University of Southern California Dissertations and Theses

Generation and characterization of an in vitro model of inner ear sensory hair cells using transcription factor mediated cellular reprogramming

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Generation and characterization of an in vitro model of inner ear sensory hair cells using transcription factor mediated cellular reprogramming

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Content 2020 Louise Menendez
Generation and Characterization
of an In Vitro Model of Inner Ear Sensory
Hair Cells using Transcription Factor
Mediated Cellular Reprogramming

by

Louise Madeleine Menendez

A Dissertation Presented to the
FACULTY OF THE USC GRADUATE SCHOOL
UNIVERSITY OF SOUTHERN CALIFORNIA
In Partial Fulfillment of the
Requirements for the Degree
DOCTOR OF PHILOSOPHY
NEUROSCIENCE

August 2020

ii
Dedication

This thesis is in first part dedicated to my parents, Anne and Benoit Menendez,
for their unconditional love and support. My passion for science and biology emerged at
a young age when my parents moved us to the rolling hills of Sonoma County. I am
ever thankful for a childhood where we were encouraged to play outside and explore
the curiosities of nature. I have the fondest memories building obstacle courses for back
yard Olympics, hiking through the creek behind our house to climb trees and search for
critters, and the countless camping/road trips we took to explore the world as a family
every chance we could get. My parents and family have always encouraged me to
follow my passion for science, to always aim for success and most importantly to help
others along the way. Thank you for everything you have done to support me through
this journey.
This thesis is also dedicated to my partner, Thiago, for his love, caring and
understanding throughout this process. We met when I was about to take my qualifying
exam to become a PhD candidate and he has helped me immensely over the past 5
years to reach the end of my PhD. I am forever grateful for the celebrations after
successful experiments and presentations, as well as the consoling and comforting
during the tougher times. Thank you for always helping me to carry on and to realize my
full potential as a scientist, a friend, and a family member.

iii
Acknowledgements

This PhD has been a seven-year journey and has involved the advice, effort,
feedback, time and guidance from many people along the way. Most importantly I need
to thank my mentors Dr. Justin Ichida and Dr. Neil Segil for taking me in as a very green
graduate student and helping me to finally reach the culmination of my PhD. Justin and
Neil allowed me to work on a collaborative project that was bridging the field of direct
cellular reprogramming with efforts to study the inaccessible and non-regenerative
sensory hair cells of the inner ear. The timing of this collaboration allowed me to join the
project at its conception, work diligently to develop an entirely new model of induced
sensory hair cells, and to open up the doors to many more research questions about
cell fate establishment in development and future regenerative strategies for hearing
loss.
To Justin, thank you for being my mentor and accepting me into your lab at the
very beginning of all of this. I came to USC with the idea in my head that I would work
on Drosophila models of Alzheimer. When it came time to decide on my third rotation, I
felt lost, but Dr. Dion Dickman suggested your lab and I am forever grateful. Learning to
work with cells and reprogramming my first batch of induced motor neurons was nothing
short of life changing. I was hooked on the excitement and all the potential possibilities
that in vitro cell models could provide for the future of medicine. Seeing and
experiencing your enthusiasm for this research over the years has been an incredible
motivation. Thank you for all of your guidance and encouragement along the way.
Thank you for teaching me to think independently, design proper experiments, and most
iv
importantly to present my research to any audience possible. Your success in the field
has been incredible and I can’t wait to see what is to come.
To Neil, thank you for all of your support and always fueling my research with
important fundamental questions. I will always remember my first ISSCR in 2015 when I
was the only poster studying the cells of the inner ear. I was surprised at the time but
more importantly it excited me that we were embarking on a research project that was
so novel. At the last ISSCR in 2019 there were many more labs, presentations and
posters studying the auditory system and it has been an honor to grow with the field
while working in your lab. Thank you for teaching me to always question the unknown
and pushing me to be as informed as possible about the relevant literature. I am grateful
to have generated this new model as part of your lab and hope it can answer many
more questions about the development of a hair cell, their lack of regenerative capacity
and most importantly a future for prevention and treatment of hearing loss
I would also like to thank all of the current and former lab members of both the
Ichida lab and the Segil lab for their advice and assistance, many of which have been
essential to the completion of my manuscript. I am incredibly thankful for Dr. Suhasni
Gopalakrishanan who helped to get this project off the ground and was always available
to answer my questions. This project would not have been the same without her
guidance, incredible knowledge of cell biology and her perseverance. I must also thank
Talon Trecek for all of his help with the bioinformatics portions of the project. He was an
immense help in establishing the proper controls and comparisons throughout the
collection of our sequencing data and I believe he made our manuscript much stronger
with his expertise of statistics and bioinformatics. Another key lab member in this project
v
was Juan Llamas. He always made himself available to help me with technical
assistance ranging from dissections, cell sorting and mouse breeding. I am extremely
grateful to have worked with such a talented technician. I would also like to thank Dr.
Radha Kalluri and her graduate student Alex Markowitz for their collaboration and
assistance in performing electrophysiology on our cells. Lastly, a very special thanks
goes out to the lab managers, Welly Makmura and Mickey Huang. These diligent lab
managers constantly kept up with our reagent orders, supply and demand of the entire
lab, and maintained overall lab communication. Mickey Huang also runs the Choi
Screening Facility and helped immensely with the drug screening portion of my
dissertation. I must also thank my committee members Dr. Qilong Ying and Dr. Radha
Kalluri for their guidance throughout the years, Bernadette Masinsin for all of her help in
the Flow Cytometry Core Facility and Dr. Seth Ruffins for his assistance in the
Microscopy Core Facility.

vi
Abstract

The specialized mechanoreceptive hair cells in the mammalian inner ear are
selectively vulnerable to numerous genetic and environmental insults. The lack of
regenerative capacity leads to permanent hearing loss and vestibular dysfunction when
they degenerate. The small numbers and difficult accessibility of primary hair cells limits
the search for otoprotectants and regenerative initiatives. Direct cellular reprogramming
could offer a robust route to the generation of sensory hair cells. Here we report a
combination of four transcription factors (Atoh1, Pou4f3, Gfi1 and Six1) can induce cell
fate conversion to a hair cell-like state. Induced hair cells (iHCs) exhibit robust
similarities to primary hair cells at the transcriptome and epigenome level, as well as
rudimentary stereocilia, uptake of styryl dyes, and hypersensitivity to gentamicin. Our
demonstration of direct reprogramming to iHCs provides a platform to study causes of
acquired and genetic hearing loss, high throughput drug screens to identify
otoprotectants, and regenerative initiatives.

vii
Table of Contents
Dedication .............................................................................................................................. ii
Acknowledgements ............................................................................................................... iii
Abstract ................................................................................................................................. vi
List of Figures and Tables ....................................................................................................... ix
Chapter 1: Background ........................................................................................................... 1
1.1 Inner Ear Development .............................................................................................................. 1
1.2 Sensory Hair Cell Fate ................................................................................................................. 3
1.3 Hearing Loss and Lack of Hair Cell Regeneration ......................................................................... 6
1.4 Cell Fate Reprogramming: Directed Differentiation vs Direct Reprogramming .......................... 11
1.5 Current Hair Cell Models .......................................................................................................... 18
1.6 Our Approach ........................................................................................................................... 21
Chapter 2: Generation of Induced Sensory Hair Cells using .................................................... 23
2.1 Introduction ............................................................................................................................. 23
2.2 Identification of Hair Cell Specific Transcription Factors ........................................................... 25
2.3 Transgenic Reporter Activation and Immunostaining ............................................................... 26
2.4 Transcriptional Profiling of Induced Hair Cells .......................................................................... 30
2.5 Induced Hair Cells are Distinct from other Atoh1 Dependent Lineages ..................................... 36
2.6 Chromatin Accessibility Profile of Induced Hair Cells ................................................................ 39
2.7 Reprogramming Post Natal Cell Types ...................................................................................... 42
2.8 Discussion ................................................................................................................................ 46
Chapter 3: Functional Characterization of Induced Hair Cells ................................................ 48
3.1 Induced Hair Cells Express Functionally Relevant Hair Cell Genes ............................................. 48
3.2 Co-culture of Induced Hair Cells with Dissociated Primary Organs of Corti ................................ 49
3.3 Electrophysiological Profile of Induced Hair Cells ...................................................................... 52
3.4 FM Styryl Dye Accumulation in Induced Hair Cells .................................................................... 55
3.5 Discussion ................................................................................................................................ 56
Chapter 4: Using Induced Hair Cells to Study Ototoxicity ....................................................... 58
4.1 Induced Hair Cells Selectively Accumulate Gentamicin ............................................................. 58
4.2 Survival Assay of Ototoxin Treated Induced Hair Cells .............................................................. 59
4.3 Large Scale Compound Screen on Ototoxin Treated Induced Hair Cells ..................................... 61
4.4 Kinase Inhibitor Compounds and FDA Approved Analogs ......................................................... 69
viii
4.5 Antisense Oligonucleotides and Neutralizing Antibodies for Target Validation ......................... 74
4.6 Drug Treatment of Organ of Corti Explant Cultures ................................................................... 77
4.7 Preliminary Modeling of In Vivo Cisplatin Ototoxicity ............................................................... 79
4.8 Discussion ................................................................................................................................ 81
Chapter 5: Conclusions .......................................................................................................... 83
5.1 Discussion ................................................................................................................................ 83
5.2 Significance .............................................................................................................................. 86
5.3 Future Directions ..................................................................................................................... 87
Appendix I: Materials and Methods ...................................................................................... 89
References ............................................................................................................................ 97

ix
List of Figures

Chapter 1 :
1.1. Inner Ear Development and Anatomy pg. 2
1.2. Cell Cycle Exit and Differentiation of Sensory Hair Cells pg. 4
1.3. Organization of the Cochlea for Mechanotransduction pg. 5
1.4. Hair Cell Loss in Birds Compared to Mammals pg. 7
1.5. Pie Chart Summarizing Causes of Hearing Loss pg. 8
1.6. Molecular Mechanisms of Aminoglycoside and Cisplatin Ototoxicity pg. 10
1.7. Essential Transcription Factors in Hair Cell Development pg. 11
1.8. Schematic of Waddington Landscape pg. 16
1.9. Summary of Existing In Vitro Hair Cell Models pg. 21

Chapter 2 :
2.1. Organ of Corti Organization pg. 23
2.2. Identification of Hair Cell Specific Transcription Factors pg. 26
2.3. Combinatorial Testing of Transcription Factors pg. 28
2.4. Optimal Reprogramming of iHCs with Six1, Atoh1, Pou4f3 and Gfi1 pg. 29
2.5. Induced Hair Cells Transcriptionally Resemble Primary Hair Cells pg. 31
2.6. iHCs Activate Genes Related to Hair Cell Development and Function pg. 34
2.7. Gene Ontology Gene Sets pg. 35
2.8. iHCs are Transcriptionally Distinct from other Atoh1 Dependent Lineages pg. 37
2.9. GSEA Gene Sets pg. 38
2.10. iHC Chromatin Accessibility Resembles that of Primary Hair Cells pg. 40
2.11. iHCs Successfully Open Primary Hair Cell Enhancer Regions pg. 42
2.12. Six1, Atoh1, Pou4f3 and Gfi1 can Reprogramming Post Natal Cells pg.45

Chapter 3
3.1. Induced Hair Cells Express Functionally Relevant Hair Cell Genes pg. 48
3.2. Co-cultures of iHCs with Dissociated Organs of Corti Self Organize pg. 51
3.3. iHCs show Voltage Dependent Electrophysiological Responses pg. 53
3.4. Styryl Dyes Specifically Accumulates in Primary and Induced Hair Cells pg. 55
x

Chapter 4
4.1. Gentamicin Accumulates Specifically in Primary and Induced Hair Cells pg. 59
4.2. Induced Hair Cells Recapitulate Selective Vulnerability to Gentamicin pg. 60
4.3. Summary of Gentamicin Screening Results pg. 62
4.4. Summary of Otoprotective Hits from Micro-Explant Paper pg. 63
4.5. Survival Curve of Induced Hair Cells Treated with Cisplatin pg. 65
4.6. Summary of Cisplatin Screening Results pg. 66
4.7. Table of Identified Potential Otoprotectants Against Cisplatin pg. 67
4.8. Protein Kinase Inhibitor Compound Validation pg. 72
4.9. FDA Approved Kinase Inhibitor Compound Testing pg. 73
4.10. Induced Hair Cell Survival with ASOs and nABs pg. 76
4.11. Primary Hair Cell Protection in Organ of Corti Explants pg. 78
4.12. Summary of Results for Intratympanic Cisplatin Treatment pg. 81

