Characterization of bone marrow-derived stem cells and adipose tissue-derived stem cells isolated from transgenic pigs expressing red fluorescent protein

• Main Authors: Shiu-Chung Wong, 王兆中
• Other Authors: 吳信志
• Format: Others
• Language: zh-TW
• Published: 2011
• Online Access: http://ndltd.ncl.edu.tw/handle/35350447365606689418

碩士 === 國立臺灣大學 === 動物科學技術學研究所 === 99 === Adult stem cells are undifferentiated cells found in living organisms that can selfrenew and can differentiate to yield major specialized cell types of the tissue or organ. Mesenchymal stem cells (MSCs) are adult stem cells found in various tissues, such as bone marrow, adipose tissue, umbilical cord, synovium, skeletal muscle and placenta. Although MSCs isolated from different sites have similar phenotype, they may exhibit heterogeneity in differentiation potentials, which related to the tissue sources. Several characteristics, such as multipotency and less ethical concerned make MSCs hold the promise to restore human damaged tissue or organs, but it also faces tremendous challenges on the way to reach clinical application. To elucidate all these problems, improvement of research tools should be applied to gain knowledge from basic cell culture system and model organism. To understand which tissue derivative stem cells will be proper graft materials for repairing target tissue in the future. MSCs were isolated from bone marrow and adipose tissue of transgenic pigs expressing red fluorescent proteins (RFP) gene, an ideal tracing marker used to monitor cellular processes in living systems. Herein, we aim to isolate two kinds of adult stem cells, RFP porcine bone marrow mesenchymal stem cells (rpBMMSCs) and RFP porcine adipose tissue-derived stem cells (rpADSCs) from the same RFP transgenic pigs, and verify their characterizations of proliferation, differentiation, migration, immunosupression properties and expression of surface markers. In proliferation capacity, rpADSCs showed higher colony forming unit fibroblasts (CFU-Fs) than rpBMMSCs (p < 0.05), but rpBMMSCs exhibited the same population doubling time in serial passage to p6. In contrast, proliferation ability of rpADSCs decreased in p6 (p < 0.01). The result in surface markers assay indicated that both rpBMMSCs and rpADSCs are positive in CD29, CD44 and CD90 mesenchymal stem cell surface markers and negative in CD31, CD45 and CD4a hematopoietic cell markers. In mitogen proliferation assay and mix lymphocyte reaction, peripheral blood mononuclear cell proliferation was eased by these two cell types (p < 0.01), which validated the immunomodulatory feature of mesenchymal stem cells. Wound healing assay revealed that rpADSCs migrated faster than rpBMMSCs. Differentiation potency were investigated by osteogenic and adipogenic differentiation. Extracellular calcium produced by rpBMMSCs was higher in osteogenic induction, whereas oil accumulation of induced rpADSCs was higher in adipogenic culture. Adiogenic related gene, adipocyte fatty acid binding protein (FABP4) and osteogenic related gene, osteonectin and osteocalsin gene expressions were also confirmed by RT-PCR. All of differentiated cells expressed RFP gene stably. These results might represent that characteristics of stem cells are tend to fit their original niche. Except characterizing the properties of mesenchymal stem cells for these two cell types, we also proceeded trans-differentiation experiments for rpADSCs. After neural induction medium treatment for three days, rpADSCs expressed several genes related to neural progenitor cell and neural specific βIII-tubulin could be observed in some cells. On the other hand, pancreatic islet differentiation of rpADSCs was induced by high glucose induction medium and three-dimensional culture. Several pancreatic genes, such as Ngn3, NeuroD and Nkx6.1, were detected by RT-PCR in differentiated cells. The next step is to check the function of these cells comparing with mature islet to demonstrate wether ADSCs is a good candidate cell type for islet transplantation. Taken together, both rpBMMSCs and rpADSCs possess regular mesenchymal stem cell properties that can be applied to cell tracing in model animal and some different characteristics of them may influence their future application in regenerative medicine.