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Human neural stem cell adhesion and proliferation on a biometric polyurea functionalized with RGD peptides

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Title:
Human neural stem cell adhesion and proliferation on a biometric polyurea functionalized with RGD peptides
Creator:
Laughter, Melissa ( author )
Place of Publication:
Denver, CO
Publisher:
University of Colorado Denver
Publication Date:
Language:
English
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1 electronic file (85 pages). : ;

Thesis/Dissertation Information

Degree:
Master's ( Master of Science)
Degree Grantor:
University of Colorado Denver
Degree Divisions:
Department of Bioengineering, CU Denver
Degree Disciplines:
Bioengineering

Subjects

Subjects / Keywords:
Stem cells -- Transplantation ( lcsh )
Spinal cord -- Wounds and injuries ( lcsh )
Genre:
bibliography ( marcgt )
theses ( marcgt )
non-fiction ( marcgt )

Notes

Review:
There is currently no effective cure for spinal cord injury (SCI). In hopes of finding the first restorative treatment, researchers have turned their focus to stem cell therapies due to their potential to repair or completely replace the injured cells and tissue. Although this type of research has shown a great deal of promise, there are still many complications to overcome before stem cell therapies become institution in SCI treatment. One obstacle that stands in the way is the lack of an implantable cell scaffold that can support efficient neural stem cell (NSC) differentiation and proliferation. Due to significant cell death during implantation, the initial number of cells implanted into the injury site far outweighs the surviving cells that are able to integrate into the surrounding tissue and restore function. Of course, encouraging more NSC attachment, differentiation, and proliferation through the use of a cell scaffold, would significantly increase the number of surviving cells and thus the effectiveness of these cell therapies. Therefore, the development of a cell scaffold that encourages efficient motor neuron (MN) induction and survival without alteration of the cell's DNA may be sufficient to make stem cell therapies an institution in spinal cord injury treatment. We aimed to overcome this hurdle through the use of an Arg-Gly-Asp (RGD) functionalized biomimetic polyurea, optimized to encourage efficient differentiation of NSCs without the use of any complex genetic alterations. The RGD sequence, an integrin-binding motif found in fibronectin and laminin (major components of the ECM), was found to be implicated in outside-inside cell signaling that can affect cell proliferation, migration, and cell survival in most tissues. We began by synthesizing a functionalized RGD polymer scaffold (PSHU-RGD). Poly (serinol hexamethylene urea) (PSHU) was employed due to its protein-like backbone structure and its potential to attach a large quantity of biomolecules. This is extremely beneficial for because achieving a high concentration of biomolecules for cell-biomolecule interactions plays a crucial role in stem cell survival and differentiation efficiency. Both 1H NMR, FT-IR, and HPLC were used to confirm the overall polymer structure, ensure the presence of free amine groups, and quantify the conjugation of RGD. We began by coating a 24-well plate with varying concentrations of PSHU-RGD (0.01-100 ug/ml) (deprotected-PSHU and no coating as the negative controls). After this, NSCs were seeded on top of the coatings in N2B27 media with ATRA and SAG factors. After 7 days, ATRA and SAG were replaced with BDNF and GDNF and left for another 7 days or 14 days.
Thesis:
Thesis (M.S.)--University of Colorado Denver. Bioengineering
Bibliography:
Includes bibliographic references.
System Details:
System requirements: Bioengineering.
General Note:
Department of Bioengineering
Statement of Responsibility:
by Melissa Laughter.

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University of Colorado Denver
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|Auraria Library
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All applicable rights reserved by the source institution and holding location.
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904554408 ( OCLC )
ocn904554408

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Full Text
HUMAN NEURAL STEM CELL ADHESION AND PROLIFERATION ON A
BIOMIMETIC POLYUREA FUNCTIONALIZED WITH RGD PEPTIDES
by
MELISSA LAUGHTER B.S., University of Colorado Boulder, 2012
A thesis submitted to the Faculty of the Graduate School of the University of Colorado in partial fulfillment of the requirements for the degree of Master of Science Bioengineering
2014


This thesis for the Master of Science degree by Melissa Laughter has been approved for the Bioengineering Program by
Daewon Park, Chair Michael Yeager Karin Payne
9/23/2014
11


Laughter, Melissa (M.S., Bioengineering)
Human neural stem cell adhesion and proliferation on a biomimetic polyuria
functionalized with RGD peptides
Thesis directed by Assistant Professor Daewon Park
ABSTRACT
There is currently no effective cure for spinal cord injury (SCI). In hopes of finding the first restorative treatment, researchers have turned their focus to stem cell therapies due to their potential to repair or completely replace the injured cells and tissue. Although this type of research has shown a great deal of promise, there are still many complications to overcome before stem cell therapies become institution in SCI treatment. One obstacle that stands in the way is the lack of an implantable cell scaffold that can support efficient neural stem cell (NSC) differentiation and proliferation. Due to significant cell death during implantation, the initial number of cells implanted into the injury site far outweighs the surviving cells that are able to integrate into the surrounding tissue and restore function. Of course, encouraging more NSC attachment, differentiation, and proliferation through the use of a cell scaffold, would significantly increase the number of surviving cells and thus the effectiveness of these cell therapies. Therefore, the development of a cell scaffold that encourages efficient motor neuron (MN) induction and survival without alteration of the
m


cells DNA may be sufficient to make stem cell therapies an institution in spinal cord injury treatment. We aimed to overcome this hurdle through the use of an Arg-Gly-Asp (RGD) functionalized biomimetic polyurea, optimized to encourage efficient differentiation of NSCs without the use of any complex genetic alterations. The RGD sequence, an integrin-binding motif found in fibronectin and laminin (major components of the ECM), was found to be implicated in outside-inside cell signaling that can affect cell proliferation, migration, and cell survival in most tissues.
We began by synthesizing a functionalized RGD polymer scaffold (PSHU-RGD). Poly (serinol hexamethylene urea) (PSHU) was employed due to its protein-like backbone structure and its potential to attach a large quantity of biomolecules. This is extremely beneficial for because achieving a high concentration of biomolecules for cell-biomolecule interactions plays a crucial role in stem cell survival and differentiation efficiency. Both 1H NMR, FT-IR, and HPLC were used to confirm the overall polymer structure, ensure the presence of free amine groups, and quantify the conjugation of RGD. We began by coating a 24-well plate with varying concentrations of PSHU-RGD (0.01-100 ug/ml) (deprotected-PSHU and no coating as the negative controls). After this, NSCs were seeded on top of the coatings in N2B27 media with ATRA and SAG factors. After 7 days, ATRA and SAG were replaced with BDNF and GDNF and left for another 7 days or 14 days.
IV


Images were taken every two days of the culture period to determine which PSHU-RGD polymer coating encouraged hNSC attachment and cell growth. We were able to show a clear difference in NSC attachment and proliferation between the amounts of polymer used. 10 ug of polymer was found to induce the most neurite spreading and proliferation indicating that this polymer possesses the qualities required of a NSC cell scaffold. In summary, we anticipate that the controllable properties of this synthetic polymer that improve hNSCs differentiation and survival in vitro will have great implications on future stem cell therapies for SCI treatment.
The form and content of this abstract are approved. I recommend its publication.
Approved: Daewon Park
v


ACKNOWLEDGEMENTS
I would like to thank all the people that contributed to the development of this work. I am extremely grateful to all my mentors, professors, family and friends that helped me achieve this goal. This experience has helped me not only define my interests and passions but has been amazing in terms of personal development.
First of all I would like to thank Dr. Daewon Park who has led me through this whole project and from whom Fve learned a tremendous amount. I am looking forward to working with him in the future.
At the same time, I would like to thank my committee members, Dr. Michael Yeager and Dr. Karin Payne for their encouragement and advice.
Similarly, I would like to thank Lindsay Hockensmith, Maria Bortot, and the TBRL lab for sharing this experience with me and making it fun.
I would also like to thank Dr. Young Lee and Dr. Kurt Freeds lab for their guidance with hNSC culture and testing. Dr. Lees guidance and advice were an invaluable and essential part of this work.
Finally, I dedicate this thesis to my family, to my dad for being so inspiring and encouraging me everyday to have goals and motivate me to strive for greatness. To my mom who has been with me every step of the way and who has supported me with everything I do. To my sister, who I share every moment
vi


with and who has believed in me immensely. She has always given me the confidence to continue pursuing my dreams.
Vll


TABLE OF CONTENTS
Chapter
1. Introduction............................................................1
1.1 Spinal cord injury epidemiology and background information.........1
1.2 Basic treatment of SCI.............................................2
1.3 Molecular approaches to SCI........................................3
1.4 Bioengineered therapies for spinal cord regeneration ..............5
1.4.1 Somatic cell-based therapies for SCI...........................5
1.4.2 Stem cell-based therapies for SCI...............................6
1.5 Challenges in bioengineering cell therapies with NSCs and MNs .....6
1.6 Objectives of this study............................................7
2. Literature Review......................................................10
2.1 Review of human neural stem cells culture techniques...............10
2.2 Review of hNSC monolayer differentiation techniques ...............12
2.3 Review stem cell niche.............................................15
2.4 Review RGD peptide.................................................17
2.5 Review biomimetic polymer scaffold.................................20
2.6 Review in vivo studies with NSCs and MNs...........................22
3. Individual Objectives and Experimental Approach........................24
27
viii
4. Materials and Equipment


4.1 Materials ............................................................27
4.2 Equipment............................................................27
5. Methods...................................................................29
5.1 Biomimetic poly (serinol hexamethylene urea) synthesis................29
5.2 Removal of BOC protective groups from the polymer backbone...........29
5.3 Maximum conjugation of RGD to PSHU backbone..........................30
5.4 PSHU-RGD coating optimization........................................31
5.5 Human embryonic stem cell culture conditions.........................32
5.6 Neural stem cell induction...........................................32
5.7 Motor neuron differentiation.........................................32
5.8 Varying RGD conjugation amounts......................................33
6. Results and Discussion ...................................................35
6.1 Polymer backbone characterization using NMR ..........................35
6.2 RGD conjugation confirmation using FT-IR.............................38
6.3 RGD conjugation quantification using HPLC ...........................39
6.4 Human neural stem cell phase contrast images ........................41
6.5 Human neural stem cell MTT assay ....................................49
6.6 Partial GRGDS conjugation analysis using FT-IR ......................50
6.7 Partial GRGDS conjugation quantification using HPLC..................52
7. Conclusion ...............................................................54
IX


8. Future Work
57
8.1 Analysis of varied RGD conjugation polymer system with NSCs.....57
8.2 Development of a 3D injectable scaffold.........................57
8.3 Spinal cord injury animal model.................................57
References.............................................................59
Appendix
A. RGD conjugation analysis using HPLC................................65
B. FT-IR confirmation of deprotected PSHU.............................69
C. Plans and schematic for future work.................................70
x


LIST OF FIGURES
Figure
1 Schematic of primary (acute) and secondary (chronic) phases
of SCI................................................................ 2
2 Comparison between neurosphere and monolayer culture
techniques........................................................... 11
3 Schematic of motor neuron differentiation and motor neuron
axon elongation...................................................... 14
4 Integrin binding, clustering, and signaling cascade.................. 18
5 Schematic of PSHU-RGD synthesis with the final structure of
PSHU-RGD............................................................. 35
6 *H NMR (500 MHz, CDCI3) spectrum of P SHU to confirm
overall structure of polymer chain................................... 37
7 NMR (500 MHz, CDC13) spectrum of PSHU and deprotected PSHU to confirm removal of the BOC protecting
groups during deprotection process................................... 37
8 FT-IR of dPSHU, PSHU, and PSHU-RGD.................................... 38
9 HPLC Curve of 1.3 initial molar ratio of RGD/free amine
groups............................................................... 40
10 24-well plate PSHU-RGD (93% conjugation) polymer coating
schematic............................................................ 41
11 Phase contrast images taken after 2 days of culture................... 43
12 Phase contrast images taken after 4 days of culture................... 45
13 Phase contrast images taken after 8 days of culture................... 47
xi


14 Comparison of MTT assay results between the PSHU-RGD
surface coatings and the deprotected-PSHU surface coating......... 49
15 FT-IR of dPSHU, PSHU, 40% PSHU-RGD, 60% PSHU-RGD,
80% PSHU-RGD, 100% PSHU-RGD, and 130% PSHU-RGD.................... 50
16 Graph of actual amine-RGD conjugation ratio compared to the
initial molar ratio of amine-RGD.................................. 51
A. 1 High performance liquid chromatography RGD binding
calibration curve................................................. 57
A.2 RGD surface density calculated per well on a 24-well plate........... 57
A.3 RGD surface density on a 24-well plate calculated for varied
PSHU-RGD conjugation amounts...................................... 58
A. 4 HPLC Diagrams to determine the actual conjugation amount of
RGD to the PSHU Backbone.......................................... 58
B. 1 Confirmation of free amine groups on dPSHU after
deprotection...................................................... 60
C. 1 24-well plate PSHU-RGD partial conjugation polymer coating
schematic......................................................... 61
xii


ABBREVIATIONS
Abbreviations Meaning
SCI MP CNS PNS ESC ECM NTF NSC iPSC NSPC EB MN hNSC hMN BDNF GDNF RGD GRGDS EGF FGF RA SHH SAG GAGs PEG PLA PET PLLA PLGA PSHU NMR HDI DMF EDC Spinal Cord Injury Methy lpredni sol one Central Nervous System Peripheral Nervous System Embryonic Stem Cell Extracellular Matrix Neurotrophic Factors Neural Stem Cell Induced Pluripotent Stem Cells Neural Stem/Progenitor Cells Embry oid Bodies Motor Neuron Human Neural Stem Cell Human Motor Neuron Brain Derived Neurotrophic Factor Glial-derived Neurotrophic Factor Arginylglycylaspartic acid Gly-Arg-Gly-Asp-Ser Epidermal Growth Factor Fibroblast Growth Factor Retinoic Acid Sonic Hedgehog Purmorphamine Glycoasaminoglycans Poly (ethylene glycol) Poly (lactic acid) Poly (ethylene-terephthalate) Poly(L-lactic acid) Poly(lactic-co-glycolic acid) Poly (serinol hexamethylene urea) Nuclear Magnetic Resonance Hexamethylene Diisocyanate N,N-dimethylformamide N-(3-Dimethylamino- propyl)-N'-ethylcarbodiimide hydrochloride
TFE TFA DCM 2,2,2- Trifluoroethanol Trifluoroacetic Acid Anhydrous Dichloromethane
xm


FT-IR Fourier Transform Infrared
DMSO Dimethyl Sulfoxide
MTT 3-(4,5-Dimethylthiazol-2-YL)-2,5-
Diphenyltetrazolium Bromide
NHS N-Hydroxysuccinimide
HPLC High-performance liquid
chromatography
XIV


1.
Introduction
1.1 Spinal cord injury epidemiology and general information
Over one million people in North America suffer from paralysis caused by spinal cord injury (SCI) [1], Although the severity of these injuries may vary, the recovery is often extremely difficult with full recovery being extremely rare. Most spinal cord injuries involve displacement of the bone or spinal disk into the spinal cord proper causing either contusion or compression of the cord [2], This primary or immediate damage to the spinal cord results in severance of axon connections, loss of neurons and glia, and demyelination [3], After this initial mechanical insult, the spinal cord is subjected to a secondary process involving cellular and molecular events that progress in a cascading fashion. The secondary injury phase can be grouped in several pathological events: (a) immune response, (b) apoptosis, (c) free-radical damage, (d) excitoxicity, and (e) axonal damage [4], These cellular events present themselves clinically as vascular dysfunction, edema, ischemia, and inflammation. The result of this secondary damage is additional neuronal and glial cell death, formation of glial scar tissue surrounding the initial injury site, and further demyelination. All of these occurrences present a physical and chemical barrier to the regeneration of damaged nerve connections and thus a large challenge for treatment of this injury (Figure 1) [5][6],
1


Acute stage:
1. Direct impact
2. Lacerated axons
3. Contused axons
4. Influx of inflammatory cells
\
Chronic stage:
1. Central cavity
2. Scar tissue
3. Inhibition of axonal regeneration
4. Demyelination
5. Axonal dieback
6. Influx of inflammatory cells
Treatment options:
1. Cell-, tissue transplantation
2. Blocking inhibition of axonal regrowth
3. Enhancing axonal regeneration
4. Modulating inflammatory responses
Figure 1. Schematic of primary (acute) and secondary (chronic) phases of spinal cord injury along with some treatment options [5],
1.2 Basic treatment of SCI
The first line of treatment is to prevent further spinal cord damage by immobilizing the patient. Clinicians will initially focus on restoring normal alignment, performing operative decompression, and preventing shock. All of these measures focus on minimizing the extent of the primary damage. However, additional steps are taken to reduce the damage caused by secondary injury. Considering the further damage caused by subsequent inflammation and scar formation, many treatments are focused on restricting and preventing progression of the secondary injury stage [5], One medication for this is Methylprednisolone (MP), a neuroprotective steroid agent
2


[2][7], MP works by counteracting part of the secondary injury process. Clinically, MP has been shown to improve post-traumatic spinal cord blood flow, decrease inflammation, and reduce lipid peroxidation [8], However, MP only provides limited improvement and must be administered within 8 hrs of the initial insult. In addition, there is opposition towards the use of corticosteroids after SCI due to the risks of giving this medication to a trauma patient. It has been shown that corticosteroids used in this setting can lead to increased risk of infection and gastrointestinal complications that can be fatal [7] [9], The use of this drug has decreased significantly in the recent years due to its limited efficacy and considerable side effects [1],
1.3 Molecular approaches to SCI
To this end, there has been extensive research to understand the pathophysiological mechanism of SCI and in turn develop alternative treatments to prevent secondary damage and promote axon regeneration. Compared to the peripheral nervous system (PNS), the central nervous system (CNS) has very limited neural regenerative potential. As mentioned above, nerve regeneration after SCI is also limited by the inhibitory nature of the injury site due to demyelination and scar tissue formation [6][10], Researchers have examined molecular approaches that combat both the limited intrinsic regenerative potential of the CNS and the inhibitory extrinsic environment that develops after SCI.
One approach to enhance the neural regeneration capacity of the CNS is to block inhibitors of neural regeneration. Studies have shown that myelin, the material
3


that forms a layer around axons to aid electrical impulses, contains growth-inhibiting factors that suppress neural plasticity and impede neural regeneration. Once damaged due to a spinal cord insult, myelin releases these neural growth-inhibiting factors, such as Nogo-A. Studies have shown that the administration of antibodies directed against Nogo-A resulted in improved neural regeneration and function in vivo [6], Along with inhibiting the effects of myelin, treatments have also focused on inhibiting the formation of glial scar tissue and thus preventing a physical barrier to neural regeneration. One example is the degradation of chondroitin sulfate proteoglycans, an important component in the extracellular matrix (ECM) and scar formation. Administration of chondroitinase, an enzyme for chondroitin, resulted in a decrease in scar cavity formation and gliosis [11],
Neurotrophic factors (NTF) are another area of interest that not only improve the regenerative capacity of the CNS but also mediate the secondary inflammatory process. Administration of Brain-derived neurotrophic factor (BDNF) or NT-3 following a spinal cord injury have both shown to improve neural regeneration followed by improved functional recovery in mice models [11],
Although these molecular treatments show a great deal of promise, the focus is primarily on preserving or reconnecting the undamaged endogenous nerves. However this tactic is unlikely to provide substantial or full functional recovery. For more dramatic improvement to these patients recovery, researchers have begun to investigate the use of these molecular treatments in conjunction with cell therapies
4


