Abstract
Embryonic stem cells (ESCs) are a unique cell population
that can differentiate into all three embryonic germ layers
(endoderm, mesoderm, and ectoderm), rendering them an invaluable
cell source for studying the molecular mechanisms of embryogenesis.
Signaling molecules that direct tissue patterning during embryonic
development are secreted by ESC aggregates, known as embryoid
bodies (EBs). As many of these signaling proteins interact with the
extracellular matrix (ECM), manipulation of the ESC extracellular
environment provides a means to direct differentiation. ECM
components, such as glycosaminoglycans (GAGs), play crucial roles
in cell signaling and regulation of morphogen gradients during
early development through binding and concentration of secreted
growth factors. Thus, engineered biomaterials fabricated from
highly sulfated GAGs, such as heparin, provide matrices for
manipulation and efficient capture of ESC morphogens via reversible
electrostatic and affinity interactions. Ultimately, biomaterials
designed to efficiently capture and retain morphogenic factors
offer an attractive platform to enhance the differentiation of ESCs
toward defined cell types. The overall objective
of this work was to examine the ability of microparticles
synthesized from both synthetic and naturally-derived materials to
enhance the local presentation of morphogens to direct ESC
differentiation. The overall hypothesis was
that microparticles that mimic the ECM can modulate ESC
differentiation through sequestration of endogenous morphogens
present within the EB microenvironment.
Table of Contents
ACKNOWLEDGEMENTS v
LIST OF TABLES xiv
LIST OF FIGURES xv
SUMMARY xvii
INTRODUCTION 1
BACKGROUND 4
2.1 Embryonic stem cells 4
2.2 ESC differentiation 5
2.3 Embryoid body differentiation 6
2.4 Extracellular matrix-cell interactions 8
2.4.1 Extracellular matrix in embryonic development 8
2.4.2 HS/heparin in stem cell differentiation 10
2.5 Biomaterial strategies for directed stem cell differentiation
12
2.5.1 ECM-mimetic materials that regulate growth factor activity
12
2.5.2 Microparticles for ESC differentiation 17
pNIPMAm MICROPARTICLES FOR DELIVERY OF BMP4 TO PLURIPOTENT STEM
CELL AGGREGATES 19
Introduction 19
Methods 21
Synthesis and characterization of pNIPMAm MPs 21
BMP4 loading and release from MPs 22
Alkaline phosphatase (ALP) activity assay 22
Cell culture 23
Embryoid body formation and culture 24
Gene expression analysis 24
Histology 25
Flow Cytometry 26
Confocal microscopy 26
Statistical Analysis 26
Results 27
pNIPMAm MP characterization 27
BMP4 loading and release from pNIPMAm MPs 28
Delivery of BMP4 via pNIPMAm MPs to skeletal myoblasts 31
Incorporation of pNIPMAm MPs in EBs 33
Delivery of BMP4 via pNIPMAm MPs to EBs 34
BMP Signaling in ESCs 38
Discussion 40
Conclusion 42
HEPARIN-METHACRYLAMIDE (HMAM) MICROPARTICLES ENHANCE
NEUROECTODERMAL DIFFERENTIATION OF EMBRYONIC STEM CELL AGGREGATES
43
Introduction 43
Methods 45
Cell culture 45
Embryoid body formation and culture 45
Collection and analysis of spent media 46
HMAm MP incorporation analysis 46
Gene expression analysis 46
Histology analysis and immunostaining 47
Plating of EBs 48
Statistical Analysis 49
Results 49
Incorporation of HMAm MPs in EBs 49
Analysis of growth factor concentration in spent media 52
HMAm MP incorporation results in morphological differences in EB
structure 54
Gene expression is modulated by HMAm MP incorporation 57
Protein expression is altered by HMAm MP incorporation 59
Discussion 65
Conclusion 67
Introduction 69
Methods 71
BMP4 and conditioned media loading of HMAm MPs 71
Fluorescamine assay 72
Conditioned media protein extraction and digestion 72
HMAm MPs retrieval from EBs 73
Statistical Analysis 74
Results 74
Characterization of BMP4 release from HMAm MPs using SDS-PAGE
74
Concentration and characterization of ESC CM bound to HMAm MPs
77
Mass spectrometry analysis of gels loaded with MP bound protein.
81
Direct characterization and quantification of MP bound protein
86
Retrieval of HMAm microparticles incorporated within embryoid
bodies 88
Discussion 92
Conclusions 94
CONCLUSIONS AND FUTURE DIRECTIONS 95
REFERENCES 101
About this Master's Thesis
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