1

Chapter 1: Background

1.1 Inner Ear Development
In mice the development of the inner ear begins between embryonic day 8.0 to 8.5
(E8.0-8.5) with the formation of the otic placodes, a thickening of the cranial ectoderm
(Le Douarin, 1984; Morsli et al., 1998) (Fig. 1.1A,B). These placodes invaginate to form
the otocysts or otic vesicles by E9.5 (Haddon & Lewis, 1996; Fritzsch et al., 2002; M W
Kelley, 2006; Abello & Alsina, 2007; Sanchez-Calderon et al., 2007). The otocyst
subsequently undergoes a series of morphological changes and patterning, which
include the extension and coiling of the nascent vestibular and cochlear duct structures
of the inner ear, as well as the neurons of the vestibulocochlear ganglion (Morsli et al.,
1998; Satoh & Fekete, 2005; M W Kelley, 2006; Matthew W Kelley et al., 2009) (Fig.
1.1A,B). The inner ear is comprised of two sensory systems: 1) the vestibular system
involved in sensing balance and head orientation and 2) the auditory system involved in
the perception of sound. The cochlea of the auditory system is a snail-shaped, fluid
filled duct that is divided into three compartments: scala vestibuli, scala media, and
scale tympani (Fig. 1.1C). The middle compartment is the scala media and it houses the
sensory epithelium of the cochlea, known as the organ of Corti (Fig. 1.1C). The highly
structured organ of Corti arises from a pro-sensory domain established in the
developing cochlear duct between embryonic days E12.5 and E14.5 in mice (Ruben &
Sidman, 1967; Fekete, 1996; Löwenheim et al., 1999; P Chen & Segil, 1999; Matei et
al., 2005; Y. S. Lee et al., 2006).

2

Figure 1.1: Inner Ear Development and Anatomy
A. Schematic of mouse inner ear development from otic placode at E9.5 to cochlear and vestibular
regionalization at E15.5. Adapted from (M W Kelley, 2006) B. Mouse inner ear growth depicted by paint-
filled membranous labyrinths depicting the morphological changes that occur between days E9.5 and
E17. Adapted from (Morsli et al., 1998). C. Diagram of the adult human ear divided into outer, middle and
inner ear. A cross section through the cochlea shows the three fluid filled compartments (scala vestibuli,
scala media, and scala tympani) and the sensory epithelium, the organ of Corti.

3
1.2 Sensory Hair Cell Fate
The coordination of cell proliferation, cell cycle exit and cell differentiation are
required for the development of many tissues (Zhu & Skoultchi, 2001; Brouillard &
Cremisi, 2003; Pagano & Jackson, 2004). In the developing cochlea, the prosensory
domain starts to express p27
Kip1
, a cyclin-dependent kinase inhibitor, resulting in a wave
of cell cycle exit from apex to base between E12.5 and E14.5 (P Chen & Segil, 1999; Y.
S. Lee et al., 2006) (Fig. 1.2A,B). These post-mitotic cells are the progenitors for
sensory hair cells and their adjacent supporting cells (Fekete et al., 1998; M W Kelley,
2006; Driver et al., 2013). This post-mitotic, prosensory domain differentiates in a wave
from base to apex with the activation of the basic helix-loop helix (bHLH) transcription
factor Atoh1 between E13 and E14.5 (Lanford et al., 2000; Ping Chen et al., 2002;
Lumpkin et al., 2003) (Fig. 1.2C). This spatial and temporal expression of Atoh1 is
critical for hair cell development and proper patterning of the organ of Corti. The mosaic
of hair cells and supporting cells in the developing organ of Corti is generated by Notch-
mediated lateral inhibition (Lanford et al., 1999, 2000; Kiernan, Cordes, et al., 2005; W.
Pan et al., 2010; Hartman et al., 2010; Neves et al., 2013) (Fig. 1.2D). The expression
of Atoh1 becomes restricted to the cells that will acquire a hair cell fate and this
upregulation of Atoh1 in nascent hair cells leads to lateral inhibition and the suppression
of Atoh1 in the neighboring cells fated to become supporting cells (Helms & Johnson,
1998; Bermingham et al., 1999; Ping Chen et al., 2002; Lumpkin et al., 2003) (Fig.
1.2D).

4
Figure 1.2: Cell Cycle Exit and
Differentiation of Sensory Hair
Cells
A. Lack of BrDU labeling in the
prosensory domain shows the wave
of cell cycle exit from apex to base
in the developing organ of Corti. B.
Anti-P27kip1 immunostaining in
developing organ of Corti matches
the wave of cell cycle exit seen in
Panel A. C. Activation of Atoh1
expression in developing organ of
Corti progresses as a wave from
base to apex. D. Schematic of
Notch mediated lateral inhibition
showing the Atoh1 expressing hair
cell in green and the adjacent
supporting cell in yellow where
Atoh1 is inhibited by the Notch
signaling. Panels A-C Adapted from
(Y.-S. S. Lee et al., 2006)

The differentiation of
hair cells and supporting cells
is completed during
embryonic development,
however the maturation of the
organ of Corti progresses until
the onset of hearing at approximately postnatal day 12 (P12) (Pujol & Hilding, 1973; D.
J. Lim & Anniko, 1985). The non-sensory supporting cells that surround the hair cells
are required for hair cell function, organization and physiological support (Matthew W
Kelley et al., 2009; May et al., 2013). There are several populations of supporting cells:
Border cells, Inner border cells, inner phalangeal cells, pillar cells, Deiters’ cells, and
Hensen’s cells (Wan et al., 2013). The sensory hair cells function as the essential
mechanoreceptors that convert sound vibrations into electrical signals, which are then
transmitted to the brain via the spiral ganglion neurons that innervate the hair cells
5
(Geleoc & Holt, 2003). The organ of Corti extends the length of the cochlear duct and
the hair cells are arranged into one row of inner hair cells and three rows of outer hair
cells (Fig. 1.3A). Hair cells get their name from the distinctive bundles of actin-rich
filaments at their apical surface. These membrane projections are known as stereocilia
and they house the mechanotransduction channels required for hair cell signaling
(McGrath et al., 2017) (Fig. 1.3B). The stereocilia project into the fluid of the scala
media in between the organ of Corti and the tectorial membrane such that when sound
waves pass through the fluid the stereocilia are deflected. The deflection of the
stereocilia triggers the opening of the mechanoelectrical transduction (MET) channels
allowing an influx of K+ to depolarize the hair cells (Alharazneh et al., 2011; Kawashima
et al., 2015) (Fig. 1.3B). Upon sufficient depolarization the hair cells will release
neurotransmitters to communicate with the post-synaptic afferent spiral ganglion
neurons.

Figure 1.3: Organization of the Cochlea for Mechanotransduction
A. The hair cells in the organ of Corti are organized into one row of inner hair cells and three rows of
outer hair cells. The tectorial membrane sits on top of the stereocilia in the scala media. The hair cells are
interdigitated by supporting cells and supported by the basilar membrane. Sensory neurons connect the
hair cells to the spiral ganglia, which communicate with the auditory cortex. B. Mechanotransduction
occurs when mechanical sound waves cause deflection in the hair cell stereocilia. The mechanically
gated MET channels at the tip of the stereocilia open to allow ion influx. cause the hair cell to depolarize
and release synaptic vesicles to the post-synaptic sensory neurons. Adapted from (Peng et al., 2011)

6
1.3 Hearing Loss and Lack of Hair Cell Regeneration
Loss of sensory hair cells is the leading cause of sensorineural hearing loss because
regeneration of hair cells does not occur in the mammalian organ of Corti (Corwin &
Cotanche, 1988; Cruickshanks et al., 2003; Brigande & Heller, 2009). Approximately
466 people worldwide report some degree of hearing loss and a Global Burden of
Disease Study found hearing loss to be the fourth leading cause of disability worldwide
(GBD 2015 Disease and Injury Incidence and Prevalence Collaborators, 2016; WHO,
2019). Currently the only therapies for hearing loss are hearing aids and cochlear
implants. Hearing aids only work for patients with mild to moderate hearing loss. They
are simply sound amplifying devices and rely on the remaining sensory hair cells and
intact auditory circuitry. For severe and total sensorineural hearing loss, cochlear
implants are the only option for rehabilitation. Normal hearing is not restored by
cochlear implants, but the implant can bypass the hair cells and directly stimulate the
auditory neurons.
In mammals the loss of hair cells in the organ of Corti leads to morphological
changes in the surrounding supporting cells to close up the void that the hair cells once
occupied (Lenoir et al., 1999; Raphael, 2002). Birds, in contrast to mammals, have been
shown to regenerate functional hair cells by supporting cell re-entry into the cell cycle
and giving rise to one new hair cell and one new supporting cell or by direct
transdifferentiation of the supporting cell into a new hair cell (B. M. Ryals & Rubel, 1988;
Corwin & Cotanche, 1988) (Fig. 1.4A). Although hair cells do not regenerate in the
mature mammalian cochlea (Fig. 1.4B), perinatal supporting cells have been shown to
have a transient ability to directly transdifferentiate into hair cells in response to Atoh1
7
(Kelly et al., 2012; Zhiyong Liu et al., 2012), or loss of Notch-mediated lateral inhibition
(Mizutari et al., 2013; Maass et al., 2015), however this potential is lost at very early
postnatal stages (White et al., 2006; Takebayashi et al., 2007; Doetzlhofer et al., 2009;
Z Liu et al., 2012; Cox et al., 2014; Bramhall et al., 2014).

Figure 1.4: Hair Cell Loss in Birds Compared to Mammals
A. Hair cell regeneration in birds can occur by two methods: First, the supporting cells can re-enter the
cell cycle and give rise to one new hair cell and one new supporting cell. Second, the supporting cell can
directly transdifferentiate into a new hair cell. Adapted from (Forge et al., 2020) B. Electron micrograph
image of an intact mammalian cochlea next to a damaged cochlea with no hair cell regeneration. Adapted
from House Ear Institute in Los Angeles.

The main causes of sensorineural hearing loss include genetic mutations, aging,
noise exposure, and exposure to drugs that have ototoxic side effects (Cunningham &
Tucci, 2017) (Fig. 1.5A). Fifty percent of hearing loss is caused by genetic mutations
(Morton & Nance, 2006) (Fig. 1.5A). Syndromic hearing loss refers to a combination of
disorders which includes hearing loss. Genetic hearing loss is considered non-
syndromic if the hearing loss in not associated with additional disorders. There are more
than 100 genes that have been linked to non-syndromic hearing loss (Nishio et al.,
2015; Hereditary Hearing Loss Homepage, 2020).

8

Figure 1.5: Pie Chart Summarizing Causes of Hearing Loss
A. Fifty percent of hearing loss is genetic and can be divided into syndromic and non-syndromic. Non-
syndromic hearing loss represents the majority of genetic hearing loss. Fifty percent of hearing loss is
acquired. Percentage adapted from (Choo, 2002; Cunningham & Tucci, 2017)

Acquired hearing loss can come in various forms (Fig. 1.5A). Age related hearing
loss is caused by the naturally degenerative effects of aging, as well as the
accumulated exposure to environmental causes of hearing loss. Noise induced hearing
loss is caused by mechanical stress on the cochlea which leads to the activation of
stress-induced apoptotic cellular pathways.
Ototoxicity from therapeutic drugs is another example of acquired hearing loss.
There are over 200 known ototoxins, compounds which cause hair cell damage and
degeneration, which lead to hearing loss. One group of ototoxins used in clinical
settings are aminoglycoside antibiotics. Aminoglycoside antibiotics were first discovered
in the 1940s and have since been used worldwide as an effective class of low cost
antibiotics (Waksman & Lechevalier, 1949; Umezawa et al., 1957; Weinstein et al.,
1963). Unfortunately, sensory hair cells of the inner ear are selectively vulnerable to
9
aminoglycosides, leading to hearing loss in 20-50% of patients receiving
aminoglycosides (Fausti et al., 1992; Forge & Schacht, 2000; Duggal & Sarkar, 2007).
There are also genetic susceptibilities that can make patients more vulnerable to
aminoglycoside ototoxicity (Nguyen & Jeyakumar, 2019). The aminoglycosides, such as
gentamicin, accumulate specifically in the sensory hair cells and can be visualized in the
laboratory by conjugating gentamicin to Texas Red (G. P. Richardson et al., 1997; Q.
Wang & Steyger, 2009). The entry of gentamicin into the hair cells requires a functional
MET channel (Alharazneh et al., 2011). Blocking the mechanotransduction channel
function by small molecule or genetic manipulation will inhibit the accumulation of
gentamicin in the hair cells. The ototoxic characteristics of aminoglycosides are well
characterized and there are now better alternatives in the antibiotic realm (J. Xie et al.,
2011).
Another group of ototoxins are platinum based chemotherapy drugs, such as
cisplatin (Ravi et al., 1995; Rybak & Somani, 1999; Dilruba & Kalayda, 2016). Cisplatin
is effective in treating various forms of pediatric and adult solid tumor cancers (Dilruba &
Kalayda, 2016), however, cisplatin causes some degree of hearing loss in as many as
60-80% of patients receiving cancer treatment (Langer et al., 2013). This side effect of
hearing loss if often the dose limiting factor when patients are undergoing treatment.
Unfortunately, cisplatin is very effective at treating cancers and only in the past 5-10
years have newer and more target specific chemotherapeutics come to play in an effort
to replace the more toxic cisplatin.

