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BIOENGINEERED, STEM CELL DERIVED OCULAR OUTFLOW TISSUE Item Type Dissertation Authors Tian, Yangzi Isabel Download date 22/08/2022 18:35:17 Link to Item http://hdl.12648/1647 BIOENGINEERED, STEM CELL DERIVED OCULAR OUTFLOW TISSUE By Yangzi Isabel Tian, M. A Dissertation Submitted to the SUNY Polytechnic Institute, in Partial Fulfillment of the Requirements for the Degree of Doctor of Philosophy Colleges of Nanoscale Sciences and Engineering 2018 ACKNOWLEDGEMENT ! This Ph. journey has truly been a life-changing experience. First and foremost, I would like to acknowledge my thesis advisor, Dr.

Yubing Xie, for always supporting me 200% in all my academic and career endeavors. Her patience, insight, and constant encouragements were instrumental for my growth as an independent researcher, a STEM mentor, and a science illustrator. I was fortunate to have her guidance throughout my graduate career. I would also like to thank Dr.

Karen Torrejon, for serving on my thesis committee and for her substantial contribution to my work. Torrejon piloted the research in using SU-8 scaffolds as a platform for studying the outflow tissue in vitro, which laid the foundation for my project. I have also had the pleasure of working with her research team at Glauconix Biosciences, including Dr. Andrea Unser, Dr.

Feryan Ahmed, and Dr. Xulang Zhang, all of whom provided expertise and assistance in experimental design. I would like to extend my sincere gratitude to my other committee members, Dr. Nate Cady, Dr.

John Danias, and Dr. Susan Sharfstein, for their invaluable advice and feedback. Special thanks from the bottom of my heart to all the amazing people I have had the pleasure of getting to know. Specifically, I thank Dr.

Andrea Unser, Dr. Lauren Sfakis, and Adi Leonardi for being my support network. Regular coffee dates and girl’s nights with them brought fun to graduate school life. Furthermore, I am grateful for all members of the Xie/Castracane lab (both past and present) for an enjoyable and friendly work !ii environment.

Bridget Mooney and Dr. Michael Zonca for training and stimulating discussions, Dr. Logan Butt for help with mask fabrication, and Atul Dhall for COMSOL training. I would also like to recognize all the undergraduate interns that have helped me in this work, including Gretchen Long, Aishwarya Panneerselvam, and Devina Puri.

Many thanks also go to the engineering support staff, Mr. Kyle Unser and Mr. Steve Steward, for finding and repairing essential equipment for my experiments. Finally, a big and hearty thank you to my best friend and fiancee, Joshua Holt, for always being a source of happiness even through the most stressful moments of graduate school.

His positive outlook on life is contagious and has helped motivate me through this journey. With such a cheerful and loving person by my side, I’m excited to embark on the next chapters of our life together. Last but not least, I would not be here today without the unwavering support of my parents Jingyan (Joy) Tang and Baoying (Kevin) Tian. Their unconditional love and sacrifice have provided me with the opportunity to live and study in the United States.

For all they’ve done for me, I am forever grateful. !iii ABSTRACT ! Glaucoma is one of the leading causes of irreversible blindness in the world. Despite decades of research, intraocular pressure (IOP) is the only known treatable risk factor. IOP is affected by the timely removal of aqueous humor through the conventional outflow track, which is made up of the trabecular meshwork and adjacent Schlemm’s canal.

Dysfunction in these tissues due to aging, oxidative stress, metabolic or pathological changes lead to increased flow resistance, elevated IOP, and ultimately glaucoma. Recent advances in ocular regenerative therapy have the potential to rescue glaucomatous tissue function and restore its delicate microenvironment. The possibility of using stem cell-derived trabecular meshwork and Schlemm’s canal cells to recreate a functional outflow tissue are explored in this thesis. Previously, our lab developed a well- defined, micro-porous substrate that promotes in vivo-like physiology and outflow function in primary trabecular meshwork and Schlemm’s canal cell cultures.

Using these primary cell cultures as controls, we have created 3D stem cell-derived outflow tissues, evaluated and compared their morphology, expression, outflow facility, and drug responsiveness. To explore the importance of the dynamic microenvironment in outflow function, we developed a dual-flow microfluidic chamber that mimics the basal-to-apical and shear flow of aqueous humor through the conventional outflow track. Overall, this dissertation demonstrates the promising application of stem cells in future glaucoma drug screening and treatment. !iv TABLE OF CONTENTS ! ACKNOWLEDGEMENT ii ABSTRACT iv TABLE OF CONTENTS v LIST OF ABBREVIATIONS vii LIST OF FIGURES AND TABLES x FIGURES x TABLES xiii CHAPTER 1 1 RECENT DEVELOPMENTS AND CHALLENGES IN PRIMARY OPEN ANGLE GLAUCOMA 1.