[12], Thus, future treatments of spinal cord injury might include a molecular agent to encourage a better environment for neural regeneration as well as the implantation of new cells or tissue.
1.4 Cell-based therapies for spinal cord regeneration
1.4.1 Somatic cell-based therapies for SCI
Along with molecular approaches to SCI, cell based therapies have recently become a large part of this research to further improve functional recovery. Although these cell based regenerative strategies vary significantly, they all aim to directly replace the cells lost from the injury or to support neural regeneration and protection [6], The first group of cell-based therapies are aimed at re-myelinating damaged axons and thus promoting neuronal regeneration. Glial cells can be transplanted into the injury site to remyelinate axons as well as provide neuroprotection. Oligodendrocytes, astrocytes, and motor neurons (MN) have all been used in attempts to increase function after SCI. However, studies have shown that transplanting cells alone results in poor survival and thus limited functional recovery [6] [13],
1.4.2 Stem cell-based therapies for SCI
Stem cells have recently become a major facet of cell therapy research in hopes of finding the first efficacious and complete cure for SCI. A stem cell, by definition, has the ability to continuously proliferate through asymmetrical division or generate daughter cells with a committed cell type through differentiation [4], Stem cells will initially differentiate into progenitor cells, which have restricted
5


proliferation capacity and at this point can only differentiate into certain cell types [4], There are different types of stem cells that have been examined for the treatment of SCI: embryonic stem cells (ESC), neural stem cells (NSC), and inducible pluripotent stem cells (IPSC) [4] [14], Some of these cell types, such as ESCs and IPSCs, have the capacity to differentiate into almost all cell types. Whereas NSCs are multipotent cells that primarily differentiate into neurons, oligodendrocyes, and astrocytes, making this type of stem cell of particular interest for SCI treatment. Despite their differences, all of these types of stem cells possess the ability to replace certain populations of cells lost during the primary and secondary spinal cord injury phases.
1.5 Challenges in cell therapies with NSCs and MNs
In the field of SCI research, NSCs and MNs have recently gained interest for use in cell implantation and regeneration treatments. Although research surrounding cell transplantation therapies using MNs and NSCs has shown a great deal of promise, there are still many obstacles to overcome. For cell transplantation using naive MNs, one obstacle is the lack of an efficient and straightforward method to obtain a large population of these cells for transplantation. For MN cell replacement therapy to become a viable treatment for SCI, there needs to be an efficient source for differentiating the maximum number of homogeneous MNs for implantation. So far, several protocols have reported a percent production in the range of 10%-40% of MNs from the initial embryonic stem cell population; however most of the larger
6


percentages are achieved through complicated and variable genetic alterations that may not be well-suited for cell transplantation [15],
For cell transplantation using NSCs, the key obstacle is the lack of an implantable scaffold that effectively encourages NSC differentiation, cell survival, and axonal extension. Cell transplantation therapies involving only the injection of NSCs have shown limited improvement due to significant cell death after implantation. Of course, encouraging more NSC survival through the use of an implantable cell scaffold would significantly increase the number of surviving cells and thus the effectiveness of these cell therapies. This may be due to the environment of the injury site and that the cells have limited ligands to attach to after implantation. For use in both cell transplantations or solely to provide a supportive substrate to guide existing axons across the lesion, scaffolds must posses the correct qualities that encourage neural attachment, differentiation, and proliferation. Because functional recovery of the spinal cord depends on successful differentiation and integration of the implanted NSCs into the surrounding tissue, scaffolds must provide the necessary support and cues to encourage this behavior [16], This task has proved to be extremely challenging due to the uncertainty of these signals and the difficulty of harnessing these signals within a controlled polymer system.
1.6 Objectives of this study
Stem cell therapies have become a fervent hope in the field of SCI treatment due to their potential to completely replace diseased or damaged cells and tissue.
7


These cell replacement therapies promise the ability restore the axonal connections that have been damaged after SCI to increase function and quality of life significantly more than the current SCI treatments. We hope to contribute to this research effort through the development of a synthetic polymer than can be manipulated to support human neural stem cell (hNSC) differentiation and encourage human motor neuron (hMN) growth in vitro. If successful in vitro, this polymer would possess the qualities required of an hMN or hNSC scaffold suited for implantation for SCI treatment.
The overall objective of this study is to test the ability of a polymer scaffold functionalized with Arginylglycylaspartic acid (RGD) to support hNSC differentiation and hMN growth. Through we use of a synthetic polymer, we are able to control the interactions these cells are exposed to and thus support hNSC growth and neurite extension.
1.7 Organization of this paper
Chapter I Introduction: This chapter includes the necessary background information for the rest of the paper as well as a brief overview of the current SCI treatments. Chapter II Literature Review: This chapter includes the literature review focusing on the current status of cell therapy research as well as how biomimetic polymers have been incorporated into this field.
Chapter III Experimental Approach: This chapter will provide the experimental thought process and individual benchmarks and goals for this project.
Chapter IV Materials and Equipment:
8


Chapter V Results and Discussion Chapter VI Conclusion:


2.
Literature Review
2.1 Review of hNSC cells culture techniques
Neural stem cells are self-renewing multipotent cells that can differentiate first into neural stem/progenitor cells (NSPCs) and subsequently into neurons or glial cells. NSCs differentiate during the formation of the developing brain but also account for the restricted regenerative potential in the adult CNS. In the body, NSCs are present in niches that control the self-renewal or differentiation of the stem cells [17], These niches were found to exist in the dorsal horn of the spinal cord parenchyma [18], Substantial research has been conducted to optimize protocols aimed at mimicking these stem cell niches and thus promoting self-renewal or stable differentiation. One side of this research focuses on achieving purification and expansion of NSCs in vitro. This task has proved to be extremely challenging; many of the signals that control self-renewal in vivo are poorly understood thus mimicking these signals in vitro comes with significant uncertainty. Initially researchers relied on a neurosphere culture system to maintain NSCs in vitro. Neurospheres are 3D cell aggregates composed of NSPCs, a small amount of NSCs, and a small number of cells that have already differentiated. Once neurospheres have been obtained in vitro, growth factors can be added to the culture to cause selective proliferation of the stem cells into a more homogeneous neurosphere [19], This technique has become extremely common; however, it does produce a heterogeneous population of cells with a small fraction exhibiting neurogenic potential. Therefore, these culture systems are unable
10


to produce a significant amount of neurons even with the addition of neurogenic growth factors [20], In addition to neurosphere cultures, monolayer systems have also been investigated; this culture method is able to produce a much more homogenous culture resulting in higher neurogenic potential (Figure 2). However monolayer
Figure 2. Comparison between neurosphere and monolayer culture techniques. Neurosphere cultures have less neurogenic potential than monolayer cultures and are unable to produce a large number of neurons [17],
11


systems have not been shown to have long term potential [17], More recently, it has been established that NSCs sustain long-term propagation in monolayer culture with the use of epidermal growth factor (EGF) and basic fibroblast growth factor (FGF2) [17][21], Twenty years ago, Reynolds and Weiss developed a process combining the neurosphere system along with EGF and FGF2 [1], Although this system produced a heterogeneous mixture of NSCs and NPSCs, it is still widely used today to obtain undifferentiated NSC colonies [1],
2.2 Review of hNSC monolayer differentiation techniques
Efficiently directing NSCs down a specific pathway of neuronal differentiation in vitro has become a widespread but challenging field of research. NSCs will initially differentiate into neuronal or glial progenitor cells (NSPCs). These progenitor cells are multipotent stem cells that have the ability to self-renew, but unlike NSCs, they have committed to a specific lineage (neuron, oligodentrocyte, or astrocyte). However, without outside forces, NSCs primarily differentiate into oligodendrocytes and astrocytes, with a very small proportion differentiating into neurons [22],
For this reason, researchers have begun focusing on developing efficient protocols to differentiate NSCs into NSPCs and finally into MNs [23], Motor neurons are efferent nerves that transmit signals from the spinal cord to the surrounding tissue. These nerve impulses facilitate life-dependent muscle contractions (swallowing, breathing, etc.) and gland stimulation. Motor neurons can be found in the CNS, brain
12


stem, and spinal cord [24], Because of the functions and extent of these cells, degeneration and death of motor neurons often leads to severe and sometimes fatal outcomes [25],
Researchers have determined that replacing or repairing damaged MNs could represent a potential treatment or cure for SCI. Significant efforts have been undertaken to obtain a method to encourage re-growth of endogenous MN or to completely replace the damaged or dead cells. Concerning the latter, a significant obstacle that stands in the way is the lack of an efficient source of naive spinal motor neurons (sMNs) for research purposes and cell transplantation. As mentioned above, one potential source of naive MNs is the differentiation of NSCs. So far, several protocols for differentiating NSCs into MNs have been reported [26][16]; however these methods display a limited percent production of homogenous MNs from the initial cell population.
Three different types of protocols have been reported to differentiate human stem cells into neural stem cells. The first approach uses the formation of embryoid bodies (EB) to obtain neural rosettes [27], the second approach bypasses the formation of EB and solely forms neural rosettes [15] [16], and the third approach focuses on the accelerated formation of neural rosettes from EB [28], Researchers can then induce motor neuron differentiation of these NSCs through the use of retinoic acid (RA) and sonic hedgehog (SHH) or SHH agonists. These two molecules induce the production of transcriptions factors that subsequently activate pathways leading to
13


motor neuron differentiation [24], RA is known to play an important role in the development and maintenance of the CNS. In addition, RA was found to induce the development of subtype spinal cord (cholinergic) neurons from stem cells in culture.
The exposure to SHH or SHH agonists was found to enhance the
neural rosettes | embryoid bodies
Shh/SAG, RA Motor neuron differentiation
Motor neuron
GDNF, BDNF Motor neuron axon elongation
Mature motor neuron
Figure 3. Schematic of motor neuron differentiation and motor neuron axon elongation. Starting with human stem cells (iPSCs or ESCs), cells are differentiation into neural precursors (neural rosettes or embryoid bodies) [24],
14


signals of RA and encourage the enrichment of different subtypes of neurons (dopaminergic, serotonergic, and cholingergic neurons) depending on other signaling factors involved [29], RA and SHH are usually used in conjunction with a media containing N2 or B27. N2 supplement is a serum free supplement that is often used in combination with B27 for differentiation of ES cells into neuron lineages [15][24], It has also been shown that purmorphamine (SAG), a synthetic form of SHH, can be substituted for SHH in culture [30], Research has shown that effective concentrations of RA and SHH are close to luM and 50-500 ng/ml, respectively [31], After motor neuron differentiation, motor neurons are induced to extend axons. Without axonal extension, MNs are unable to connect with their surroundings to conduct signals that will restore functionality. Neurite outgrowth may be stimulated in vitro through neurotrophic growth factors [10], The most commonly used growth factors are brain-derived neurotrophic factor (BDNF) and glial-derived neurotrophic factor (GDNF) [32], It is also important to stop or lower the concentration of RA and SHH at this point; continued use can inhibit maturation of motor neurons [30],
2.3 Review stem cell niche
Not only is it important for NSC to differentiate into the appropriate cell type, but also for the NSCs to efficiently differentiate into a stable cell type. Stem cells will not differentiate into a stable cell type without the proper balance of signals either from the cell itself (intrinsic) or environmental factors (extrinsic). Incorrect signals
15


can cause stem cells to differentiate into a heterogeneous population and severely decrease the number of differentiated cells achieved [33], This will force researchers to perform cell sorting or use a larger number of initial stem cells to achieve the desired results. As far as clinical applications, stem cells that receive incorrect types or amounts of signals have a high risk of becoming cancerous and present a dangerous result if implanted clinically [14], For this reason, researchers have focused on eliminating this risk by investigating the signals that control stem cell self-renewal and differentiation in vivo. Understanding stem cell differentiation in vivo would provide great insight on how to manipulate and control stem cell fate in vitro [14] [34], In addition, understanding how stem cells are controlled through a stem cell niche provides valuable information for the design of cell scaffolds. With this information, cell scaffolds can be manipulated to imitate the stem cell environment in vivo and interact properly with the cells and surrounding tissue once implanted [35],
It has been accepted that in vivo stem cell self-renewal and differentiation are controlled through a balance of signals coordinated in special stem cell niches. Stem cells within these niches must have sufficient intrinsic and extrinsic signals to encourage self-renewal or instead differentiate into a chosen cell type. These stem cell niches are tissue-specific but are all controlled through similar types of signals. Some of the signals stem cells are exposed to are paracrine signals, growth factors, cytokine gradients, glycoasaminoglycans (GAGs), and the physiochemical environment [36], However all of these signals can be grouped into the categories:
16


cell-cell interactions, cell-ECM interactions, and cell-biomolecule interactions. Understanding how neural stem cells and their niches interact has become a great focus in research in order to understand what gives these cells neurogenic capacity. This information could provide potential new treatments for tissue regeneration following a spinal cord injury.
Although we may know the general makeup of neural stem cell niches, few specific details are known about the in situ neural stem cell niches that exist within the adult spinal cord. Researchers have proposed two models to depict the localization and function of these stem cell niches and hopefully provide more information in order to isolate these cells or mimic the function of these niches for SCI treatment [18], The first model describes slowly proliferating neural stem cells that exist in the ependymal later of the spinal cord [37], Relating to the first model, researchers have successfully isolated and cultured cells from this ependymal layer indicating the presence of stem cells. In addition, researchers described the properties of an identified stem cell niche that resides in the central canal ependymal [38], In this work, Hamilton et al identified some of the similarities and differences between this spinal cord stem cell niche and the well-documented stem cell niche located in the forebrain subventricular zone (SVZ) [38], They were able to determine that unlike in the SVZ, stem cells within the ependymal layer mainly self-renew and show little evidence of lineage relationship further showing the limited regenerative capacity of the CNS [38],
17


Not only is there little information about the localization, function, and signals of neural stem cell niches within the adult spinal cord, but after SCI, the ECM in the stem cell niche will be altered [39], These changes in the ECM components are thought to induce signals for NSC activation [39], Following mechanical insult to the spinal cord, the limited proliferation capacity of the adult NSCs is increased. In these cases, NSCs can be induced to divide, migrate, and mature into spinal cord progenitor cells [40],
2.4 Review RGD peptide
Polymers can be synthesized to provide inherent biocompatible and mechanical support for cells similar to the role of a stem cell niche. However, polymers alone do not have bioactive molecules that can bind to cells and encourage cell growth and spreading [35], To improve the interaction between polymers and cells, researchers have begun conjugating cell-binding proteins to the polymer structure. In 1984, Pierschbacher and Ruoslahti used reduction techniques to isolate the cell-binding motif on fibronectin (common integrin-binding protein in the ECM) [41], They discovered that the majority of the cell-binding activity was achieved through the peptide, RGD. Further research showed that this cell-binding binding motif was also present on many other ECM proteins including laminin, collagen, and vitronectin [42], Because of its extensive presence and its highly effective cell binding capacity, RGD has become the most commonly employed peptide used to enhance cell attachment to a polymers surface.
18


RGD is the principle integrin-binding domain, capable of binding to a plethora of cell adhesion receptors found throughout the body. However, the most numerous and multipurpose group of cell adhesion receptors is the integrin family. This group of adhesion molecules is not only responsible for cell attachment, but they also play an important role in cell differentiation, embryogenesis, proliferation, and gene expression. Binding of the RGD peptide to integrins causes a cascade of signals that can influence cell behavior in different ways (Figure 2) [42], The first stage is ligand binding of the integrin and partial attachment of the cell body integrin-binding ECM. At this point, attachment is weak and can only withstand gentle agitations. In the
Figure 4. Integrin binding, clustering, and signaling cascade. This schematic provides some examples to the signaling pathways that can be triggered through integrin
binding.
19


second stage, cell attachment becomes stronger and the integrins begin to cluster together. Once the integrins are clustered, focal adhesion-associated proteins (focal adhesion kinase, tensin, syndecans etc.) are recruited to the binding site [43], Thirdly, stress fibers composed of microfilament bundles begin to form. Finally, the cell becomes physically anchored through focal adhesions and also experiences signal transduction through the focal adhesion-associated proteins [44], As detailed before, focal adhesion formation is conducive to cell survival signals and proliferation. Research has shown that focal adhesion-associated proteins trigger the expression of anti-apoptotic protein Bcl-2 and the focal adhesion kinase pathway [45], These two pathways along with focal adhesion formation play an important role in cell survival, proliferation, and cell spreading.
It was also determined that disrupting the interaction between integrins and cell-binding motifs can induce apoptosis and cell death [46], This integrin-mediated death is caused by the lack of an integrin substrate to bind the ligand leading to the recruitment of caspase and the cell death cascade [46], Relating this finding to a cellular interaction with the ECM, we can see that the absence of integrin-binding ligands in a microenvironment can cause the cell to undergo apoptosis [46],
To this end, cells grown or differentiated in a 2D culture are typically provided with some sort of ligand to simulate these essential cell attachment and survival signals. NSCs, specifically, are usually cultured on surfaces coated with
20


