established six TBPC lines, two per trimester. Their identity was determined by immunolocalization of a suite of antigens. Function was assessed via Matrigel invasion and co-culture with explants of the human smooth chorion. An siRNA approach was used to down-regulate HMGA2 and GATA4 expression and the results were confirmed by immunoblotting and quantitative reverse transcription-polymerase chain reaction (qRT-PCR) analyses. The endpoints analyzed included proliferation, as determined by 5-bromo-2'-deoxyuridine (BrDU) incorporation, and the expression of stage-specific antigens and hormones, as determined by qRT-PCR and immunostaining approaches.
  
  
  
  As with the original cell lines, the progenitors expressed a combination of human embryonic stem cell and TB markers. Upon differentiation, they primarily formed CTBs, which were capable of Matrigel invasion. Co-culture of the cells with smooth chorion explants enabled their migration through the mesenchyme after which they intercalated within the chorionic CTB layer. Down-regulation of HMGA2 showed that this DNA-binding protein governed their self-renewal. Both HMGA2 and GATA4 had pleitropic effects on the cells' progenitor state and TB identity.
  
  
  
  This study supported our hypothesis that TBPCs from the chorionic mesenchyme can contribute to the subpopulation of CTBs that reside in the smooth chorion. In the absence of in vivo data, which is difficult to obtain in humans, the results have the limitations common to all in vitro studies.
  
  
  
  The accepted view is that progenitors reside among the villous CTB subpopulation. Here, we show that TBPCs also reside in the mesenchymal layer of the smooth chorion throughout gestation. We theorize that they can contribute to the CTB layer in this region. This phenomenon may be particularly important in pathological situations when CTBs of the smooth chorion might provide a functional reserve for CTBs of the placenta proper.
  
  
  
  Research reported in this publication was supported by the Eunice Kennedy Shriver National Institute of Child Health and Human Development of the National Institutes of Health under award P50HD055764. O.G., N.L., K.O., A.P., T.G.-G., M.K., A.B., M.G. have nothing to disclose. S.J.F. received licensing fees and royalties from SeraCare Life Sciences for trisomic TBPC lines that were derived according to the methods described in this manuscript.
  
  
  
  N/A.
authors.affiliation: Center for Reproductive Sciences, University of California San Francisco, San Francisco, CA 94143, USA Department of Obstetrics, Gynecology, and Reproductive Sciences, University of California San Francisco, San Francisco, CA 94143, USA.
authors.forename: O
authors.initials: O
authors.lastname: Genbacev
authors.affiliation: Center for Reproductive Sciences, University of California San Francisco, San Francisco, CA 94143, USA Department of Obstetrics, Gynecology, and Reproductive Sciences, University of California San Francisco, San Francisco, CA 94143, USA.
authors.forename: N
authors.initials: N
authors.lastname: Larocque
authors.affiliation: Center for Reproductive Sciences, University of California San Francisco, San Francisco, CA 94143, USA Department of Obstetrics, Gynecology, and Reproductive Sciences, University of California San Francisco, San Francisco, CA 94143, USA.
authors.forename: K
authors.initials: K
authors.lastname: Ona
authors.affiliation: Center for Reproductive Sciences, University of California San Francisco, San Francisco, CA 94143, USA Department of Obstetrics, Gynecology, and Reproductive Sciences, University of California San Francisco, San Francisco, CA 94143, USA.
authors.forename: A
authors.initials: A
authors.lastname: Prakobphol
authors.affiliation: Center for Reproductive Sciences, University of California San Francisco, San Francisco, CA 94143, USA Department of Obstetrics, Gynecology, and Reproductive Sciences, University of California San Francisco, San Francisco, CA 94143, USA Department of Obstetrics and Gynecology, Fundacion IVI, Instituto Universitario IVI, School of Medicine, Universidad de Valencia, INCLIVA, Valencia, Spain.
authors.forename: T
authors.initials: T
authors.lastname: Garrido-Gomez
authors.affiliation: Center for Reproductive Sciences, University of California San Francisco, San Francisco, CA 94143, USA Department of Obstetrics, Gynecology, and Reproductive Sciences, University of California San Francisco, San Francisco, CA 94143, USA.
authors.forename: M
authors.initials: M
authors.lastname: Kapidzic
authors.affiliation: Center for Reproductive Sciences, University of California San Francisco, San Francisco, CA 94143, USA Department of Obstetrics, Gynecology, and Reproductive Sciences, University of California San Francisco, San Francisco, CA 94143, USA.
authors.forename: A
authors.initials: A
authors.lastname: Bárcena
authors.affiliation: Center for Reproductive Sciences, University of California San Francisco, San Francisco, CA 94143, USA Department of Obstetrics, Gynecology, and Reproductive Sciences, University of California San Francisco, San Francisco, CA 94143, USA.
authors.forename: M
authors.initials: M
authors.lastname: Gormley
authors.affiliation: Center for Reproductive Sciences, University of California San Francisco, San Francisco, CA 94143, USA Department of Obstetrics, Gynecology, and Reproductive Sciences, University of California San Francisco, San Francisco, CA 94143, USA The Eli and Edythe Broad Center for Regeneration Medicine and Stem Cell Research, University of California San Francisco, San Francisco, CA 94143, USA Department of Anatomy, University of California San Francisco, San Francisco, CA 94143, USA sfisher@cgl.ucsf.edu norma.mccormack@ucsf.edu.
authors.forename: S J
authors.initials: SJ
authors.lastname: Fisher
mesh_terms: D002454:Cell Differentiation; D002460:Cell Line; D002823:Chorion / Q000166:cytology / Q000378:metabolism; D018920:Coculture Techniques; D005434:Flow Cytometry; D050991:GATA4 Transcription Factor / Q000235:genetics / Q000378:metabolism / Q000502:physiology*; D018507:Gene Expression Regulation, Developmental; D025743:HMGA2 Protein / Q000235:genetics / Q000378:metabolism / Q000502:physiology; D006801:Humans; D039441:Integrin alpha4 / Q000235:genetics / Q000378:metabolism*; D050436:Regulatory Elements, Transcriptional; D014327:Trophoblasts / Q000378:metabolism*
chemical_list: D050991:GATA4 Transcription Factor; C484170:GATA4 protein, human; D025743:HMGA2 Protein; D039441:Integrin alpha4
keywords: GATA4; HMGA2; progenitor cells; self-renewal; smooth chorion; trophoblast