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In Vitro and In Vivo Development of Horse Cloned Embryos Generated with iPSCs, Mesenchymal Stromal Cells and Fetal or Adult Fibroblasts as Nuclear Donors.
In Vitro and In Vivo Development of Horse Cloned Embryos Generated with iPSCs, Mesenchymal Stromal Cells and Fetal or Adult Fibroblasts as Nuclear Donors.
- Source :
-
PloS one [PLoS One] 2016 Oct 12; Vol. 11 (10), pp. e0164049. Date of Electronic Publication: 2016 Oct 12 (Print Publication: 2016). - Publication Year :
- 2016
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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.<br />Competing Interests: The authors have declared that no competing interests exist.
- Subjects :
- Animals
Blastocyst cytology
Cell Differentiation
Cell Lineage
Cells, Cultured
Embryo Transfer
Female
Horses
Humans
Induced Pluripotent Stem Cells cytology
Kruppel-Like Factor 4
Mesenchymal Stem Cells cytology
Microinjections
Nuclear Transfer Techniques
Oocytes cytology
Plasmids genetics
Plasmids metabolism
Pregnancy
Transcription Factors genetics
Transcription Factors metabolism
Umbilical Cord cytology
Cell Nucleus physiology
Fetus cytology
Fibroblasts cytology
Induced Pluripotent Stem Cells metabolism
Mesenchymal Stem Cells metabolism
Subjects
Details
- Language :
- English
- ISSN :
- 1932-6203
- Volume :
- 11
- Issue :
- 10
- Database :
- MEDLINE
- Journal :
- PloS one
- Publication Type :
- Academic Journal
- Accession number :
- 27732616
- Full Text :
- https://doi.org/10.1371/journal.pone.0164049