title: In Vitro and In Vivo Development of Horse Cloned Embryos Generated with iPSCs, Mesenchymal Stromal Cells and Fetal or Adult Fibroblasts as Nuclear Donors.
abstract: The demand for equine cloning as a tool to preserve high genetic value is growing worldwide; however, nuclear transfer efficiency is still very low. To address this issue, we first evaluated the effects of time from cell fusion to activation (<1h, n = 1261; 1-2h, n = 1773; 2-3h, n = 1647) on in vitro and in vivo development of equine embryos generated by cloning. Then, we evaluated the effects of using different nuclear donor cell types in two successive experiments: I) induced pluripotent stem cells (iPSCs) vs. adult fibroblasts (AF) fused to ooplasts injected with the pluripotency-inducing genes OCT4, SOX2, MYC and KLF4, vs. AF alone as controls; II) umbilical cord-derived mesenchymal stromal cells (UC-MSCs) vs. fetal fibroblasts derived from an unborn cloned foetus (FF) vs. AF from the original individual. In the first experiment, both blastocyst production and pregnancy rates were higher in the 2-3h group (11.5% and 9.5%, respectively), respect to <1h (5.2% and 2%, respectively) and 1-2h (5.6% and 4.7%, respectively) groups (P<0.05). However, percentages of born foals/pregnancies were similar when intervals of 2-3h (35.2%) or 1-2h (35.7%) were used. In contrast to AF, the iPSCs did not generate any blastocyst-stage embryos. Moreover, injection of oocytes with the pluripotency-inducing genes did not improve blastocyst production nor pregnancy rates respect to AF controls. Finally, higher blastocyst production was obtained using UC-MSC (15.6%) than using FF (8.9%) or AF (9.3%), (P<0.05). Despite pregnancy rates were similar for these 3 groups (17.6%, 18.2% and 22%, respectively), viable foals (two) were obtained only by using FF. In summary, optimum blastocyst production rates can be obtained using a 2-3h interval between cell fusion and activation as well as using UC-MSCs as nuclear donors. Moreover, FF line can improve the efficiency of an inefficient AF line. Overall, 24 healthy foals were obtained from a total of 29 born foals.
authors.affiliation: KHEIRON S.A Laboratory, Pilar, Buenos Aires, Argentina.
authors.forename: Ramiro
authors.initials: R
authors.lastname: Olivera
authors.affiliation: Laboratory of Biology of Cell Development, LIAN-Unit associated with CONICET, FLENI, Belen de Escobar, Buenos Aires, Argentina.
authors.forename: Lucia Natalia
authors.initials: LN
authors.lastname: Moro
authors.affiliation: KHEIRON S.A Laboratory, Pilar, Buenos Aires, Argentina.
authors.forename: Roberto
authors.initials: R
authors.lastname: Jordan
authors.affiliation: Laboratory of Biology of Cell Development, LIAN-Unit associated with CONICET, FLENI, Belen de Escobar, Buenos Aires, Argentina.
authors.forename: Carlos
authors.initials: C
authors.lastname: Luzzani
authors.affiliation: Laboratory of Biology of Cell Development, LIAN-Unit associated with CONICET, FLENI, Belen de Escobar, Buenos Aires, Argentina.
authors.forename: Santiago
authors.initials: S
authors.lastname: Miriuka
authors.affiliation: Laboratory of Neruogenetic and Molecular Cytogentic, School of Sciences, National University of San Martin, CONICET, Buenos Aires, Argentina.
authors.forename: Martin
authors.initials: M
authors.lastname: Radrizzani
authors.affiliation: The Roslin Institute and Royal School of Veterinary Studies, University of Edinburgh, Easter Bush, Midlothian, United Kingdom.|Euan MacDonald Centre for MND Research, University of Edinburgh, Edinburgh, United Kingdom.
authors.forename: F Xavier
authors.initials: FX
authors.lastname: Donadeu
authors.affiliation: KHEIRON S.A Laboratory, Pilar, Buenos Aires, Argentina.
authors.forename: Gabriel
authors.initials: G
authors.lastname: Vichera
mesh_terms: D000818:Animals; D001755:Blastocyst / Q000166:cytology; D002454:Cell Differentiation; D019070:Cell Lineage; D002467:Cell Nucleus / Q000502:physiology*; D002478:Cells, Cultured; D004624:Embryo Transfer; D005260:Female; D005333:Fetus / Q000166:cytology*; D005347:Fibroblasts / Q000166:cytology*; D006736:Horses; D006801:Humans; D057026:Induced Pluripotent Stem Cells / Q000166:cytology / Q000378:metabolism*; D000090062:Kruppel-Like Factor 4; D059630:Mesenchymal Stem Cells / Q000166:cytology / Q000378:metabolism*; D008845:Microinjections; D053652:Nuclear Transfer Techniques; D009865:Oocytes / Q000166:cytology; D010957:Plasmids / Q000235:genetics / Q000378:metabolism; D011247:Pregnancy; D014157:Transcription Factors / Q000235:genetics / Q000378:metabolism; D014470:Umbilical Cord / Q000166:cytology
chemical_list: C000715467:KLF4 protein, human; D000090062:Kruppel-Like Factor 4; D014157:Transcription Factors