재생의학 기술 개발을 위한 세포접착 제어를 통한 중간엽 줄기세포의 기능 조절 연구
: Modulation of mesenchymal stem cell function by control of cell adhesion mechanism for technical development of regeneration medicine

 
저자 강정미
형태사항 ; 26 cm
일반주기 지도교수: 김상헌
학위논문사항 Thesis(doctoral)-- 과학기술연합대학원대학교 : 의공학(BiomedicalEngineering) 2016. 8
발행국 대한민국
언어 영어
출판년 2016
소장기관 과학기술연합대학원대학교

 
초록
This study fabricates novel biofunctional matrices (MBP fusion protein surfaces) with exact cell-matrix adhesion mechanisms that are mediated by heparin sulfate proteoglycan (HSPG) as the engineered extracellular microenvironments. MBP-fused proteins are designed to form cell adhesion matrices for mesenchymal stem cells (hMSCs), in order to examine cellular responses related to cell adhesion signaling. Primary approach is based on assessing cell adhesion behavior and the differentiation capacities that control cell adhesion mechanisms. In addition, MBP fusion proteins are used to promote a three-dimensional (3D) clustering of hMSCs by weakening cell-matrix adhesion for tissue regeneration purpose. This study is composed of three segments, wherein the design and characterization of MBP-fused protein surfaces, the control of hMSC phenotype and differentiation, and a three-dimensional (3D) clustering of hMSCs on a MBP-fused protein and their application are investigated consecutively. 1. In this study, anchor various bioactive proteins (basic FGF (bFGF), VEGF, HBD) linked to maltose-binding protein (MBP) was designed as a biofunctional matrix for cell adhesion. The maltose-binding protein (MBP), which possesses a large number of exposed hydrophobic zones, can be used as a link for the immobilization of growth factors. MBP-fused proteins were immobilized on polystyrene (PS) surfaces by spontaneous adsorption. I analyzed XPS spectra and AFM images to determine the binding energy and topography, respectively, of the MBP-fusion protein-coated surfaces. The adsorption of MBP-fused proteins on PS surfaces was investigated using the nitrogen 1s (N1s) signal intensity in the XPS spectra, a similar approach had been applied to PS surfaces with adsorbed albumin and AFM was used to characterize the topology of surfaces coated with MBP-fusion protein. The amount of MBP-fused proteins immobilized on the PS surface increased with increasing protein concentration. In addition, I sought to determine the adhesive properties of human adipose-derived stem cell (hASC) for extracellular matrix proteins. The adhesion of hASCs to MBP-bFGF immobilized on a PS surface (PS-MBP-bFGF) was inhibited by heparin. Integrin signaling and cell spreading of hASC on PS-MBP-bFGF were down-regulated compared with those on fibronectin-coated surfaces or tissue culture polystyrene (TCP). hASC differentiated into adipocytes, which stained positive for lipid vacuoles with Oil Red, more readily on PS-MBP-bFGF than on TCP. In contrast, hASC hardly differentiated into osteoblast on PS-MBP-bFGF or on TCP. These results suggest that the mechanism of hASC adhesion to PS-MBP-bFGF is mediated by a specific interaction between bFGF and heparin, and that the adhesion mechanism might provide an insight into the design of biomaterials to control the fate of stem cells. 2. Control of cell-matrix adhesion has become an important issue in the regulation of stem cell function. In this study, PS-MBP-bFGF was applied as an artificial matrix to regulate integrin-mediated signaling. I sought to characterize human bone marrow mesenchymal stem cell (hBMSC) behavior in response to two different mechanisms of cell adhesion; (i) bFGF-heparan sulphate proteoglycan (HSPG)-mediated adhesion vs. (ii) fibronectin (FN)-integrin-mediated adhesion. Heparin inhibited hBMSC adhesion to PS-MBP-bFGF but not to FN-coated surface. The phosphorylation of focal adhesion kinase, cytoskeletal re-organisation, and cell proliferation were restricted in hBMSC adhering to PS-MBP-bFGF compared to FN-coated surface. Expression of MSC markers, such as CD105, CD90 and CD166, decreased in hBMSC expanded on PS-MBP-bFGF compared to expression in cells expanded on FN-coated surface. hBMSC that were expanded on FN-coated surface differentiated into osteogenic and adipogenic cells more readily than those that were expanded on PS-MBP-bFGF. Furthermore, we characterised the N-linked glycan structures of hBMSC depending on the cell adhesion mechanism using mass spectrometry (MS)-based quantitative techniques. MS analysis revealed that 2,3-sialylated glycans, a potential marker of stem cell function, were more abundant on hBMSC expanded on FN-coated surface than on those expanded on PS-MBP-bFGF. Thus, the differentiation potential of hBMSC is controlled by the type of adhesion substrate that might provide an idea for the design of biomaterials to control stem cell fate. Elucidation of the glycan structure on the cell membrane may help characterize hBMSC function. In addition, it is essential to characterize the biological properties of MSC populations to maintain quality specifications and control in regenerative medicine. In this study, I also characterize cell adhesion dependent cellular behaviors of hASCs and hBMSCs. I used a PS-MBP-bFGF and a FN-coated surface to restrict and support, respectively, integrin-mediated adhesion. The cells adhered to PS-MBP-bFGF exhibited restricted actin cytoskeleton organization and focal adhesion kinase phosphorylation. The hASCs and hBMSCs exhibited different cytoplasmic projection morphologies on PS-MBP-bFGF. Both hASCs and hBMSCs differentiated more dominantly into osteogenic cells on FN than on PS-MBP-bFGF. In contrast, hASCs differentiated more dominantly into adipogenic cells on PS-MBP-bFGF than on FN, whereas hBMSCs differentiated predominantly into adipogenic cells on FN. The results indicate that hASCs exhibit a competitive differentiation potential (osteogenesis vs. adipogenesis) that depends on the cell adhesion matrix, whereas hBMSCs exhibit both adipogenesis and osteogenesis in integrin-mediated adhesion and thus hBMSCs have non-competitive differentiation potential. I suggest that our approach has potential for development of classification models that use biofunctional materials to classify hMSCs by characterizing their adhesion behaviors and differentiation potentials. 3. hMSCs are an attractive cell source in regenerative

