In Vitro and In Vivo Development of Horse Cloned Embryos Generated with iPSCs, Mesenchymal Stromal Cells and Fetal or Adult Fibroblasts as Nuclear Donors.

This research study investigates ways to improve the efficiency of cloning horses, exploring the impact of different timings from cell fusion to activation and using various types of cells as nuclear donors, including induced pluripotent stem cells and mesenchymal stromal cells. The researchers identified an optimum timing interval for cell fusion and activation and determined that using certain types of nuclear donors can enhance the production rate of blastocysts.

Experiment Design

The scientists conducted a series of tests to optimize the process of equine cloning. They began by assessing the effects of various time intervals from cell fusion to activation on horse embryos created through cloning. The timings under examination were less than 1 hour, 1-2 hours, and 2-3 hours. The next step involved evaluating the impact of using diverse types of nuclear donor cells in two chained experiments. The cells used were:

• Induced pluripotent stem cells (iPSCs)
• Adult fibroblasts (AF)
• Ooplasts injected with pluripotency-inducing genes (OCT4, SOX2, MYC, and KLF4)
• Umbilical cord-derived mesenchymal stromal cells (UC-MSCs)
• Fetal fibroblasts (FF) derived from an unborn cloned foetus

Results of Time Interval Test

According to the study, the success rate of both blastocyst production and pregnancy was highest for the 2-3 hours group. There were no significant differences in the number of foals born per pregnancy when the 2-3 hours and the 1-2 hours timespan were employed.

Results of Different Cell Types Test

During the testing of varying cell types, it was observed that iPSCs did not generate any embryos at the blastocyst stage, and that the injection of oocytes with pluripotency-inducing genes did not necessarily lead to higher rates of blastocyst production or pregnancy. However, the researchers found that the use of UC-MSCs resulted in a significantly higher blastocyst production compared to using FF or AF.

Conclusions Drawn

Following these experiments, the study concluded that optimal blastocyst production can be achieved by employing a 2-3-hour gap between cell fusion and activation. Additionally, utilizing UC-MSCs as nuclear donors significantly improved the efficiency of blastocyst production. The study also suggested that the FF line might be used to improve the efficacy of an otherwise inefficient AF line. In total, out of 29 foals born, 24 healthy foals were obtained, indicating the potential application of these strategies in boosting the efficiency of equine cloning.