ECM proteins such as laminin, a major protein in the basal lamina, which possesses this RGD binding site [47], Along these lines, studies have shown that cell behavior and growth can vary drastically by altering the amount of RGD ligands present [48], More specifically, research has shown that RGD may have the ability to regulate neural stem cell survival and differentiation in a dose-dependent manner [49], Research surrounding stem cell therapies and potential implantable scaffolds can benefit drastically from this finding. Knowing that there may be an optimum RGD concentration to encourage NSC differentiation and survival would allow researchers the ability to fine-tune cell scaffolds to encourage this behavior.
2.5 Review biomimetic polymer scaffold
Scaffold design and fabrication has become a major field in tissue engineering. Research has shown that cell survivability when merely injected into tissue is low and must be increased to have significant therapeutic effects. Scaffolds implanted along with the cells can provide the necessary support and signals by acting as a substrate on which cell populations can attach and grow [13],
A subcategory of polymers that have been of particular interest in tissue engineering is biomimetic polymers. These polymers combine the controllability of a synthetic polymer with the biocompatibility of natural material found in the body. Biomimetic polymers can be manipulated to prompt a specific cellular response or direct new tissue formation. Extensive work has been conducted to create biomimetic polymers suitable for cell transplantation therapies. One of the most prominent
21


methods to create a biomimetic material is through surface modification of common ECM proteins or short peptide binding motifs, such as RGD [50], As mentioned in the previous section, RGD is the most commonly used peptide for surface modifications and has been incorporated into polymers for research in NSC proliferation and differentiation in vitro [51],
Different types of polymers have been conjugated with RGD and examined for the desired cellular response in vitro. Poly (ethylene glycol) (PEG) [52], poly (lactic acid) (PLA) [53], poly (ethylene-terephthalate) (PET) [35] and others have been studied for use in conjunction with RGD in NSC cultures. In one in vitro study, it was shown that NSCs grown on a two-dimensional RGD functionalized lipid bilayer produced single adhered cells comparable to monolayer growth seen on laminin surfaces. NSCs in monolayer growth vs. neurospheres have enhanced proliferation and are able to generate a higher percentage of neurons from the initial population [54], This observation shows that RGD functionalized materials may be extremely useful in synthetic culture systems for neural stem cells [55],
Very recently, work has been done to create three-dimensional scaffolds for NSC growth in vitro and thus bridge the gap between these two-dimensional studies and in vivo experiments. One research group used a naturally derived 3D polymer scaffold (collagen and hyaluronan) to culture neural stem and progenitor cells successfully. However these naturally derived polymers are difficult to control and may present residual unknown components [56], Another research group was able to
22


show NSC differentiation and neurite out-growth on a 3D scaffold made from poly(L-lactic acid) PLLA electrospun fibers [57], This cell-scaffold interaction relied mostly on the porosity and modulus of the material. However, because this type of scaffold presents very limited cellular recognition motifs, the cell-scaffold interaction and subsequently NSC attachment and differentiation were restricted [57], Despite the promising results of incorporating RGD in two-dimensional culture conditions to increase the interactions between the cells and the material, few efforts have been taken to incorporate RGD in three-dimensional scaffolds for NSCs. However, one research group created a 3D self-assembling peptide scaffold functionalized with RGD that was able to support NSC proliferation and differentiation [58], This study displays the viability of an RGD functionalized three-dimensional scaffold used to culture NSCs and provides proof that this type of polymer system can be applied to in vivo SCI regeneration studies.
For this study we wished to test the ability of biomimetic poly (serinol hexamethylene urea) (PSHU) conjugated with RGD to promote hNSC survival and spreading and thus be a promising cell scaffold for stem cell SCI treatment. PSHU was employed due to its protein-like backbone structure, which gives this polymer an inherent biomimetic quality. This characteristic is extremely beneficial to increase the biocompatibility and interaction with the cells. In addition, PSHU has the potential to attach a large quantity of biomolecules and thus increases the adjustability of this polymer system.
23


2.6 Review in vivo studies with NSCs and MNs
Recently, SCI research has moved to in vivo studies to determine if these cell therapies are viable for SCI treatment. In vivo studies have been conducted using the implantation of NSCs or NSPCs in an attempt to regenerate the damaged nerves. However, there are very few reports that describe satisfactory differentiation into neurons and integration of these neurons into the surrounding tissue [4], As stated previously, a critical challenge seen in these studies is the propensity of NSCs to differentiate primarily into glial cells as well as moderate cell survival after transplantation. Typically less than 10% of transplanted cells survive [3], To this end researchers have turned to 3D scaffolds to use in conjugation with NSCs for in vivo studies. One group designed a blended poly(lactic-co-glycolic acid) (PLGA) scaffold seeded with NSCs to implant into a SCI rat model. They were able to show possible neural regeneration and long term functional recovery suggesting increased NSC survival and tissue integration [3], In another study, researchers compared the survival of ESC-derived NSCs when transplanted alone and when encapsulated in a fibrin scaffold containing growth factors. They were able to show higher cell survival when implanted with the fibrin scaffold as well as an increase in the number of neurons present after 8 weeks of transplantation [1], More recently, pre-differentiated NSPCs were seeded in a fibrin scaffold and implanted in a fully transected rat model. Increased recovery and better cell survival was seen with the use of this scaffold compared to cell injection alone [59], Together, these studies represent the benefits of
24


a cell scaffold for improving NSC survival, integration, and most importantly
functional recovery after SCI.
25


3. Individual objectives and experimental approach
(1) Synthesize and characterize a highly functionalizable biomimetic polymer:
Significant work has been done to create functionalizable polymers for use in bioengineering. However, most of these polymer systems have limited conjugation capacity and thus may not contain sufficient molecules to induce certain cellular behavior. We believe we can achieve a much higher concentration of biomolecules through the synthesis of a highly functionalizable polymer, capable of conjugating larger amounts of molecules. Characterization of this polymer is one of the most important aspects of this work. We first need to ensure that this polymer possesses the expected peptide-like backbone structure and number of functionable groups.
(2) Synthesize a biomimetic polymer by conjugating RGD and determine the extent of cell attachment to the polymer scaffold:
Not only will this polymer have biomimetic properties from its peptide-like backbone but also through the conjugation of RGD. As mentioned earlier, one of the most common methods to create a biomimetic polymer is through the conjugation of biomolecules. We decided to use RGD (GRGDS) because it is known to induce attachment and spreading of NSCs. We believe that this improved attachment and spreading will in turn improve the differentiation efficiency and axonal extension of the NSCs. First, we wanted to characterize the polymer further through the quantification of the amount of RGD successfully conjugated to the polymer backbone. Previous works have shown that the amount of RGD may affect cell
26


behavior in a dose-dependent manner. Therefore, it is extremely important to determine the amount of RGD conjugated to the polymer backbone as it will have great effects on the cellular behavior and determine the direction of this project.
(3) Evaluate the survival and health of human neural stem cells when seeded on RGD functionalized biomimetic polymer:
The first step to test the viability of this biomimetic polymer as a NSC scaffold is to examine its ability to encourage hNSC survival and proliferation. As stated above, cells implanted alone frequently have a low survival rate. One of the roles of this polymer system is to encourage cell survival and prevent death of the implanted cells.
(4) Evaluate the differentiation of human neural stem cells into mature motor neurons when seeded in RGD polymer scaffold:
Once we have shown that this polymer scaffold encourages cell growth and survival, we can test the ability of this polymer scaffold to support NSC differentiation. For stem cell therapies with NSCs, the implantable polymer scaffold should support and encourage differentiation and attachment of NSCs. For cell therapies with MNs, this scaffold could aid in the differentiation of MN from NSCs in vitro and thus could increase the number of MNs produced for implantation
(5) Synthesize and quantify varying conjugation amounts of RGD to the polymer backbone:
Once we have collected data and observations from the polymer scaffold with close to 100% RGD conjugation, we can begin to determine the optimum amount of RGD
27


to conjugate. As stated earlier, the amount of RGD may have a dose-dependent affect on cell survival and NSC differentiation. Thus there may be an optimum amount of RGD to induce the maximum amount of NSC survival and differentiation. We hope to synthesize polymer systems each with different RGD conjugation amounts to determine the optimum RGD surface density without varying the amount of polymer present.
(6) Determine the optimum RGD conjugation ratio in the scaffold to promote maximum differentiation and maturation:
Finally, we will test each of these polymer systems and continue work with the optimum amount of conjugated RGD. We will conduct this test again by determining the polymer system that promotes the most NSC survival and differentiation.
28


4. Materials and Equipment
4.1 Materials
N-BOC-Serinol, urea, hexamethylene diisocyanate (HDI), anhydrous chloroform, and anhydrous N,N-dimethylformamide (DMF) were purchased from Sigma-Aldrich (St. Louis, MO, USA). N-(3-Dimethylamino- propyl)-N'-ethylcarbodiimide hydrochloride (EDC), N-hydroxysuccinimide (NHS), 2,2,2-trifluoroethanol (TFE), and trifluoroacetic acid (TFA) were purchased from Alfa Aesar (Ward Hill, MA, USA). Anhydrous diethyl ether was purchased from Fisher Scientific (Pittsburgh, PA, USA). Anhydrous dichloromethane (DCM) was purchased from JT Baker (Phillipsburg, NJ, USA). The pentapeptide Gly-Arg-Gly-Asp-Ser (GRGDS) was purchased from Biomatik (Wilmington, DE).
4.2 Equipment
Infrared spectra (2 wt.% sample in polyethylene IR sample card) were recorded on a Nicolet 6700 FT-IR spectrometer from ThermoElectron Corporation. 1H-NMR spectra were recorded on a Varian-500 NMR (500 MHz, Varian) with CDC13 as the solvent at 25C. Chemical shifts are in ppm using the solvent peak as the internal reference. RGD-conjugation was determined using Agilent Zorbax 300SB C8 HPLC system. The analytical column size is 2.1x150 mm with a flow rate of 0.3 ml/min. The buffers used were 1) 0.2% TFA/water and 2) 0.18% TFA/AcN (acetonitrile). A 6 min isocratic run was used first and then 6 min at a linear gradient 1% B/min. The detection was taken at 210 nm. All in vitro cell morphologies were examined on a
29


Nikon DIAPHOT 300 equipped with CCD camera (SPOT RT 2.3.0, Diagnostic Instruments) using SPOT Advanced software for post-hoc analysis.
30


5. Methods
5.1 Biomimetic poly (serinol hexamethylene urea) synthesis
We began by synthesizing N-Boc Serinol using Serinol (1.959 g, 21.50 mmol) and Di-Tert-Butyl-Dicarbonate (5.973 ml, 26.0 mmol). N-Boc-Serinol will be incorporated into the polymer backbone and protect the free amine groups on the polymer backbone from reacting inappropriately. Next, Poly (serinol hexamethylene urea) (PSHU) was synthesized by combining HDI (1.928 ml, 12 mmol), N-BOC-Serinol (1.147 g, 6 mmol), and urea (0.360 g, 6 mmol) in 6 ml of anhydrous DMF. This reaction was maintained at 90 deg C for 7 days. After the 7-day reaction period, the solution was cooled to room temperature and roto-evaporated at 70 deg C to remove the DMF. Next, the product mixture was re-dissolved in a small amount of DMF (2-3 ml) and precipitated into cooled anhydrous diethyl ether (100 ml). The purification process was repeated 2 times and finally the solution was washed overnight in excess ether (100 ml) to remove any unreacted products and remaining solvent. Finally, the product was roto-evaporated at 45 deg C until dry and stored at room temperature until conjugation with RGD.
5.2 Removal of BOC protective groups from the polymer backbone
To begin, the protective BOC groups from the polymer backbone were removed. This will provide free amines to conjugate the RGD-COOH molecules. Hydrogenation by a strong acid was used to deprotect the amine group and obtain PSHU-NH2. PSHU was first dissolved (lg, 1.96 mmol) in methylene chloride (15
31


ml). This solution was then mixed and trifluoroacetic acid (15 ml) was added dropwise to the stirring solution. This deprotection reaction took place at room temperature for 45 minutes. The TFA/PSHU/Methylene Chloride mixture was roto-evaporated at 45 degrees C until the TFA is removed (5-10 min) and the product solution was dissolved in anhydrous DMF (1 ml). Once fully dissolved in DMF, this solution was purified by precipitation in excess cooled ether (100 ml). The product was purified further by dissolving it in 2,2,2- trifluoroethanol (TFE) and precipitating this mixture in excess ether. Finally, the product was roto-evaporated at 45 degrees C, dried, and stored at room temperature.
5.3 Maximum conjugation of RGD to PSHU backbone
After deprotection of PSHU, GRGDS-COOH may be conjugated to the free amine groups on the backbone of PSHU. Each molecule of deprotected PSHU possesses 18 free amine groups that may be functionalized with GRGDS-COOH. Note that GRGDS was used instead of RGD in order to preserve the integrity of the entire RGD binding motif. To start, 100 mg of deprotected PSHU was measured (0.01 mmol PSHU, .1956 mmol amine groups). From the molar amount of the free amine groups, the moles of GRGDS-COOH were calculated based on the desired ratio of conjugation. For this study we used a molar ratio of 1.3 (moles of GRGDS-COOH/amine groups). This was intended to conjugate close to 100% of the free amine groups on the PSHU backbone. Once the appropriate amounts of deprotected PSHU and GRGDS-COOH were obtained, GRGDS-COOH was dissolved in DMF (1
32


ml) and combined with N'-ethylcarbodiimide hydrochloride (EDC) (3X moles GRGDS-COOH) and N-hydroxysuccinimide (NHS) (3X moles GRGDS-COOH). This NHS-ester activation reaction was allowed to carry on for 2 hours and then combined to 100 mg of deprotected PSHU dissolved in DMF (1 ml). The conjugation reaction rook place overnight at room temperature and was finally precipitated in excess ether and rotary evaporated to remove the unreacted by products and solvent. The final product was stored away from light at 4 deg C until use.
5.4 PSHU-GRGDS coating optimization
The optimal amount of PSHU-GRGDS coating was determined by examining the affects of each amount on the proliferation and differentiation of the hNSCs. First PSHU-GRGDS was dissolved in TFE with varying amounts: O.Olug, 0.1 ug, 1 ug, 10 ug, 100 ug. In a 24 well plate, three wells for each PSHU-GRGDS amount were filled with 0.1 ml of TFE and the corresponding amount of polymer. Knowing the actual conjugation ratio of GRGDS and the amount of PSHU-RGD in each well, we were able to calculate the amount of PSHU and RGD in each well (Figure A.2). After the 24-well plate was filled with the PSHU-GRGDS in solution, the plate was placed on a mechanical agitator. While on the agitator, the solvent TFE will evaporate from the well leaving the polymer coated to the bottom of the well. The constant agitation provided while the solvent evaporates provided a more even coating of the polymer on the bottom of the well. This method produces less variability of the polymer amount deposited on different sections of the well.
33


5.5 Human embryonic stem cell culture conditions
Human embryonic stem cells (ESCs), UCSF-4 were cultured in mTesR media (STEMCELL) on Cellstart (Invitrogen) coating plate. For passaging, UCSF-4 cell colonies were cut into small squares using StemPro EZPassage Disposable Stem Cell Passaging Tool (Gibco) at a dilution of 1:10.
5.6 Neural stem cell induction
hESCs (UCSF-4) were treated with 10 ng/ml hLIF(Milipore), 3 pMCHIR.99021 (Cellagentech), and 2 pM SB431542 (Cellagentech) in neural induction media, N2B27, containing DMEM/F12: Neurobasal (1:1), 0.5xN2, lxB27, 1% Glutmax, for 10 d. The culture was then split 1:3 for the next passages using Accutase and expanded in neural induction media supplemented with lOng/ml hLIF, 3 pM CHIR99021, and 2 pM SB431542 on Cellstart coating plate.
5.7 Motor neuron differentiation
Motor neurons were differentiated from neural stem cells in N2B27 media with treatment of 1 pM Retinoic acid (Sigma) and 100 nM SAG (Smoothened agonist, SHH Activator, EMD Chemicals) for 7 days on poly-D-lysine(Sigma) and laminin coating surface or different concentration of PSHU or PSHU-RGD coating plate, and then the cells were terminally differentiated in the presence of 10 ng/ml BDNF and 10 ng/ml GDNF(R&D systems) in N2B27 media for another 14 days.
5.8 Varying RGD conjugation amounts
34


Next, we wished to synthesize polymer and vary the conjugation ratio of RGD to the polymer backbone. To do this, we began by removing all of the protective BOC groups from the polymer backbone. This will expose the free amine groups and allow conjugation of GRGDS-COOH. We chose to use molar ratios of GRGDS-COOH/free amine groups of 0.4, 0.6, 0.8 and 1.0. Based on the previous results using a molar ratio of 1.3, we anticipated that these molar ratios would produce a similar actual conjugation ratio to the initial molar ratio. To begin, the appropriate amounts of GRGDS-COOH, EDC (1.2x molar amount of GRGDS-COOH), and NHS (1.5x molar amount of GRGDS-COOH) were weighed and allowed to mix in 1 ml of DMF for two hours. This initial reaction period is meant to create a more stable, amine-reactive intermediate. After two hours, 100 mg of deprotected PSHU dissolved in 1 ml of DMF was added to each of the four samples. The deprotected PSHU and activated GRGDS molecules were allowed to react for 24 hours at room temperature. Next, the solution was precipitated in cold ether twice to remove most of the DMF. The remainder of the DMF was removed during rotoevaporation and finally through dialysis of the solution. After dialysis, the RGD conjugated polymer was lyophilized and stored wrapped in foil at 4 degrees Celsius to prevent degradation and decrease in RGD bioactivity.
5.9 Statistical analysis
35


All results are expressed as means standard deviation. All quantitative results were analysed using analysis of variance (ANOVA) and, if necessary, followup analysis by Tukeys test. Statistical significance was considered at p < 0.05.
36


6. Results and Discussion
6.1 Polymer backbone characterization using NMR
H
N=C
O > / \\ / O x + h2n'nh2 o H^ Vc ) NH + WO'
C = N
Hexamethylene Diisocyanate Urea N-BOC Serinol
X
O^NH
PSHU Deprotection, 45 min, RT H H iv Y^ O O O
^ N N N N H H H H
Y-
Poly (serinol hexamethylene urea), Protected PSHU
PSHLIGRGDS-COOH Conjugation, ON, RT
NH2
rS
tV Yn-
o o o
XkAkA
N N N H H H
Poly (serinol hexamethylene urea), Deprotected PSHU
PSHU Synthesis, 7 days, 90C
GRGDS
GRGD:
O
O O
O O O
N|X X X
N N N H H H
Poly (serinol hexamethylene urea) (PSHU)-GRGDS
Figure 5. Schematic of PSHU-RGD synthesis with the final structure of PSHU-RGD. The first stage is PSHU synthesis followed by the removal of the N-Boc groups from the PSHU backbone and finally RGD conjugation. Note that the value of n was determined to be ~18 with RGD attached to a certain percentage of monomers depending on desired RGD conjugation ratio.
37


In this study, we aimed to test this biomimetic polymers ability to efficiently promote NSC survival, proliferation, and differentiation into mature motor neurons. We began by confirming and characterizing the synthesis process of Poly (serinol hexamethylene urea). NMR was used to confirm the overall polymer structure as well as the removal of the N-BOC groups from the primary amines. The synthesis progression of Poly (serinol hexamethylene urea) (PSHU) can been seen in Figure 5 and the NMR analysis of the polymer structure can be seen in Figure 6. The NMR spectrum of PSHU confirmed the expected copolymer structure, with peaks at 1.3 (-CH2-), 1.5 (-NH-CH2-CH2-), and 3.2 (-NH-CH2-) associated with HD I, at 1.4 (-C-(CH3)3), and 4.1 (-CH-NH-) associated withN-BOC-serinol.
NMR analysis was also used to confirm complete removal of the BOC-protecting groups from the polymer backbone. This process is extremely important, as the free amines will later be used to conjugate RGD; residual BOC-protecting groups will alter the expected conjugation amount of RGD. The presence of BOC-protecting groups will produce a peak on the NMR spectrum at 1.4 (Figure 7). The top spectrum in Figure 7 shows the disappearance of peak B (methyl groups N-BOC) indicating the complete removal of the BOC-protecting groups. Once the BOC-protecting groups are removed from the polymer backbone, the exposed free amine groups can be conjugated with various biomolecules (RGD in this case).
38


