Segunda M N, Bahamonde J, Muñoz I, Sepulveda S, Cortez J, De Los Reyes M, Palomino J, Torres C G, Peralta O A
Department of Animal Production Sciences, Faculty of Veterinary and Animal Sciences, University of Chile, Santa Rosa, 11735, Santiago, Chile.
Department of Animal Production Sciences, Faculty of Veterinary and Animal Sciences, University of Chile, Santa Rosa, 11735, Santiago, Chile; Department of Biomedical Sciences and Pathobiology, Virginia-Maryland Regional College of Veterinary Medicine, 205 Duck Pond Drive, Blacksburg, VA, USA.
Theriogenology. 2019 May;130:8-18. doi: 10.1016/j.theriogenology.2019.02.034. Epub 2019 Feb 28.
In vitro gamete derivation based on differentiation of germ cells (GC) from stem cells has emerged as a potential new strategy for the treatment of male infertility. This technology also has potential applications in animal reproduction as an alternative method for dissemination of elite animal genetics, production of transgenic animals, and conservation of endangered species. Mesenchymal stem cells (MSC) are multipotent progenitor cells defined by their ability to differentiate into mesodermal lineages. Under the effect of selected bioactive factors, MSC upregulate expression of pluripotent and GC specific-markers revealing their potential for GC differentiation. In addition to the effect of trophic factors, cell-to-cell interaction with Sertoli cells (SC) may be required to guide the sequential differentiation of MSC into GC. Thus, the aim of the present study was to investigate the effect of coculture with SC on the potential for in vitro GC differentiation of bovine fetal MSC (bfMSC) derived from bone marrow (BM-MSC) and adipose tissue (AT-MSC). bfMSC were isolated from male bovine fetuses and SC were collected from adult bull testes. The effect of SC interaction with BM-MSC or AT-MSC was analyzed on the expression of pluripotent factors OCT4 and NANOG, GC genes FRAGILLIS, STELLA and VASA and male GC markers DAZL, PIWIL2, STRA8 and SCP3 at Day 14 of coculture. Flow cytometry analyses detected that the majority (95,5% ± 2.5; P < 0.05) of the isolated population of SC cultures were positive for SC-specific marker WT1. Levels of mRNA of WT1 in BM-MSC and AT-MSC were lower (P < 0.05) compared to SC; whereas, WT1 expression was not detected in bovine fetal fibroblasts (FB). Cocultures of BM-MSC and AT-MSC with SC had higher (P < 0.05) OCT4 mRNA levels compared to monocultures of BM-MSC, AT-MSC and SC. Moreover, cocultures of BM-MSC with SC had higher (P < 0.05) proportion of cells positive for Oct4 and Nanog compared to monocultures of BM-MSC and SC. Levels of mRNA of DAZL, PIWIL2 and SCP3 were upregulated in cocultures of AT-MSC with SC compared to monocultures of AT-MSC and SC. Accordingly, the proportion of cells positive for Dazl were higher (P < 0.05) in cocultures of AT-MSC with SC compared to monocultures of AT-MSC and SC. Changes in gene expression profiles during coculture of SC with AT-MSC suggest that cell-to-cell interaction or bioactive factors provided by SC may induce progression of AT-MSC into early stages of GC differentiation.
基于干细胞分化为生殖细胞（GC）的体外配子衍生技术已成为治疗男性不育症的一种潜在新策略。该技术在动物繁殖中也有潜在应用，可作为传播优良动物基因、生产转基因动物和保护濒危物种的替代方法。间充质干细胞（MSC）是多能祖细胞，其定义为能够分化为中胚层谱系。在选定的生物活性因子作用下，MSC上调多能和GC特异性标志物的表达，揭示其向GC分化的潜力。除了营养因子的作用外，可能还需要与支持细胞（SC）进行细胞间相互作用，以引导MSC依次分化为GC。因此，本研究的目的是探讨与SC共培养对源自骨髓（BM-MSC）和脂肪组织（AT-MSC）的牛胎儿MSC（bfMSC）体外GC分化潜力的影响。bfMSC从雄性牛胎儿中分离，SC从成年公牛睾丸中收集。在共培养第14天，分析SC与BM-MSC或AT-MSC相互作用对多能因子OCT4和NANOG、GC基因FRAGILLIS、STELLA和VASA以及雄性GC标志物DAZL、PIWIL2、STRA8和SCP3表达的影响。流式细胞术分析检测到，分离的SC培养物群体中大多数（95.5%±2.5；P<0.05）对SC特异性标志物WT1呈阳性。与SC相比，BM-MSC和AT-MSC中WT1的mRNA水平较低（P<0.05）；而在牛胎儿成纤维细胞（FB）中未检测到WT1表达。与BM-MSC、AT-MSC和SC的单培养相比，BM-MSC和AT-MSC与SC的共培养具有更高（P<0.05）的OCT4 mRNA水平。此外，与BM-MSC和SC的单培养相比，BM-MSC与SC的共培养中Oct4和Nanog阳性细胞的比例更高（P<0.05）。与AT-MSC和SC的单培养相比，AT-MSC与SC的共培养中DAZL、PIWIL2和SCP3的mRNA水平上调。因此，与AT-MSC和SC的单培养相比，AT-MSC与SC的共培养中Dazl阳性细胞的比例更高（P<0.05）。SC与AT-MSC共培养期间基因表达谱的变化表明，SC提供的细胞间相互作用或生物活性因子可能诱导AT-MSC向GC分化的早期阶段进展。
