Myocardial infarction (MI) progresses from the acute death of cardiomyocytes and the infiltration of inflammatory cells into granulation, followed by scars. Recently, cell transplantation via local intramuscular injection is a promising therapy for patients with MI. However, the time required to process and expand autologous cells in vitro makes applying these cells in patients with an acute MI in a timely fashion difficult. Moreover, following intramuscular injection, retention of the dissociated cells in the transplanted area remains problematic. These facts can be deleterious to cell transplantation therapy.
The purpose of study I to evaluate using human amniotic fluid stem cells (hAFSCs) derived from second-trimester amniocentesis for the therapeutic potential of cardiac repair. Whether hAFSCs can be differentiated into cardiomyogenic cells and toward the maturation of endothelial cell lineage was investigated in vitro using mimicking differentiation milieu. Test samples were xenogenically transplanted into the peri-ischemia area of an immune-suppressed rat model at 1 week after MI induction. There were three treatment groups (n ≥ 10): sham; saline; dissociated hAFSCs. After 4 weeks, hAFSCs-treated animals showed a preservation of the infracted thickness, an attenuation of left ventricle remodeling, a higher vascular density, and thus an improvement in cardiac function.
Study II examined the hypothesis that the thermoreversible methylcellulose hydrogel was used as a coating on TCPS dishes and developed for harvesting living hAFSCs sheets. The optioned hAFSCs sheets preserved the intercellular junctions and endogenous extracellular matrix (ECM) and retained the cell phenotype. Test samples were xenogenically transplanted into the peri-ischemia area of an immune-suppressed rat model at 1 week after MI induction. There were four treatment groups (n ≥ 10): sham; saline; dissociated hAFSCs; and hAFSCs sheet fragments. The results obtained in the echocardiography revealed that the group treated with hAFSCs-sheet-fragment had a superior heart function to those treated with saline and dissociated hAFSCs group (P < 0.05). Due to their inherent ECM, hAFSCs sheet fragments had a better ability of cell retention and proliferation than dissociated hAFSCs upon transplantation to the host myocardium. Additionally, transplantation of the hAFSCs sheet fragments stimulated a signification increase in vascular density, consequently contributing towards improved wall thickness and a reduction in the infarct size. Our histological findings and qPCR analyses suggest that the transplanted hAFSCs can be differentiated into cardiomyocyte-like cells and cell of endothelial lineaged and modulate expression of multiple angiogenic cytokines and cardiac protective factor with the potential to promote neo-vascularization, which evidently contributed to the improvement of ventricular function. These results suggest that the use of fragments of cell sheets can serve as a cell-delivery vehicle by providing an adequate physical size and a favorable ECM environment to retain the transplanted hAFSCs in the infracted myocardium. The benefit effects of hAFSCs may be mediated not only through their ability to differentiate into cardiomyocyte-like cells and cells of endothelial lineages but also through the growth factor-mediated paracrine regulation.
In our previous study, the number of cells on the 50-mesh-sized for 27 gauge optimize injection condition is 65 cells/fragment. The ideal number of cell transplantation for clinical use is around from 1×106 to 1×107. Recently, the use of three dimensional multicellular aggregates (cell bodies) for myocardium infarction was investigated. For the human amniotic fluid stem cells, 1×104 cells on the methylcellulose hydrogel can form the cell body and the diameter can reach to 350 μm. However, it has been often reported that cell necrosis occurs due to the insufficient supply of oxygen/nutrients and the accumulation of metabolic wastes, especially at the center of the cell bodies. In order to address this problem, the injectable of three dimensional porous micro-scaffolds were developed to enhance the transportation of oxygen/nutrients and wastes. In the study III, uniform porous poly(D, L-lactide-co-glycolide) (PLGA) beads with controlled pore diameters were prepared based on an emulsion templating method in a simple fluidic device. Unstable water-in-oil (W-O) emulsion, being in the course of phase-separation, was introduced into the fluidic device to form water-in-oil-in-water (W-O-W) droplets. The resultant W-O-W droplets evolved into porous PLGA beads with controlled pore diameters at the inside and surface of the beads after the organic solvent had evaporated. Porous beads prepared using the upper part of the phase-separated emulsion exhibited the large inner and surface pores as well as high interconnectivity compared to the lower part of the emulsions. Fluorescence microscopy imaging and 3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide (MTT) assay confirmed the more uniform distribution and higher viability of the cells in the bead with large pores. These results suggest that the bead with large pores could provide favorable environment for cells and thus be potentially used for both tissue engineering and cell delivery system.

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

Chapter 1. Introduction 1

Chapter 2.
Cellular Cardiomyoplasty with Human Amniotic FluidStem Cells: In Vitro and In Vivo Studies
2-1 Introduction ----------6
2-2 Materials and Methods 7
2-2.1. Characterization of hAFSC 7
2-2.2. Differentiation of hAFSC into endothelial-like cells in vitro-8
2-2.3. Cardiomyogenic differentiation of hAFSC in vitro 8
2-2.4. Animal study 11
2-2.5. LV function assessment by echocardiography 12
2-2.6. Histological examinations 13
2-2.7. Statistical analyses 15
2-3 Results 15
2-3.1. Charscterization of hAFSC 15
2-3.2. Differentiation of hAFSC into endothelial-like cells in vitro
--------------------------------------------------------------------------------16
2-3.3. Cardiomyogenic differentiation of hAFSC in vitro 17
2-3.4. Animal study 20
2-3.5.Cardiac function 20
2-3.6.Morphological observations 23
2-3.7.Histological findings 24
2-4. Discussion -----------------------------------------------------------------28
2-5. Conclusions 32

Chapter 3.
Cardiac Repair with Injectable Cell Sheet Fragments of Human Amniotic Fluid Stem Cells in an Immune-Suppressed Rat Model
3-1. Introduciton ----------------------------------------------------------------33
3-2. Materials and Methods 36
3-2.1. Characterization of isolated hAFSCs--------------------------36
3-2.2. Construction and characterization of hAFSC sheet fragments
-------------------------------------------------------------------------------37
3-2.3. Cell transplantations----------------------------------------------38
3-2.4. Left ventricular function assessment by echocardiography
------------------------------------------------------------------------------39
3-2.5. Histological examinations---------------------------------------40
3-2.6. Quantitative real-time polymerase chain reaction (qPCR) analyses------------------------------------------------------------------------42
3-2.7. Statistical analysis 42
3-3 Results and Discussion 42
3-3.1. Characterization of isolated hAFSCs 43
3-3.2. Characteristics of hAFSCs sheet fragments 43
3-3.3. Animal study 46
3-3.4. LV function 46
3-3.5. Morphological observations 48
3-3.6. Histological findings 50
3-3.7. qPCR analyses 55
3-4 Conclusions 58

Chapter 4.
Uniform PLGA Beads with Controllable Pore Sizes for Biomedical Applications
4-1 Introduction 59
4-2 Materials and Methods 61
4-2.1. Materials 61
4-2.2. Preparation and characterization of porous beads using the fluidic device 62
4-2.3 In Vitro cell culture study 63
4-2.4 Cell viability 64
4-3 Results and Discussion 65
4-3.1 Fabrication of porous beads using fluidic device 65
4-3.2 Characterization of porous beads using fluidic device 68
4-3.3 Cell culture with porous beads of different pore sizes 72
4-4 Conclusions 78

References 79

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