10
Figure 1.6: Molecular Mechanisms
of Aminoglycoside and Cisplatin
Ototoxicity
A. Schematic of aminoglycosides
(AG) ototoxicity. 1) AG enters hair
cells through MET channel. 2) AG-
Iron complexes (AG-Fe) cause the
formation of reactive oxygen species
(ROS). 3) ROS activation of the JNK
signaling pathway. 4) Signaling
cascade initiates transcription of
genes in cell death pathway. 5)
Initiation of mitochondrial apoptosis.
6) Cytochrome-C release from
mitochondria triggers caspases. 7)
caspase activated apoptosis. B.
Schematic of Cisplatin based (CP)
ototoxicity. 1) CP enters hair cell
through MET channel. 2) Formation
of highly reactive Cisplatin
monohydrate complex (CP-MHC)
activates NADPH oxidase 3 (NOX-3).
3) NOX-3 mediated accumulation of
ROS. 4-7) same as AG described
above. Figure adapted from
(Mahmoudian-sani et al., 2019)

In the case of both gentamicin and cisplatin a main culprit for the ototoxicity is the
production of reactive oxygen species (ROS) (Fig. 1.6A,B). ROS levels have been
shown to increase after treatment with aminoglycosides in vitro and in vivo (Priuska &
Schacht, 1995; Clerici et al., 1996). The elevated levels of ROS are sufficient to activate
cell death signaling cascades and the intrinsic mitochondrial apoptosis pathway (Forge
& Li, 2000; Sadowitz et al., 2002). Similarly, cisplatin causes oxidative stress by
accumulation of ROS (Clerici et al., 1996; Hellberg et al., 2009), depletion of antioxidant
enzymes in the cochlea (Rybak et al., 2000), and cisplatin-DNA adducts which block
DNA replication and transcription (D. Wang & Lippard, 2005; van Ruijven et al., 2005).
Though the ototoxicity of these drugs is well known, the full realm of cellular
mechanisms that cause hair cell degeneration have not been identified due to limited
availability of primary hair cells in laboratory models.
11

1.4 Cell Fate Reprogramming: Directed Differentiation vs Direct Reprogramming

Transcription Factors in Hair Cell Differentiation
Transcription factors (TFs) regulate the temporal and spatial patterns of gene
expression within the cells of complex tissues, establishing cell fate specification, and
ultimately determining their morphological and functional properties (Lemon & Tjian,
2000; Levine & Tjian, 2003; W. Zhang et al., 2004). There are up to 2,600 identified TFs
in the human genome identified by their DNA-binding domains, which is approximately
10% of genes in genome (van Nimwegen, 2003). These TFs bind to specific sequences
in enhancer or promoter regions of DNA in order to regulate gene expression and
downstream gene regulatory networks (Babu et al., 2004). The combinatorial use of TFs
in different cell types can account for the unique regulation of each gene in each cell of
a developing human. When studying the inner ear, several TFs have been identified as
critical players in the development, differentiation, and maintenance of hair cells (Fig.
1.7A).

Figure 1.7: Essential Transcription
Factors in Hair Cell Development
A. The transcription factors (TFs)
Sox2, Six1 and Eya1 are expressed in
the prosensory progenitors of the
developing cochlear duct. These TFs
are required for induction of the hair
cell master TF, Atoh1. Following Atoh1
expression, Pou4f3 and Gfi1 are TFs
required for hair cell survival. Network
adapted from (Schimmang, 2013)

12
Sox2
The Sox family of transcription factors play a role in maintaining progenitors in a
state of pluripotency and self-renewal (Wegner & Stolt, 2005; Takahashi & Yamanaka,
2006). The transcription factor Sox2 is involved in various aspects of vertebrate
neurogenesis (Graham et al., 2003; Bylund et al., 2003). The Sox2 expressing cells in
the developing inner ear give rise to the prosensory domain and Sox2 is required for
prosensory development (Dabdoub et al., 2008). The loss of Sox2 results in a
shortened cochlea and lack of hair cells and supporting cells (Kiernan, Pelling, et al.,
2005; Puligilla & Kelley, 2017).

Six1/Eya1
The TFs Six1 and Eya1 act as co-factors and play critical roles in the
development of the otic placode, the otic vesicle, and the prosensory progenitors (P. X.
Xu et al., 1999; W. Zheng et al., 2003; Zou et al., 2004). These cofactors have been
shown to promote proliferation of the sensory progenitors as well as sensory cell
differentiation (Zou et al., 2004).
Loss of Eya1 in mice leads to developmental arrest at the otocyst stage and
failure of all inner ear formation (P. X. Xu et al., 1999). Loss of Six1 also leads to early
arrest of inner ear development at the otocyst stage, no cochlea or an uncoiled cochlea,
and absence or truncation of vestibular organs (W. Zheng et al., 2003; Ozaki et al.,
2004). Mutations in Six1 and Eya1 have been found to cause syndromic forms of
sensorineural hearing loss in humans (Ruf et al., 2004; Song et al., 2013; Klingbeil et
al., 2017; Kari et al., 2019).
13

Atoh1
Another transcription factor, Atoh1, is essential for hair cell fate determination.
The expression of Atoh1 is dependent on Sox2 expression (Kiernan, Pelling, et al.,
2005; Neves et al., 2012). Additionally, Atoh1 has been shown to be transcriptionally
activated by direct Six1/Eya1 binding to its enhancer. In fact, the expression gradient of
Six1 precedes Atoh1 expression and matches the normal process of hair cell
differentiation (Ahmed et al., 2012). Within the inner ear, expression of Atoh1, a bHLH
class transcription factor (Lo et al., 1991; Ross et al., 2003) is both necessary and
sufficient for the induction of sensory hair cells in the embryonic and neonatal cochlea,
and ultimately plays an integral role in initiating the hair cell gene expression program
(Bermingham et al., 1999; J. L. Zheng & Gao, 2000; Woods et al., 2004; Kelly et al.,
2012; Chonko et al., 2013; Tiantian Cai et al., 2013; Ryan et al., 2015; Scheffer et al.,
2015; Z P Stojanova et al., 2016; Costa et al., 2017).
Manipulations of Atoh1 in various studies have demonstrated its essential role in
hair cell differentiation. Atoh1 knock out mice do not form hair cells (Bermingham et al.,
1999). Conversely, ectopic expression of Atoh1 can directly convert supporting cells
into ectopic hair cells in an age dependent manner (Kelly et al., 2012; Zhiyong Liu et al.,
2012) and induce the formation of ectopic hair cells in the greater epithelial ridge (J. L.
Zheng & Gao, 2000; Woods et al., 2004). Additionally, disrupting Notch signaling leads
to upregulation of Atoh1 and direct transdifferentiation of supporting cells to a hair cell-
like state in embryonic and early postnatal stages of the mouse cochlea (Takebayashi
et al., 2007; Doetzlhofer et al., 2009; Mizutari et al., 2013). However, previous studies
14
have shown that Atoh1 expression alone is not sufficient to induce hair cell
differentiation in somatic cells (Izumikawa et al., 2008; Costa et al., 2015; Abdolazimi et
al., 2016), or mature supporting cells of the organ of Corti (Kelly et al., 2012; Zhiyong
Liu et al., 2012).

Pou4f3/Gfi1
Two additional transcription factors, Gfi1 and Pou4f3, are required for hair cell
differentiation (Xiang et al., 1998; Wallis et al., 2003). Atoh1 expression in the
developing hair cells induces Pou4f3 to promote further hair cell differentiation (Ahmed
et al., 2012). Pou4f3 has been shown to be a direct target of Atoh1 and is required for
hair cell maturation and survival (Xiang et al., 1998; Masuda et al., 2011). Gfi1 is likely a
downstream target of Pou4f3 and the co-expression of these transcription factors is a
marker for a more differentiated hair cell (Hertzano et al., 2004).
Loss of Pou4f3 or Gfi1 causes hair cells to die shortly after differentiation
demonstrating their critical roles in the maintenance of hair cell survival (Xiang et al.,
1998; Wallis et al., 2003; Hertzano et al., 2004). The deletion of Pou4f3 in mice leads to
loss of hair cells by E17 and deafness at birth (Erkman et al., 1996). In humans, various
different mutations in Pou4f3 are linked to autosomal dominant non-syndromic
progressive hearing loss (Vahava et al., 1998; Freitas et al., 2014; C. Zhang et al.,
2016).

Transcription Factors in Cellular Reprogramming

Cell fate acquisition in development has been commonly depicted using a
Waddington landscape (Fig. 1.8A). A pluripotent stem cell at the top of the mountain
15
must make gene expression choices as it differentiates and further restricts its cell fate
to a particular lineage and final differentiated cell type. Only in recent years have
research studies made it clear that cells are much more plastic than previously thought.
Several critical studies in the mid 20
th
century proved that cell fate can be manipulated
and even reversed with the discovery of the frog embryo “organizer” that directs cell fate
(Spemann & Mangold, 2001), nuclear totipotency demonstrated by somatic cell nuclear
transfer (Briggs & King, 1952; King & Briggs, 1955) and the experiments showing that
differentiated, somatic nuclei could return to a totipotent state and contained all the
genetic information needed to give rise to a fully developed frog (Gurdon et al., 1958;
Gurdon, 1962). Thirty years later, cell fate was manipulated in mammalian model
organisms with the cloning of Dolly the sheep and the first cloned laboratory mice
(Campbell et al., 1996; Loi et al., 1997; Wakayama et al., 1998; Wilmut et al., 2007).
More recently, in vitro cell fate manipulation was achieved with the introduction of direct
cellular reprogramming, or transdifferentiation from one cell fate to another (Fig.
1.8B,C). The first demonstration of cellular transdifferentiation was the generation of
myoblasts from fibroblasts (Davis et al., 1987). This was followed by the ground
breaking generation of induced pluripotent stem cells (iPSCs) from fibroblasts by
overexpression of a core set of genes associated with pluripotency (Takahashi &
Yamanaka, 2006) (Fig1.8B).
These findings of cell fate plasticity have opened up many doors to address
question in the fields of biology and medicine. Previously, if a lab wanted to study a
particular cell type, they would need to isolate that exact cell type from laboratory model
organisms or from postmortem human tissues. Now with the ability to reprogram cells in
16
vitro, labs have access to infinitely more cell types and more specifically to the cell types
that are rare and difficult to access from primary sources. Another advantage to these in
vitro systems is the ability to use human cells in order to address clear biological
species differences between humans and commonly used laboratory model organisms.
The use of in vitro cell models has become an indispensable tool for the fields of biology
and medicine.

Figure 1.8: Schematic of Waddington Landscape
A. In normal development, a pluripotent stem cell (yellow) “rolls” down the hill of development and
changes fate as it reaches valleys. Often there are intermediate progenitors (orange) prior to reaching a
terminally differentiated state (red). B. Terminally differentiated cells (red) can be reprogrammed to
pluripotency (yellow) using the Yamanaka factors. C. One terminally differentiated cell (red) can be
converted into another differentiated cell (green) using direct reprogramming, also known as
transdifferentiation. D. Schematic of several direct reprogramming strategies that have successfully
generated induced cell types using defined transcription factors sets. Adapted from (Graf, 2011).