B
Poly (serinol hexamethylene urea). Protected PSHU
DM SO
i----i--'-i--'--i----i----i----i---i----i--1--i----i----1 i
Chemical Shift (ppm)
Figure 6. 'HNMR (500 MHz, CDCI3) spectmm of PSHU to confirm overall
structure of polymer chain.
Removal of HOC Groups
Disappearance of peak at 1.4
Poly (serinol hc\umcth)lcnc urea). I>epralcetod PSHU
Ptil) (MTinol hciumrlhvlcnr urea). Pmtcvtcd IM It
B
Figure 7. 'HNMR (500 MHz, CDCI3) spectrum of PSHU and deprotected PSHU to confirm removal of the BOC protecting groups during deprotection process.
39


6.2 RGD conjugation confirmation using FT-IR
After PSHU was successfully characterized, RGD was conjugated to the PSHU backbone intended to further improve differentiation and spreading of the motor neurons. As mentioned above, we initially chose a molar excess of 1.3 moles of RGD to the moles of free amine groups. FT-IR was used to confirm that RGD-COOH was successfully conjugated to the free amine groups on the
Wavenumber (cm-1)
Figure 8. FT-IR of dPSHU, PSHU, and PSHU-RGD. Confirmation of free amine groups on dPSHU after deprotection (B). Shift in carbonyl absorbance to confirm attachment of RGD to the polymer backbone (A).
40


PSHU backbone. We can first confirm the presence of free amine groups on DPSHU (Deprotected-PSHU) by examining the region around 798 cm'1. This wavenumber corresponds to N-H primary amines and can be seen very clearly in the FT-IR figure below (B). Next we can confirm the conjugation of RGD to the polymer backbone by viewing the region of 1630 to 1680 cm'1. This region is associated with the carbonyl groups found within the polymer backbone as well as the carbonyl groups found within pepide bonds of RGD. The wavenumber correlated to the carbonyl groups of RGD are slightly lower than that of the carbonyl groups in the polymer backbone. In the PSHU-RGD spectrum, we can observe an obvious shift in this carbonyl peak towards the lower end of the spectrum, indicating the presence of carbonyl groups in the RGD peptide. Thus we can confirm the presence of RGD conjugated to the PSHU polymer backbone.
6.3 RGD conjugation quantification using HPLC
HPLC was used to quantify the amount of GRGDS-COOH that was successfully conjugated to the free amine groups on the PSHU backbone. A calibration curve was first constructed using known concentrations of GRGDS-COOH (0.78-200 ug/ml) (Figure A.l). Each of these concentrations produced a corresponding HPLC peak area. For this study, a 1.3 molar ratio of GRGDS-COOH/free amine groups was used in order to obtain close to 100% conjugation of the free amine groups. The calibration curve constructed previously was then applied to HPLC of this polymer sample to determine the actual conjugation ratio of
41


GRGDS-COOH to the polymer backbone (Figure 9). We were able to show that using a 1.3 molar ratio of GRGDS-COOH/free amine groups produced 93% conjugation of RGD to the free amine groups on the polymer backbone. The theoretical molar ratio of 1.3 was expected to produce a conjugation value close to 100%. Previous studies have demonstrated that the amount of RGD present can have a significant effect on cellular behavior; therefore this analysis provided vital information on optimizing the conditions of this polymer scaffold [49],
Minutes
ROD Calibration tune
leooc
14000
(RUI <*1 all
Figure 9. HPLC Curve of 1.3 initial molar ratio of RGD/free amine groups. Using the calibration curve on the right we were able to shown this molar ratio produced close to 100% conjugation of RGD to the free amine groups on the polymer backbone.
42


6.4 Human neural stem cell phase contrast images
After quantifying the amount of conjugated RGD to the polymer backbone (93%), PSHU-RGD was coated to the bottom of a 24-well plate using the method described in section 5.4. The plate was coated using varying amounts of polymer and three replicates of each amount. These variations of PSHU-RGD amounts were chosen to begin to narrow down the optimum amount of RGD to encourage maximum hNSC growth. Because the conjugation ratio and mass of PSHU is known, we can calculate the total amount of RGD coated on the well (Figure A.2). Deprotected PSHU and no-coating were used as negative controls. The schematic of the 24-well plate can be seen in figure 10.
1 2 3 4 5
A ( 0.01 ug ) ( 0.1 ug ) ( "lug )( 10ug )( 100ug
B (01
Figure 10. 24-well plate PSHU-RGD (93% conjugation) polymer coating schematic.
43


After coating the 24-well plate with the appropriate amount of polymer, hNSCs were plated using the culture techniques described in section 5.7. Every two days, phase contrast images of the cell cultures were taken to compare the behavior of the cells. The lowest amounts of PSHU-RGD (O.Olug and O.lug) cultures show no cell attachment as can be seen from the rounded cell morphology and bright cell border. Unfortunately the 0.1 ug PSHU-RGD polymer coating was displaying some bubbling or buckling of the polymer, which may affect cell attachment and health. In the next two amount of PSHU-RGD (1 ug and 10 ug), cell attachment is already observed. More strikingly, there appears to be more cell attachment and neurite outgrowth in the 10 ug culture vs. the lug culture. On the other hand, the hNSCs cultured on the 100 ug PSHU-RGD coating show initial cell attachment, but no cell spreading or neurite outgrowth. Interestingly, there appears to be a level of RGD that becomes toxic and impedes not only hNSC survival but also axon extension. This observation indicates that the cells initially attached to the lOOug PSHU-RGD coating but were unable to spread and extend axons. This is likely due to oversaturation of RGD on the culture surface causing excessive focal adhesions that inhibit cell movement [54], With the inability to spread, these cells will be unable to differentiate into functional neurites and will instead proceed to apoptosis [48],
As a control for this polymer system, we evaluated hNSC cell growth and axon spreading on deprotected-PSHU (polymer w/out RGD conjugation) and no surface coating. After 2 days, the hNSCs cultured on the deprotected-PSHU showed
44


no attachment but no immediate cell death (i.e. floating cells with ruptured membrane and cell debris) indicated that deprotected-PSHU is non-toxic to the cells however does not permit cell attachment. The cells cultured on no surface coating also showed little attachment but no immediate cell death. We could see that the cells were not attached to the bottom of the plate by moving the plate and examining the cells. In addition, a brighter ring around the rounded cells indicates floating cells in the culture. However both deprotected-PSHU and no-coating cultures show rounded cell morphology corresponding to poor cell health and the induction of apoptosis [60],
45


Polymer
lug PSHU-RGD_______ lOug PSHU-RGD

Polymer
lOOug PSHU-RGD
Figure 11. Phase contrast images taken after 2 days of culture.
Phase contrast images of the cell cultures were taken again after 4 days. At this point, the cells grown on the two lowest polymer concentrations (0.01 ug and 0.1
46


ug) show neurosphere formation. As discussed before (Section 2.1), neurospheres are heterogeneous free-floating aggregates of neural stem cells. Although the cells in these two cultures are surviving, the formation of neurospheres signifies a heterogeneous population of cells with limited neurogenic potential and poor cell attachment. In contrast, the cells grown on the next two polymer concentrations (1 ug and 10 ug) show substantial cell attachment and axon extension. Additionally, after 4 days of culture the difference between the 1 ug and 10 ug polymer coatings is much more apparent. The NSCs culture on lOug of PSHU-RGD show significantly more attachment and proliferation. This finding indicates that there may be a sweet spot surface density of RGD to induce the maximum amount of NSC attachment and axon extension.
47


Figure 12. Phase contrast images taken after 4 days of culture.
After 8 days of culture, there is still NSC proliferation and attachment on the 10 ug PSHU-RGD coating. However, we began to see neurosphere formation on the
48


1 ug PSHU-RGD polymer coating. We believe this is due to slight detachment of the polymer coating from the surface of the plate. The detachment of the polymer would cause the detachment of the cells or induce weaker attachment of the cells and thus the formation of neurospheres. This can be avoided in the future by increasing the chain-length and thus decreasing the solubility of the polymer in water or by allowing more time for the polymer coating to dry and fully adhere to the plate surface. Regardless, there was still extensive NSC proliferation and attachment on the 10 ug PSHU-RGD coating. This further indicates that the 10 ug PSHU-RGD surface density of RGD and polymer may be close to the optimum amount in order to induce maximum attachment and proliferation of NSCs. However, in the future we would need to compare the hNSC cultures on our polymer system with a positive control. It is important to see what healthy hNSCs in a 2D culture look like to determine if our polymer can support cell health and growth. We propose the use of PDL-Laminin, a standard coating for stem cell proliferation and differentiation [47],
8 Days
No Surface Coating__________________Deprotected PSHU
49


Controls
Polymer
Polymer
lOOug PSHU-RGD


Figure 13. Phase contrast images taken after 8 days of culture.
6.5 Human neural stem cell MTT assay
After 14 days, an MTT colorimetric assay was used to measure the cell viability of each culture. MTT, a yellow tetrazole, is reduced to purple formazan in the presence of NAD(P)H and thus provides a technique to measure cellular metabolic activity. An MTT assay provides one of the most straightforward methods to compare the concentration of live cells between samples. We used an MTT assay to compare the cell viability of the cells cultured on the different surfaces (Figure 14). This MTT assay confirmed the observations from the phase contrast images shown previously. There is a significant difference between the cell viability on the lowest PSHU-RGD amounts (0.01 ug and 0.1 ug) and the two next PSHU-RGD amounts (1 ug and 10 ug). More importantly, there is also a significant difference between the cell viability of the cells cultured on lug and lOug of polymer. This difference was also seen after just 2 days of culture with phase contrast images. This observation further confirms that RGD may have a dose-dependent effect on NSC attachment and
51


viability. It is important to note that although an MTT assay may provide information about the relative cell metabolism between the samples, it does not provide any information about the actual cell count. In order to obtain this information we would have to compare the samples metabolism to MTT measurement of known cell numbers. In addition, it is also important to compare the values of this MTT assay to a positive control for NSC health. As mentioned above, in the future we would plan to use PDL-Laminin to compare to our polymer coatings.
0.2
0.18
0.16
0.14
0.12
0.1
0.08
0.06
0.04
0.02
0
*

0.01 0.1 1 10
PSHU-RGD Amount (ug)
i
100 DP
Figure 14. Comparison of MTT assay results between the PSHU-RGD surface coatings and the deprotected-PSHU surface coating. There is a significant difference between the cell viability of the cultures grown on the lug and lOug amounts of
polymer (* p-value<0.01).
52


6.6 Partial RGD conjugation analysis using FT-IR
In the previous experiment we varied the amount of RGD present by varying the amount of PSHU-RGD. However while doing this we also inadvertently varied the amount of polymer present in each culture. To ensure that the differences in cell survival and attachment are attributed to the surface density of RGD, we must keep the amount of polymer present constant in each culture. After synthesis of the different polymer systems with varied RGD conjugation amounts (section 5.8), FT-IR was employed to show the differences in RGD present between each polymer. Although FT-IR did not provide a quantitative analysis of the amount of RGD present, it provided preliminary data that each polymer system had varied RGD conjugation amounts. As detailed before, increasing the amount of RGD present should show an increased shift in the carbonyl peak towards 1630 cm'1.
---dPSHU 0.4PSHURGD
---PSHU ----1.3PSHURGD
---0.6PSHURGD -----0.8PSHURGD
1 .OPSHURGD
1550
1850
1750
1650
Wave Number (crn-1)
53


Figure 15. FT-IR of dPSHU, PSHU, 40% PSHU-RGD, 60% PSHU-RGD, 80% PSHU-RGD, 100% PSHU-RGD, and 130% PSHU-RGD. Shift in carbonyl absorbance to confirms attachment of RGD to the polymer backbone and provides information on the relative difference between the polymer systems.
6.7 Partial RGD conjugation quantification Using HPLC
Next, HPLC was used to determine the percentage of free amines that were actually conjugated with RGD. As stated previously, we used initial molar ratios of 0.4, 0.6, 0.8, 1.0, and 1.3 to achieve various amount of RGD conjugation. However, we needed to determine the actual percentage of free amines that were conjugated with RGD in order determine the optimum amount of RGD for NSC survival.
Figure 16. Graph of actual amine-RGD conjugation ratio compared to the initial molar ratio of amine-RGD. This graph was constructed by performing HPLC on each polymer sample and using the previously constructed HPLC calibration curve.
54


HPLC was performed on each polymer sample (Figure A.4). Using the calibration curve constructed previously (Figure A.l), we were able to determine the actual amine-RGD conjugation ratio (Figure 14). With this information, the amount of RGD that the cells were exposed to in culture could be quantified and used for future work (Figure A.3).
55


7. Conclusion
This study was conducted with the ultimate goal of encouraging attachment and survival of human neural stem cells through the use of a controlled biomimetic polymer. Although there have been a number of experiments elucidating the potential of NSCs in SCI treatment, there are still a few obstacles that may prevent the progression of these therapies. We chose to combat two of the most significant obstacles 1) limited cell localization and 2) limited cell survival of implanted cells.
We began by successfully synthesizing and verifying the structure of a highly functionalizable biomimetic polymer that is capable of attaching a large quantity of biomolecules. Achieving a high concentration of biomolecules for cell-biomolecule interactions plays a large role in stem cell survival and differentiation efficiency [61], Substantial research has been conducted to determine which biomolecules in the ECM play an important role in regulating NSC function in vivo and thus could be used to mimic the cell-biomolecule interactions and guide stem cell fate. The RGD sequence, an integrin-binding motif found in laminin (a large component of the ECM), was found to be implicated in outside-inside cell signaling that can affect cell proliferation, migration, and cell survival in different types of tissues [62], It has also been determined that integrin binding motifs, specifically RGD, are involved in supporting attachment, spreading, proliferation, and differentiation of NSC in a dose-dependent fashion [63][49], Therefore incorporating enough of this RGD sequence
56


into synthetic polymers to produce a biomimetic synthetic scaffold has the potential to increase NSC differentiation and proliferation [55], Not only do cell-biomolecule interactions play a role in directing stem cell fate, but also cell-ECM interactions can help to modulate neural stem cell behavior and differentiation [64], To cater to these interactions, we engineered this polymer to also possess multiple peptide-mimicking bonds to increase the biocompatibility of the polymer.
The next stage was to determine if this polymer coating would encourage hNSC attachment and spreading in vitro. We were able to show that 10 ug PSHU-RGD coating amount, displayed increased hNSC attachment and spreading and higher cell viability after 14 days. However, it was also observed that the lower amounts of PSHU-RGD and the highest amount of PSHU-RGD showed neurosphere formation and no cell growth respectively. This indicated that certain amounts of PSHU-RGD are able to encourage NSC attachment and proliferation while insufficient or excessive amount of PSHU-RGD prevented NSC attachment and survival. To further optimize this biomimetic polymer system, we next varied the conjugation amount of RGD to the polymer backbone. This would allow us to hold the amount of polymer constant and just examine the affects of RGD concentration on hNSC behavior. We believe that this experiment will elucidate an optimum amount of RGD conjugated to this biomimetic polymer to induce the maximum amount of hNSC attachment and survival.
57


In closing, we successfully designed a controllable biomimetic polymer that encourages spreading and survival of hNSCs. In addition, we anticipate that the properties of this polymer system that improve hNSC spreading and survival in vitro will also be conducive to increased cell survival and integration in vivo. Therefore, not only does this polymer may be a promising scaffold to support cell survival and integration during cell transplantation.
58


8. Future Work
8.1 Analysis of varied RGD conjugation polymer system with NSCs
The next stage of this project is to further optimize the PSHU-RGD polymer system. As mentioned above, we have successfully synthesized partially conjugated PSHU-RGD polymer systems. NSCs will then be cultured on these polymer systems using the same culture methods as before (Figure C. 1). We anticipate that the largest percent conjugation will produce comparable results to the lOOug PSHU-RGD sample shown above. However the smaller percent conjugations should produce NSC attachment and proliferation and uncover the optimum RGD conjugation percentage (Figure A.3).
8.2 Development of a 3D injectable scaffold
The next stage of this project is to synthesize an injectable scaffold using the optimized PSHU-RGD. Following SCI, further surgery to implant a cell scaffold can cause further nerve damage and loss of function. In these cases, the use of an injectable scaffold can be loaded with cellular and molecular components and injected into the lesion [65], We plan to accomplish this by conjugating a reverse therogel polymer to the PSHU-RGD polymer system. This will create a polymer system that encourages the correct cellular behavior while also avoiding the need for surgical implantation.
8.3 Spinal cord injury animal model
59


After synthesis and optimization of this injectable biomimetic NSC scaffold, we plan to examine the results of injecting this scaffold in a SCI rat model. Using behavior analysis and tissue histology we can determine if this scaffold successfully encourages regeneration of neurons and functional recovery.
60


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Appendix A
RGD conjugation analysis using high performance liquid chromatography
Concentration RGD (ug/rnl)
Figure A.l: High performance liquid chromatography RGD binding calibration curve. This graph was constructed by measuring the area under the HPLC curve related to known concentrations of RGD. This calibration curve allowed quantification of polymer samples to determine the RGD conjugation amount.
Polymer Name Amount of Polymer On Surface (ug) Percent RGD Conjugation Total amount of RGD (umol) Density of RGD on Surface (pmole/cm2)
No coating N/A N/A N/A N/A
dPSHU 100 N/A N/A N/A
0.01 PSHU-RGD 0.01 93% 0.0000182 9.58
0.1 PSHU-RGD 0.1 93% 0.000182 95.8
1 PSHU-RGD 1 93% 0.00182 958.8
10 PSHU-RGD 10 93% 0.0182 9578.94
100PSHU- 100 93% 0.182 95789.47
66


RGD | III
Figure A.2. RGD surface density calculated per well on a 24-well plate. Using a surface or growth area of 1.9 cm2 per well. However, it is important to note that not all of the RGD ligands conjugated to the polymer backbone will be in the correct
orientation to bind integrins.
Polymer Name Amount of Polymer On Surface (ug) Percent RGD Conjugation Total amount of RGD (umol) Density of RGD on Surface (pmole/cm2)
No coating N/A N/A N/A N/A
dPSHU 100 N/A N/A N/A
20% PSHU-RGD 100 -10% 0.0196 10315.79
40% PSHU-RGD 100 -30% 0.0587 30894.74
60% PSHU-RGD 100 -50% 0.0978 51473.68
80% PSHU-RGD 100 -70% 0.1369 72052.63
100% PSHU-RGD 100 -90% 0.1760 92631.57
130% PSHU-RGD 100 93% 0.182 95789.47
Figure A.3. RGD surface density on a 24-well plate calculated for varied PSHU-RGD conjugation amounts. Using a surface or growth area of 1.9 cm2 per well. However, it is important to note that not all of the RGD ligands conjugated to the polymer backbone will be in the correct orientation to bind integrins.
Initial RGD Molar Ratio 1.3 1.0
Area Under Curve 1.621 -104 1.428 104
Conjugation Ratio 93% 89%
67


