Potential of Adipose-Derived Mesenchymal Stem Cells and Skeletal Muscle-Derived Satellite Cells for Somatic Cell Nuclear Transfer Mediated Transgenesis in Arbas Cashmere Goats

Abstract

Somatic cell nuclear transfer is used to generate genetic models for research and new, genetically modified livestock varieties. Goat fetal fibroblast cells (gFFCs) are the predominant nuclear donors in Cashmere goat transgenic cloning, but have disadvantages. We evaluated the potential of goat adipose-derived mesenchymal stem cells (gADSCs) and goat skeletal muscle-derived satellite cells (gMDSCs) for somatic cell nuclear transfer, evaluating their proliferation, pluripotency, transfection efficiency and capacity to support full term development of embryos after additive gene transfer or homologous recombination. gADSCs and gMDSCs were isolated by enzyme digestion and differentiated into neurocytes, myotube cells and insulin-producing cells. Neuron-specific enolase, fast muscle myosin and insulin expression were determined by immunohistochemistry. Following somatic cell nuclear transfer with donor cells derived from gADSCs, gMDSCs and gFFCs, transfection and cloning efficiencies were compared. Red fluorescent protein levels were determined by quantitative PCR and western blotting. 5-Methylcytosine, H4K5, H4K12 and H3K18 were determined immunohistochemically. gADSCs and gMDSCs were maintained in culture for up to 65 passages, whereas gFFCs could be passaged barely more than 15 times. gADSCs and gMDSCs had higher fluorescent colony forming efficiency and greater convergence (20%) and cleavage (10%) rates than gFFCs, and exhibited differing H4K5 histone modification patterns after somatic cell nuclear transfer and in vitro cultivation. After transfection with a pDsRed2-1 expression plasmid, the integrated exogenous genes did not influence the pluripotency of gADSCs–pDsRed2-1 or gMDSCs–pDsRed2-1. DsRed2 mRNA expression by cloned embryos derived from gADSCs–pDsRed2-1 or gMDSCs–pDsRed2-1 was more than twice that of gFFCs–pDsRed2-1 embryos (P<0.01). Pregnancy rates of gADSCs–pDsRed2-1 and gMDSCs–pDsRed2-1 recipients were higher than those of gFFCs–pDsRed2-1 recipients (P<0.01). With their high proliferative capacity and transfection efficiency, gADSCs and gMDSCs are a valuable cell source for breeding new, genetically modified varieties of livestock by somatic cell nuclear transfer.

Hypoxia enhances buffalo adipose‐derived mesenchymal stem cells proliferation, stemness, and reprogramming into induced pluripotent stem cells

“…In particular, biPSCs exhibiting packed dome morphology Because of their multipotent nature, capacity for self-renewal, easy collection, and amplification in vitro, ASCs have become an ideal candidate for regenerative medicine, and donor cells in SCNT and iPSCs research (Abouhamzeh et al, 2015;Ren et al, 2014;Wei et al, 2015;Y. Zhang et al, 2014).…”

Fibroblast Growth Factor 1-Transfected Adipose-Derived Mesenchymal Stem Cells Promote Angiogenic Proliferation

“…The day (day 5) in which all the cells were dead was determined. The optimal concentration of G418 for selection (100 mg/mL) was defined as the lowest dose at which all the cells died (Moore et al, 2010;Ren et al, 2014).…”

“…Briefly, on sixth day posttransfection, the medium was replaced by DMEM containing G418 (100 mg/mL) and this treatment was continued for six consecutive days. The detached untransfected cells were removed by changing the medium every other day (Moore et al, 2010;Ren et al, 2014). The selected cells were then maintained and expanded in the G418-free DMEM and used for confirmation of FGF1 mRNA expression.…”

Impact of the source and serial passaging of goat mesenchymal stem cells on osteogenic differentiation potential: implications for bone tissue engineering

“…Goat adipose-derived MSCs were isolated and expanded in tissue culture using methods previously reported for equine and goat fat [ 11 , 25 ]. Briefly, 5 g of subQ adipose tissue was harvested from the inguinal area of a 2 - year - old mixed breed goat.…”

“…Adult stem cells can be conveniently sampled from an extensive array of sources bypassing the ethical controversy associated with the embryonic stem cells, which is increasingly affecting the use of stem cell research and therapy. Even though, MSCs can be isolated from a variety of tissues and species, there is significant variation in their biological properties (morphological and functional changes) [ 11 – 17 ]. These changes can be due to the source of MSCs, age-dependent donor-to-donor variation, and due to the signaling pathways that each cell type undergoes during differentiation.…”