Research On Generation Of Arbas Cashmere Goat Induced Pluripotent Stem Cells

Arbas Cashmere goat is an important livestock specie in Inner Mongolia with excellent quality of cashmere and meat. The cashmere is considered as soft gold, the meat as ginseng. Arbas Cashmere goat plays an important role in the genetic breeding of animal husbandry in China. The generation of Arbas Cashmere goat pluripotent stem cells with modern biotechnology could not only provide theoretical basis for the development of related disciplines, but also has significant values in genetic breeding. At present, the embryonic stem cell line of livestock has not been established successfully, although a lot of research achievement has been obtained in the field. Recently, it is possible to reprogram somatic cell to pluripotent stem cells with some specific factors with the application of the induced pluripotent stem cell (iPSC) technique.In this study, Arbas Cashmere goat fetal fibroblasts (GFFs) were reprogrammed to pluripotent stem cells (giPSCs) by defined factors. Then the expression of embryonic stem cells markers and its karyotype of giPSCs were observed. Pluripotency of these cells was confirmed by embryoid body formation in vitro and teratoma formation assays which generated all three germ layers.The global gene expression profiling of giPSCs was analysed to reveal reprogramming and molecular mechanism. The results of this study provided experimental basis for the establishement of the Cashmere goat embryonic stem cells.1 Establishment of giPSC line of Arbas Cashmere goat1.1 Generation of giPSCs of Arbas Cashmere goatDefined factors (Oct4, Sox2, c-Myc and Klf4) were transducted into Arbas Cashmere goat fetal fibroblast cells with the doxycycline inducible system, and the cells were reprogrammed to pluripotent stem cells. About 20 days after transduction, visible embryonic stem cell clones like mouse embryonic stem cells formed. The Cashmere goat induced pluripotent stem cells had characteristics as stereoscopic growth, elliptic shape, smooth surface and strong refraction. At 28 days after transduction, about 80% of the clones were positive for AP staining.1.2 Pluripotency detection of giPSCsImmunofluorescence staining results showed that giPSCs expressed embryonic stem cell markers as OCT4, SOX2, NANOG, SSEA-1 and TRA-1-60 and TRA-1-81, without the expression of SSEA-3 and SSEA-4. RT-PCR analyses showed that giPSCs expressed embryonic stem cell gene markers as OCT4, SOX2, NANOG, KLF4, LIN28, REX1. giPSCs differentiated and formed embryoid bodies in vitro, and Ⅲβ-Tubulin for ectoderm, desmin for mesoderm and cytokeratin for entoderm were positive after immunofluorescence staining. giPSCs formed the teratoma with three germ layers in vivo. The differentiation of gland-like tissues (endoderm), muscle tissues (mesoderm) and nerve tissues (ectoderm) were observed afer tissue slice staining. Karyotyping assay revealed that the giPSCs had normal karyotype with 58+XX.2 Optimization of induction and culture conditions of giPSCs2.1 Optimization of transcription factors and culture mediumThe combinations of OSMK (4 facotrs in one vector), Oct4+Sox2+Klf4+c-Myc and Oct4+Sox2+Klf4 were used to induce the formation of giPSCs from fetal fibroblasts.22.8±2.9 AP positive clones/5×104 cells were obtained by induction system of OSMK, with higher reprogramming degree and low ability to differentian. The endogenous genes of OCT4 and NANOG were significantly upregulated (P<0.01).23.8±2.2 AP positive clones/5×104 cells were obtained by induction system of Oct4+Sox2+Klf4+c-Myc with high expression of SOX2. The endogenous genes of SOX2 was significantly upregulated (P<0.01). The clones with high passages differentiated easily.5.0±1.0 AP positive clones/5×104 cells were obtained by induction system of Oct4+ Sox2+Klf4 with low expression of OCT4, SOX2 and NANOG. The clones differentiated easily. The endogenous genes of OCT4, SOX2 and NANOG were significantly downregulated (P<0.01). The pluripotent stem cell medium supplemented with small molecular compounds Vc, VPA and LiCl effectively improved the reprogramming efficiency.17.5±1.3 AP positive clones/5×104 cells were obtained in pluripotent stem cell medium supplemented with Vc. The endogenous genes of OCT4, SOX2 and NANOG were significantly upregulated compared with the cells cultured in the medium without the supplemented of Vc(P<0.01).24.8±2.1 AP positive clones/5×104 cells were obtained in pluripotent stem cell medium supplemented with VPA