Bone marrow mesenchymal stem cells as nuclear donors improve viability and health of cloned horses

Abstract

Introduction: Cell plasticity is crucial in cloning to allow an efficient nuclear reprogramming and healthy offspring. Hence, cells with high plasticity, such as multipotent mesenchymal stem cells (MSCs), may be a promising alternative for horse cloning. In this study, we evaluated the use of bone marrow-MSCs (BM-MSCs) as nuclear donors in horse cloning, and we compared the in vitro and in vivo embryo development with respect to fibroblasts.

Materials and methods: Zona-free nuclear transfer was performed using BM-MSCs (MSC group, n=3432) or adult fibroblasts (AF group, n=4527). Embryos produced by artificial insemination (AI) recovered by uterine flushing and transferred to recipient mares were used as controls (AI group).

Results: Blastocyst development was higher in the MSC group than in the AF group (18.1% vs 10.9%, respectively; p<0.05). However, pregnancy rates and delivery rates were similar in both cloning groups, although they were lower than in the AI group (pregnancy rates: 17.7% [41/232] for MSC, 12.5% [37/297] for AF and 80.7% [71/88] for AI; delivery rates: 56.8% [21/37], 41.5% [17/41] and 90.1% [64/71], respectively). Remarkably, the gestation length of the AF group was significantly longer than the control (361.7±10.9 vs 333.9±8.7 days), in contrast to the MSC group (340.6±8.89 days). Of the total deliveries, 95.2% (20/21) of the MSC-foals were viable, compared to 52.9% (9/17) of the AF-foals (p<0.05). In addition, the AF-foals had more physiological abnormalities at birth than the MSC-foals; 90.5% (19/21) of the MSC-delivered foals were completely normal and healthy, compared to 35.3% (6/17) in the AF group. The abnormalities included flexural or angular limb deformities, umbilical cord enlargement, placental alterations and signs of syndrome of neonatal maladjustment, which were treated in most cases.

Conclusion: In summary, we obtained 29 viable cloned foals and found that MSCs are suitable donor cells in horse cloning. Even more, these cells could be more efficiently reprogrammed compared to fibroblasts.

Keywords: MSC; SCNT; cloning; equine.

Figure 1

Evaluation of the mesenchymal status of the bone marrow–derived cells. Notes: (A) Characterization of cell surface markers CD29, CD44, CD90 and CD105 of mesenchymal origin and CD34 of hematopoietic origin by flow cytometry in two different MSC lines. The histograms show cell fluorescence intensity on the X-axis and cell frequency distribution on the Y-axis. The bone marrow isolated cells were positive for the four mesenchymal markers and negative for the hematopoietic marker. (B) Evaluation of multipotent capability of bone marrow–derived cells into (a) chondrogenic, (b) adipogenic and (c) osteogenic lineages. The isolated cells were able to differentiate into these three lineages. Abbreviation: BM-MSC, bone marrow–derived mesenchymal stem cell.

Figure 2

Eight cloned polo horses derived from the same MSC line, born in August, September and October 2016. Abbreviation: MSC, mesenchymal stem cell.

Figure 3

Weight at birth and gestational length of the AF and MSC cloned groups. Notes: The green dashed lines are delimiting the range of normality according to information obtained from artificial insemination foals. Both weight at birth and gestational length were higher in the AF group compared to the MSC group (Student’s t-test, *p<0.05; ***p<0.001). Abbreviations: AF, adult fibroblasts; MSC, mesenchymal stem cell.