Initial RGD Molar Ratio
Area Under Curve
Conjugation Ratio
HPLC Curve
0.8
5.873 103
67.1%
0.6
4.038 104
51.1%
0.4
Area Under Curve 4.773 103
Conjugation Ratio 30%
HPLC Curve i
t" u

68


Figure A.4. HPLC Diagrams to determine the actual conjugation amount of RGD to the PSHU Backbone. Both theoretical conjugation percentage and actual conjugation
percentages are provided.
69


Appendix B
FT-IR confirmation of deprotected PSHU
900 870 840 810 780
Wave Number (cm-1)
Figure B.l. Confirmation of free amine groups on dPSHU after deprotection (B). This FT-IR was performed before conjugating varying amounts of RGD in order to prevent residual N-BOC groups from altering the reaction kinetics and
outcome.
70


Appendix C
Plans and schematics for future work
1 2 3 4 5 6
Figure C.l. 24-well plate PSHU-RGD partial conjugation polymer coating schematic.
71


Full Text

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HUMAN NEURAL STEM CELL ADHESION AND PROLIFERATION ON A BIOMIMETIC POLYUREA FUNCTIONALIZED WITH RGD PEPTIDES by MELISSA LAUGHTER B.S., University of Colorado Boulder, 2012 A thesis submitted to the Faculty of the Graduate School of the University of Colorado in partial fulfillment of the requirements for the degree of Master of Science Bioengineering 2014

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! ii This thesis for the Master of Science degree by Melissa Laughter has been approved for the Bioengineering Program by Daewon Park Chair Michael Yeager Karin Payne 9/23/2014

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! iii Laughter, Melissa (M.S., Bioengineering) Human neural stem cell adhesion and proliferation on a biomimetic polyuria functionalized with RGD peptides Thesis directed by Assistant Professor Daewon Park ABSTRACT There is curre nt ly no effective cure for spinal cord injury (SCI). In hopes of finding the first restorative treatment, researchers have turned their focus t o stem cell therapies due to their potential to repair or completely replace the injured cells and tissue. Although this type of research has shown a great deal of promise, there are still many complications to overcome before stem cell therapies become institution in SCI treatment. One obstacle that stands in the way is the lack of an implantable cell scaffold that can support efficient neural stem cell ( NSC ) differentiation and proliferation. Due to significant cell death during implantation, the initial number of cells implanted into the injury site far outweighs the surviving cells that are able to integrate into the surrounding tissue and restore functio n. Of course, encouraging more NSC attachment, differentiation, and proliferation through the use of a cell scaffold, would significantly increase the number of surviving cells and thus the effectiveness of these cell therapies. Therefore, the development of a cell scaffold that encourages efficient motor neuron ( MN ) ind uction and survival without alteration of the

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! iv cell's DNA may be sufficient to make stem cell therapies an institution in spinal cord injury treatment. We aimed to overcome this hurdle through the use of an Arg Gly Asp (RGD) functionalized biomimetic polyurea, optimized to encourage efficient differentiation of NSC s without the use of any complex genetic alterations. The RGD sequence, an integrin binding motif found in fibronectin and laminin (m ajor components of the ECM), was found to be implicated in outside inside cell signaling that can affect cell proliferation, migration, and cell survival in most tissues We began by synthesizing a functionalized RGD polymer scaffold (PSHU RGD). Poly (seri nol hexamethylene urea) (PSHU) was employed due to its protein like backbone structure and its potential to attach a large quantity of biomolecules. This is extremely beneficial for because achieving a high concentration of biomolecules for cell biomolecul e interactions plays a crucial role in stem cell survival and differentiation efficiency Both 1H NMR FT IR, and HPLC were used to confir m the overall polymer structure, ensure the presence of free amine groups and quantify the conjugation of RGD. W e began by coating a 24 well plate with varying concentrations o f PSHU RGD ( 0.01 100 ug/ml) (dep rotected PSHU and no coating as the negative controls ) After this, NSCs were seeded on top of the coatings in N2B27 media with ATRA and SAG factors. After 7 days ATR A and SAG were replaced with BDNF and GDNF and left for another 7 days or 14 days.

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! v I mages were taken every two days of the culture period to determine which PSHU RGD polymer coating encoura ged hNSC attachment and cell growth. We were able to show a clear difference in NSC attachment and proliferation between the amounts of polymer used. 10 ug of polymer was found to induce the most neurite spreading and proliferation indicating that this polymer possesses the qualities required of a NSC cell scaffold In summary, we anticipate that the controllable properties of this synthetic polymer that improve hNSCs differentiation and survival in vitro will have great implications on future stem cell therapies for SCI treatment. The form and content of this abstr act are approved. I recommend its publication. Approved: Daewon Park

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! vi ACKNOWLEDGEMENTS I would like to thank all the people that contributed to the development of this work. I am extremely grateful to all my mentors, professors, family and friends that helped me achieve this goal. This experience has helped me not only define my interests and passions but has been amazing in terms of personal development. First of all I would like to thank Dr. Daew on Park who has le d me through this whol e project and from whom I ve learned a tremendous amount. I am looking forward to working with him in the f uture. At the same time, I would like to thank my committee members, Dr. Michael Yea ger and D r Karin Payne for their encouragement and advice. Simil arly, I would like to thank Lind say Hockensmith, Maria Bortot, and the TBRL lab for sharing this experience with me and making it fun. I would also like to thank Dr. Young Lee and Dr. Kurt Freed's lab for their guidance with h NSC culture and testing. Dr. L ee's guidance and advice were an invaluable and essential part of this work. Finally, I dedicate this thesis to my family, to my dad for being so inspiring and encouraging me everyday to have goals and motivate me to strive for greatness. To my mom who has been with me every step of the way and who has supported me with everything I do. To my sister, who I share every moment

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! vii with and who has believed in me immensely. She has always given me the confidence to continue pursuing my dreams. ! ! ! ! ! ! ! ! ! ! ! ! ! ! ! !

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! viii TABLE OF CONTENTS "#$%&'( 1. Introduction ................................ ................................ ................................ .......... 1 1.1 Spinal cord i njury epidemiology and background i nformation ..................... 1 1.2 Basic t reatment of SCI ................................ ................................ .................. 2 1.3 Molecular a pproaches to SCI ................................ ................................ ........ 3 1.4 Bioengineered therapies for spinal cord r egeneration ................................ .. 5 1.4.1 Somatic cell based t herapies for SCI ................................ ..................... 5 1.4.2 Stem cell based t herapies for S CI ................................ ........................... 6 1.5 Challenges in bioengineering cell t herapies with NSCs and MNs ............... 6 1.6 Objectives of this study ................................ ................................ ................. 7 2. Literature Review ................................ ................................ ............................... 1 0 2.1 Review of human neural stem c ell s culture t echniques ............................... 10 2.2 Review of hNSC monolayer differentiation t echniques ............................. 12 2 .3 Review stem cell n iche ................................ ................................ ................ 15 2 .4 Review RGD peptide ................................ ................................ ................... 17 2 .5 Review b iomimetic polymer scaffold ................................ .......................... 2 0 2 .6 Review in v ivo studies with NSCs and MN s ................................ ............... 2 2 3. Individual Objectives and Experimental Approach ................................ ........... 24 4. Materials and Equipment ................................ ................................ .................. 27

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! ix 4 .1 Materials ................................ ................................ ................................ ..... 2 7 4 .2 Equipment ................................ ................................ ................................ .... 2 7 5. Methods ................................ ................................ ................................ ............. 29 5 .1 Biomimetic p ol y (serinol hexamethylene urea) s ynthesis ........................... 29 5 .2 Removal of BOC protective g roups from the polymer backbone ............... 29 5.3 Maximum c onjugation of RGD to PSHU backbone ................................ ... 30 5.4 PSHU RGD coating o ptimization ................................ ............................... 3 1 5.5 Human embryonic stem cell culture c onditions ................................ ........... 32 5.6 Neural stem cell i nduction ................................ ................................ ........... 32 5.7 Motor neuron d ifferentiation ................................ ................................ ....... 32 5.8 Varying RGD conjugation a mounts ................................ ............................ 33 6. Results and Discussion ................................ ................................ ..................... 35 6 .1 Polymer backbone characterization u sing NMR ................................ ........ 35 6 .2 RGD conjugation c onfirmation u sing FT IR ................................ .............. 38 6.3 RGD conjugation q uantification u sing HPLC ................................ ............ 39 6.4 Human neural stem cell phase contrast i mages ................................ .......... 4 1 6.5 Human neural stem cell MTT a ssay ................................ ........................... 49 6.6 Partial G RGD S c onjugation a nalysis u sing FT IR ................................ ..... 50 6.7 Partial GRGDS conjugation q uantification u sing HPLC ............................ 5 2 7. Conclusion ................................ ................................ ................................ ........ 54

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! x 8. Future Work ................................ ................................ ................................ ...... 57 8.1 Analysis of varied RGD conjugation polymer system with NSCs .............. 5 7 8.2 Development of a 3D injectable scaffold ................................ ..................... 57 8. 3 Spinal cord injury animal model ................................ ................................ .. 57 )'*'('+,'! ................................ ................................ ................................ ................................ ... /0 ! 1%%'+234 A. RGD conjugation analysis u sing HPLC ................................ ........................... 65 B. FT IR confirmation of d eprotected PSHU ................................ ........................ 69 C. Plans and schematic for future w ork ................................ ................................ 70 ! ! ! ! !

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! xi LIST OF FIGURES Figure 1 Schematic of primary (acute) and secondary (chronic) phases of SCI ................................ ................................ ................................ ............. 2 2 Comparison between neurosphere and monolayer culture techniques ................................ ................................ ................................ ...... 11 3 Schematic of motor neuron differentiation and motor neuron axon elongation ................................ ................................ .............................. 14 4 Integrin binding, clustering, and signaling cascade ................................ ....... 18 5 Schematic of PSHU RGD synthesis with the final structure of PSH U RGD ................................ ................................ ................................ ... 35 6 1 H NMR (500 MHz, CDCl 3 ) spectrum of PSHU to confirm overall structure of polymer chain ................................ ................................ 37 7 1 H NMR (500 MHz, CDCl 3 ) spectrum of PSHU and deprotected PSHU to confirm removal of the BOC protecting groups during deprotection process. ................................ .............................. 37 8 FT IR of dPS HU, PSHU, and PSHU RGD ................................ ................... 38 9 HPLC Curve of 1.3 initial molar ratio of RGD/free amine groups ................................ ................................ ................................ ............. 40 10 24 well plate PSHU RGD (93% conjugation) polymer coating schematic ................................ ................................ ................................ ........ 41 11 Phase contrast images taken after 2 days of culture ................................ ...... 43 12 Phase contrast images taken after 4 days of culture ................................ ...... 45 13 Phase contrast images taken after 8 days of culture ................................ ...... 47

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! xii 14 Comparison of MTT assay results between the PSHU RGD surface coatings and the deprotected PSHU surface coating ........................ 49 15 FT IR of dPSHU, PSHU, 40% PSHU RGD, 60% PSHU RGD, 80% PSHU RGD, 100% PSHU RGD, and 130% PSHU RGD .................... 50 16 Graph of actual amine RGD conjugation ratio compared to the initial molar ratio of amine RGD ................................ ................................ ... 51 A.1 High performance liquid chromatography RGD binding calibration curve ................................ ................................ ............................. 57 A.2 RGD surface density calculated per well on a 24 w ell plate ......................... 57 A.3 RGD surface density on a 24 well plate calculated for varied PSHU RGD conjugation amounts ................................ ................................ 58 A.4 HPLC Diagrams to determine the actual conjugation amount of RGD to the PSHU Backbone ................................ ................................ ......... 58 B.1 Confirmation of free amine groups on dPSHU after deprotecti on ................................ ................................ ................................ .... 60 C.1 24 well plate PSHU RGD partial conjugation polymer coating schematic ................................ ................................ ................................ ........ 61

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! xiii ABBREVIATIONS Abbreviations Meaning SCI Spinal Cord Injury MP Methylprednisolone CNS Central Nervous System PNS Peripheral Nervous System ESC Embryonic Stem Cell ECM Extracellular Matrix NTF Neurotrophic Factors NSC Neural Stem Cell iPSC Induced Pluripotent Stem Cells NSPC Neural Stem/Progenitor Cells EB Embryoid Bodies MN Motor Neuron hNSC Human Neural Stem Cell hMN Human Motor Neuron BDNF Brain Derived Neurotrophic Factor GDNF Glial derived Neurotrophic Factor RGD Arginylglycylaspartic acid GRGDS Gly Arg Gly Asp Ser EGF Epidermal Growth F actor FGF Fibroblast Growth F actor RA Retinoic Acid SHH Sonic H edgehog SAG Purmorphamine GAGs Glycoasaminoglycans PEG Poly (ethylene glycol) PLA Poly (lactic acid) PET Poly (ethylene terephthalate) PLLA Poly(L lactic acid) PLGA P oly(lactic co glycolic acid) PSHU Poly (serinol hexamethylene urea) NMR Nuclear Magnetic Resonance HDI Hexamethylene D iisocyanate DMF N,N dimethylformamide EDC N (3 Dimethylamino propyl) N ethylcarbodiimide hydrochloride TFE 2,2,2 T rifluoroethanol TFA Trifluoroacetic A cid DCM Anhydrous D ichloromethane

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! xiv FT IR Fourier Transform Infrared DMSO Dimethyl Sulfoxide MTT 3 (4,5 Dimethylthiazol 2 YL) 2,5 Diphenyltetrazolium Bromide NHS N Hydroxysuccinimide HPLC High performance liquid chromatography

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! 1 1. Introduction 1.1 Spinal cord i njury e pidemiology and general i nformation Over one million people in North America suffer from paralysis caused by spinal cord injury (SCI) [1] Although the severity of these injuries may vary, the recovery is often extremely difficult with ful l recovery being extremely rare Most spinal cord injuries involve displacement of the bone or spinal d isk into the spinal cord proper causing either contusion or compression of the cord [2] This primary or immediate d amage to the spinal cord results in severance of axon connections, loss of neurons and glia, and demyelination [3] After this initial mechanical insult, the spinal cord is subjected to a secondary process involving cellular and mo lecular events that pr ogress in a cascading fashion. The secondary injury phase can be grouped in several pathological events: (a) immune response, (b) apoptosis, (c) free radical damage, (d) exci toxicity, and (e) axonal damage [4] These cellular events present themselves clinically a s vascular dysfunction, edema, i schemia, and inflammation The result of this seco ndary damage is additional neuronal and glial cell death, formation of glial scar tissue surrounding the initial injury site, and further demyelination All of these occurrences present a physical and chemical barrier to the regeneration of dam aged nerve c onnection s and thus a large cha llenge for treatment of this injury (Figure 1) [5] [6]

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! 2 Figure 1 Schematic of primary (acute) and secondary (chroni c) phases of spinal cord injury along with some treatment options [5] 1.2 Basic t reatment of SCI The first line of treatment is to prevent further spinal cord damage by immobilizing the patient Clinicians will initially focus on restoring normal alignment, performing operative decompre ssion and preventing shock All of these measure s focus on minimizing the extent of the primary damage. How ever additional steps are taken to reduce the damage caused by secondary injury. Considering the further damage caused by subsequent inflammation and scar formation, many treatments are focused on restricting and preventing progression of the secondary in jury stage [5] One medication for this is Methylprednisolone (MP) a neuroprotective steroid agent

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! 3 [2] [7] M P works by counteracting part of the secondary injury process. Clinically MP has b een shown to improve post traumatic spinal cord blood flow, decrease inflammation, and reduce lipid peroxidation [8] However, MP only provides limited improvement and must be administered within 8 hr s of the initial insult In addition, there is opposition towards the use of corticosteroids after SCI due to the risks of giving this medication to a trauma patie nt. It has been shown that corticosteroids used in this setting can lead to increased risk of infection and gastrointestinal complications that can be fatal [7] [9] The use of this drug has decreased significantly in the recent years due to its limited efficacy and considerable side effects [1] 1.3 Molecular a pproaches to SCI To this end there ha s been extensive research to understand the pathophysiological mechanism of SCI and in turn develop alternative treatment s to prevent secondary damage and promote axon regeneration. Compared to the peripheral nervous system (PNS), th e central nervous system ( CNS ) has very limited neural regenerati v e potential As mentioned above n erve regeneration after SCI is also limited by the inhibitory nature of the injury si t e due to de myelination and scar tissue formation [6] [10] Researchers have examined molecular approaches that combat both the limi ted intrinsic regenerative potential of the CNS and the inhibitor y extrinsic environment that develops after SCI One approach to enhance the neural regeneration capacity of the CNS is to block inhibitors of neural regeneration. Studies have shown that my elin, the material

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! 4 that forms a layer around axons to aid electrical impulses, contains growth inhibiting factors that suppress neural plasticity and impede neural regeneration. Once damaged due to a spinal cord insult, myelin releases the se ne ural growth inhibiting factors, such as Nogo A Studies have shown that the administration of antibodies directed against Nogo A resulted in improved neural regeneration and function in vivo [6] Along with inhibiting the effects o f myelin, treatments have also focused on inhibiting the formation of glial scar tissue and thus preventing a physical barrier to neural regeneration One example is the degradation of chondroitin sulfate proteoglycans, an important component in the extrac ellular matrix (ECM) a nd scar formation Administration of chondroitinase, an enzyme for chondroitin, resulted in a decrease in scar cavity formation and gliosis [11] Neurotrophic factors (NTF) are another are a of interest that not only improve the regenerative capacity of the CNS but also mediate the secondary inflammatory process Administration of Brain derived neu rotrophic factor (BDNF) or NT 3 following a spinal cord injury have both shown to improve neural regeneration followed by improved functional recovery in mice models [11] Although these molecular treatments show a great deal of promise, the focus is primarily on preservin g or reconnecting the undamaged endogenous nerves. However this tact ic is unlikely to provide substantial or full functional recovery. Fo r more dramatic improvement to these patient's recovery researchers have begun to investigate the use of these molecular treatments in conjunction with cell therapies