17
There are currently two main methods for generating cells of interest in vitro: 1)
directed differentiation and 2) direct cellular reprogramming. Using directed
differentiation, the starting cell type is a pluripotent cell, which in turn can be
differentiated by treatment with morphogens and/or small molecules with temporal
precision to mimic the natural development of the cell of interest. Directed differentiation
employs the use of a ‘blank’ cell in order to get to a desired cell of interest, however this
comes with some caveats. Often these protocols are months long and there is immense
heterogeneity of the final cells across batch to batch and cell line to cell line (Hiler et al.,
2015; Mellough et al., 2019; Yoon et al., 2019). Additionally, the use of a pluripotent cell
in the in vitro model limits their clinical translation for fear of graft-versus-host disease
and potential for tumor formation.
Using direct cellular reprogramming can address many of the caveats that arise
with directed differentiation. The Nobel prize winning work by Yamanaka and colleagues
showed that cells can be directly reprogrammed from a differentiated, somatic cell to an
induced pluripotent stem cell (iPSC) (Yamanaka & Takahashi, 2006) (Fig. 1.8B,C).
Direct reprogramming is the cell fate conversion of one somatic cell type into another
differentiated cell type (Fig. 1.8C). This direct reprogramming bypasses normal
developmental cues and employs the use of forced expression of transcription factors
and gene regulatory networks to change the cell fate. Direct reprogramming has been
successful in making many different cell types in vitro, such as macrophages, beta islet
cells, cardiomyocytes and neurons (H. Xie et al., 2004; Zhou et al., 2008; Vierbuchen et
al., 2010; Ieda et al., 2010) (Fig. 1.8D). Most importantly there is no need for a
pluripotent stage during direct reprogramming and the forced expression of select
18
transcription factors causes cell fate conversion on a much more rapid timeline than
directed differentiation.

1.5 Current Hair Cell Models
Since the inner ear is located deep within the temporal bone of the skull it is difficult
to study the cells of the cochlea from older mouse models (Sobkowicz et al., 1993).
Even if the temporal bone is decalcified postmortem it is still nearly impossible to dissect
out an intact adult organ of Corti. The organ of Corti can be dissected and cultured at
very early postnatal stages of the mouse (P1-8), however there are only a small number
of hair cells per cochlea making high-throughput studies difficult. Due to all of these
limitations in studying mammalian inner ear hair cells there has been a large push
towards in vitro models.
Most of the current approaches to generate hair cells use directed differentiation of
mouse or human embryonic stem cells to mimic normal development and differentiation
in vitro. This method employs the use of known morphogens and signaling activators
and/or repressors to follow the temporal stages of inner ear development that have
been studied extensively in model organisms. As mentioned previously, these protocols
are often time consuming, cell line dependent and require the use of pluripotent cells
making them less clinically relevant (Fig. 1.9A).
In 2003, the first in vitro model of sensory hair cells came from directed
differentiation mouse embryonic stem cells (H. Li et al., 2003) (Fig. 1.9A). The protocol
consisted of aggregating the embryonic stem cells into three-dimensional embryoid
bodies and culturing them in the presence of growth factors known to induce otic
19
placode and otic vesicle formation. The first round of culturing gave rise to otic
progenitor cells, as characterized by relevant gene expression. Subsequently, the otic
progenitor cells were differentiated into more mature cell types by withdrawal of growth
factors. They validated the loss of otic progenitor gene expression, as well as activated
expression of genes present in differentiated hair cells, such as Atoh1 and MyosinVIIa.
In 2010, this group was able to demonstrate that their protocol also worked on iPSCs
derived from mouse embryonic fibroblasts and could generate differentiated hair cells
with stereocilia bundles that were responsive to mechanical stimulation (Oshima et al.,
2010). Shortly afterwards they successfully generated hair cell-like cells from directed
differentiation of human embryonic stem cells, however the protocol took twice as long
in human cells (Ronaghi et al., 2014).
Around the same time, another group was able to generate otic vesicles and
sensory hair cells using directed differentiation of mouse embryonic stem cells (K R
Koehler et al., 2013) (Fig. 1.9A). The protocol was refined to more specific timing of
addition and removal of growth factors which increased the efficiency and specificity of
differentiation, however the full length protocol still took almost a month (Karl R Koehler
& Hashino, 2014). The hair cells generated were able to self-organize in the embryoid
bodies and develop stereocilia bundles. The hair cells were further characterized and
demonstrated hair cell-like electrophysiogical responses to mechanical stimulation,
which ultimately resembled the functionality of primary vestibular hair cells (X. P. Liu et
al., 2016). In 2017, they were also able to demonstrate that the protocol could
differentiate human iPSCs into inner ear embryoid bodies with hair cells (K R Koehler et
al., 2017).
20
The next group to generate an in vitro model of hair cells used a combination of
directed differentiation and transcription factors over expression to generate hair cell-
like cells in vitro (Costa et al., 2015) (Fig. 1.9A). The protocol used transgenic mouse
embryonic stem (ES) cells which had an inducible construct containing either the
transcription factor Atoh1 alone or the combination of transcription factors Atoh1,
Pou4f3 and Gfi1. The mouse ES cells were differentiated in embryoid bodies for four
days towards otic progenitors and then the transcription factor expression was induced,
and additional growth factors were introduced. They reported that the generation of hair
cell-like cells was more robust with the combination of three transcription factors
compared to Atoh1 alone. The induced hair cells expressed known hair cell markers,
developed stereocilia bundles and polarized within the embryoid bodies. The induced
hair cells were transcriptionally profiled by microarray, but not properly compared to a
primary hair cell transcriptome (Costa & Henrique, 2015).
All of the in vitro models mentioned above use directed differentiation and three
dimensional cultures of embryonic or induced pluripotent stem cells. A newer
publication from 2018 reported the use of direct reprogramming of human primary
fibroblasts into induced hair cells (Duran Alonso et al., 2018) (Fig. 1.9A). This group
used the three transcription factors Atoh1, Pou4f3 and Gfi1 identified in one of the
directed differentiation models (Costa et al., 2015). The transcription factors were
delivered to the human fibroblasts via lentiviral infection and allowed to reprogram for
two weeks. They reported activation of hair cell gene expression, such as MyosinVIIa,
Pou4f3 and Espin. They also performed RNA sequencing and reported upregulation of
inner ear and hair cell development GO terms. Taken together these results are
21
substantial as they are the first published results of direct reprogramming towards
induced hair cell-like cells, however more detailed characterization at the molecular and
functional level will be required to validate the usefulness of the model.

Figure 1.9: Summary of Existing In Vitro Hair Cell Models
A. Table summarizing the existing protocols for generating induced hair cells in vitro. The protocols are
organized in chronological order. For each protocol the cell type, timeline and extent of characterization
done on the induced hair cells is listed.

1.6 Our Approach
There is currently no well-established in vitro model of direct cellular reprogramming
to an induced sensory hair cell fate. Due to the need for high throughput studies in the
field of hearing loss, it was important to us that we could generate and thoroughly
characterize a novel model of induced hair cells. In the work described in this thesis, we
aimed to generate an in vitro model of sensory hair cells using direct reprogramming
from somatic cells. This began with the identification of a core set of hair cell specific
transcription factors (TFs) and combinatorial testing of these TFs using transgenic
mouse embryonic fibroblasts with a hair cell specific fluorescent reporter. Based on the
transgenic reporter activation accompanied by immunocytochemistry for additional hair
cell markers, we identified a set of four TFs that were sufficient to induce a hair cell-like
state. These induced hair cells (iHCs) were then characterized extensively at a
22
transcriptional and epigenetic level in order to compare iHCs to their primary hair cell
counterparts. The iHCs were also examined in several functional assays and
importantly, were able to recapitulate sensitivity to known ototoxins. Lastly, iHCs were
used in a large-scale drug screen to identify novel otoprotectants against cisplatin. This
screen resulted in several key candidates that were tested in various ways in vitro.
These candidates remain to be further examined and validated as the molecular
mechanisms of protection have not been studied and their effects in vivo need to be
tested.

23
Chapter 2: Generation of Induced Sensory Hair Cells using

2.1 Introduction
Hearing loss is the most common sensory deficit with estimates of around 466
million people affected worldwide (WHO, 2019). Loss of sensory hair cells of the inner
ear is the primary cause of sensorineural hearing loss (Bohne & Harding, 2000;
Hinojosa et al., 2001; Geleoc & Holt, 2014; Wong & Ryan, 2015). Sensory hair cells are
located in both the auditory and vestibular portions of the inner ear (Fig. 2.1A). The hair
cells within the organ of Corti are precisely arranged into one row of inner hair cells and
three rows of outer hair cells, interdigitating with a variety of supporting cells; inner
border, inner phalangeal, pillar cells, Deiters’ cells and Hensen’s cells (Fig. 2.1A).

Figure 2.1: Organ of
Corti Organization
A. Diagram of the
mouse inner ear shows
vestibular system
(green) and the cochlea
of the auditory system
(red). Cross section
through one turn of the
cochlea shows
organization in the
organ of Corti as a
mosaic of sensory hair
cells (one row of inner
hair cells and three rows of outer hair cells) interdigitated by various supporting cell populations labeled
from left to right (Inner border/phalangeal, Piller, Dieters’ and Hensen’s).

Hair cells are susceptible to degeneration by a variety of genetic mutations and
environmental stressors, such as exposure to loud noise, ototoxic drugs including
cancer chemotherapy and aminoglycoside antibiotics, aging and over 200 known
syndromic and non-syndromic genetic loci conferring predispositions to hearing loss
24
(Matsui et al., 2004; Cheng et al., 2005; Bodmer, 2008; Langer et al., 2013; Atkinson et
al., 2015; Wong & Ryan, 2015; Vaisbuch & Santa Maria, 2018). In mammals hearing
and balance are dependent on the maintenance of hair cells present at birth (Groves,
2010; Geleoc & Holt, 2014), since hair cells do not spontaneously regenerate
(Roberson & Rubel, 1994; Chardin & Romand, 1995; Forge et al., 1998), and so their
death leads to lifelong hearing loss and balance disorders.
The paucity and inaccessibility of primary inner ear hair cells have limited the
identification of effective otoprotective and regenerative strategies. Recent studies have
demonstrated the in vitro formation of hair cells from murine pluripotent stem cells either
by directed differentiation (Oshima et al., 2010; K R Koehler et al., 2013), human
embryonic stem cells (H. Li et al., 2003; Ronaghi et al., 2014), or in a combination of
directed differentiation to an ectodermal, non-neural, placodal cell type, followed by
transcription factor induction to a hair cell-like state (Costa et al., 2015). However,
these elegant approaches require three-dimensional culture conditions that complicate
high-throughput studies, for instance screening for otoprotectants. In contrast to
morphogen-based directed differentiation of pluripotent stem cells, transcription factor
(TF) -mediated lineage conversion of somatic cells enables the rapid production of
neurons and other cell types at microtiter scale with the reproducibility and homogeneity
required for high-throughput phenotypic screening (J. Xu et al., 2015; Babos et al.,
2019). Thus, the identification of a transcription factor cocktail that can convert somatic
cells into sensory hair cells could enable screening for new otoprotective targets.
Moreover, delivery of such a cocktail in vivo would enable regenerative medicine
25
strategies for hair cell replacement in situ, which have thus far been ineffective
(Izumikawa et al., 2005; R. T. Richardson & Atkinson, 2015; Roccio et al., 2015).
To this end, we have identified a cocktail of four transcription factors, Six1,
Atoh1, Pou4f3, and Gfi1 (SAPG), capable of converting mouse embryonic fibroblasts,
adult tail tip fibroblasts, and postnatal mouse supporting cells into induced hair cells
(iHCs). iHCs are highly similar to primary hair cells in terms of global gene expression
and chromatin accessibility profiles, morphological features, and electrophysiological
properties. In addition, we established a robotic imaging platform with automated
analysis to track iHC survival and show that like primary hair cells, iHCs are selectively
sensitive to gentamicin toxicity. These findings show that iHCs make a valuable in vitro
model to study hair cell regeneration, maturation, function and susceptibility to
ototoxins.