Primary open angle glaucoma 2 1. Conventional outflow pathway 4 1. Pathogenesis of POAG 7 1. Potential of stem cell in POAG 13 1.

Construction of stem cell-derived outflow tissue 17 1. References 20 CHAPTER 2 25 INDUCED PLURIPOTENT STEM CELL-DERIVED TRABECULAR MESHWORK AS A BIOENGINEERED DRUG TESTING PLATFORM 2. Material and Methods 28 2. References 50 CHAPTER 3 53 STEM CELL-DERIVED SCHLEMM’S CANAL-LIKE CELLS FOR IN VITRO GLAUCOMA DRUG SCREENING 3.

Material and Methods 58 3. References 86 CHAPTER 4 91 STEM CELL-DERIVED SCHLEMM’S CANAL-LIKE CELLS FOR IN VITRO GLAUCOMA DRUG SCREENING 4. Material and Methods 94 4. References 113 SUMMARY 115 !vi LIST OF ABBREVIATIONS oC Degrees Celsius 3D Three-dimensional A2P L-Ascorbic acid 2-phosphate ADSC Adipose-derived stem cell AH Aqueous humor ANOVA Analysis of variance BtA Basal-to-apical CD-31 (PECAM1) Platelet endothelial cell adhesion molecule-1 CLAN Cross-linked actin network CO2 Carbon dioxide ColIV Collagen type IV DAPI 4’,6-diamidino-2-phenylindole DEX Dexamethasone DMEM Dulbecco’s Modified Eagle’s Medium DMSO Dimethyl sulfide ECM Extracellular matrix eNOS Endothelial nitric oxide synthase FBS Fetal bovine serum FITC Fluorescein isothiocyanate GAPDH Glyceraldehyde 3-phosphate dehydrogenate GC Glucocorticoids HMDS Hexamethyldisilazane HSC Human schlemm’s canal HTM Human trabecular meshwork ICC Immunocytochemistry !vii IOP Intraocular pressure iPSC Induced pluripotent stem cell iPSC-TM iPSC-derived trabecular meshwork JCT Juxtacanalicular connective tissue KLF4 Kruppel-like factor 4 MEM Minimum Essential Media MYOC Myocilin NO Nitric oxide OCT 3/4 Octamer-binding transciption factor 3/4 ONH Optic nerve head Pa Pascal PBS Phosphate buffered saline PCR Polymerase chain reaction PDMS Polydimethylsiloxane PFA Paraformaldehyde PMMA Poly(methyl methacrylate) POAG Primary open-angle glaucoma PROX1 Prospero homeobox protein 1 q-PCR Quantitative polymerase chain reaction RGC Retinal ganglion cell RNA Ribonucleic acid ROCK Rho-associated protein kinase ROS Reactive oxygen species SD Standard deviation SDS-PAGE Sodium dodecyl sulfate polyacrylamide gel electrophoresis SEM Scanning electron microscopy SU-8 SU-8 negative photoresist !viii TGF-β2 Transforming growth factor-beta 2 UV Ultraviolet VEGF Vascular endothelial growth factor VEGFR Vascular endothelial growth factor receptor α-SMA Alpha-smooth muscle actin !ix LIST OF FIGURES AND TABLES Figures Chapter 1 Figure 1.

Global prevalence of primary open-angle glaucoma. Pathophysiology of glaucoma. Aqueous humor production and flow. Schematics of the fabrication process for micropaterned, porous SU-8 scaffolds via photolithography.

Schematics of the flow system constructed perfusion studies. Primary HTM and iPSC-TM cell morphology on conventional 2D culture flask and 3D SU-8 scaffolds. Confocal images showed expression of HTM markers of myocilin and α- SMA in 3D cultured trabecular meshwork cells. Confocal images showed expression of HTM markers of fibronectin and f- actin in 3D cultured trabecular meshwork cells.

Cell morphology of primary HTM and iPSC-TM cells after treatment with dexamethasone. Quantification of nuclei size after DEX-treatment in HTM and iPSC-TM cells. Effect of dexamethasone on CLAN formation in HTM and iPSC-TM after 7 day treatment. Effect of dexamethasone on phagocytosis in HTM and iPSC-TM after 3 hours of incubation with 488-conjugated dextran beads.

Confocal analysis of protein expression in IPSC-TM with and without DEX treatment. Western blot analysis of myocilin and Oct3/4 expression in HTM and IPSC- TM before and after DEX treatment. Confocal analysis of extracellular matrix proteins deposition by 3D iPSC- TM. Effect of dexamethasone on outflow facility of HTM and iPSC-TM.

