幹細胞球體於心肌再生醫學之應用｜國立清華大學博碩士論文庫

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研究生：	李文毓
論文名稱：	幹細胞球體於心肌再生醫學之應用 Development of Cell Body Systems for Cellular Therapy in Myocardial Regenerative Medicine
指導教授：	宋信文
口試委員:	許明照張燕黃效民林維文王俊杰宋信文
學位類別：	博士 Doctor
系所名稱：	工學院 - 化學工程學系 Department of Chemical Engineering
論文出版年：	2011
畢業學年度：	99
語文別：	英文
論文頁數：	99
中文關鍵詞：	甲基纖維素、細胞球體、心肌梗塞、細胞移植、血管新生
相關次數：	點閱：1 下載：0
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ABSTRACT
Myocardial infarction (MI) represents one of the major causes of morbidity and mortality worldwide. Following MI, cardiomyocytes undergo apoptosis due to the reduced or obstructed blood flow, thus leading to impaired cardiac functions. Stem cell transplantation is a promising therapeutic strategy for ischemic heart diseases; however, retention of the transplanted cells at the sites of the cell graft is frequently limited.
In study I, we developed a methylcellulose hydrogel system to cultivate spherically-symmetric cell bodies with enriched endogenous extracellular matrices (ECM). The obtained cell bodies were transplanted into the skeletal muscle of rats via local injection. It was found that the cell aggregates can provide an adequate physical size to entrap into the muscular interstices and offer a favorable ECM environment to enhance retention of the transplanted cells at the sites of the cell graft. The obtained results indicated that the spherically-symmetric cell aggregates developed in the study may serve as a cell-delivery vehicle for therapeutic applications.
Our previous study found that human amniotic-fluid stem cells (hAFSCs) are potential to differentiate into endothelial and cardiomyogenic lineages, thus being a suitable cell source for myocardial regeneration. In study II, we cultivated hAFSC bodies for cellular cardiomyoplasty in a rat MI model. Quantitative analyses revealed that, when compared to dissociated hAFSCs, the transplanted hAFSC bodies significantly promoted short-term cell retention and enhanced long-term engraftment, thus improving cardiac function.
Rapid vascularization is essential for the success of treating ischemic tissues. The formation of mature and functional vascular networks requires the cooperation of endothelial cells (ECs) and perivascular cells. In study III, we fabricated core-shell cell bodies composed of cord-blood mesenchymal stem cells (cbMSCs) and human umbilical vascular ECs (HUVECs) for functional vasculogenesis. The in vitro Matrigel tube formation assay demonstrated the inherent abilities of cbMSC/HUVEC core-shell bodies in forming mature and stable tubular networks, showing that the cored cbMSCs can function as perivascular cells to stabilize the elongated vascular networks established by the shelled HUVECs. When embedded in Matrigel and implanted subcutaneously in nude mice, the cbMSC/HUVEC bodies could form visible blood-filled vessels within the matrix. In this study, we establish a new approach by using core-shell bodies of cbMSCs/HUVEVs for functional vascularization, providing a step forward in clinical applications.
The vasculogenic cbMSC/HUVEC bodies were further xeno-transplanted in an experimentally-created MI rat model. Saline, HUVEC bodies and cbMSC bodies were used as controls. Four week after transplantation, rats received cbMSC/HUVEC bodies exhibited significant increased vessel densities and restored heart function. The study demonstrates a new concept of cellular cardiomyoplasy by implanting core-shell bodies of pericytes and ECs for vasculogenesis, thus enhancing blood perfusion and restoring impaired cardiac function

ABSTRACT    I
TABLE OF CONTENT    III
LIST OF FIGURES AND TABLES    VI

Chapter 1. Introduction    1
Chapter 2. Study II    6
2.1. Materials and Methods    6
    2.1.1. Preparation of the cell-aggregate culture system    6
2.1.2. Cultivation of cell aggregates    8
2.1.3. Characterization of cell aggregates    8
2.1.4. Animal study    9
2.1.5.    Statistical analysis    11
2.2. Results and Discussion    12
2.2.1. Construction of cell aggregates    12
2.2.2. Characterization of cell aggregates    13
2.2.3. Animal study    20
2.3. Conclusions    22