2.2 Identification of Hair Cell Specific Transcription Factors
To identify a group of TFs needed to convert somatic cells into induced hair cells,
we analyzed the transcriptome of postnatal day 1 (P1) cochlear hair cells that had been
FACS-purified from a transgenic mouse expressing GFP in nascent hair cells under the
control of an Atoh1 3’ enhancer (Lumpkin et al., 2003). We compared the primary P1
cochlear hair cell transcriptome to a reference transcriptome of the FACS-purified GFP-
negative cells from the same organ of Corti preparations (Fig. 2.2A). We identified 16
candidate TFs that were highly enriched in P1 hair cells (Atoh1::nGFP+), some of which
are known to have essential roles in hair cell development (Li et al., 2003; Wallis et al.,
26
2003; Qian et al., 2006; Hume et al., 2007; Ahmed et al., 2012; Chonko et al., 2013; Liu
et al., 2014a; Cai et al., 2015; Scheffer et al., 2015).

Figure 2.2 : Identification of Hair Cell Specific Transcription Factors
A. Heat map of RNA sequencing data from primary P1 Atoh1::nGFP+ cochlear hair cells compared to the
Atoh1::nGFP- population. 16 transcription factors (TFs) were identified as only and/or highly expressed in
the Atoh1::nGFP+ hair cell population. (n=3 replicates per sample). B. Schematic of experimental design
for transcription factor mediated reprogramming. Mouse embryonic fibroblasts (MEFs) were isolated from
Atoh1::nGFP transgenic reporter mice. MEFs were plated at a density of 5000 cells per well of a 96 well
plate, infected with retroviral transcription factors and allowed to reprogram for 14 days prior to analysis.

2.3 Transgenic Reporter Activation and Immunostaining
Using retroviral delivery, we transduced the TFs into mouse embryonic
fibroblasts (MEFs) from the Atoh1::nGFP reporter mouse (Fig. 2.2B). MEFs transduced
with a control virus (dsRed) did not express the Atoh1::nGFP transgene after 14 days
(Fig. 2.3A). In contrast, overexpression of all 16 TFs led to Atoh1::nGFP activation in
1.7% (+/- 0.3) of MEFs at 14 days post infection (Fig. 2.3B). Reprogramming efficiency
was calculated as a percent of Atoh1::nGFP-positive MEFs out of the starting MEF
number (5000 cells per well). This result indicated that within this initial group were
individual transcription factors, or combinations thereof, able to reprogram MEFs to a
27
hair cell-like state. The low level of reprogramming efficiency is expected when large
numbers of factors are infected simultaneously, since only a subset of factors is
expected to infect any given cell (Phan & Wodarz, 2015; Mistry et al., 2018), and since
using large numbers of factors, and/or virus, is likely to challenge cellular
transcription/translational machinery, thus further reducing efficiency (Babos et al.,
2019).
To identify the TFs critical for the Atoh1::nGFP reporter activation in MEFs, we
tested the efficiency of Atoh1 and each of the other 15 TFs separately (Fig. 2.3B). We
observed that Atoh1 alone activated the Atoh1::nGFP reporter in 5.8% (+/- 1.5) of
starting MEFs, while Pou4f3-alone only did so in 0.15% (+/- 0.03) of the starting MEFs
(Fig. 2.3B). None of the other 14 factors alone activated the Atoh1::nGFP reporter. We
then tested the reprogramming efficiency of Atoh1 in combination with each of the other
15 TFs (Fig. 2.3C). The most significant reporter activation came from a combination of
Atoh1 and Pou4f3, which provided 17.5% (+/- 4.4) reprogramming efficiency (Fig. 2.3C).
We then tested the addition of each remaining individual factor to the combination of
Atoh1 and Pou4f3 (AP) (Fig. 2.3D). Gfi1 combined with AP (APG) increased the
reporter activation to 26.9% (+/- 5.6) reprogramming efficiency (Fig. 2.3D). A
subsequent round of addition of individual TFs the this three-factor combination showed
that the addition of Six1 to Atoh1, Pou4f3, and Gfi1 (SAPG) further increased the
reporter activation to reach 35.2% (+/- 1.8) reprogramming efficiency (Fig. 2.3E).
Addition of the remaining individual factors to the cocktail of SAPG did not increase
reprogramming efficiency (Fig. 2.3F).

28

Figure 2.3: Combinatorial Testing of Transcription Factors
A. MEFs isolated from the Atoh1::nGFP transgenic mouse do not express the transgenic reporter. MEFs
were infected with a dsRed viral control for visualization. Scale bar represents 100 um. B. All
quantification of reporter activation was done at 14 days post infection (dpi). Reprogramming efficiency
was calculated as the number of Atoh1::nGFP positive cells divided by the 5000 MEFs plated per well.
Reprogramming efficiency with all 16 TFs was 1.7% (+/- 0.3) and single factor infections only gave
reporter activation with Atoh1 alone (5.8% +/-1.5) and Pou4f3 alone (0.15% +/- 0.03). C. Reprogramming
efficiency with Atoh1 alone (5.8% +/-1.5) graphed alongside single factor add-on to Atoh1. Addition of
Pou4f3 significantly increased reprogramming efficiency to 17.5% (+/- 4.4). D. Reprogramming efficiency
with Atoh1 and Pou4f3 (AP, 17.5% +/- 4.4) graphed alongside single factor add-on to AP. Addition of Gfi1
significantly increased reprogramming efficiency to 26.9% (+/- 5.6). E. Reprogramming efficiency with
Atoh1, Pou4f3 and Gfi1 (APG, 26.9% +/- 5.6) graphed alongside single factor add-on to APG. Addition of
Six1 significantly increased reprogramming efficiency to 35.2% (+/- 1.8). F. Reprogramming efficiency
with Six1, Atoh1, Pou4f3, and Gfi1 (SAPG, 35.2% +/- 1.8) graphed alongside single factor add-on to
SAPG. No single factor addition gave a significant increase in reprogramming efficiency. (C-G: N=3
independent experiments per condition, n=3 replicates per condition per experiment; numbers reported as
mean ± SEM; one-way ANOVA *p<0.05, **p<0.01, ***p<0.001, ****p<0.0001)

29
Since Atoh1 is expressed in other cell types and lineages (Klisch et al., 2011; T.-
H. H. Kim et al., 2014; Ostrowski et al., 2015), we performed immunostaining for
MyosinVIIa and Parvalbumin, two additional markers that are more specific to a hair cell
fate (Eybalin & Ripoll, 1990; Demêmes et al., 1993; Pack & Slepecky, 1995; Hasson et
al., 1997; Sahly et al., 1997; G. P. Richardson et al., 1997, 1999; Boëda et al., 2002).
The majority of SAPG-transduced cells that activated Atoh1::nGFP also expressed
MyosinVIIa and Parvalbumin (78.4% +/- 1.9) (Fig. 2.4A,B). Overall, SAPG transduction
activated Atoh1::nGFP with a 35% efficiency, and nearly 80% of all Atoh1::nGFP
positive cells also expressed MyosinVIIa and Parvalbumin (Fig. 2.4B). In contrast, only
50% of the Atoh1::nGFP+ cells generated by AP or APG expressed MyosinVIIa and
Parvalbumin, indicating that most Atoh1::nGFP+ cells generated from these alternative
cocktails were not hair cell-like (Fig. 2.4B). Our results support the importance of Six1,
Atoh1, Pou4f3 and Gfi1 in direct reprogramming of somatic cells to a hair cell-like state,
with high efficiency, purity, and reproducibility.
Figure 2.4: Optimal Reprogramming
of iHCs with Six1, Atoh1, Pou4f3
and Gfi1
A. Images of MEFs reprogrammed
with SAPG fixed at 14 dpi.
Atoh1::nGFP reporter activation
(green) and immunostaining for anti-
MyosinVIIa (red) and anti-Parvalbumin
(cyan). Scale bar represents 50 um in
length. B. All quantification was
performed at 14 dpi. Reporter
activation and immunostaining for
anti-MyosinVIIa and anti-Parvalbumin
was used to quantify triple positive
cells (Atoh1::nGFP+/MyosinVIIa+/
Parvalbumin+). A= Atoh1, P=Pou4f3,
G=Gfi1, S=Six1. The combination
SAPG gave 35% (+/- 1.8)
reprogramming efficiency and 78%
(+/-1.9) of Atoh1::nGFP+ cells were triple positive. Statistics shown are for the comparison of triple
positive cells in each condition. (N=3 independent experiments per condition, n=3 replicates per condition
per experiment; mean ± SEM; one-way ANOVA *p<0.05, **p<0.01, ***p<0.001, ****p<0.0001)
30

2.4 Transcriptional Profiling of Induced Hair Cells
Direct lineage reprogramming relies on the forced expression of transcription
factors to induce a molecular rewiring of the transcriptional programs that characterize
specialized cells (Takahashi & Yamanaka, 2006; Takahashi et al., 2007). This involves
both upregulation of the target cell-specific genes, in this case of primary cochlear hair
cells, and downregulation of the starting cell-specific genes, in this case of MEFs. To
assay the extent to which induced hair cells replicate the mouse primary cochlear hair
cell gene expression program, we performed RNA-sequencing on FACS-purified
Atoh1::nGFP+ cells generated by overexpression of Six1, Atoh1, Pou4f3, and Gfi1
(SAPG) at 14 days post infection (dpi)(hereafter referred to as iHCs). We compared the
gene expression of the iHCs to FACS-purified Atoh1::nGFP+ primary cochlear hair cells
at postnatal day 1 (P1; hereafter referred to as P1 HCs), and MEFs infected with a
control retrovirus expressing a fluorescent protein (dsRed; hereafter referred to as
MEFs) (Fig. 2.5A,B). PCA analysis of bulk RNA-seq data from MEFs, compared to
either primary P1 HCs or iHCs show the relative difference between these populations
(Fig. 2.5A). We categorized the gene expression in iHCs as either “successfully
reprogrammed”, “not-reprogrammed” or “inappropriately expressed” and divided the
categories into those genes that are normally expressed in P1 HCs, but not in MEFs
(P1 HC genes, black bar), and those that are normally expressed in MEFs, but not in
primary hair cells (MEF genes, black bar)(Fig. 2.5B). From this analysis we determined
that the iHCs had transcriptionally activated a hair cell-like signature by successfully
upregulating 64% of P1 HC genes, while simultaneously becoming distinct from the
31
starting MEF population by successfully downregulating 69% of MEF genes (Fig. 2.5B).
These percentages are comparable to those achieved in the TF-induced direct
conversion of MEFs into spinal motor neurons, as well as those attained in MEF-to-
cardiomyocyte and hepatocyte-to-neuron direct conversion (Ieda et al., 2010; Marro et
al., 2011; Gopalakrishnan et al., 2017; Justin K. Ichida et al., 2018). These results
suggest that iHCs largely resemble primary P1 cochlear hair cells at the transcriptional
level.

Figure 2.5: Induced Hair Cells Transcriptionally Resemble Primary Hair Cells
A. Principle component analysis (PCA) shows RNA expression profiles for MEFs, P1 cochlear hair cells
(P1 HCs), and induced hair cells (iHCs). (n=3 replicates for each cell type). B. Categorical heat-map
comparing gene expression (RNA-seq) mouse embryonic fibroblasts (MEFs), P1 cochlear hair cells (P1
HCs), and induced hair cells (iHCs). (n=3 replicates for each cell type). Venn diagrams show percent of
correctly reprogrammed genes. 64% of uniquely expressed P1 HC genes are correctly upregulated
during reprogramming, and 69% of inappropriately expressed MEF genes are downregulated.
32

Nonetheless, a number of genes did not respond to the SAPG group of
transcription factors used for reprogramming. Of the 1,506 genes expressed in P1 HCs,
but not MEFs, 36% were not successfully upregulated in the iHCs, and 118 genes were
inappropriately upregulated. Of the 939 genes that are expressed in MEFs, but not in
P1 HCs, and thus need to be downregulated during reprogramming, 31% failed to
downregulate, and 142 genes were inappropriately downregulated.
Gene Ontology (GO) analysis showed that the genes successfully upregulated
during reprogramming were significantly enriched for “sensory perception of sound” and
“detection of mechanical stimulus”, which revealed as three clusters of genes (Fig.
2.6A). The first cluster was enriched for development-related GO terms such as “inner
ear receptor cell development”, “mechanoreceptor differentiation”, and “hair cell
differentiation” (Fig. 2.6A). The second cluster was enriched for stereocilia-related GO
terms such as “plasma membrane bound cell projection assembly”, “cilium organization”
and “cilium movement” (Fig. 2.6A). The third cluster was enriched for synaptic signaling
GO terms such as “establishment of synaptic vesicle localization”, “synaptic vesicle
cycle” and “neurotransmitter secretion” (Fig. 2.6A). These three clusters of GO terms
were used to generate cluster-specific gene sets driving the GO designation (Fig. 2.7A).
Expression levels of each GO cluster-specific gene set, in each cell type, were
plotted to visualize the statistically significant iHC divergence from MEFs, and iHC
convergence towards a P1 HC expression profile (Fig. 2.6B). This analysis also
revealed a significant difference in the level of gene expression (p<0.05) between iHCs
and P1 HCs (Fig. 2.6B). This difference may be explained by the maturity level of the
33
iHCs as well as the presence of the residual MEF transcriptional profile. However,
further investigation of the FPKM values for several key genes in each GO cluster
demonstrated that the iHCs efficiently upregulated important hair cell genes including
Whirlin (Whrn) involved in cell polarity, Cadherin23 (Cdh23) and Espin (Espn) important
for stereocilia organization and functionality, as well as Bassoon (Bsn) and Otoferlin
(Otof) required for synaptic scaffolding and synaptic vesicle signaling (Fig. 2.6C). After
looking at the expression of key hair cell genes, we determined that the iHCs had also
activated all of the initial transcription factors included in the set of 16 candidate factors,
with the exception of Zfp503 (Fig. 2.6D). Together, the RNA sequencing results indicate
that the iHCs generated by Six1, Atoh1, Pou4f3, and Gfi1 (SAPG) overexpression are
capable of repressing most of the initial MEF gene signature, while simultaneously
adopting a gene expression signature similar to primary P1 HCs.