 
목차
I . Fabrication and Characterization of Novel Bio-functional Matrix and Cellular Analysis during Adhesion and Differentiation
1. Background
2. Materials and methods
2.1. Materials
2.2. Expression of recombinant MBP fusion proteins 
2.3. Preparation of cell adhesion substrates
2.4. Characterization of PS surfaces coated with MBP-fused proteins
2.5. Isolation and culture of human adipose-derived stem cells
2.6. Binding of biotinylated heparin to MBP fusion protein surfaces
2.7. hASC adhesion assay
2.8. Adipogenesis and osteogenesis of hASCs on PS-MBP-bFGF
2.9. Quantitative analysis of differentiation
2.10. Statistical analysis
3. Results
3.1. Preparation of MBP fusion proteins
3.2. Characterization of recombinant MBP fusion protein surfaces
3.3. Adhesion mechanism of hASCs on PS-MBP-bFGF
3.4. hASCs differentiation on PS-MBP-bFGF
4. Discussion 

II. Bio-functional Matrices Characterize Cell Function (Signaling,Morphology, and Differentiation Potential)
5. Background 
6. Materials and methods
6.1. Cells culture
6.2. Preparation of cell adhesion surfaces
6.3. Flow cytometry analysis6.4. hMSCs adhesion assay
6.5. hBMSCs proliferation assay
6.6. Western blot analysis
6.7. Immunofluorescence staining
6.8. Fractionation,deglycosylation, and membrane proteins of hMSCs
6.9. Permethylation of N-linked glycans
6.10. MALD-TOF MS analysis 
6.11. Scanning electron microscope (SEM) 
6.12. Adipogenesis and osteogenesis of hMSCs
6.13. Quantitative real-time polymerase chain reaction (qRT-PCR) 
6.14. Statistical analysis
7. Results 
7.1. Control of integrin-mediated behavior in hBMSCs on PS-MBP-bFGF 
7.2. Functional analysis of hBMSCs on PS-MBP-bFGF
7.3. Relative quantitation of N-linked glycans on hMSCs
7.4. Analysis of sialylated N-linked glycans
7.5. Characterization of hMSCs on PS-MBP-bFGF
7.6. Cytoskeletal organization of hMSCs on surfaces
7.7. Cytoplasmic projection morphology of hMSCs on adhesion surfaces
7.8. Differentiation properties of hMSCs on MBP-bFGF and FN surfaces
8. Discussion 

III. Three-Dimensional (3D) clustering of hMSCs cultured on PS-MBP-bFGF enhances Stem Cell function 
III-1. A synergistic therapeutic effect of AngioclusterTM with angiopoietin-1 in ischemic hindlimbs
9. Background 
10. Materials and methods
10.1. hASCs culture and formation of Angiocluster TM
10.2. Cell viability assay
10.3. Flow cytometry analysis
10.4. Gene expression using polymerase chain reaction (PCR) 
10.5. Western blot analysis 
10.6. Human angiogenic protein analysis 
10.7. Preparation of the experimental mouse ischemia model) 
10.8. Treatment of limb ischemia
10.9. Immunofluorescence staining for angiogenesis of implants in ischemia mice 
10.10. Histological examination for ischemic hindlimb muscles 
10.11. Statistical analysis 
11. Results 
11.1. Characterization of monolayer hASC and AngioclusterTM 
11.2. Hypoxic induction and survival of AC
11.3. Production angiogenic factors by hASCs in AC
11.4. Improvement of ischemic limb salvage by transplantation of AC with Ang-1
11.5. Angiogenic and arteriogenic efficacy in ischemic limbs 
11.6. ACs with Ang-1 stimulates angiogenesis via improvement of the ability to paracrine and differentiation into vessel cells, in ischemic areas
11.7. The synergistic angiogenic effect of AC with Ang-1
12. Discussion  

III-2. Chondrogenesis potential in hBMSCs 3D in vitro model
13. Background
14. Materials and met
14.1. Cell culture and chondrogenic differentiation
14.2. Biochemical analysis
14.3. Quantitative real-time RT-PCR
14.4. Histology and immune florescent staining
14.5. Statistical analysis
15. Results 
15.1. Formation of 3D cell mass vs. pellet
15.2. Effects of 3D cell mass vs. pellet on cell proliferation and extracellular matrix synthesis 
15.3. Influences of 3DCM vs. pellet on gene expression profile of differentiating hBMSCs
16. References

주제어
Mesenchymal stem cell, Recombinant protein, Growth factor immobilization, Cell signaling, Cell adhesion, Cell differentiation, 3D cell mass (3DCM), Angiogenesis