Isolation, Culture, Differentiation, and Nuclear Reprogramming of Mongolian Sheep Fetal Bone Marrow–Derived Mesenchymal Stem Cells

Abstract

We have characterized the differentiation potentiality and the developmental potential of cloned embryos of fetal bone marrow mesenchymal stem cells (BMSCs) isolated from Mongolian sheep. BMSCs were harvested by centrifuging after the explants method and the mononuclear cells obtained were cultured. The isolated BMSCs were uniform, with a fibroblast-like spindle or stellate appearance, and we confirmed expression of OCT4, SOX2, and NANOG genes at passage 3 (P3) by RT-PCR. We measured the growth of the passage 1, 5, and 10 cultures and found exponential growth with a population doubling time of 29.7±0.05 h. We cultured the P3 BMSCs in vitro under inductive environments and were able to induce them to undergo neurogenesis and form cardiomyocytes and adipocytes. Donor cells at passages 3-4 were used for nuclear transfer (NT). We found the BMSCs could be expanded in vitro and used as nuclear donors for somatic cell nuclear transfer (SCNT). Thus, BMSCs are an attractive cell type for large-animal autologous studies and will be valuable material for somatic cell cloning and future transgenic research.

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

“…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 ].…”

PAX3-FOXO1 escapes miR-495 regulation during muscle differentiation

“…PAX3-FOXO1 is also not found in sheep, pig, or horse muscle differentiation systems Given the pro-proliferate activity of PAX3-FOXO1, we then reasoned that larger mammals with higher skeletal muscle mass may have developed a need for the fusion. We have established bone marrow-derived MSC cells from pig and sheep, and ASC cells from horse [11,12]. We then induced them into skeletal muscle differentiation for up to 20 days.…”

“…Mouse bone marrow-derived mesenchymal stem cells D1-MSCs, and adipose-derived stromal/stem cells (ASC) were gifts from Dr. Gary Balian (University of Virginia). Sheep MSC and horse ASC cells were established by us previously [11,12]. Pig MSCs, mouse myoblast cell line C2C12, mouse MSCs, and mouse ASCs were maintained in Dulbecco's modified Eagle's medium (DMEM) containing 10% FBS (Gibco).…”

Atividade dos genes relacionados à pluripotência em ovinos

“…Roxo: Payen et al, 1997Liu et al, 2014;Parte et al, 2014. Cinza: Bao et al, 2011Shi et al, 2015;Su et al, 2015. Funções dos genes relacionados à pluripotência em ovinos O gene OCT4 foi o primeiro GRPs caracterizado em mamíferos (Schöler et al, 1989;Rosner et al, 1990). Além disso, OCT4 permanece como o principal GRPs estudado e o único indispensável para manutenção da pluripotência em camundongos e humanos (Nichols et al, 1998;Wu e Schöler, 2014;Zeineddine et al, 2014).…”