34

Figure 2.6: iHCs Activate Genes Related to Hair Cell Development and Function
A. Gene Ontology analysis of successfully upregulated genes in iHCs categorized into three relevant
gene clusters: Development, Stereocilia and Synapse. B. Violin plots show relative RNA expression of
the gene sets associated with each GO cluster: Development (81 genes), stereocilia cluster (165 genes)
and Synapse cluster (149 genes). C. Gene expression (FPKM) in MEFs, iHCs and P1 HCs for selected
hair cell-enriched genes in each of the three clusters. All genes are significantly upregulated in iHCs
compared to MEFs with p<0.05 and FDR<0.01. (n=3 replicates per cell type; mean ± SEM). D. Gene
Expression (FPKM) in MEFs, iHCs and P1 HCs for the initial set of 16 transcription factors (TFs) identified
for reprogramming. iHCs are able to activate expression of all TFs with the exception of Zfp503. (n=3
replicates for MEFs, n=3 replicates for P1 HCs, n=3 replicates for iHCs; mean ± SEM).

35

Figure 2.7: Gene Ontology Gene Sets
Gene Ontology (GO) analysis showed that the genes successfully upregulated during reprogramming
were revealed as three clusters of GO terms: development-related GO terms, stereocilia-related GO
terms, and synapse-related GO terms. These three clusters of GO terms were used to generate cluster-
specific gene sets driving the GO designation. Development GO terms represented 81 genes. Stereocilia
GO terms represented 165 genes. Synapse GO terms represented 149 genes.
36

2.5 Induced Hair Cells are Distinct from other Atoh1 Dependent Lineages
Expression of Atoh1 is necessary and sufficient for hair cell differentiation in the
context of the inner ear primordium (Bermingham et al., 1999; Ping Chen et al., 2002;
Woods et al., 2004; Chonko et al., 2013; Tiantian Cai et al., 2013), however several
other lineages including cerebellar granule cell progenitors (Klisch et al., 2011), Merkel
cells (Ostrowski et al., 2015), and the secretory cell lineage of the gut (T.-H. H. Kim et
al., 2014) rely on Atoh1 expression for differentiation. To characterize the specificity of
our reprogramming to the hair cell-like state, we analyzed RNA sequencing data from
FACS-purified iHCs relative to other Atoh1-dependent lineages including cerebellar
granule precursors (CGP), secretory cells of the gut (GUT), and Merkel cells (MC).
Principle component analysis (PCA) indicated that iHCs were more similar to primary
P1 cochlear hair cells (P1 HC) than to either cerebellar granule cell precursors (CGP)
(Fig. 2.8A), secretory cells of the gut (GUT) (Fig. 2.8B), or Merkel cells (MC) (Fig. 2.8C),
showing that they established a hair cell-specific transcriptional program and have not
adopted the transcriptional profile of other Atoh1-dependent lineages.

37
Figure 2.8: iHCs are
Transcriptionally Distinct
from other Atoh1
Dependent Lineages
A-C. Principle component
analysis showing the
differences in transcriptional
profiles between P1
cochlear hair cells (P1
HCs), induced hair cells
(iHCs), control mouse
embryonic fibroblasts
(MEFs), and cerebellar
granule precursor cells
(CGP), secretory cells of the
gut (GUT), and Merkel cells
(MC), respectively. D. Gene
Set Enrichment Analysis
(GSEA) comparing iHC
expression profile to the
unique gene sets of MEFs,
HCs, CGPs, GUT cells, and
MCs. Table reports
normalized enrichment
score (NES), p values, and
number of genes in each
gene set. Normalized
Enrichment Score (NES)
shows the strongest correlation between gene sets was between iHCs and the primary hair cell (HC)
gene set. Gene signature lists in Figure 3 Supplement.

As an additional test, we compared iHCs to the other Atoh1-dependent lineages
using Gene Set Enrichment Analysis (GSEA) (Subramanian et al., 2005). By comparing
the transcriptomes in MEFs, P1 hair cells (HC), P1 Cerebellar granule precursors
(CGP), adult Gut secretory cells (GUT), and P1 Merkel cells (MC), we defined groups of
genes as part of a specific signature for each cell type (Fig. 2.9). The GSEA identified
gene lists specific to each cell type, which were not expressed in any of the other cell
types. We calculated Normalized Enrichment Scores (NES) (Subramanian et al., 2005)
for each cell type in comparison to iHCs. The largest NES was for the comparison of
iHCs to P1 hair cells (HC), indicating an enrichment for the HC gene signature, while
showing lower enrichment scores, and even negative enrichment scores, for the other
38
cell type comparisons (Fig. 2.8D). Thus, reprogramming with SAPG establishes a hair
cell-like transcriptional program without adopting the transcriptional profiles of other
Atoh1-dependent lineages.

39
Figure 2.9: GSEA Gene Sets
Gene Set Enrichment Analysis (GSEA) (Subramanian et al., 2005) was used to compare the
transcriptomes in MEFs, P1 hair cells (HC), P1 Cerebellar granule precursors (CGP), adult Gut secretory
cells (GUT), and P1 Merkel cells (MC). We defined groups of genes as part of a specific signature for
each cell type. MEF signature represents 188 genes. HC signature represents 109 genes. MC signature
represents 43 genes. CGP signature represents 69 genes. GUT signature represents 113 genes. These
gene signatures were used to calculate Normalized Enrichment Scores (NES) (Subramanian et al., 2005)
and p-values for each cell type in comparison to iHCs (table, Fig. 2.8D).

2.6 Chromatin Accessibility Profile of Induced Hair Cells
Chromatin structure controls the accessibility of genes for either activation or
repression in response to developmental and environmental signaling (Volpi et al.,
2000; Kozubek et al., 2002; Goetze et al., 2007; Jason D Buenrostro et al., 2015; X.
Chen et al., 2016; Sijacic et al., 2018), and as such, is an important regulator of cell
type-specific gene expression. We used an Assay of Transposase Accessible
Chromatin (ATAC) sequencing (J D Buenrostro et al., 2015; X. Chen et al., 2016) to
analyze the regions of open/accessible chromatin in MEFs (MEF peaks), primary P1
hair cells (P1 HC peaks), and iHCs (Fig. 2.10A). As in our analysis of gene expression
(Fig. 2.5), we characterized the open chromatin regions into those that are present in
primary P1 HCs, but not in MEFs (P1 HC peaks, black bar), and those that are open in
MEFs, but not primary HCs (MEF peaks, black bar), as analyzed by ATAC-seq
accessibility (Fig. 2.10A). We defined these groups, as in Figure 2.5, as either
“successfully reprogrammed”, “not reprogrammed”, and “inappropriately opened/closed”
(i.e. not matching either P1 HC peaks or MEF peaks).
The iHCs show robust opening of de novo distal element regions of the
chromatin that are open in P1 HCs, as well as large-scale chromatin closing in regions
of the genome that were accessible in the starting MEF population. The hair cell-
appropriate changes in chromatin accessibility of iHCs are also accompanied by a
40
proportion of inappropriate opening of closing of chromatin regions. Of the 13,390 peaks
present uniquely in P1 HCs, 73% were successfully opened during reprogramming,
while 27% were not opened during reprogramming, and an additional 18,084 peaks
opened inappropriately in iHCs (Fig. 2.10A). Conversely, of the 26,847 peaks unique to
MEFs, 84% of peaks were successfully closed during reprogramming, 16% were not
closed during reprogramming, and an additional 833 peaks were inappropriately closed
during reprogramming (Fig. 2.10A).

Figure 2.10: iHC Chromatin Accessibility Resembles that of Primary Hair Cells
A. Heat map comparing genome wide chromatin accessibility profiles of MEFs, P1 HCs, and iHCs.
Accessibility is divided into 7 clusters: 1) successfully reprogrammed HC peaks, 2) not reprogrammed HC
peaks, 3) inappropriately opened peaks, 4) successfully closed MEF peaks, 5) not reprogrammed MEF
peaks, 6) inappropriately closed peaks, and 7) successfully unchanged peaks. Scale of each sample
column is +/- 2.5 Kb from ATAC peak. Venn diagrams show percent of correctly reprogrammed chromatin
regions. 73% of unique P1 HC chromatin regions are successfully opened during reprogramming, and
84% of MEF chromatin regions are successfully closed in reprogramming.

41
Since most distal accessible elements are not active enhancers in a given cell
type (Heintzman et al., 2007), we analyzed the H3K27ac-state of the distal elements
present in P1 HCs, a marker of active enhancers (Creyghton et al., 2010) (Fig. 2.11A).
These results show that most of the enhancers identified in P1 HCs are opened in iHCs.
Global enhancer targets have not been analyzed in these cell types due to small
numbers, so we arbitrarily assigned putative gene targets to each P1 HC enhancer by
identifying the closest transcriptional start site. This is expected to identify 27-47% of
genuine targets, based on chromosome conformation capture experiments performed in
other cell types (Sanyal et al., 2012). Based on our RNA-seq data, we found that the
genes defined as putative targets of P1 HC-specific enhancers had significantly higher
expression in both P1 HCs and iHCs compared to MEFs (Fig. 2.11B).
To visualize the ATAC-seq and ChIP-seq data at specific loci we used the
Integrative Genomics Viewer (IGV) (Robinson et al., 2011). We chose the four known
hair cell loci, Pou4f3, Mreg, Rasd2, and Barhl1, that exemplify the changes in chromatin
structure at H3K27ac-defined enhancers between P1 HCs, iHCs and MEFs (Fig.
2.11C). These results indicate that robust and hair cell-appropriate global changes in
chromatin accessibility accompany the large shift in the transcriptional profile of iHCs.

42

Figure 2.11: iHCs Successfully Open Primary Hair Cell Enhancer Regions
A. Heat map comparing chromatin accessibility at primary cochlear hair cell enhancers. Enhancers were
identified as regions with open chromatin ATAC peaks and H3K27ac in P1 HCs. ChIP data for H3K27ac
shown as green heat map and ATAC chromatin accessibility data of the respective regions shown as blue
heat maps. Heat maps ordered from low to high information content. iHCs successfully open P1 HC
enhancer regions that were closed in the starting MEF population. Scale of each sample column is +/- 2.5
Kb from ATAC peak. B. Expression levels (Log2(RPKM)) of 3,082 putative primary hair cell enhancer
targets. Enhancer targets were identified by mapping to the nearest transcription start site for each
enhancer and the expression of each putative target was acquired from the RNA-seq results. iHCs
significantly upregulate the expression of the putative primary hair cell enhancer targets. C. Integrative
Genomics Viewer (Robinson et al., 2011) tracks show primary hair cell H3K27ac profile alongside
chromatin accessibility profiles of P1 HCs, iHCs and MEFs. Chromatin accessibility changes at specific
hair cell enhancers for Pou4f3, Mreg, Rasd2 and Barhl1 are highlighted in grey boxes.

