Studies on the Establishment of Optimal Culture Conditions of Chicken Sox9 Positive Cells and the Exploration of Putative Bioactive Substances

학위논문 (박사)-- 서울대학교 대학원 : 농업생명과학대학 농생명공학부(바이오모듈레이션전공), 2018. 8. 임정묵.Bone marrow cells are considered as the prominent cell source for clinical application to injuries and degenerative diseases. For cell-based therapies, the optimal cell source must be readily accessible with easily isolated and large-scale expanded cells maintenance is important for clinical application. Optimizing manipulation protocol of chicken bone marrow cells have not been reported because of their heterogeneous phenotype and mixed cell population. In this study, I conducted a study on the cell characteristics and the culture environment for a mass culture environment using chicken Sox9 positive cells. And the possibility of applying the treatment confirmed through consideration of the application in terms of regenerative medicine using the cell secretions. First, I observed the histology of the long bone and cellular properties of bone marrow cells retrieved from 4-day-old neonatal chicks. Long bones, such as the femur, have been a major source of bone marrow cells in which endochondral ossification is responsible for bone formation. Histology, cell culture outcomes, specific marker expression, and differentiation potential were evaluated the academic and industrial feasibility of chick bone marrow cells as a new biomaterial. The results of this study clearly demonstrated that bone marrow tissue of neonatal chicks was premature at least up to 4 days after hatching. This tissue contains a variety of cells involved in endochondral ossification as well as mature bone marrow cells. The majority of these cells became cuboidal until the end of primary culture, and more than 80% of cells were Sox9-positive. The results of the osteogenic and chondrogenic differentiation ability suggest the feasibility of Sox9 for regenerating bone marrow tissues. Next, I investigated the necessity culture regime for enhancing cell maintenance, expanding the Sox9-rich neonatal bone marrow cells and differentiation through a suspended culture. Neonatal bone marrow cells retrieved from 4-day-old white leghorn (WL) were subcultured in high glucose DMEM, low glucose DMEM or DMEM/F12. Use of DMEM/F12 as a basic medium for chicken bone marrow cell culture was found suitable for the culture environment from the results of this study. In retrieved cells from 4-day-old chicken, osteogenic and chondrogenic related gene expression were expressed regardless types of culture media. But, changing of basic medium to DMEM/F12 mainly serves as the activator of cell proliferation. Also, this beneficial function directly enhances osteogenic or chondrogenic differentiations potential of sox9 positive cells using DMEM/F12 media although mixed population of the cells involving endochondral ossification. In conclusion, the 4-day-old chicken bone marrow cells culture in vitro amplifies Sox9- and Col II-positive cells, and optimized culture environment of DMEM/F12 was useful for customizing culture regime for cell maintenance and differentiation. Third study is the analysis on effectiveness of basic fibroblast growth factor (bFGF) and stem cell culture medium like primordial germ cell (PGC) culture media for expanding germness and pluripotent related expression. First, In DMEM/F12 culture media with different concentrations of bFGF (0ng/ml, 1ng/ml, 5ng/ml, 10ng/ml) are added to the optimized sox9 positive cell culture medium. Basic fibroblast growth factor (bFGF) is a member of the FGF family and is a critical component of stem cell culture field. However, the role and optimized concentration of bFGF on promoting the chicken derived sox9 positive cells has not been reported. To monitor the influence of bFGF on retrieved cells in vitro, proliferative capacity, cell morphology, gene expression, immunophenotype and differentiation potential and functional tests like cell migration and invasion assay were evaluated as experimental parameters. Second, the PCG culture medium is similar to the medium condition of embryonic stem cells, which has been amplification of the germness and pluripotent gene expression of primary cells in 4-day-old whole bone marrow. In conclusion, the results of this study suggested that addition of 5 ng/ml bFGF in vitro on sox9 positive cells was effective for proliferation, differentiation, migration and invasion ability. Also using PGC media for culture of isolated cells shows different cell characteristics. In cell specific marker expression, stromal cell marker (CD44) expressed on PGC culture media and in differentiation assay shows adipogenic differentiation ability on PGC medium cultured cell population. Finally, I applied putative bioactive substances substance to human adipose derived mesenchymal stromal cells (hADSC) that is the origin of cell secretion and explored the effect of regenerative medicine such as bone differentiation potential. Bone marrow cells from four-day-old white leghorn chicks differentiated into osteogenic lineages. After 3weeks differentiation time, media changes serum-free fresh culture media. These Collected media were concentrated until total protein concentration reached to 50, 100 or 200 ug/ml. Using these concentration soup, osteogenesis was induced either by routine protocol or by the protocol using at different concentrations of total protein. From the results of this experimentation suggests that certain substrates secreted by chick bone marrow cells stimulate osteogenic differentiation of hADSCs.CHAPTER 1 : General Introduction 1 CHAPTER 2 : Literature Review 11 1. Chicken as an experimental animal 12 2. Difference in developmental stages between aves and mammals 13 3. Bone formation 14 4. Stem cells 15 5. Bone marrow cells 16 5.1. Chicken cell culture medium 21 5.2. Culture conditions of osteogenic differentiation 23 5.3. Characterization of Bone marrow-derived cells 25 6. Sox9 positive cells 28 6.1. Characterization and Signal Pathway 30 7. Extracellular matrix and cell-to-cell interaction 33 7.1. Hyaluronan 35 7.2. Chondroitin Sulfate 36 7.3. ECM and the Stem Cell Niche 37 8. Conditioned media 39 9. Future directions, Perspective 40 9.1. Limitation of regenerative medicine using stem cell therapy 40 9.2. Use of chicken bone marrow-derived progenitor cells in regenerative medicine 42 CHAPTER 3 : General materials and methods 44 CHAPTER 4 : Characterization of Bone Marrow Cells Retrieved from Neonatal ChicksDocto

