title: Trophoblast stem cell marker gene expression in inner cell mass-derived cells from parthenogenetic equine embryos.
abstract: Although putative horse embryonic stem (ES)-like cell lines have been obtained recently from in vivo-derived embryos, it is currently not known whether it is possible to obtain ES cell (ESC) lines from somatic cell nuclear transfer (SCNT) and parthenogenetic (PA) embryos. Our aim is to establish culture conditions for the derivation of autologous ESC lines for cell therapy studies in an equine model. Our results indicate that both the use of early-stage blastocysts with a clearly visible inner cell mass (ICM) and the use of pronase to dissect the ICM allow the derivation of a higher proportion of primary ICM outgrowths from PA and SCNT embryos. Primary ICM outgrowths express the molecular markers of pluripotency POU class 5 homeobox 1 (POU5F1) and (sex determining region-Y)-box2 (SOX2), and in some cases, NANOG. Cells obtained after the passages of PA primary ICM outgrowths display alkaline phosphatase (AP) activity and POU5F1, SOX2, caudal-related homeobox-2 (CDX2) and eomesodermin (EOMES) expression, but may lose NANOG. Cystic embryoid body-like structures expressing POU5F1, CDX2 and EOMES were produced from these cells. Immunohistochemical analysis of equine embryos reveals the presence of POU5F1 in trophectoderm, primitive endoderm and ICM. These results suggest that cells obtained after passages of primary ICM outgrowths are positive for trophoblast stem cell markers while expressing POU5F1 and displaying AP activity. Therefore, these cells most likely represent trophoblast cells rather than true ESCs. This study represents an important first step towards the production of autologous equine ESCs for pre-clinical cell therapy studies on large animal models.
authors.affiliation: Department of Veterinary Biomedicine, Faculty of Veterinary Medicine, Centre de Recherche en Reproduction Animale, University of Montreal, 3200 Sicotte, St-Hyacinthe, Quebec J2S 7C6, Canada.
authors.forename: Joëlle A
authors.initials: JA
authors.lastname: Desmarais
authors.forename: Simon-Pierre
authors.initials: SP
authors.lastname: Demers
authors.forename: Joao
authors.initials: J
authors.lastname: Suzuki
authors.forename: Simon
authors.initials: S
authors.lastname: Laflamme
authors.forename: Patrick
authors.initials: P
authors.lastname: Vincent
authors.forename: Sheila
authors.initials: S
authors.lastname: Laverty
authors.forename: Lawrence C
authors.initials: LC
authors.lastname: Smith
mesh_terms: D000818:Animals; D015415:Biomarkers / Q000032:analysis / Q000378:metabolism*; D053624:Blastocyst Inner Cell Mass / Q000166:cytology / Q000378:metabolism* / Q000502:physiology; D002417:Cattle; D018929:Cell Culture Techniques; D048708:Cell Growth Processes / Q000502:physiology; D048430:Cell Shape; D002478:Cells, Cultured; D004622:Embryo, Mammalian; D050993:GATA6 Transcription Factor / Q000235:genetics / Q000378:metabolism; D015870:Gene Expression; D006736:Horses / Q000235:genetics / Q000378:metabolism / Q000502:physiology; D053652:Nuclear Transfer Techniques; D050814:Octamer Transcription Factor-3 / Q000235:genetics / Q000378:metabolism; D010312:Parthenogenesis / Q000235:genetics* / Q000502:physiology; D013234:Stem Cells / Q000166:cytology / Q000378:metabolism*; D014327:Trophoblasts / Q000166:cytology / Q000378:metabolism*
chemical_list: D015415:Biomarkers; D050993:GATA6 Transcription Factor; D050814:Octamer Transcription Factor-3