Chapter 3. Study III    21
3.1. Materials and Methods    21
3.1.1. Isolation, characterization and transfection of hAFSCs    21
3.1.2. Construction and characterization of hAFSCs bodies    24
3.1.3. Cell transplantation    24
3.1.4. Bioluminescence imaging    25
3.1.5. Echocardiography    25
3.1.6. Magnetic resonance imaging    25
3.1.7. Morphometric and histological analyses    26
3.1.8. Real-time polymerase chain reaction    27
3.1.9. Statistical analysis    28
3.2. Results and Discussion    29
3.2.1. Effects of lentiviral transfection on hAFSCs    30
3.2.2. CharacteristicsofhAFSCbodies    31
      3.2.3. Evaluation of engrafted cells by BLI and real-time PCR    33
      3.2.4. Assessment of LV function by echocardiography and MRI    35
      3.2.5. Morphometric and histological analyses    39
      3.2.6. Differentiation of transplanted hAFSC bodies    41
      3.2.7. Gene expression analysis    42
3.3. Conclusions    44

Chapter 4. Study III    45
4.1. Materials and Methods    45
4.1.1. Cell culture    45
4.1.2. Cultivation of cell bodies    46
4.1.3. Immunostaining of test cells and cell bodies    46
4.1.4. Real-time polymerase chain reaction    47
4.1.5. Tubeformationassay    48
4.1.6. In vivo vasculogenesis    48
4.1.7. Histological analyses    49
4.1.8. Statistical analysis    49
4.2. Results and Discussion    50
    4.2.1. Construction of core-shell bodies    50
    4.2.2. Tube formation assay    53
    4.2.3. Optimization of the ratio of cbMSCs to HUVECs    55

  4.2.4. Colocalization of cbMSCs with HUVECs in forming tubular
       networks    57
  4.2.5. In vivo vascularization    59
   4.3. Conclusions    65

Chapter 5. Study IV    66
   5.1. Materials and Methods    66
          5.1.1. Cell culture    66
  5.1.2. Cultivation of cell bodies    67
  5.1.3. Tube formation assay    67
  5.1.4. Real-time polymerase chain reaction    68
  5.1.5. Transplantation of cell bodies    69
  5.1.6. Echocardiography    69
  5.1.7. Magnetic resonance imaging    70
  5.1.8. Morphometric and histological analyses    70
  5.1.9. Statistical analysis    71
   5.2. Results and Discussion    72
  5.2.1. Cultivation of cbMSC/HUVEC bodies    72
  5.2.2. In vitro tube formation assaym    74
  5.2.3. Gene expression    76
  5.2.4. Cardiac function    77
  5.2.5. Morphometric and histological analyses    80
   5.3. Conclusions    84

References    85
Publication    97

LIST OF FIGURES
Figure 2-1.    (a) Schematic illustrations of the process used for the construction of spherically-symmetric cell aggregates inherent with the endogenous extracellular matrices for local intramuscular injection. (b) Physical structures of the MC hydrogel formed in the wells of a 96-well.    7
Figure 2-2.    Fluorescent images of the formation of a cell aggregate in the 96-well methylcellulose hydrogel system incubated in a living chamber. After cells sank to the bottom of the well, they began to self-assemble and constrict around the well and formed a single cell aggregate within 24 h.    14
Figure 2-3.    Fluorescent images of cell aggregates grown at different cell seeding densities. A single cell aggregate was observed in each well of the 96-well hydrogel system, except for the case with a cell seeding density of 5 × 103 cells/well.    15
Figure 2-4.    Sizes of cell aggregates grown at different cell seeding densities
    15
Figure 2-5.    Live/dead staining images of 4 optical sections of a cell aggregate grown at a cell seeding density of 1.0 × 104 cells/well
    17
Figure 2-6.    Immunofluorescence images of a cell aggregate grown at a cell seeding density of 1.0 × 104 cells/well.    18
Figure 2-7.    (a) Photomicrographs and fluorescent images and (b) immunofluorescence images of a cell aggregate in time sequence, after injection through a needle and seeding onto a 12-well culture plate.    19


Figure 2-8.    Immunofluorescence images of test samples obtained from the groups treated with dissociated cells or cell aggregates retrieved at day 1 or 4 weeks postoperatively.    22
Figure 3-1.    (a, b) Cell morphology and (c) growth of hAFSCs before and after transfection with Fluc gene; (d, e) results of BLI analysis of Fluc-transfected hAFSCs showed increasing bioluminescence signals with cell numbers (r2 = 0.99).    29
Figure 3-2.    (a) Photometric images and (b) diameters of hAFSC bodies grown at different cell densities; (c) immunofluorescence images of hAFSC bodies grown at a cell seeding density of 5 × 103 cells/well; (d) bioluminescence images of grown hAFSC bodies.    32
Figure 3-3.    (a) Representative bioluminescence images and (b) bioluminescence signals of animals treated with dissociated hAFSCs or hAFSC bodies at days 1, 7, 14 and 28 postoperatively; (c) results of real-time PCR quantification of the cell retention rate in the hearts injected with dissociated hAFSCs or hAFSC bodies at day 1 and week 4 after cell transplantation.    34
Figure 3-4.    (a) Representative M-mode echocardiographic images of LV of all test groups recorded at baseline and 4 weeks after cell transplantation; (b) LVFS and (c) changes in LVFS measured by echocardiography at baseline and 4 weeks postoperatively.
    37
Figure 3-5.    (a) Representative MR images of hearts of all test groups in end-systolic and end-diastolic phases; (b) LVEF and (c) infarcted wall thickening fraction assessed by MRI at 4 weeks after transplantation.    38