Schematics of differentiation strategy to induce ADSCs into SC-like cells using a combination of VEGF-C, shear stress and co-culture with HTM cells on the SU-8 scaffold. Differentiation timeline of ADSCs into HSC-like cells through VEGF-C and shear stress treatments. Optical images of endothelial differentiation of ADSCs induced by 3-day and 11-day treatment with VEGF-A and shear stress. Expression of endothelial markers in differentiated ADSCs induced by 11-day treatment with VEGF-A and shear stress.

qPCR analysis of endothelial markers expressions in differentiated ADSCs induced by 11-day treatment of VEGF-A and shear stress. Differentiation of ADSCs into Schlemm’s canal-like cells induced by VEGF- C and shear stress. Expression of characteristic HSC markers demonstrated that ADSCs exhibited HSC-like differentiation after 11-day treatment with VEGF-C and shear stress. Western blot analysis of HSC marker in ADSCs after 11-day treatment with VEGF-C and shear stress.

Confocal analysis of HSC marker expression in ADSCs treated with VEGF-C and shear stress. Confocal analysis of HSC marker expression in ADSCs treated with VEGF- C and shear stress. Confocal analysis of stem cell marker expression in ADSCs treated with VEGF-C and shear stress. Co-culture with 3D HTM-scaffold constructs facilitate HSC-like differentiation of ADSCs.

Confocal analysis of HTM markers expression in 3D HTM, HTM/HSC and HTM/ADSC-derived HSC-like constructs after perfusion studies. Confocal analysis of HSC markers expression in 3D HTM, HTM/HSC and HTM/ADSC-derived HSC-like constructs after perfusion studies. DEX-induced responses in HTM/HSC and HTM/ADSC-derived HSC-like constructs. DEX-induced responses in HTM/HSC and HTM/ADSC-derived HSC-like constructs.

Perfusion studies of the outflow facility of HTM/HSC and HTM/ADSC- derived HSC-like constructs after (A) 3-days and (B) 7-day of DEX treatment. Cross-sectional view of the dual-flow perfusion chamber. Schematic of the dual-flow perfusion system setup. COMSOL model of the dual-flow perfusion device.

Velocity profile of the dual-flow perfusion device. Parameter sweep of shear chamber height and flow rates to determine the wall shear stress experienced at the SU-8 interface. Composite result of parameter sweep. Experimental validation of simulation data using latex beads perfused through a shear chamber with 200 µm height.

Confocal imaging of HSC construct under various shear stress. ImageJ quantification of immunocytochemistry analysis of HSC cells. Perfusion of HSC-scaffold construct at 0 (static), 0. Perfusion of co-cultured HTM/HSC-scaffold construct at 0 (static), 0.

!xii Table Table 1. Available types of IOP-lowering medications for POAG (Zhang et al., 2012 with modifications) Table 3. qPCR primer sequences used in this study. RECENT DEVELOPMENTS AND CHALLENGES IN PRIMARY OPEN ANGLE GLAUCOMA 1.1 Introduction Glaucoma is a debilitating neurodegenerative eye disease that has lead to visual impairment in over 64 million people worldwide (Tamm et al.

Due to its tendency to remain asymptomatic until advanced stage, glaucoma is the primary cause of irreversible blindness (Weinreb et al. Patients living with advanced glaucoma not only suffer physically and emotionally, but are also met with the financial burden of disease management (Carreras et al. Currently, there is no cure for glaucoma, and the only known, modifiable risk factor is intraocular pressure (IOP). Typical management plan involves medication(s) and/or surgery to reduce IOP, and regular clinic visits to monitor treatment efficacy and disease progression (Chae et al.

In the United States alone, the annual economic burden of glaucoma-related disorders is $5.5 billion, and will continue to grow with the increase in elderly population (Frick et al. Moreover, a recent meta-analysis estimates that the global prevalence of glaucoma will exceed 111 million in 2040, with Africa and Asia affected more than the rest of the world (Fig.1; Weinreb et al., 2016; Tham et al. Therefore, there is an urgency to develop a more permanent and cost-effective treatment strategy for glaucoma through a better understanding of its pathophysiology. Global prevalence of primary open-angle glaucoma.

For individuals >40 years of age, the population valence data (%) in 2015 per region are color-coded. Reprinted by permission from Springer Nature: Primary open-angle glaucoma, Weinreb et al.2 Primary Open Angle Glaucoma Primary open angle glaucoma (POAG) is the most common type of glaucoma, responsible for 90% of all reported cases. Among the few well-recognized risk factors for POAG, such as age, race, gender, and family history, elevated IOP is the only modifiable risk factor during disease development.