Incorporation of Bio-Inspired Microparticles within Embryonic Stem Cell Aggregates for Directed Differentiation Open Access

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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School	Laney Graduate School
Department	Biomedical Engineering
Degree	MS
Submission	Master's Thesis
Language	English
Research Field	Engineering, Biomedical
Keyword	Microparticles Stem Cell Engineering
Committee Chair / Thesis Advisor	McDevitt, Todd, Georgia Tech
Committee Members	Jane, Lebkowski, Asterias Biotherapeutics Temenoff, Johnna, Georgia Tech

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