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! 5 [12] Thus, future treatments of spinal cord injury might include a molecular agen t to encourage a better environment for neural regeneration as well as the implantation of new cells or tissue. 1.4 Cell based therapies for spinal cord r egeneration 1.4.1 Somatic c ell based t herapies for SCI Along with molecular approaches to SCI c ell based therapies have recently become a large part of this research to further improve functional recovery Although these cell based regenerative strategies vary significantly, they all aim to directly replace the cells lost from the injury or to support neural regeneration and protection [6] The first group of cell based therapies are aimed at re myelinating damaged axons and thus promoting neuronal regeneration. Glial cells can be trans planted into the i njury site to remyelinate axons as well as provide neur oprotection. Oligodendrocytes, astrocytes, and motor neurons (MN) have all been used in attempts to increase function after SCI. However, studies have shown that transpla n ting cells alone results in poor survival and thus limited function al recovery [6] [13] 1.4.2 Stem cell based t herapies for SCI Stem cell s have recently become a major facet of cell therapy research in hopes of finding the first efficacious and complete cure for SCI A stem cell, by definition, has the ability to continuously proliferate through asymmetrical division or generate daughter cells with a committed cell type through differentiation [4] Stem cells will i nitially differentiate into progenitor cells, which have restricted

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! 6 proliferation capacity and at this point can only differentiate into certain cell types [4] There are different types of stem cells that have been examined for the treatment of SCI : embryonic stem cells (ESC), neur al stem cells (NSC), and inducible pluripotent stem cells (IPSC) [4] [14] Some of these cell types, such as ESCs and IPSCs have the capa city to differentiate into almost all cell types. Whereas NSCs are multipotent cells that primarily differentiate into neurons, oligodendrocyes and astrocytes making this type of stem cell of particular interest for SCI treatment Despite their differences, all of these types of stem cells possess the ability to re place certain populations of cells lost during the primar y and secondary spinal cord injury phases. 1.5 Challenges in cell t herapies with NSCs and MNs In the field of SCI research, NSCs and MNs have recently gained interest for use in cell implantation and regeneration treatments. Although research surrounding cell transpl antation therapies using MNs and NSCs has shown a great deal of promise, there are still many obstacles to overcome. For cell transplantation using na•ve MNs, one obstacle is the lack of an eff icient and straightforward method to obtain a large population of these cells for transplantation For MN cell replacement therapy to become a viable treatment for SCI there needs to be an efficient source for differentiating the maximum number of homogeneous MNs for implantation. So far, several protocols have reported a percent production in the range of 10% 40% of MNs from the initial embryonic stem cell population; however most of the larger

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! 7 percentages are achieved through complicated and variable genetic alterations that may not be well suite d for cell transplantation [15] For cell transplantation using NSCs, the key obstacle is the lack of an imp lantable scaffold that effectively encourages NSC differentiation, cell survival, and axonal extension. Cell transplantation therapies involving only the injection of NSCs have shown limited improvement due to significant cell death after implantation Of course, encouraging more NSC survival through the use of an implantable cell scaffold would significantl y increase the number of surviving cells and thus the effectiveness of these cell therapies This may be due to the environment of the injury site and that the cells have limited ligands to attach to after implantation. For use in both cell transplantation s or solely to provide a supportive substrate to guide existing axons across the lesion, scaffolds must posses the correct qualities that encourage neural attachment differentiation and proliferation Because f unctional recovery of the spinal cord depends on successful differentiation and integration of the implanted NSCs into the surrounding tissue scaffolds must provide the necessary support and cues to encourage this behavior [16] This task has proved to be extremely challenging due to the uncertainty of these signals and the difficulty of harnessing these signals within a controlled polym er system. 1.6 Objective s of this s tudy Stem cell therapies have become a fervent hope in the field of SCI treatment due to their potential to completely replace diseased or damaged cells and tissue

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! 8 These cell replacement therapies promise the ability restore the axonal connections that have been damaged after SCI to increase function and quality of life significantly more than the current SCI treatments. We hope to contribute to this research effort through the development of a synthetic p olymer than can be manipulated to support human neural stem cell (h NSC ) differentiation and encourage human motor neuron (h MN ) growth in vitro. If successful in vitro this polymer would possess the qualities required of an h MN or h NSC scaffold suited for implantation for SCI treatment. The overall objective of this study is to test the ability of a polymer scaffold functionalized with Arginylglycylaspartic acid ( RGD ) to support hNSC differentiation and hMN growth. Through we use of a s ynthetic polymer we are able to control the interactions these cells are exposed to and thus support hNSC growth and neurite extension 1.7 Organization of this p aper Chapter I Introduction: This chapter includes the necessary background information for the rest of the paper as well as a brief overview of the current SCI treatments. Chapter II Literature Review: This chapter includes the literature review focusing on the current status of cell therapy research as well as how biomimetic polymers hav e been incorporated into this field. Chapter III Experimental Approach : This chapter will provide the experimental thought process and individual benchmarks and goals for this project. Chapter IV Materials and Equipment:

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! 9 Chapter V Results and Discussion : Chapter VI Conclusion:

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! 10 2. Literature Review 2.1 Review of hNSC c ells c ulture t echniques Neural stem cells are self renew ing multipotent cells that can differentiate first into neural stem/progenitor cells (NSPCs) and subsequently into neurons or glial cells. NSCs diffe rentiate during the formation of the developing brain but also account for the restricted regenerative potential in the ad ult CNS. In the body, NSCs are present in niches that control the self renewal or differentia tion of the stem cells [17] These niches were found to exist in the dorsal horn of the spinal cord parenchyma [18] Substantial research has been conducted to optimize protocols aimed at mimicking these stem cell niches and thus promoting self renewal or stable differentiation. One side of this research focuses o n achieving purification and expansion of NSCs in vitro This task has proved to be extremely challenging; many of the signals that control self renewal in vivo are poorly understood thus mimicking these signals in vitro comes with significant uncertainty Initially researchers relied on a neurosphere culture system to maintain NSC s in vitro Neurospheres are 3D cell aggregates composed of NSPCs, a small amount of NSCs, and a small number of cells that have already differentiated Once neurospheres have be en obtained in vitro, growth factors can be added to the culture to cause selective proliferation of the stem cells into a more homogeneous neurosphere [19] This techni que has become extremely common; h owever it does produce a heterogeneous population of cell s with a small fraction exhibiting neurogenic potential Therefore, these culture systems are unable

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! 11 to produce a significant amount of neurons even with the addition of n eurogenic growth factors [20] In addition to neurosphere cultures, monolayer systems have also been investigated ; this culture method is able to produce a much more homogenous culture resulting in higher neurog eni c potential (Figure 2). H owever monolayer Figure 2. Comparison between neurosphere and monolayer culture techniques. Neurosphere cultures have less neurogenic potential than monolayer cultures and are unable to produce a large number of neurons [17]

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! 12 systems have not been shown to have long term potential [17] More recently, it has been established that NSC s sustain lon g term propagation in monolayer cult ure with the use of epidermal growth factor (EGF) and basic fibroblast growth factor ( FGF2 ) [17] [21] Twenty years ago, Reynolds and Weiss developed a process c ombining the neurosphere system along with EGF and FGF2 [1] Although this system produced a heterogeneo us mixture of NSCs and NPSCs, it is still widely used today to obtain undifferentiated NSC colonies [1] 2.2 Review of hNSC m onolayer differentiation t echniques Efficiently d irecting NSCs down a specific pathway of neuronal differentiation in vitro has become a widespread but challenging field of research NSCs will initially differentiate into neuronal or glial progenitor cells (NSPCs) These progenitor cells are multipotent stem cells that have the ability to self renew, but unlike NSC s they have committed to a specific linea ge (neuron, oligodentrocyte, or astrocyte) However, without outside forces, NS Cs primarily differentiate into oligodendrocytes and astrocytes with a very small proportion differentiating into neurons [22] For this reason r esearchers have beg un focusing on developing efficient protocols to differentiate NSCs into NSPCs and finally into MNs [23] Motor neurons are efferent nerves that transmit signals from the spinal cord to the surro unding tissue. These nerve impulses facilitate life dependent muscle contraction s (swallowing, breathing, etc.) and gland stimulation. Motor neurons can be found in the CNS, brain

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! 13 stem, and spinal cord [2 4] Because of the functions and extent of these cells, degeneration and death of motor neurons often leads to severe and sometimes fatal outcomes [25] Researchers have determined that replacing or repairing damaged MNs could represent a po tential treatment or cure for SC I. Significant efforts have been undertaken to obtain a method to encourage re grow th of endogenous MN or to completely replace the damaged or dead cells. Concerning the la t ter, a significant obstacle that stands in the way is the lack of an efficient source of na•ve spinal motor n eurons (sMNs) for research purposes and cell transplantation As mentioned above, one potential so urce of na•ve MN s is the differentiation of NSCs So far, several protocols for differentiating NSCs into MNs have been reported [26] [16] ; however these methods display a limited per cent production of homogenous MNs from the initial cell population. Three different types of protocols have been reported to differentiate human stem cells into neural stem cells. The first approach uses the formation of embryoid bodies (EB) to obtain neural rosettes [27] the second approach bypasses the formation of E B and solely forms neural rosettes [15] [16] and the third approach focuses on the accelerated formation of neural rosettes from E B [28] Researchers can then induce motor neuron differentiation of these NSCs through the use of retinoic acid (RA) and sonic hedgehog (SHH) or SHH agonists These two molecules induce the production of transcriptions factors that subsequently activate pathways leading to

PAGE 28

! 14 motor neuron differentiation [24] RA is known to play an important role in the development and maintenance of the CNS. In addition, RA was found to induce the development of subtype spinal cord (cholinergic) neurons from stem cells in culture The exposure to S HH or SHH agonists was found to enhance the Figure 3 Schematic of motor neuron differentiation and motor neuron axon elongation. Starting with human stem cells (iPSCs or ESCs), cells are differentiation into neural precursors (neural rosettes or embryoid bodies) [24]

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! 15 signals of RA and encourage the enrichment of different subtypes of neurons (dopaminergic, serotonergic, and cholingergic neurons) depending on other signaling facto rs involved [29] RA and SHH are usually used in conjunction with a media containing N2 or B27 N2 supplement is a serum free supplement that is often used in combination with B27 for differentiation of ES cells into neuron lineages [15] [24 ] It has also been shown that purmorphamine (SAG), a synthetic form of SHH, can be substituted for SHH in culture [30] Research has shown that effective concentrations of RA and SHH are close to 1uM and 50 500 ng/ml, respectively [31] After motor neuron differentiation, motor neur ons are induced to extend axons. Without axonal extension, MNs are unable to connect with their surroundings to conduct signal s that will restore functionality Neurite outgrowth may be stimulated in vitro through neurotr o phic growth factors [10] The most commonly used growth factors are brain deriv ed neurotrophic factor ( BDNF ) and glial derived neurotrophic factor (GDNF) [32] It is also important to stop or lower the concentration of RA and SHH at this point; continued use can inhibit maturation of motor neurons [30] 2.3 Review stem cell n iche Not only is it important for NSC to differentiate into the appropriate cell type, but also for the NSCs to efficiently differentiate into a stable cell type. Stem ce lls will not differentiate into a stable cell type without the proper balance of signals either from the cell itself (intrinsic) or environmental factors (extrinsic) I ncorrect signals

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! 16 can cause st em cells to differentiate into a heterogeneous population and severely decrease the number of differentiated cells achieved [33] Thi s will f orce researche r s to perform cell sorting or use a larger number of initial stem cells to achieve the desired results. As far as clinical applications, stem cells that receive incorrect types or amounts of signals have a high risk of becoming cancer ous and present a dangerous result if implanted clinically [14] For this reason, researchers have focused on eliminating this risk by investigating the signals that control stem cell self renewal and differentiation in vivo. Understanding stem cell differentiation in vivo would provide great insi ght on how to manipulate and control stem cell fate in vitro [14] [34] In addition, understanding how stem cells are controlled th rough a stem cell niche provides valuable i nformation for the design of c ell scaffolds With this information, cell scaffolds can be manipulated to imitate the stem cell environment in vivo and interact properly with the cells and surrounding tissue once implanted [35] It has been accepted that in vivo stem cell self renewal and differentiation are controlled through a balance of signals coordinated in spe cial stem cell niches. Stem cells within these niches must have sufficient intrinsic and extrinsic signals to encourage self renewal or instead differentiate into a chosen cell type. These stem cell niches are tissue specific but are all controlled throu gh similar types of signals. Some of the signals stem cells are exposed to are paracrine signals, growth factors, cytokine gradients, glycoasaminoglycans (GAGs), and the physiochemical environment [36] However a ll of these signals can be grouped into the categories:

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! 17 cell cell interactions, cell ECM interactions, and cell biomolecule interactions. Understanding how neural stem cells and their niches interact has become a great focus in research in order to understand what gives these cells neurogenic capacity. This information could provide potential new treatments for tissue regeneration following a spinal cord injury. Although we may know the general makeup of neural stem cell niches few specific details are known about the in situ neural stem cell niches that exist within the adult spinal cord. Researchers have proposed two models to depict the localization and function of these stem cell niches and hopefully provide more information in order to isolate these cells or mimic the function of these niches for SCI treatment [18] The first model describes slowly proliferating neural stem cells that exist in the ependymal later of the spinal cord [37] Relating to the first model, researchers have su ccessfully isolated and cultured cells from this ependymal layer indicating the presence of stem cells. In addition, researchers described the properties of an identified stem ce ll niche that resides in the central canal ependyma l [38] In this work, Hamilton et al identified some of the similarities and differences between this spinal cord stem cell niche and the well documented stem cell niche located in the forebrain subventricular zone (SVZ) [38] They were able to determine that unlike in the SVZ, stem cells within the ependymal layer mainly self renew and show little evidence of lineage relationship further showing the limited regenerative capacity of the CNS [38]

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! 18 Not only is th ere little information ab out the localization, function, and signals of neural stem cell niches within the adult spinal cord, but after SCI the ECM in the stem cell niche will be altered [39] These changes in the ECM components are thought to induce signals for NSC activation [39] Following mechanical insult to the spinal cord, the limited proliferation capacity of the adult NSCs i s increased. In these cases, NSCs can be induced to divide, migrate, and mature into spinal cord progenitor cells [40] 2.4 R eview RGD peptide P olymers can be synthesized to provide inherent biocompatible a nd mechanical support for cells similar to the role of a stem cell niche However p olymers alone do not have bioactive molecules that can bind to cells and encourage cell growth and spreading [35] To improve the interaction between polymer s and cells researchers have begun conjugating cell binding proteins to the polymer structure. In 19 84, Pierschbacher and Ruoslahti used reduction techniques to isolate the cell binding motif on fibronect i n (common integrin binding protein in the ECM) [41] They discovered that the majority of the cell binding activity was ach ieved through the peptide, RGD. Further research showed that this cell binding binding motif was also present on many other ECM proteins including laminin, collagen, and vitronectin [42] Because of its extensive presence and its highly effective cell binding capacity RGD has become the most commonly employed peptide used to enhance cell attachment to a polymer's surface.

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! 19 RGD is the principle integrin binding domain, capable of binding t o a plethora of cell adhesion receptors found throughout the body. However, the most numerous and multipurpose group of cell adhesion receptors is the integrin family This group of adhesion molecules is n ot only responsible for cell attachment, but they a lso play an important role in cell differentiation, embryogenesis, proliferation, and gene expression. Binding of the RGD peptide to integrins causes a cascade of signals that can influence cell behavior in different ways (Figure 2) [42] The first stage is ligand binding of the integrin and partial attachment of the cell bo dy integrin binding ECM At this point, attachment is weak and can only withstand gentle agitations. In the Figure 4 Integrin binding, clustering, and signalin g cascade. This schematic provides some examples to the signaling pathways that can be triggered through integrin binding.

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! 20 second stage, cell attachment becomes stronger and the integrins begin to clus ter together. Once the integrins are clustered, focal adhesion associated proteins (focal adhesion kinase, tensin, syndecans etc.) are recruited to the binding site [43] Thirdly, stress fibers composed of microfilament bundles begin to form. Finally the cell becomes physically anchored through focal adhesions an d also experienc es signal transduction through the focal adhesion associated proteins [44] As detailed before, focal adhesion formation is conducive to cell survival signals and proliferation. Research has shown that focal adhesion associated proteins trigger the expression of anti apoptotic protein Bcl 2 and the f ocal adhesion kinase pathway [45] These two pathways along with focal adhesion formation play an important role in cell survival, proliferation, and cell spreading. It was also determined that disrupting the interaction between integrins and cell binding motifs can induce apop tosis and cell death [46] This integrin mediated death' is caused by the lack of a n integrin substrate to bind the li gand leading to the recruitment of caspase and the cell death cascade [46] Rel ating this finding to a cellular interaction with the ECM, we can see that the absence of integrin binding ligands in a micro environment can cause the cell to undergo apoptosis [46] To this end, cells grown or differentiated in a 2D culture are typically provided with some sort of ligand to simulate these essential cell attachment and survival signals. NS Cs, specifically, are usually cultured on surfaces coated with

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! 21 ECM proteins such as laminin a major protein in the basal lamina, which possess es this RGD binding site [47] Along th ese lines, studies have shown that cell behavior and growth can vary drastical ly by altering the amount of RGD ligands present [48] More specifically, research has shown that RGD may have the ability to regulate neural stem cell survival and differentiation in a dose dependent manner [49] Research surrounding stem cell therapies and potential implantable scaffold s can benefit drastically from this finding. Knowing that there may be an optimum RGD concentration to encourage NSC differentiation and survival w ould allow researchers the ability to fine tune cell scaffolds to encourage this behavior. 2.5 Review b iomimetic polymer scaffold Scaffold design and fabrication has become a major field in tissue engineerin g. Research has shown that cell survivability when merely injected into tissue is low and must be increased to have significant therapeutic effects. Scaffold s implanted along with the cells can provide the necessary support and signals by act ing as a substrate on which cell populations can attach and grow [13] A subcategory of polymers that have b een of particular interest in tissue engineering is biomimetic polymers. These polymers combine the controllability of a synthetic polymer with the biocompatibility of natural mater ial found in the body. Biomimetic polymers can be manipulated to prompt a specific cellular response or direct new tissue formation Extensive work has been conducted to create biomimetic polymers suitable for cell transplantation therapies. One of the most prominent

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! 22 methods to create a biomimetic material is through surface mod ification of common ECM proteins or short peptide b inding motifs, such as RGD [50] As mentioned in the previous section, RGD is the most commonly used peptide for surface modification s and has been incorporated into polymers for research in NSC proliferation and differentiation in vitro [51] Different types of polymers have been conjugated with RGD and examined for the desired cellular response in vitro Poly (ethylene glycol) (PEG) [52] poly (lactic acid) (PLA) [53] poly (ethylene terephthalate) (PET) [35] and others have been studied for use in conjun ction with RGD in NSC cultures In one in vitro study, it was shown that NSC s grown on a two dimensional RGD functionalized lipid bilayer produced single adhered cells comparable to monolayer gr owth seen on laminin surfaces. NSCs in monolayer growth vs. ne urospheres have enhanced proliferation and are able to generate a higher percentage of neurons from the initial population [54] This observation shows that RGD functionalized materials may be extremely useful in synthetic culture systems for neural stem cells [55] Very r ecently, work has been done to create three dimensional scaffolds for NSC growth in vitro and thus bridge the gap between these two dimensional studies and in vivo experiments. One research group used a naturally derived 3D polymer scaffold (collagen and hyaluronan) to culture neural stem and progenitor cells successfully. However these naturally derived polymers are difficult to control and may present residual unknown components [56] Another research group was able to

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! 23 show NSC differentiation and neurite out growth on a 3D scaffold made from poly(L lactic acid) PLLA electrospun fibers [57] This cell scaffold interaction relied mostly on the porosity and modulus of the material. However, because this type of scaffold presents very limited cellular recognition motifs, th e cell scaffold interaction and subsequently NSC attachment and differentiation were restricted [57] Despite the promising results of incorporating RGD in two dimensional culture conditions to increase the interactions bet ween the cells and the material few efforts have been taken to incorporate RGD in three dimensional scaffolds for NSCs. However, one research group created a 3D self assembling peptide scaffold functionalized with RGD that was able to support NSC prolifer ation and differentiation [58] This study displays the viability of an RGD functionalized three dimensional scaffold used to culture NSC s and provides proof that this type of polymer system can be applied to in vivo SCI regeneration studies For this study we wished to test the ability of biomimetic poly (serinol hexamethylene urea) (PSHU) co njugated with RGD to promote hNSC survival and spreading and thus be a promising cell scaffold for stem cell SCI treatment. PSHU was employed due to its protein like backbone structure, which gives this polymer an inherent biomimetic quality. This characte ristic is extremely beneficial to increase the biocompatibility and interaction with the cells. In addition, PSHU has the potential to attach a large quantity of biomolecules and thus increases the adjustability of this polymer system.