닭 골수 유래 세포의 확립과 조작기법 향상에 관한 연구

학위논문 (석사)-- 서울대학교 대학원 : 농생명공학부, 2016. 8. 임정묵.Bone marrow-derived cells have an enormous value for the research on mesenchymal stem cell and cell transformation. And also Mesenchymal-derived, multipotent cells have become a valuable resource for cell-to-tissue regeneration and experimental modelling for differentiation and reprogramming. In the chicken, bone marrow-derived cells can induce somatic chimerism and if their pluripotency is confirmed, they could be used as resources to expand the applications of chicken pluripotent cells for various purposes. Bone marrow cells are considered as the basic material for clinical application to injuries and degenerative diseases. For clinical implementation of bone marrow cells, their stable maintenance is important for optimizing manipulation protocol of chicken bone marrow cells and the large-scale expanded culture system has been used to high reproducibility of bone marrow cells. And also no information on cell culture properties have been reported because of their heterogeneous phenotype and mixed cell population. In this study, optimized and improved manipulation system of chicken bone marrow derived cells was established and by evaluating the effect of addition of growth factors on the growth and maintenance of chicken bone marrow derived cells. First, I observed two different populations were maintained: a spindle shape dominant population consisting mainly of fibroblast-like cells and a cuboidal shape dominant population. So, I investigated A significant correlation affecting cell retrieval was detected between the parameters of body weight and leg length in the spindle shape cell-dominant and cuboidal shape cell-dominant populations. Additionally, analysis of cell kinetics, protein marker expression of the chicken bone marrow cells at passage 5. As the results, based on these observations, we conclude that chicken bone marrow cells possess different cellular characteristics compared with the bone marrow cells of mice and humans, and that a different approach and culture regimen may be necessary for manipulating chicken bone marrow-derived multifunctional cells. Next, I investigated the necessity of growth factors and different basic media to modify the culture system of chicken bone marrow cells and the change of cellular properties in chicken bone marrow cells through a suspended culture. Four-day-old white leghorn chicks were employed for experimental animal and the isolated bone marrow derived cells were cultured in different four types media(High glucose DMEM, Low glucose DMEM, F-12/DMEM, αMEM) to which 5 ng/ml bFGF and 500 unit/ml LIF were supplemented or not. To monitor of the effect of growth factors on the capacity of cell maintenance in vitro, primary cell attachment, CFU-F colony number, proliferative activity was evaluated as experimental parameters. As a results, the number of CFU-F-positive colonies was higher (p<0.05) after the bFGF and LIF addition than after no addition, which resulted improved primary cell attachment and also better proliferative activity was detected in the growth factor supplementation group. Additionally, analysis of pluripotency- or differentiation-related gene expression and protein marker expression in the chicken bone marrow derived cells were detected. Different expression profile in the expression of several genes was detected after the supplementation or not, which differed from that of primordial germ cells and embryonic fibroblasts. And also, flow cytometry analysis data shows more expression rate detected such as CD44, CD105, MCAM in growth factors supplementation group. In conclusion, the combined addition of bFGF and LIF to culture medium improved the culture efficiency of chicken BMCs of mixed population, which may contain various reprogrammable cells.CHAPTER 1 : General Introduction 1 CHAPTER 2 : Literature Review 8 1. Bone marrow derived cells 9 2. Mesenchymal stem cells differentiation pathways 11 2.1 Mesoderm differentiation 12 2.2 Ectoderm differentiation 13 3. Mesenchymal stem cells isolation 14 3.1 Mesenchymal stem cell isolation methods 16 3.2 Leukemia inhibitory factor (LIF) signaling 18 3.3 Fibroblast growth factor-2 (FGF-2) signaling 20 4. Mesenchymal stem cells characterization 22 5. Mesenchymal stem cell clinical application 24 CHAPTER 3 : Culture and Subsequent Characterization of Bone Marrow Cells Retrieved from Neonatal White Leg Horn of Different Physical Profiles 29 1. Introduction 30 2. Material and Method 32 3. Result 37 4. Discussion 49 CHAPTER 4 : Optimizaition of Medium Composition for the Expansion of Bone Marrow-derived Cells in Chicken 52 1. Introduction 53 2. Material and Method 55 3. Result 62 4. Discussion 84 CHAPTER 5 : Improvement of Medium Composition by the Addition of Growth Factors for the Expansion of Bone Marrow-derived Cells in Chicken 88 1. Introduction 89 2. Material and Method 91 3. Result 97 4. Discussion 112 CHAPTER 6 : General Disccussion and Conclusion 114 REFERENCE 119 SUMMARY IN KOREAN 141Maste