2.7 Reprogramming Post Natal Cell Types
We have used mouse embryonic fibroblasts (MEFs) as a starting cell type for our
reprogramming efforts in the experiments described thus far. However, MEFs are an
embryonic and relatively heterogeneous cell population (Singhal et al., 2016). To
determine if the SAPG transcription factors are able to reprogram adult fibroblasts, we
43
virally transduced Atoh1::nGFP transgenic adult tail tip fibroblasts (TTFs) with Six1,
Atoh1, Pou4f3, and Gfi1 (Fig. 2.12A,C). The TTFs proved difficult to expand and infect
in vitro with high efficiency. In separate experiments, we demonstrated that the addition
of 10% Fetal Bovine Serum (FBS) and RepSox, a tissue culture additive that increases
reprogramming into iPSCs (J K Ichida et al., 2009) and induced motor neurons (Shi et
al., 2018; Babos et al., 2019), significantly increased the efficiency of reprogramming
TTFs into iHCs (Fig. 2.12A). Although less efficient than in MEFs, SAPG activated the
Atoh1::nGFP reporter in adult tail tip fibroblasts (Fig. 2.12C). In addition, the SAPG
catalyzed the expression of MyosinVIIa and Parvalbumin, indicating that they can
convert adult tail tip fibroblasts into Atoh1::nGFP+/ MyosinVIIa+/ Parvalbumin+ iHCs
(Fig. 2.12C,D).
Supporting cells are an attractive target for gene therapy approaches to hair cell
regeneration, due to their known role in regeneration in non-mammalian vertebrates (B.
M. Ryals & Rubel, 1988; Corwin & Cotanche, 1988; Stone & Cotanche, 2007; Brignull et
al., 2009), their having a common progenitor with hair cells (Fekete et al., 1998; M W
Kelley, 2006; Driver et al., 2013), and their survival in long-deafened mice (Oesterle &
Campbell, 2009). Although hair cells do not regenerate in the mature mammalian
cochlea, perinatal supporting cells have been shown to have a transient ability to
directly transdifferentiate into hair cells in response to Atoh1 (Kelly et al., 2012; Zhiyong
Liu et al., 2012), or loss of Notch-mediated lateral inhibition (Mizutari et al., 2013; Maass
et al., 2015), however this potential is lost at very early postnatal stages (White et al.,
2006; Takebayashi et al., 2007; Doetzlhofer et al., 2009; Z Liu et al., 2012; Cox et al.,
2014; Bramhall et al., 2014).
44
One plausible route to the in vivo regeneration of hair cells in the organ of Corti
would be the conversion of supporting cells into hair cells. Atoh1 alone can inefficiently
convert perinatal supporting cells into hair cells (Kelly et al., 2012; Zhiyong Liu et al.,
2012; Yang et al., 2013), but transdifferentiation potential decreases rapidly thereafter,
such that by two weeks of age, Atoh1 expression, or induction of transdifferentiation by
Notch-inhibition, cannot induce the formation of new hair cells (Maass et al., 2015; Jiang
et al., 2016). To determine if Six1, Atoh1, Pou4f3, and Gfi1 (SAPG) are able to convert
supporting cells into hair cells, we labeled supporting cells using a Lfng-
CreERt2::tdTomato transgene which permanently labels supporting cells (Fig. 2.12B).
We dissociated organs of Corti from Lfng-CreERt2::tdTomato mice at P8, and
transduced them with virus encoding Atoh1 alone, or the combination of four factors,
SAPG. The supporting cells were used from P8 organs in order to bypass the
spontaneous transdifferentiation that occurs at earlier post-natal stages. Cells were
infected and allowed to reprogram for two weeks before immunostaining for MyosinVIIa
and Parvalbumin (Fig. 2.12E). Lfng::tdTomato-positive P8 supporting cells (P8 SC)
transduced with the SAPG produced significantly more cells that activated MyosinVIIa
and Parvalbumin than Atoh1-transduced supporting cells (Fig. 2.12F). Since the
Lfng::tdTomato reporter is independent of the viral SAPG, the percent of triple positive
(Lfng::tdTomato+/MyosinVIIa+/Parvalbumin+) iHCs was calculated from the total
number of Lfng::tdTomato-positive cells per well. These results indicate that the
combination of Six1, Atoh1, Pou4f3, and Gfi1 can convert adult tail tip fibroblasts and
P8 supporting cells into induced hair cells at a significantly greater rate than Atoh1
alone.
45

Figure 2.12: Six1, Atoh1, Pou4f3 and Gfi1 can Reprogramming Post Natal Cells
A. Adult mouse tail tip fibroblasts (TTFs) were infected with SAPG and allowed to reprogram for 14 days.
Regular hair cell media (HCM) was not optimal to culture TTFs post-infection and caused severe loss of
cells. TTF survival and reprogramming was best with the addition on 10% FBS and RepSox to the Hair
Cell Media (HCM). (N=3 experiments, n=3 replicates per experiment, mean ± SEM; Student’s t-test
*p<0.05, **p<0.01, ***p<0.001, ****p<0.0001). B. Schematic of transgenes used for lineage tracing of
Lunatic fringe (Lfng) positive supporting cells C. TTFs were infected with SAPG and allowed to reprogram
for 14 days. TTFs are able to activate the Atoh1::nGFP reporter and stain for anti-MyosinVIIa (red) and
ant-Parvalbumin (grey). Merged image includes Hoechst nuclear stain (blue). Scale bar represents 50
um. D. Quantification of Atoh1::nGFP+ cells and triple positive cells (Atoh1::nGFP+/MyosinVIIa+/
Parvalbumin+) in TTFs infected with Atoh1 alone or SAPG. TTFs infected with SAPG generate
significantly more Atoh1::nGFP+ cells and 48.6% (+/- 12) of the Atoh1::nGFP+ cells are triple positive.
(N=3 experiments, n=3 replicates per experiment, mean ± SEM; Statistics shown for the comparison of
triple positive cells in each condition; Student’s t-test *p<0.05, **p<0.01, ***p<0.001, ****p<0.0001). E.
Dissociated organs of Corti from P8 transgenic mice with lineage traced supporting cells
(Lfng::tdTomato+ SC) were infected with SAPG, reprogrammed for 14 days and immunostained for anti-
MyosinVIIa (green) and anti-Parvalbumin (grey). P8 Lfng::tdTomato+ SCs infected with SAPG are able to
activate primary hair cell markers MyosinVIIa and Parvalbumin. Scale bar represents 50 um. F.
Quantification of the percent of triple positive cells (Lfng::tdTomato+/MyosinVIIa+/Parvalbumin+) out of
the total number of Lfng::tdTomato+ supporting cells per well in cultures infected with Atoh1 alone or
SAPG. Presence of the Lfng::tdTomato reporter is independent of the viral infection.. (n=7 replicates,
mean ± SEM; Student’s t-test *p<0.05, **p<0.01, ***p<0.001, ****p<0.0001)
46

2.8 Discussion
Direct cellular reprogramming has become an invaluable research tool to study
mechanisms of cell fate determination, cell fate plasticity, and to create access to
previously unattainable cells. The inability to access large amounts of primary sensory
hair cells from laboratory model organisms is a motivating factor to establish new
models of sensory hair cells, however these new models need to be thoroughly
characterized in order to prove their worth as bona fide hair cell-like cells. In order to
study the mechanisms of hearing loss and sensory hair cell susceptibility we decided to
use transcription factor mediated reprogramming to generate a hair cell model in vitro.
In this study so far, we have succeeded in identifying a cocktail of transcription
factors, Six1, Atoh1, Pou4f3, and Gfi1 (SAPG), that is able to reprogram somatic cells to
a sensory hair cell-like fate using alternately, mouse embryonic fibroblasts, adult tail tip
fibroblasts, and postnatal day 8 supporting cells. The reprogramming efficiency using
this SAPG cocktail (35%) to make iHCs is similar and in some cases higher than the
efficiencies of the best reprogramming strategies for other cell types such as
cardiomyocytes with 20% efficiency (Ieda et al., 2010) and motor neurons with 10-40%
efficiency (J K Ichida et al., 2009; Babos et al., 2019). This efficiency of reprogramming
allowed us to generate a large number of iHCs and purify them for detailed
characterization.
The RNA sequencing profile of iHCs has largely diverged from the starting MEF
transcriptional profile and converged towards a primary P1 cochlear hair cell
transcriptional profile. We summarized the transcriptional changes as successfully
47
upregulated and successfully downregulated genes, while not ignoring the genes that
fails to change in expression or change incorrectly. These are important feature of the
iHCs that can be used to improve the reprogramming. Similarly, we categorized the
epigenetic changes as successfully opened or closed, failed to open or close, and
incorrectly opened or closed. This epigenetic information can be useful in identifying
chromatin remodelers or additional transcription factors that can help rearrange the
chromatin accessibility to more faithfully recapitulate the P1 cochlear hair cell profile. All
of this characterization has highlighted the robust amount of cell fate reprogramming
that we are able to achieve using our transcription factor cocktail SAPG, as well as the
important improvements we can focus on to make iHCs more hair cell-like.
Lastly, we showed that our transcription factor cocktail SAPG is capable of
inducing the expression of hair cell markers in cells other than mouse embryonic
fibroblasts. Direct reprogramming has numerous applications for clinical research, and
we knew it would be important to show reprogramming of more mature cell types. To
this end, we showed that SAPG can induce adult mouse tail tip fibroblasts (TTFs) and
postnatal day 8 supporting cells to express two common hair cell markers, MyosinVIIa
and Parvalbumin. Though these mature reprogrammed cells remain to be characterized
in detail by transcriptional and epigenetic profiling, these results show that SAPG can be
used to reprogram multiple cell types. In the future we can use primary human cells in
order to further test our reprogramming factors in the most clinically relevant starting
cells. Although, undoubtedly the reprogramming will need to be optimized and refined in
order to generate human induced hair cells.
48
Chapter 3: Functional Characterization of Induced Hair Cells

3.1 Induced Hair Cells Express Functionally Relevant Hair Cell Genes
Using the transcriptional data described in Chapter 2, we were able to look more
closely at the expression levels of genes relevant to hair cell functionality and
maturation. We looked at a list of 80 genes that fell into categories for stereocilia,
synaptic signaling, mechanotransduction and ion channels. This list was refined from a
publication of hair cell transcriptomics (H. Liu et al., 2018).
Figure 3.1:
Induced Hair
Cells
Express
Functionally
Relevant Hair
Cell Genes
FPKM bar
charts
comparing
average
expression
levels of key
genes
between
MEFs, iHCs
and P1 HCs.
A-B.
Stereocilia
genes listed
alphabetically.
They were
divided into
two graphs
due to
different axes.
C. Synaptic
transmission
gene expression. D. Mechanotransduction gene expression. E-G. Ion channel gene expression for
Potassium channels, Chloride channels and Calcium channels respectively. (n=3 replicates per cell type;
error bars represent standard deviation)

49
The expression levels in FPKM for each of the 80 genes was compared between
MEFs, iHCs and P1 cochlear hair cells (Fig. 3.1). The gene list we examined covers
genes that are essential for: 1) stereocilia formation, organization and maturation (Fig.
3.1A,B), 2) synaptic vesicle trafficking, release and recycling (Fig. 3.1C), 3)
mechanotransduction machinery (Fig. 3.1D), 4) potassium, chlorine and calcium ion
channel composition required for depolarization and electrophysiological responses
(Fig. 3.1E-G). The results show that most of the genes (37 out of 80) had FPKM values
of less than 1 in MEFs and these genes were able to be largely upregulated in response
to the SAPG reprogramming factors. Induced hair cells were capable of upregulating a
majority of the genes to a comparable level with primary P1 cochlear hair cells, however
for approximately 10-20% of the genes, iHCs were not able to reach the level of
expression present in P1 hair cells. The overall success in upregulating key functional
hair cell genes prompted us to perform a preliminary analysis of the functionality of iHCs
compared to their primary counter parts.