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! 24 2.6 Review in v ivo s tudies with NSCs and MN s Recently, SCI research has moved to in vivo studies to determine if these cell therapies are viable for SCI treatment. In vivo studies have been conducted using the implantation of NSCs or NSPCs in an attempt to regenerate the da maged nerves. However, there are very few reports that describe satisfactory differentiation into neurons and integration of these neurons into the surrounding tissue [4] As stated previously, a critical challenge seen in these studies is the propensity of NSCs to differentiate primarily into glial cells as well as moderate cell survival after transplantation. Typically less than 10% of transplanted cells survive [3] To th is end researchers have turned to 3D scaffolds to use in conjugation with NSCs for in vivo studies. One group designed a blended poly(lactic co glycolic acid) (PLGA) scaffold seeded with NSCs to implant into a SCI rat model. They were able to show possible neural regeneration and long term functional recovery suggesting increased NSC survival and tissue integration [3] In another study, researchers compared the survival of ESC derived NSCs when transplanted alone and when encapsulated in a fibrin scaffold containing growth factors. They were able to show higher cell survival w hen implanted with the fibrin scaffold as well as an increase in the number of neurons present after 8 weeks of transplantation [1] More recently, pre differentiated NSPCs were seeded in a fibrin scaffold and implanted in a fully transected rat model. Increased recovery an d better cell survival was seen with the use of this scaffold compared to cell injection alone [59] Together, these studies represent the benefits of

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! 25 a cell scaffold for improving NSC survival, integration, and most importantly function al recovery after SCI

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! 26 3. Individual objectives and experimental approach (1) Synthesize and characterize a highly functionalizable biomimetic polymer : Significant work has been done to create functionaliz a ble polymers for use in bioengineering. However, most of these polymer systems have limited conjugation capacity and thus may not contain sufficient molecules to induce certain cellular behavior. We believe we can achieve a much higher concentration of bio molecules through the synthesis of a highly functionalizable polymer, capable of conjugating larger amounts of molecules. Characterization of this polymer is one of the most important aspects of this work. We first need to ensure that this polymer possess e s the expected peptide like backbone structure and number of functionable groups. (2) Synthesize a biomime tic polymer by conjugating RGD and d etermine the extent of cell attachment to the polymer scaffold : Not only will this polymer have biomimetic properties from its peptide like backbone but also through the conjugation of RGD. As mentioned earlier, one of the most common methods to create a biomimetic polymer is through the conjugation of biomolecules. We decided to use RGD (GRGDS) because it is known to induce attachment and spreading of NSCs. We believe that this improved attachment and spreading will in turn improve t he differentiation efficiency and axonal extension of the NSCs. First, we wanted to cha racterize the polymer further through the quantification of the amount of RGD successfully conjugated to the polymer backbone. Previous works have shown that the amount of RGD may affect cell

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! 27 behavior in a dose dependent manner. Therefore, i t is extremely important to determine the amount of RGD conjugated to the polymer backbone as it will have great effects on the cellular behavior and determine the direction of this project. (3) Evaluate the survival and health of human neural stem cells when seeded on R GD functionalized biomimetic polymer : The first step to test the viability of this biomimetic polymer as a NSC scaffold is to examine its ability to encourage hNSC survival and proliferation. As stated above, cells implanted alone frequently have a low survival rate. One of the roles of this polymer system is to encourage cell survival and prevent death of the implanted cells. (4) Evaluate the differentiation of human neural stem cells into ma ture motor neurons when seeded in RGD polymer scaffold : Once we have shown that this polymer scaffold encourages cell growth and survival, we can test the ability of this polymer scaffol d to support NSC differentiation For stem cell therapies with NSCs, the implantable polymer scaffold should support and encourage differentiation and attachment of NSCs For cell therapies with MN s this scaffold could aid in the differentiation of MN from NSCs in vitro and thus could inc rease the number of MN s produced for implantation (5) Synthesize and quantify varying conjugation amounts of RGD to the polymer backbone : Once we have collected data and observations from the polymer scaffold with close to 100% RGD conjugation, we can begin to determine the optimum amount of RGD

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! 28 to conjugate. As stated earlier, the amount of RGD may have a dose dependent affect on cell survi val and NSC differentiation. Thus there may be an optimum amount of RGD to induce the maximum amount of NSC survival and differentiation. We hope to synthesize polymer systems each with different RGD conjugation amounts to determine the optimum RGD surface density without varying the amount of polymer present. (6) Determine the optimum RGD conjugation ratio in the scaffold to promote maximum differentiation and maturation: Finally, we will test each of these polymer systems and continue work with the optim um amount of conjugated RGD. We will conduct this test again by determining t he polymer system that promotes the most NSC survival and differentiation.

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! 29 4. Materials and Equipment 4 .1 Materials N BOC Serinol, urea, hexamethylene diisocyanate (HDI), anhydrous chloroform, and anhydrous N,N dimethylformamide (DMF) were purchased from Sigma Aldrich (St. Louis, MO, USA). N (3 Dimethylamino propyl) N ethylcarbodiimide hydrochloride (EDC), N hydroxysuccinimide (NHS), 2,2,2 trifluoroet hanol (TFE), and trifluoroacetic acid (TFA) were purchased from Alfa Aesar (Ward Hill, MA, USA). Anhydrous diethyl ether was purchased from Fisher Scientific (Pittsburgh, PA, USA). Anhydrous dichloromethane (DCM) was purchased from JT Baker (Phillipsburg, NJ, USA). The pentapeptide Gly Arg Gly Asp Ser (GRGDS) was purchased from Biomatik (Wilmington, DE). 4 .2 Equipment I nfrared spectra (2 wt.% sample in polyethylene IR sample card) were recorded on a Nicolet 6700 FT IR spectrometer from ThermoElectron Corp oration. 1H NMR spectra were recorded on a Varian 500 NMR (500 MHz, Varian) with CDCl3 as the solvent at 25¡C. Chemical shifts are in ppm using the solvent peak as the internal reference. RGD conjugation was determined using Agilent Zorbax 300SB C8 HPLC sy stem. The analytical column size is 2.1x150 mm with a flow rate of 0.3 ml/min. The buffers used were 1) 0.2% TFA/water and 2) 0.18% TFA/AcN (acetonitrile). A 6 min isocratic run was used first and then 6 min at a linear gradient 1% B/min. The detection was taken at 210 nm. All in vitro cell morphologies were examined on a

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! 30 Nikon DIAPHOT 300 equipped with CCD camera (SPOT RT 2.3.0, Diagnostic Instruments) using SPOT Advanced software for post hoc analysis. ! ! ! ! ! ! ! ! ! ! ! ! ! ! ! ! ! !

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! 31 5. Methods 5 .1 Biomimetic p ol y (serinol hexamethylene urea) s ynthesis We began by synthesizing N Boc Serinol using Serinol ( 1.959 g, 21.50 mmol) and Di Tert Butyl Dicarbonate (5.973 ml, 26.0 mmol). N Boc Serinol will be incorporated into the polymer backbone and protect the free amine groups on the polymer backbone from reacting inappropriately. Next, Poly (serinol hexamethylene urea) (PSHU) was synthesized by combining HDI (1.928 ml, 12 mmol), N BOC Serinol (1.147 g, 6 mmol), and urea (0.360 g, 6 mmol) in 6 ml of anhydr ous DMF. This reaction was maintained at 90 deg C for 7 days. After the 7 day reaction period, the solution was cooled to room temperature and roto evaporated at 70 deg C to remove the DMF. Next, the product mixture was re dissolved in a small amount of D MF (2 3 ml) and precipitated into cooled anhydrous diethyl ether (100 ml). The purification process was repeated 2 times and finally the solution was washed overnight in excess ether (100 ml) to remove any unreacted products and remaining solvent. Finally, the product was roto ev aporated at 45 deg C until dry and stored at room temperature until conjugation with RGD. 5 .2 Removal of BOC protective g roups from the polymer backbone To begin, the protective BOC groups from the polymer backbone were removed. Th is will provide free amines to conjugate the RGD COOH molecules. Hydrogenation by a strong acid was used to deprotect the amine group and obtain PSHU NH2. PSHU was first dissolved (1g, 1.96 mmol) in methylene chloride (15

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! 32 ml). This solution was then mixed and trifluoroacetic acid (15 ml) was added dropwise to the stirring solution. This deprotection reaction took place at room temperature for 45 minutes. The TFA/PSHU/Methylene Chloride mixture was roto evaporated at 45 degrees C until the TFA is remo ved (5 10 min) and the product solution was dissolved in anhydrous DMF (1 ml). Once fully dissolved in DMF, this solution was purified by precipitation in excess cooled ether (100 ml). The product was purified further by dissolving it in 2,2,2 trifluoroe thanol ( TFE) and precipitating this mixture in excess ether. Finally, the product was roto evaporated at 45 degrees C, dried, and stored at room temperature. 5 .3 Maximum c onjugation of RGD to PSHU backbone After deprotection of PSHU, GRGDS COOH may be conjugated to the free amine groups on the backbone of PSHU. Each molecule of deprotected PSHU possesses 18 free amine groups that may be functionalized with GRGDS COOH. Note that GRGDS was used instead of RGD in order to preserve the integrity of the entire RGD binding motif To start, 100 mg of deprotected PSHU was measured (0.01 mmol PSHU, .1956 mmol amine groups). From the molar amount of the free amine groups, the moles of GRGDS COOH were calculated based o n the desired ratio of conjugation. For this study we used a molar ratio of 1.3 (moles of GRGDS COOH/amine groups). This was intended to conjugate close to 100% of the free amine groups on the PSHU backbone. Once the appropriate amounts of deprotected PSHU and GRGDS COOH were obtained, GRGDS COOH was dissolved in DMF (1

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! 33 ml) and combined with N ethylcarbodiimide hydrochloride (EDC) (3X moles GRGDS COOH) and N hydroxysuccinimide (NHS) (3X moles GRGDS COOH). This NHS ester activation reaction was allowed to c arry on for 2 hours and then combined to 100 mg of deprotected PSHU dissolved in DMF (1 ml). Th e conjugation reaction rook place overnight at room temperature and was finally precipitated in excess ether and rotary evaporated to remove the unreacted by pro ducts and solvent. The final product was stored away from light at 4 deg C until use. 5 .4 PSHU GRGDS coating o ptimization The optimal amount of PSHU GRGDS coating was determined by examining the affects of each amount on the proliferation and differentiation of the hNSCs. First PSHU GRGDS was dissolved in TFE with varying amounts: 0.01ug, 0.1 ug, 1 ug, 10 ug, 100 ug In a 24 well plate, three wells for each PSHU GRGDS amount were filled with 0.1 ml of TFE and the corre sponding amount of polymer. Knowing the ac tual conjugation ratio of GRGDS and the amount of PSHU RGD in each well, we were able to calculate the amount of PSHU and RGD in each well ( Figure A.2 ). After the 24 well plate was filled with the PSHU GRGDS in sol ution, the plate was placed on a mechanical agitator. While on the agitator, the solvent TFE will evaporate from the well leaving the polymer coated to the bottom of the well. The constant agitation provided while the solvent evaporates provided a more eve n coating of the polymer on the bottom of the well. This method produces less variability of the polymer amount deposited on different sections of the well.

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! 34 5 .5 Human embryonic stem cell culture c onditions Human embryonic stem cells (ESCs), UCSF 4 were cu ltured in mTesR media (STEMCELL) on Cellstart (Invitrogen) coating plate. For passaging, UCSF 4 cell colonies were cut into small squares using StemPro EZPassage Disposable Stem Cell Passaging Tool (Gibco) at a dilution of 1:10. 5 .6 Neural stem cell i nduction hESCs (UCSF 4) were treated with 10 ng/ml hLIF(Milipore), 3 MCHIR99021 (Cellagentech), and 2 M SB431542 (Cellagentech) in neural induction media, N2B27, containing DMEM/F12: Neurobasal (1:1), 0.5xN2, 1xB27, 1% Glutmax, for 10 d. The culture was then split 1:3 for the next passages using Accutase and expanded in neural induction media supplemented with 10ng/ml hLIF, 3 M CHIR99021, and 2 M SB431542 on Cellstart coating plate. 5 .7 Motor neuron d ifferentiation Mo tor neurons were differentiated from neural stem cells in N2B27 media with treatment of 1 M Retinoic acid (Sigma) and 100 nM SAG (Smoothened agonist, SHH Activator, EMD Chemicals) for 7 days on poly D lysine(Sigma) and laminin coating surface or different concentration of PSHU or PSHU RGD coating plate, and then the cells were terminally differentiated in the presence of 10 ng/ml BDNF and 10 ng/ml GDNF(R&D systems) in N2B27 media for another 14 days. 5 .8 Varying RGD conjugation a mounts

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! 35 Next, we wished to synthesize polymer and vary the conjugation ratio of RGD to the polymer backbone. To do this, we began b y removing all of the protective BOC groups from the polymer backbone. This will expose the free amine groups and allow conjugation of GRGDS COOH We chose to use molar ratios of G RGDS COOH/free amine groups of 0.4, 0.6, 0.8 and 1.0. Based on the previ ous results using a molar ratio of 1.3, we anticipated that these molar ratios would produce a similar actual conjugation ratio to the initial molar ratio. To begin, the appropriate am ounts of GRGDS COOH, EDC (1.2x molar amount of GRGDS COOH), and NHS (1.5x molar amount of GRGDS COOH) were weighed and all owed to mix in 1 ml of DMF for two hours. This initial reaction period is meant to create a more stable, amine reactive intermediate. After two hours, 100 mg of deprotected PSHU dissolved in 1 ml of DMF was added to each of the four samples. The deprotected PSHU and activated GRGDS molecules were allowed to react for 24 hours at room temperature. Next, the solution was precipitated in co ld ether twice to remove most of the DMF. The remainder of the DMF was removed during rotoevaporation and finally through dialysis of the solution. After dialysis, the RGD conjugated polyme r was lyophilized and stored wrapped in foil at 4 degrees Celsius t o prevent degradation and decrease in RGD bioactivity. 5.9 Statistical analysis

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! 36 All results are expressed as means standard deviation. All quantitative results were analysed using analysis of variance (ANOVA) and, if necessary, follow up analysis by Tuk ey's test. Statistical significance was considered at p < 0.05.

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! 37 O C N N C O Hexamethylene Diisocyanate H 2 N NH 2 O Urea HO NH O O HO PSHU Synthesis, 7 days, 90 ¡ C H N O O O NH O O H N O N H N H O N H O N H O Poly (serinol hexamethylene urea), Protected PSHU PSHU Deprotection, 45 min, RT H N O O O NH 2 H N O N H N H O N H O N H O H N O O O NH H N O N H N H O N H O N H O GRGDS PSHU GRGDS-COOH Conjugation, ON, RT OH O O Poly (serinol hexamethylene urea), Deprotected PSHU Poly (serinol hexamethylene urea) (PSHU)-GRGDS n n GRGDS n N-BOC Serinol n 6. Results and Discussion 6 1 Polymer b ackbone c haracterization u sing NMR Figure 5 Schematic of PSHU RGD synthesis with the final structure of PSHU RGD. The first stage is PSHU synthesis followed by the removal of the N Boc groups from the PSHU backbone and finally RGD conjugation. Note that the value of n was determined to be ~18 with RGD attached to a certain percentage of monomers depending on desired RGD conjugation ratio.

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! 38 In this study, we aimed to test this biomimetic polymer's ability to efficiently promote NSC survival, proliferation, and differentiation into mature motor neurons. We began by confirming and characterizing the synthesis process of Poly (serinol hexamethylene urea). NMR was used to confirm the overall polymer structure as well as the removal of the N BOC groups from the primary amines The synthesis progression of Pol y (serinol hexamethylene urea) (PSHU) can been seen in F igure 5 and the NMR analysis of the polymer s tructure can be seen in F igur e 6 The NMR spectrum of PSHU confirmed the expected copolymer structure, with peaks at 1.3 ( CH2 ), 1.5 ( NH CH2 CH2 ), and 3 .2 ( NH CH2 ) associated with HDI, at 1.4 ( C (CH3)3), and 4.1 ( CH NH ) associated with N BOC serinol. NMR analysis was also used to confirm complete removal of the BOC protecting groups from the polymer backbone. This process is extre mely important, as the free amines will later be used to conjugate RGD; residual BOC protecting groups will alter the expected conjugation amount of RGD. The presence of BOC protecting groups will produce a peak on t he NMR spectrum at 1.4 (Figure 7 ). The t op spectrum in Figure 7 shows the disappearance of peak B (methyl groups N BOC) indicating the complete removal of the BOC protecting groups. Once the BOC protecting groups are removed from the polymer backbone, the exposed free amine groups can be conjugated with vario us biomolecules (RGD in this case). !

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! 39 Figure 6 1 H NMR (500 MHz, CDCl 3 ) spectrum of PSHU to confirm overall structure of polymer chain Figure 7 1 H NMR (500 MHz, CDCl 3 ) spectrum of PSHU and deprotected PSHU to confirm removal of the BOC protecting groups during deprotection process.

