Comparison of <i>In Vitro</i> Developmental Competence of Cloned Caprine Embryos Using Donor Karyoplasts from Adult Bone Marrow Mesenchymal Stem Cells vs Ear Fibroblast Cells

Kwong, Phek Jin; Nam, Hui Yin; Khadijah, W. E. Wan; Kamarul, Tunku; Abdullah, Ramli

doi:10.1111/rda.12262

Cited by 14 publications

(6 citation statements)

References 27 publications

(47 reference statements)

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“…To the best of our knowledge, this study is the first report on the use of MSCs in equine NT, but their potential as nuclear donors has been demonstrated before in other mammalian species. As previously reported, higher in vitro preimplantation development was observed in bovine, 12 porcine 38 and goat 14 with MSCs as nuclear donors compared to fibroblasts. In the porcine, embryos reconstructed with adipose tissue MSCs (aMSCs) resulted in higher blastocyst rates compared to peripheral blood MSCs or fibroblast-reconstructed embryos, 11 which reflects the variability among different MSC sources.…”

Section: Discussionsupporting

confidence: 77%

“…Skin fibroblasts are the most common cells used, but cells derived from liver, 2 kidney, 3 granulosa, 4 , 5 and mesenchymal stem cells (MSCs) have also been tested. In the last years, cloning with MSCs has been evaluated in different farm animal species, such as porcine, 6 – 11 bovine, 12 , 13 caprine, 14 ovine, 15 and equine. 16 Although higher blastocyst rates were observed by using MSCs instead of fibroblasts in most reports, only porcine and bovine offspring has been obtained from MSC cloning procedures.…”

Section: Introductionmentioning

confidence: 99%

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

Olivera¹,

Moro²,

Jordan³

et al. 2018

SCCAA

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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.

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Section: Discussionsupporting

confidence: 77%

Section: Introductionmentioning

confidence: 99%

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

Olivera¹,

Moro²,

Jordan³

et al. 2018

SCCAA

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“…We also compared the capacity of UC-MSCs and FFs to develop in vitro and in vivo after nuclear transfer. To our knowledge, there are no reports using MSCs as nuclear donors in horse cloning, but there are some in other species [ 56 , 57 , 58 , 59 , 60 , 61 ]. One healthy clone derived from adult MSCs in the bovine was generated before, however offspring production rates were not better than those obtained with other somatic cell types [ 62 ], consistent with another report [ 63 ].…”

Section: Discussionmentioning

confidence: 99%

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

Olivera¹,

Moro

Jordan³

et al. 2016

PLoS ONE

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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.

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“…In general, as observed in other mammals (Guan et al, 2010;Kwong et al, 2014), establishment of an adequate cell line is a prerequisite step for the success of cloning and producing induced pluripotent cells . For these techniques, fibroblasts and epithelial cells derived from the skin have been widely used (Jyotsana et al, 2016;Siengdee et al, 2018).…”

Section: Introductionmentioning

confidence: 89%

Isolation, characterization, and cryopreservation of collared peccary skin-derived fibroblast cell lines

Borges

Lira

Nascimento

et al. 2020

PeerJ

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Background Biobanking of cell lines is a promising tool of support for wildlife conservation. In particular, the ability to preserve fibroblast cell lines derived from collared peccaries is of significance as these wild mammals are unique to the Americas and play a large role in maintaining the ecosystem. We identified collared peccary fibroblasts by immunofluorescence and evaluated their morphology, growth and adherence capacity. Further, we monitored the viability and metabolic activity of the fibroblasts to determine the effects of passage number and cryopreservation on establishment of cell lines. Methods Skin biopsies were collected from the peripheral ear region from five adult animals in captivity. Initially, cells were isolated from fragments and cultured in the Dulbecco’s modified Eagle medium supplemented with 10% fetal bovine serum and 2% antibiotic–antimycotic solution under a controlled atmosphere (38.5 °C, 5% CO2). We evaluated the maintenance of primary cells for morphology, adherence capacity of explants, explants in subconfluence, cell growth and absence of contamination. Moreover, we identified the fibroblast cells by immunofluorescence. Additionally, to evaluate the influence of the number of passages (first, third and tenth passage) and cryopreservation on establishment of cell lines, fibroblasts were analysed for the viability, metabolic activity, population doubling time (PDT), levels of reactive oxygen species (ROS), and mitochondrial membrane potential (ΔΨm). Results All explants (20/20) adhered to the dish in 2.4 days ± 0.5 with growth around the explants in 4.6 days ± 0.7, and subconfluence was observed within 7.8 days ± 1.0. Moreover, by morphology and immunocytochemistry analyses, cells were identified as fibroblasts which presented oval nuclei, a fusiform shape and positive vimentin staining. No contamination was observed after culture without antibiotics and antifungals for 30 days. While there was no difference observed for cell viability after the passages (first vs. third: P = 0.98; first vs. tenth: P = 0.76; third vs. tenth: P = 0.85), metabolic activity was found to be reduced in the tenth passage (23.2 ± 12.1%) when compared to that in the first and third passage (100.0 ± 24.4%, P = 0.006). Moreover, the cryopreservation did not influence the viability (P = 0.11), metabolic activity (P = 0.77), or PDT (P = 0.11). Nevertheless, a greater ΔΨm (P = 0.0001) was observed for the cryopreserved cells (2.12 ± 0.14) when compared to that in the non-cryopreserved cells (1.00 ± 0.05). Additionally, the cryopreserved cells showed greater levels of intracellular ROS after thawing (1.69 ± 0.38 vs. 1.00 ± 0.22, P = 0.04). Conclusions This study is the first report on isolation, characterization and cryopreservation of fibroblasts from collared peccaries. We showed that adherent cultures were efficient for obtaining fibroblasts, which can be used as donor cells for nuclei for species cloning and other applications.

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Comparison of In Vitro Developmental Competence of Cloned Caprine Embryos Using Donor Karyoplasts from Adult Bone Marrow Mesenchymal Stem Cells vs Ear Fibroblast Cells

Cited by 14 publications

References 27 publications

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

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

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

Isolation, characterization, and cryopreservation of collared peccary skin-derived fibroblast cell lines

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