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

Abstract

IntroductionCell 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 methodsZona-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).ResultsBlastocyst 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.ConclusionIn 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.

MicroRNA characterization in equine induced pluripotent stem cells

“…Somatic cell nuclear transfer and embryo transfer were performed as previously described by Olivera et al [35, 36]. Briefly, equine oocytes were in vitro matured and enucleated by micromanipulation.…”

Generation of myostatin edited horse embryos using CRISPR/Cas9 technology and somatic cell nuclear transfer

“…Then, in addition to the low blastocyst rates reported for horse cloning, this factor might explain the lower embryo developmental rate of the edited cloned embryos compared to controls. To increase horse cloning development, MSCs could be used as nuclear donors 1 . However, it was demonstrated that MSCs undergo senescence at early passages, showing alterations in cellular morphology, telomere shortening and proliferation arrest after 30 population doublings or 7-10 passages 53 , which makes the generation of edited clonal cell lines rather difficult.…”

“…597, Río Cuarto, Córdoba, Argentina, ZIP code: 5805) and oocytes were matured for 24 h. Matured oocytes were then treated with pronase (#P-8811, Sigma Aldrich Co., USA) to remove the zona pellucida, enucleated by micromanipulation and electrically fused with a donor cell (wild type fibroblast, G1-1 µg-C23, G2-5 µg-C13 or G2-1 µg-C02 cell). In order to incorporate a positive control, we performed the same procedure using MSCs (with the same genomic background as the HFFs) as nuclear donors, considering the enhanced effectiveness of using this type of cells in horse cloning 1 . After 2.5 h fusion, reconstructed embryos were activated with 8.7 μM ionomycin (#I24222; Invitrogen, CA, USA) for 4 min followed by individual culture in a combination of 1 mM 6-dimethylaminopurine (6-DMAP; # D2629, Sigma Aldrich Co., MO, USA) and 5 mg/ml cycloheximide (CHX; #C7698, Sigma Aldrich Co., MO, USA) in 5 µl drops of DMEM/F12 (#D8062, Gibco, Grand Island, NY, USA) for 4 h. Reconstructed embryos were cultured in DMEM/F12 containing 10% FBS, and 1% penicillin-streptomycin in the Well-of-the-Well system, three embryos together per well.…”

“…In the last 17 years, horse cloning has focused on multiplying valuable individuals mainly because of commercial interests 1 , Kheiron S.A ( www.kheiron-biotech.com ), ViaGen ( www.viagen.com )]. The strongest advantage of this technique is the genome conservative property, so that the cloned foals invariably “inherit” the original genotype.…”

Assisted reproductive techniques in mares

“…Recently, work from Argentina has suggested that use of bone‐marrow derived mesenchymal stem cells as donor cells for nuclear transfer may increase the likelihood of production of normally healthy foals (Olivera et al., , ). The possibility exists that cloning could be performed using somatic cells isolated from frozen‐thawed semen, which would allow production of clones in cases in which frozen semen was the only existing source of cells from an individual.…”