Mançanares Ana Carolina Furlanetto, Cabezas Joel, Manríquez José, de Oliveira Vanessa Cristina, Wong Alvaro Yat Sen, Rojas Daniela, Navarrete Aguirre Felipe, Rodriguez-Alvarez Lleretny, Castro Fidel Ovidio
Department of Animal Science, Faculty of Veterinary Science, Universidad de Concepción, Campus Chillan, Chillán 3780000, Chile.
Department of Veterinary Medicine, Faculty of Animal Science and Food Engineering, University of São Paulo, Pirassununga, São Paulo 13630-000, Brazil.
Animals (Basel). 2020 Jun 23;10(6):1078. doi: 10.3390/ani10061078.
In mesenchymal stem cells (MSCs), it has been reported that prostaglandin E2 (PGE2) stimulation of EP2 and EP4 receptors triggers processes such as migration, self-renewal, survival, and proliferation, and their activation is involved in homing. The aim of this work was to establish a genetically modified adipose (aMSC) model in which receptor genes EP2 and EP4 were edited separately using the CRISPR/Cas9 system. After edition, the genes were evaluated as to if the expression of MSC surface markers was affected, as well as the migration capacity in vitro of the generated cells. Adipose MSCs were obtained from Chilean breed horses and cultured in DMEM High Glucose with 10% fetal bovine serum (FBS). sgRNA were cloned into a linearized LentiCRISPRv2GFP vector and transfected into HEK293FT cells for producing viral particles that were used to transduce aMSCs. GFP-expressing cells were separated by sorting to obtain individual clones. Genomic DNA was amplified, and the site-directed mutation frequency was assessed by T7E1, followed by Sanger sequencing. We selected 11 clones of EP2 and 10 clones of EP4, and by Sanger sequencing we confirmed 1 clone knock-out to aMSC/EP2 and one heterozygous mutant clone of aMSC/EP4. Both edited cells had decreased expression of EP2 and EP4 receptors when compared to the wild type, and the edition of EP2 and EP4 did not affect the expression of MSC surface markers, showing the same pattern in filling the scratch. We can conclude that the edition of these receptors in aMSCs does not affect their surface marker phenotype and migration ability when compared to wild-type cells.
在间充质干细胞（MSC）中，据报道前列腺素E2（PGE2）刺激EP2和EP4受体可触发迁移、自我更新、存活和增殖等过程，且它们的激活参与归巢。本研究的目的是建立一种基因编辑的脂肪间充质干细胞（aMSC）模型，其中使用CRISPR/Cas9系统分别编辑受体基因EP2和EP4。编辑后，评估基因是否影响MSC表面标志物的表达以及所产生细胞的体外迁移能力。从智利品种马获取脂肪间充质干细胞，并在含10%胎牛血清（FBS）的高糖DMEM中培养。将sgRNA克隆到线性化的LentiCRISPRv2GFP载体中，并转染到HEK293FT细胞中以产生用于转导aMSC的病毒颗粒。通过分选分离出表达GFP的细胞以获得单个克隆。扩增基因组DNA，并通过T7E1评估定点突变频率，随后进行Sanger测序。我们选择了11个EP2克隆和10个EP4克隆，通过Sanger测序我们确认了1个aMSC/EP2基因敲除克隆和1个aMSC/EP4杂合突变克隆。与野生型相比，这两种编辑细胞的EP2和EP4受体表达均降低，并且EP2和EP4的编辑不影响MSC表面标志物的表达，在划痕修复中显示出相同的模式。我们可以得出结论，与野生型细胞相比，aMSC中这些受体的编辑不影响其表面标志物表型和迁移能力。