Figure 3-6.    (a) Representative Masson’s trichrome-stained myocardial sections of all test groups, retrieved at 4 weeks postoperatively; (b) results of quantitative analysis of LV morphometric parameters; (c) the numbers of capillaries and arterioles observed at the infarct and peri-infarct areas in the groups treated with saline, dissociated hAFACs, or hAFSC bodies.    40
Figure 3-7.    Immunofluorescence images of hAFSC bodies transplanted in infarcted hearts observed at 4 weeks after transplantation: (a, d) vWF; (b, e) SMA; and (c) cTnI. Area defined by a square is shown at a higher magnification in the inset.    42
Figure 3-8.    Gene expression of putative paracrine factors using test specimens retrieved at 4 weeks postoperatively.    43
Figure 4-1.    Schematic illustrations showing the processes used in the cultivation of cbMSC/HUVEC core-shell bodies.    47
Figure 4-2.    Representative photomicrographs of core-shell bodies composed of cbMSCs and HUVECs.    51
Figure 4-3.    (a) CLSM images and (b) gene expression levels of HUVECs and cbMSCs after having been cultivated in culture dishes for 24 h.    52
Figure 4-4.    Representative photomicrographs of tube formations on Matrigel.  (a) HUVEC, (b) cbMSC and (c) cbMSC/HUVEC bodies.    54
Figure 4-5.    (a) Photomicrographs of the tubular structures formed on Matrigel by core-shell bodies at different ratios of cbMSCs to HUVECs; (b) their corresponding spreading diameters and (c) numbers of branching points.    56
Figure 4-6.    Representative immunofluorescence CLSM images of tubular networks formed by cbMSC/HUVEC bodies on Matrigel.    58

Figure 4-7.    Representative in situ and ex vivo photomicrographs showing vascularization of different test samples and their corresponding H&E-stained images.    60
Figure 4-8.    Representative immunohistological and immunofluorescence images of the implant containing cbMSC/HUVEC bodies and its forming vessel densities.    63
Figure 5-1.    Schematic illustrations of the process used for the construction of cbMSC/HUVEC core-shell bodies for local intramuscular myocardial injection.    68
Figure 5-2.    Representative photomicrographs immunofluorescence image of of (a) RFP-transfected cbMSCs, (b) HUVECs and (c, d) the cultivated core-shell bodies.    73
Figure 5-3.    (a) Photomicrographs and (b) immunofluorescence images of the tubular structures formed on Matrigel by core-shell bodies.
    75
Figure 5-4.    Gene expression levels of tubular networks harvested at 1, 3, 5 and 7 days after seeding on Matrigel.    77
Figure 5-5.    (a) LVFS and (b) changes in LVFS measured by echocardiography at baseline and 4 weeks postoperatively; (c) representative delayed-enhancement MR images of all studied groups, (d) LVEF and (e) Gd-enhanced area assessed by MRI at 4 weeks after transplantation.    79
Figure 5-6.    (a) Representative Masson’s trichrome-stained myocardial sections of all studied groups, retrieved at 4 weeks postoperatively; (b) results of quantitative analysis of LV morphometric parameters.    80



Figure 5-7.    (a, b) Representative immunohistological images of the heart received cbMSC/HUVEC bodies; (c, d) Vessel densities observed in the at the infarct and peri-infarct areas in all studied groups.    82
Figure 5-8.    Immunofluorescence images of cbMSC/HUVEC bodies transplanted treated hearts observed at 4 weeks after transplantation.    83






















LIST OF TABLES
Table 3-1.     Primer list used for quantitative real-time polymerase chain reaction analyses.    28
Table 3-2.  Results of flowcytometric analysis of Fluc-transfected hAFSCs.
        30
Table 4-1.     Primer list used for quantitative real-time polymerase chain reaction analyses.    48
Table 5-1.     Primer list used for quantitative real-time polymerase chain reaction analyses.    69

                                

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