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! 40 6 .2 RGD c onjugation c onfirmation u sing FT IR After PSHU was successfully characterized, RGD was conjugated to the PSHU backbone intended to further improve differentiation and spreading of the motor neurons. As mentioned above, we initially chose a molar excess of 1.3 moles of RGD to the moles of fre e amine groups. FT IR was use d to confirm that RGD COOH was successfully conjugated to the free amine groups on the Figure 8 FT IR of dPSHU, PSHU, and PSHU RGD. Confirmation of free amine groups on dPSHU after deprotection (B). Shift in carbonyl absorbance to confirm attachment of RGD to the polymer backbone (A). A B

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! 41 PSHU backbone. We can first confirm the presence of free amine groups on DPSHU (Deprotected PSHU) by examining the region around 798 cm 1 This wavenumber correspo nds to N H primary amines and can be seen very clearly in the FT IR figure below (B). Next we can confirm the conjugation of RGD to the polymer backbone by viewing the region of 1630 to 1680 cm 1 This region is associated with the carbonyl groups found w it hin the polymer backbone as well as the carbonyl groups found within pepide bonds of RGD. The wavenumber correlate d to the carbonyl groups of RGD are slightly lower than that of the carbonyl groups in the polymer backbone. In the PSHU RGD spectrum, we ca n observe an obvious shift in this carbonyl peak towards the lower end of the spectrum, indicating the presence of carbonyl groups in the RGD peptide. Thus we can confirm the presence of RGD conjugated to the PSHU polymer backbone. 6 .3 RGD conjugation quantification u sing HPLC HPLC was used to quantify the amount of GRGDS COOH that was successfully conjugated to the free amine groups on the PSHU backbone. A calibration curve was first con s tructed using known concentrations of GRGDS COOH (0.78 200 ug/ml ) (Figure A.1 ). Each of these concentrations produced a corresponding HPLC peak area. For this study, a 1.3 molar ratio of GRGDS COOH/free amine groups was used in order to obtain close to 100% conjugation of the free amine groups. The calibration curve con structed prev iously was then applied to HPLC of this polymer sample to determine the actual conjugation ratio of

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! 42 GRGDS COOH to the polymer backbone (Figure 9 ) We were able to sh ow that using a 1.3 molar ratio of GRGDS C OOH/free amine groups produced 93% conjugation of RGD to the free amine groups on the polymer backbone. The theoretical molar ratio of 1.3 was expected to produce a conjugation value close to 100%. P revious studies have demonstrated that the amount of RGD pres ent can have a significant effe ct on cellular behavior; therefore this analysis provided vital information on optimizing the conditions of this polymer scaffold [49] Figure 9 HPLC Curve of 1.3 initial molar ratio of RGD/free amine groups Using the calibration curve on the right we were able to shown this molar ratio produced close to 100% conjugation of RGD to the free amine groups on the polymer backbone.

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! 43 6 .4 Human neural stem cell phase contrast i mages After quantifying the amount of conjugated RGD to the polymer backbone (93%) PSHU RGD was coated to the bottom of a 24 well plate using t he method described in section 5.4 The plate was coated using varying amounts of polymer and three replicates of each amount These variations of PSHU RGD amounts were chosen to begin to narrow down the o ptimum amount of RGD to encourage maximum hNSC growth. Because the conjugation ratio and mass of PSHU is known, we can calculate the total amount of RGD coated on the well (Figure A.2). Deprotected PSHU and no coating were used as negative controls. The sc hematic of the 24 we ll plate can be seen in figure 10 Figure 10 24 well plate PSHU RGD (93% conjugation) polymer coating schematic

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! 44 After coating the 24 well plate with the appropriate amount of polymer, hNSCs were plated using the culture t echniques described in section 5.7 Every two days, phase contrast images of the cell cultures were taken to compare the behavior of the cells. The lowest amounts of PSHU RGD (0.01ug and 0.1ug) cultures show no cell attachment as can be seen from the round ed cell morphology and bright cell border. Unfortunately the 0.1 ug PSHU RGD polymer coating was displaying some bubbling or buckling of the polymer, which may affect cell attachment and health. In the next two amount of PSHU RGD (1 ug and 10 ug), cell att achment is already observed. More strikingly there appears to be more cell attachment and neurite outgrowth in the 10 ug culture vs. the 1ug culture. On the other hand, the hNSCs cultured on the 100 ug PSHU RGD coating show initial cell attachment, but no cell spreading or neurite outgrowth. Interestingly, there appears to be a level of RGD that becomes toxic and impedes not only hNSC survival but also axon extension. This observation indicates that the cells initially attached to the 100ug PSHU RGD coatin g but were unable to spread and extend axons. This is likely due to oversaturation of RGD on the culture surface causing excessive foc al adhesions that inhibit cell movement [54] With the inability to spread, these cells will be unable to differentiate into functional neurites and will instead proceed to apoptosis [48] As a control for thi s polymer system, we evaluated hNSC cell growth and axon spreading on deprotected PSHU (polymer w/out RGD conjugation) and no surface coating. After 2 days, the hNSCs cultured on the deprotected PSHU showed

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! 45 no attachment but no immediate cell death (i.e. floating cells with ruptured membrane and cell debris) indicated that deprotected PSHU is non toxic to the cells however does not permit cel l at tachment. The cells cultured on no surface coating also showed little attachment but no immediate cell death. We could see that the cells were not attached to the bottom of the plate by moving the plate an d exam in ing the cells. In addition, a brighter ring around the rounded cells indicates floating cells in the culture. However both deprotected PSHU and no coating cultures show r ou nded cell morphology corresponding to poor cell health and the induction of apoptosis [60] 2 Days No Surface Coating Deprotected PSHU Controls 0.01ug PSHU RGD 0.1ug PSHU RGD

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! 46 Polymer 1ug PSHU RGD 10ug PSHU RGD Polymer 100ug PSHU RGD Polymer Figure 1 1 Phase contrast images taken after 2 days of culture Phase contrast images of the cell cultures were taken again after 4 days. At this point, t he cells grown on the two lowest polymer concentrations (0.01 ug and 0.1

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! 47 ug) show neurosphere formation. As discussed before (Section 2.1 ), neurospheres are heterogeneous free floating aggregates of neural stem cells. Although the cells in these two cultures are surviving, the formation of neurospheres signifies a heter ogeneous population of cells with limited neurogenic potential and poor cell attach ment In contrast, the cells grown on the next two polymer concentrations (1 ug and 10 ug) show substantial cell attachment and axon extension. Additionally, after 4 days of culture the difference between the 1 ug and 10 ug polymer coatings is much more apparent. The NSCs culture on 10ug of PSHU RGD show significantly more attachment and proliferation This finding indicates that there may be a "sweet spot" surface density of RGD to induce the maximum amount of NSC attachment and axon extension. 4 Days No Surface Coating Deprotected PSHU Controls 0.01ug PSHU RGD 0.1ug PSHU RGD

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! 48 Polymer 1ug PSHU RGD 10ug PSHU RGD Polymer 100ug PSHU RGD Polymer Figure 1 2 Phase contrast images taken after 4 days of culture After 8 days of culture, there is still NSC proliferation and attachment on the 10 ug PSHU RGD coating. However, we began to see neurosphere formation on the

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! 49 1 ug PSHU RGD polymer coating. We believe this is due to slight detachment of the polymer coating from the surface of the plate. The detachment of the polymer would cause the detachment of the cells or induce weaker attachment of the cells and thus the forma tion of neurospheres. This can be avoided in the future by increasing the chain length and thus d ecreasing the solubility of the polymer in water or by allowing more time for the polymer coating to dry and fully adhere to the plate surface. Regardless, the re was still extensive NSC proliferation and attachment on the 10 ug PSHU RGD coating. This further indicates that the 10 ug PSHU RGD surface density of RGD and polymer may be close to the optimum amount in order to induce maximum attachment and proliferat ion of NSCs. However, in the future we would need to compare the hNSC cultures on our polymer system with a positive control. It is important to see what healthy hNSCs in a 2D culture look like to determine if our polymer can support cell health and growth We propose the use of PDL Laminin, a standard coating for stem cell proliferation and differentiation [47] 8 Days No Surface Coating Deprotected PSHU

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! 50 Controls 0.01ug PSHU RGD 0.1ug PSHU RGD Polymer 1ug PSHU RGD 10ug PSHU RGD Polymer 100ug PSHU RGD

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! 51 Polymer Figure 1 3 Phase contrast images taken after 8 days of culture 6 .5 Human neural stem cell MTT a ssay After 14 days, an MTT colorimetric assay was used to measure the cell viability of each culture. MTT, a yellow tetrazole, is reduced to purple formazan in the presence of NAD(P)H and thus provides a technique to measure cellular metabolic activity. An MTT assay provides one of the most straightforward methods to compare the concentration of live cells between samples We used an MTT assay to compare the cell viability of the cells cultured on the different surfaces (Figure 14 ). This MTT assay confirmed the observations from the phase contrast images shown previously. There is a significant difference between the cell viability on the lowest PSHU RGD amounts (0.01 ug and 0.1 ug) and the two next PSHU RGD amounts (1 ug and 10 ug). More importantly, there is also a significant difference between the cell vi ability of the cells cultured on 1ug and 10ug of polymer. This difference was also seen after just 2 days of cult ure with phase contrast images. This observation further confirms that RGD may have a dose dep endent effect on NSC attachment and

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! 52 viability It is important to note that although an MTT assay may provide information about the relative cell metabolism between the samples, it does not provide any information about the actual cell count. In order to o btain this information we would have to compare the samples metabolism to MTT measurement of known cell numbers. In addition, it is also important to compare the values of this MTT assay to a positive control for NSC health. As mentioned above, in the futu re we would plan to use PDL Laminin to compare to our polymer coatings. Figure 1 4 Comparison of MTT assay results between the PSHU RGD surface coatings and the deprotected PSHU surface coating. There is a significant difference between the cell vi ability of the cultures grown on the 1ug and 10ug amounts of polymer (* p value<0.01). 5

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! 53 6 .6 Partial RGD c onjugation a nalysis u sing FT IR In the previous experiment we varied the amount of RGD pr esent by varying the amount of PSHU RGD. However while doing this we also inadvertently varied the amount of polymer present in each culture. To ensure that the differences in cell survival and attachment are attributed to the surface density of RGD, we mu st keep the amount of polymer present constant in each culture. After synthesis of the different polymer systems with varied RGD c onjugation amounts (section 5.8 ), FT IR was employed to show the differences in RGD present between each polymer. Although FT IR did not provide a quantitative analysis of the amount of RGD present, it provided preliminary data that each polymer system had varied RGD conjugation amounts. As detailed before, increasing the amount of RGD present should show an increase d shift in t he carbonyl peak towards 1630 cm 1

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! 54 Figure 1 5 FT IR of dPSHU, PSHU, 40% PSHU RGD, 60% PSHU RGD, 80% PSHU RGD, 100% PSHU RGD, and 130% PSHU RGD Shift in carbonyl absorbance to confirm s attachment of RGD to the polymer backbone and provides information on the relative differe nce between the polymer systems 6 .7 Partial R GD conjugation q uantification Using HPLC Next, HPLC was used to determine the percentage of free amines that were actually conjugated with RGD. As stated previously, we used initial molar ratios of 0.4, 0.6, 0.8, 1.0, and 1.3 to achieve various amount of RGD conjugation. However, we needed to determine the actual percentage of free am ines that were conjugated with RGD in order determine the optimum amount o f RGD for NSC survival Figure 1 6 Graph of actual amine RGD conjugation ratio compared to the initial molar ratio of amine RGD. This graph was constructed by performing HPLC on each polymer sample and using the previously constructed HPLC calibration curve.

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! 55 HPLC was performed on each polymer sample (Figure A.4). Using the calibration curve constructed previously (Figure A.1), we were able to determine the actual amine RGD conjugation ratio (Figure 14). With this information, the amount of RGD that the cells were exposed to in culture could be quantified and used for future work (Figure A.3). ! !

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! 56 7 Conclusion This study was conducted with the ultimate goal of encouraging attachment and survival of human neural stem cells t hrough the use of a controlled biomimetic polymer. Although there have been a number of experiments elucidating the potential of NSCs in SCI treatment, there are still a few obstacles that may prevent the progression of these therapies. We chos e to combat two of the most significant obstacles 1) limited cell localization and 2) limited cell survival of implanted cells. We began by successfully synthesizing and verifying the structure of a highly functionalizable biomimetic polymer that is capa ble of attaching a large quantity of biomolecules. Achieving a high concentration of biomolecules for cell biomolecule interactions plays a large role in stem cell survival and differentiation efficiency [61] Substantial research has been conducted to determine which biomolecules in the ECM play an important role in regulating NSC function in vivo and thus could be used to mimic the cell biomolecule interactions and guide stem cell fate. The RGD sequence, an integrin binding motif found in laminin (a large component of the ECM), was found to be implicated in outside inside cell signaling that can affect cell proliferation, migration, and cell survival in different types of tissues [62] It has also been determined that integrin binding motifs, specifically RGD, are involved in supporting attachment, spreading, proliferation, and differentiation of NSC in a dose dependent fashion [63] [49] Therefore incorporating enough of this RGD sequence

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! 57 into synthetic polymers to produce a biomimetic synthetic scaffold has the potential to increase NSC differentiation and proliferation [55] Not only do cell biomolecule interactions play a role in dir ecting stem cell fate, but also cell ECM interactions can help to modulate neural stem cell behavior and differentiation [64] To cater to these interactions, we engineered this polymer to also possess multiple peptide mimicking bonds to increase the biocompatibility of the polymer. The next stage was to determine if this polymer coating would encourage hNSC attachment and spreading in vitro. We were able to show that 1 0 ug PSHU RGD coating amount, disp layed increased hNSC attachment and spreading and higher cell viability after 14 days However, it was also observed that the lower amounts of PSHU RGD and the highest amount of PSHU RGD showed neurosphere formation and no cell growth respectively. This in dicated that certain amounts of PSHU RGD are able to encourage NSC attachment and proliferation while insufficient or excessive amount of PSHU RGD prevented NSC attachment and survival To further optimize this biomimetic polymer system, we next varied the conjugation amount of RGD to the polymer backbone. This would allow us to hold the amount of polymer constant and just examine the affects of RGD concentration on hNSC behavior. We beli eve that this experiment will elucidate an optimum amount of RGD conjugated to this biomimetic polymer to induce the maximum amount of hNSC attachment and survival

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! 58 In closing we successfully designed a controllable biomimetic polymer that encourages spr eading and survival of hNSC s In addition, we anticip ate that the properties of this polymer system that improve hNSC spreading and survival in vitro will also be conducive to increased cell survival and integration in vivo. Therefore, not only does this p olymer may be a promising scaffold to support cell survival and integration during cell transplantation.

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! 59 8. Future Work 8 .1 Analysis of varied RGD conjugation polymer s ystem with NSCs The next stage of this project is to further optimize the PSHU RGD polymer system. As mentioned above, we have successfully synthesized partially conjugated PSHU RGD polymer systems. NSCs will then be cultured on these polymer systems using the same culture methods as before (Figure C.1) We anticipate that the largest percent conjugation will produce comparable results to the 100ug PSHU RGD sample shown above. However the smaller percent conjugations should produce NSC attachment and proliferation and uncover the optimum RGD conjugation perce ntage (Figure A.3) 8 .2 Development of a 3D injectable scaffold The next stage of this project is to synthesize an injectable scaffold using the optimized PSHU RGD. Following SCI, further surgery to implant a cell scaffold can cause further nerve damage and loss of function. In these cases, the use of an injectable scaffold can be loaded with cellular and molecular components and injected into the lesion [65] We plan to accomplish this by conjugating a reverse therogel polymer to the PSHU RGD polymer system. This will create a polymer system that encourages the correct cellular behavior while also avoiding the need for surgical implantation. 8 .3 Spinal cord injury animal m odel

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! 60 After synthesis and optimization of this injectable biomimetic NSC scaffold, we plan to examine the results of injecting thi s scaffold in a SCI rat model. Using behavior analysis and tissue histology we can determine if this scaffold s uccessfully encourages regeneration of neurons and functional recovery.

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! 66 Appendix A RGD conjugation analysis using high performance liquid chromatography Figure A.1 : High performance liquid chromatography RGD binding calibration curve. This graph was constructed by measuring the area under the HPLC curve related to known concentrations of RGD. This calibration curve allowed quantification of polymer samples to determ ine the RGD conjugation amount. Polymer Name Amount of Polymer On Surface (ug) Percent RGD Conjugation Total amount of RGD (umol) Density of RGD on Surface (pmole/cm 2 ) No coating N/A N/A N/A N/A dPSHU 100 N/A N/A N/A 0.01 PSHU RGD 0.01 93% 0.0000182 9.58 0.1 PSHU RGD 0.1 93% 0.000182 95.8 1 PSHU RGD 1 93% 0.00182 958.8 10 PSHU RGD 10 93% 0.0182 9578.94 100 PSHU 100 93% 0.182 95789.47

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! 67 RGD Figure A.2 RGD surface density calculated per well on a 24 well plate. Using a surface or growth area of 1.9 cm 2 per well. However, it is important to note that not all of the RGD ligands conjugated to the polymer backbone will be in the correct orientation to bind integrins. Polymer Name Amount of Polymer On Surface (ug) Percent RGD Conjugation Total amount of RGD (umol) Density of RGD on Surface (pmole/cm 2 ) No coating N/A N/A N/A N/A dPSHU 100 N/A N/A N/A 20% PSHU RGD 100 ~10% 0.0196 10315.79 40% PSHU RGD 100 ~30% 0.0587 30894.74 60% PSHU RGD 100 ~50% 0.0978 51473.68 80% PSHU RGD 100 ~70% 0.1369 72052.63 100% PSHU RGD 100 ~90% 0.1760 92631.57 130% PSHU RGD 100 93% 0.182 95789.47 Figure A.3 RGD surface density on a 24 well plate calculated for varied PSHU RGD conjugation amounts. Using a surface or growth area of 1.9 cm 2 per well. However, it is important to note that not all of the RGD ligands conjugated to the polymer backbone will be in the correct orientation to bind integrins. Initial RGD Molar Ratio 1.3 1.0 Area Under Curve ! !"# !" ! !"# !" Conjugation Ratio 93% 89%

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! 68 HPLC Curve Initial RGD Molar Ratio 0.8 0.6 Area Under Curve ! !"# !" ! !"# !" Conjugation Ratio 67.1% 51.1% HPLC Curve Initial RGD Molar Ratio 0.4 Area Under Curve ! !!" !" Conjugation Ratio 30% HPLC Curve

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! 6 9 Figure A.4 HPLC Diagrams to determine the actual conjugation amount of RGD to the PSHU Backbone. Both theoretical conjugation percentage and actual conjugation percentages are provided.

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! 70 Appendix B FT IR confirmation of deprotected PSHU Figure B.1 Confirmation of free amine groups on dPSHU after deprotection (B). This FT IR was performed before conjugating varying amounts of RGD in order to prevent residual N BOC groups from altering the reaction kinetics and outcome.

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! 71 Appendix C Plans and schematics for future work Figure C.1 24 well plate PSHU RGD partial conjugation polymer coating schematic