and LiCl. The endogenous genes of OCT4 and NANOG were significantly upregulatedcompared with the cells cultured in the medium without the supplemented of VPA and LiCl (P<0.01).28.9±0.75 AP positive clones/5×104 cells were obtained in pluripotent stem cell medium supplemented with Vc, VPA and LiCl. The endogenous genes of OCT4, Sox2 and NANOG were significantly highlyest upregulated compared with the cells cultured in the medium without the supplemented of Vc, VPA and LiCl. The supplemented of small molecular compounds Vc, VPA and LiCl effectively improved the reprogramming efficiency.2.2 Selection of feeder layer cells and target cellsKM mouse fetal fibroblasts (MEF), cashmere goat fetal fibroblasts (GFF), the mixture of goat fetal fibroblasts cells and mouse fetal fibroblasts (MEF+GFF,1:1) were chosed as giPSCs feeder cells respectively. When the giPSCs were seeded onto MEF, the cells proliferated slowly and differentiated easily with poor state as dim refraction and low expression level of endogenous gene OCT4, SOX2, NANOG(P<0.05). When the giPSCs were seeded onto GFF, the clones proliferated quickly with better state as elliptic shape, smooth surface, strong refraction and high expression level of endogenous gene OCT4, SOX2, NANOG (P<0.05). The mixture of mouse fetal fibroblasts and goat embryonic fibroblasts had the same effects with the feeder cells from mouse fetal fibroblasts.The results showed that the GEF with same origin was suitable for the proliferation of giPSCs. Cashmere goat fetal fibroblasts (GFFs), lung mesenchymal stem cells (LMSCs), bone marrow mesenchymal stem cells (BMSCs), primary dermal papilla cells (PHFDPCs), secondary dermal papilla cells (SHFDPCs) and adult fibroblasts (GEFs) were used respectively to obtain giPSCs. The reprogramming efficiency of GEFs was the highest, and 24.7±2.1 AP positive clones /5×104 cells were obtained, followed by PHFDPCs,20.8±1.7 AP positive clones/5×104 cells, and SHFDPCs generated only 15.3±1.5 AP positive clones. The reprogramming efficiency of both mesenchymal stem cells is lower than that of GFFs and PHFDPCs, with LMSCs of 17.4±1.2 AP positive clones and BMSCs of 18.7±2.2 AP positive clones. The reprogramming efficiency of adult ear fiber cells was low, with 10.4±1.8 AP positive clones /5×104 cells. The results showed that PHFDPCs and GFFs fit the reprogramming of giPSCs.3 Microarray gene expression profiling of giPSCs3.1 Differential gene expression of giPSCsThe microarray gene expression profiling of differential gene expression of giPSCs (GFF-giPSCs), fetal fibroblasts and arbas cashmare goat embryonic stem cell-like cells (ESCLCs) showed that the gene expression among giPSCs, GFFs and ESCLCs had significant differences. iPSCs had entered the reprogramming procedure, but had a certain gap with the ESCLCs. The stem cell pluripotency related genes had expressed, but the expression level compared with ESCLCs was still lower, and the pluripotency was weak. The expression of 20 selected genes related with pluripotency as OCT4, SOX2, NANOG, DNMT3b, KLF5, PODXL, REX-1 were compared among giPSCs, GFFs and ESCLCs.The results showed that the stem cell related genes had begun to express or gene expression increased significantly and the fiber marker gene was significantly reduced in giPSCs.giPSCs had entered the reprogramming procedure. The stem cell related genes in giPSCs was lower than that in ESCLCs. These all showed that the giPSCs already had pluripotency, but with weaker self renewal ability than that of ESCLCs.3.2 Analyses of the signal pathway of stem cell proliferation and pluripotencyThe results of KEGG enrichment analyses of pluripotency and proliferation related signal pathways showed that the gene expression pattern of the pathway of Wnt, MAPK, PI3K-AKT, Cell cycle and DNA replication in giPSCs had significant difference with GFFs, but similar with ESCLCs.The signal pathway of MAPK and PI3K-AKT were positive in giPSCs and improved the pluripotency and self renewal of giPSCs. The stabilization of GSK3β in Wnt pathway upregulated the level of β-catenin, activated the TCF/LEF core transcriptional pathway, then promoted the giPSCs self renewal. The expression of CDK2, CYCA, CYCD, MCM2 in cell cycle and DNA replication were upregulated in giPSCs. The expression of MCM3, MCM4, MCM6, MCM7, CDC20, CDK1 in giPSCs were the same as that in GFFs. The expression of MCM5 was downregulated in giPSCs, indicated that DNA replication activity in giPSC was lower, and the proliferation speed was slow.