Isolation Of Embryonic Stem Cells (ESCs) Derived From Porcine Fetal Mesenchymal Stem Cell By Somatic Cell Nuclear Transfer (SCNT)

This study was performed to develop a technique system for producing porcine embryos in vitro by somatic cell nuclear transfer (SCNT), which will be used to clone pigs and isolate embryonic stem cells (ESCs). Different types of porcine cells were used as donor cells, and in vitro matured enucleated oocytes were employed as recipient oocytes. The five parts of this thesis is as follows:1. Preparation of donor cellsWe established 4 porcine fetal fibroblasts (pFF) cell lines from 4 porcine fetuses, one porcine ear skin fibroblasts (pESF) cell line from neonatal porcine era skin tissues by tissue culturing methods and 3 porcine fetal mesenchymal stem cells (pFMSCs) lines from bone marrow of 3 porcine fetuses respectively. pFF, pESF and pFMSCs were cytopreserved from passage one to passage 5 respectively. The results showed that there was no obvious change in morphology and cell viability after thawing. pFMSCs exhibited a stretched fibroblast-like phenotype, and maintained a stable fibroblast-like morphology for 13 passages in culture. Growth delitescence of subcultivated pFMSCs was about 1-2 days and then the cells entered the stage of logarithmic growth. Cells stayed at the stage of growth platform after culturing 7 days. Transmission electron microscopic observation demonstrated that cytoplasmic structures of pFMSCs were rather simple with a high nuclear to cytoplasmic ratio, containing a few organelles. The number of chromosomes counted in pFMSCs of passage 5 showed a normal karyotype, being a male 38 XY with no visible abnormality. Flow cytometry analysis of isolating pFMSCs demonstrated expression of CD44,CD166,CD105,and CD117, and absence of CD45,CD90,CD11a, and CD14. pFMSCs cultured in media containing 5 mMβ- mercaptoechanol differentiated into neuron-like cells, which were Nestin positive. Cell cycle distribution in G0/G1 was 86.72% and 88.44% respectively by flow cytometry detecting after treatment with serum starvation and contact inhibition. Establishment of these porcine cell lines is benefitial to related study for porcine somatic cell nuclear transfer.2. Establishment a system for porcine oocytes in vitro maturation (IVM)Porcine ovaries were obtained from a local abattoir and transported to laboratory in...