3.2 Co-culture of Induced Hair Cells with Dissociated Primary Organs of Corti
Sensory hair cells have a very distinct morphology. As their name suggests,
these specialized cells possess hair-like actin-based apical membrane protrusions,
called stereocilia, that contain at their tips the mechanically gated ion channels required
for mechanotransduction (Kawashima et al., 2011; B. Pan et al., 2013; Holt et al., 2014).
Development of stereocilia involves the elaboration of a single primary, tubulin-based,
cilium, known as the kinocilium, centered on a cuticular plate of F-actin filaments from
which the stereocilia arise as elongated bundles of microvilli (Cotanche & Corwin, 1991;
50
Troutt et al., 1994; Leibovici et al., 2005; Jianbo Wang et al., 2005; B Tarchini et al.,
2016; McGrath et al., 2017).
To assess the morphological properties of iHCs we performed immunostaining at
14 days post-infection following SAPG reprogramming, which is approximately 10 days
after initial Atoh1::nGFP detection. At this time, iHCs exhibited highly polarized F-actin
staining as observed by well-defined labeling of Phalloidin-Rhodamine near the apical
surface and a primary cilium that labels with antibody to acetylated tubulin, and is
centered on the nascent cuticular plate (Fig. 3.2A). This highly polarized pattern is
reminiscent of hair cells in both the developing cochlear and vestibular systems
(Cotanche & Corwin, 1991; Troutt et al., 1994; Leibovici et al., 2005; Jianbo Wang et al.,
2005; Basile Tarchini et al., 2016; McGrath et al., 2017).
Previous work has demonstrated that mixing dissociated cells from embryonic or
perinatal organ of Corti with periotic mesenchyme, allows them to rapidly self-organize
and differentiate in vitro into epithelial island-like structures (Doetzlhofer et al., 2004;
White et al., 2006). To determine if iHCs are capable of integrating appropriately into
these sensory epithelial-like structures which contain both primary hair cells and
supporting cells, we FACS-purified Atoh1::nGFP+ iHCs and mixed them with
dissociated primary embryonic (E13.5) sensory epithelium, containing primary hair cells,
primary supporting cells, and a portion of the surrounding periotic mesenchyme (Fig.
3.2B-E). After two weeks of co-culture, iHCs contained polarized cuticular plates (F-
actin) and stereocilia (espin-positive), and were found incorporated into the epithelial
islands containing native hair cells and supporting cells (Fig. 3.2E). Greater than 80% of
iHCs that engrafted in epithelial islands exhibited highly polarized F-actin staining (data
51
not shown). Thus, iHCs morphologically resemble primary hair cells and possess
properties required for the proper structural integration with primary hair cells and
supporting cells.

Figure 3.2: Co-cultures of
iHCs with Dissociated
Organs of Corti Self
Organize
A. Monolayer iHCs also show
polarized F-actin by Phalloidin
labeling (red) and a kinocilium
by anti-acetylated Tubulin
labeling (cyan). Scale bar
represents 10 um. B. iHCs
(Atoh1::nGFP+/
MyosinVIIa+) co-cultured with
dissociated primary E13.5
organs of Corti organize into
epithelial islands alongside
primary hair cells
(Atoh1::nGFP-/MyosinVIIa+)
and primary supporting cells
(Sox2+). Scale bar represents
100 um. C. Co-cultured iHCs
show F-actin polarization by
Phalloidin labeling (grey) and
express MyosinVIIa (red).
Scale bar represents 20 um. D.
Co-cultured iHCs show F-actin
rich projections by Phalloidin
labeling (red) and express
MyosinVIIa (cyan). Scale bar
represents 20 um. E. Co-
cultured iHCs show an F-actin
rich cuticular plate by
Phalloidin labeling (red) and
stereocilia by anti-Espin
labeling (grey). Scale bar
represents 20 um. (A-E. All
merged images include
Hoechst nuclear stain.)

52
3.3 Electrophysiological Profile of Induced Hair Cells
To determine if iHCs possess electrophysiological properties similar to those of
primary hair cells, we performed whole-cell patch-clamp recordings. We measured the
biophysical properties of our cells in voltage-clamp and current-clamp to analyze the
voltage-gated currents and passive membrane properties of these cells (Fig. 3.3). We
compared primary hair cells (n=5) with Atoh1::nGFP+ iHCs in two experimental
conditions: monolayer-cultured iHCs (n=10) and iHCs co-cultured with dissociated
organ of Corti (n=10). Within co-cultures, the presence of the Atoh1::nGFP reporter
enabled specific patch clamp analysis of iHCs.
Current-clamp was used to measure the passive membrane properties of primary
hair cells, co-cultured iHCs and monolayer-cultured iHCs (Fig. 3.3A). The properties
measured included the resting potential, membrane capacitance, and input resistance
(Fig. 3.3B). The mean resting potentials for primary hair cells, cocultured iHCs, and
monolayer-cultured iHCs were -58.6 mV (+/- 6.9), -54.8 mV (+/- 4.1), and -50.8 mV (+/-
2.4), respectively (Fig. 3.3B). These values are comparable to previously reported
primary hair cell resting potentials (Dallos, 1985; Oliver et al., 2003). The input
resistances were measured to infer the total ion channel composition of the cell. Higher
input resistance values indicate the cell may have fewer ion channels to allow current to
flow in and out of the plasma membrane. The input resistance was highest in
monolayer-cultured iHCs (3878 +/- 557MΩ). However, the input resistance of co-
cultured iHCs (1432 +/- 327 MΩ) was comparable to that of primary hair cells (1950 +/-
755MΩ) (Fig. 3.3B). Lastly, the capacitance, which can be used to infer the surface area
53
of the cell, was highest in primary hair cells (8.4 +/- 3.1pF), followed by co-cultured iHCs
(5.6 +/- 2.2pF) and then monolayer-cultured iHCs (4.2 +/- 1.2pF) (Fig. 3.3B).

Figure 3.3: iHCs Show Voltage Dependent Electrophysiological Responses
A. Whole cell patch clamping was performed on P1 HCs from a dissociated organ of Corti, co-cultured
iHCs and monolayer-cultured iHCs. Results from current clamp show the change in cell voltage as a
response to an applied current. Dashed red line represents -60 mV. Current clamp protocol shows steps
from -10 to +150 pA in 20 pA increments. Scale bars represent 50 mV on X-axis and 250 ms on Y-axis.
B. Basic membrane properties were calculated from the current clamp data to report resting membrane
potential (Vm), membrane capacitance (Cm) and input resistance (Rin). Table reports mean (SEM) for
each value. C. Results from voltage clamp shows the current output of the cell as a response to applied
voltage for primary HCs, co-cultured iHCs and monolayer-cultured iHCs. Dashed red line represents 0
pA. Voltage clamp protocol shows steps from -120 to +70 mV in 10 mV increments. Scale bars represent
1 nA on X-axis and 125 ms on Y-axis. D. IV curve plotting current density (normalized for cell size) as a
function of applied voltage for primary HCs, co-cultured iHCs and monolayer-cultured iHCs. Co-cultured
iHCs show similar current output to P1 primary hair cells. E. Exponential fits to the voltage clamp traces
were used to calculate the current activation time constants for primary HCs, co-cultured iHCs and
monolayer-cultured iHCs. Dashed red line represents 0 pA. Solid red line shows exponential fit to outward
currents when clamped from -120 mV to +70 mV. Scale bars represent 1000 pA on X-axis and 12.5 ms of
Y-axis. F. Current activation time constants reported for P1 HCs, cocultured iHCs and monolayer-cultured
iHCs. Co-cultured iHCs show similar current activation kinetics to P1 HCs.

54
In addition, we performed voltage-clamp to measure the magnitude and time
dependent activity of the whole-cell currents in primary hair cells, co-cultured iHCs and
monolayer-cultured iHCs (Fig. 3.3C). In response to the applied voltage, both primary
hair cells and iHCs produced positive-outward currents (Fig. 3.3C). However, the
monolayer-cultured iHCs produced relatively small whole-cell currents that rapidly
inactivated (Fig. 3.3C). In contrast, primary hair cells and co-cultured iHCs displayed
robust outward currents that more slowly inactivated over the course of the protocol
(Fig. 3.3C). We measured the steady-state outward current as a function of the voltage-
clamp potential and normalized the current magnitude by the cell’s capacitance to
analyze current densities. Monolayer-cultured iHCs showed small current densities
while the co-cultured iHCs and primary hair cells displayed overlapping magnitudes of
voltage-dependent current densities (Fig. 3.3D).
A prominent voltage-clamp feature in primary hair cells is a delayed onset of a
slow-activating outward current (Housley & Ashmore, 1992; Marcotti & Kros, 1999). In
order to measure the kinetic properties of this slow-activating outward current, we fit a
single exponential at the onset of the current (Fig. 3.3E) to compare the mean time
constants when the cells were clamped from -10mV to 70mV (Fig. 3.3F). The delayed
onset current of monolayer-cultured iHCs displayed fast time constants (Fig. 3.3F). In
contrast, the co-cultured iHCs and primary hair cells showed similarly longer time
constants, indicating that their outward currents have similar activation kinetics (Fig.
3.3F). Together, these electrophysiological data suggest that when iHCs are co-cultured
with dissociated organ of Corti, the size, passive membrane properties and ion channel
function of iHCs are similar to those of primary hair cells.
55

3.4 FM Styryl Dye Accumulation in Induced Hair Cells
Primary sensory hair cells acquire distinct functional properties early in
development in order to properly convert mechanical sound waves into neurotransmitter
signaling (Wu et al., 2017). Mechanotransduction relies on the organization of
stereocilia, the assembly of tip links, and insertion of mechanically gated ion channels at
the tip of each stereocilia (Kawashima et al., 2011; B. Pan et al., 2013).
Mechanotransduction channels are highly permeable to styryl dyes, and their
accumulation in hair cells occurs with much faster kinetics than most other cells (Gale et
al., 2001).

Figure 3.4: Styryl Dye Specifically Accumulates in Primary and Induced Hair Cells
A. Organ of Corti explants from Atoh1::nGFP transgenic mice accumulate the styryl dye FM4-64
specifically in the hair cells. Image taken after 30 second of incubation with FM4-64. Merged image
includes Hoechst nuclear stain. Scale bar represents 100 um. B. Dissociated primary hair cells
expressing the Atoh1::nGFP reporter accumulate the styryl dye FM4-64. Image taken after 30 seconds of
incubation with FM4-64. Hoechst nuclear stain in blue. Scale bar represents 25 um. C. MEFs infected
with eGFP control virus do not accumulate the styryl dye FM4-64. Image taken after 30 seconds of
incubation with FM4-64. Merged image includes Hoechst nuclear stain. Scale bar represents 50 um. D.
iHCs expressing the Atoh1::nGFP reporter accumulate the styryl dye FM4-64. Image taken after 30
seconds of incubation with FM4-64. Hoechst nuclear stain in blue. Scale bar represents 25 um.

Primary hair cells within the intact organ of Corti rapidly and selectively
accumulate the styryl dye FM4-64 within seconds, a time frame consistent with entry of
56
the dye through mechanotransduction channels rather than endocytosis (Lelli et al.,
2009) (Fig. 3.4A). Similarly, primary hair cells from dissociated P1 organ of Corti
preparations also accumulate FM4-64 (Fig. 3.4B). In contrast, MEFs failed to
incorporate FM4-64 within the 30 second time frame (Fig. 3.4C). However, iHCs rapidly
incorporated FM4-64 to high levels within a 30 second time course (Fig. 3.4D)
demonstrating that iHCs possess rudimentary mechanotransduction channels with
similar styryl dye uptake as primary hair cells.

3.5 Discussion
Our results show that iHCs are capable of upregulating a large number of genes
that are important for primary hair cell development, maturation and function. The list of
genes we compared in our transcriptional data included genes important for stereocilia
organization, mechanotransduction, ion channels important for the depolarization of hair
cells in response to the opening mechanically gated ion channels, and synaptic vesicle
signaling. Many of these genes have been implicated in genetic forms of hearing loss so
it was important to identify their expression in iHCs. Though the expression of these
genes is not sufficient to say that the proteins in question are properly localized and
functional in the iHCs, it is a good indication that the iHCs are on their way to becoming
functional hair cells, so we investigated their functionality.
We found that co-culturing of iHCs with dissociated primary organs of Corti
prompted robust self-organization of the cultures. In these co-cultures, the iHCs
organize alongside the primary hair cells and supporting cells. We also found from the
electrophysiology recordings that the co-culture of iHCs caused a maturation of the
57
cellular membrane properties and the voltage dependent responses measured in whole
cell patch clamping. Lastly, we showed that iHCs are capable of rapidly and selectively
accumulating the styryl dye FM4-64. The primary hair cells of the intact organ of Corti or
in dissociated organ of Corti preparations were used as positive controls and the iHCs
showed selective accumulation of FM4-64 within seconds. The negative control MEFs
did not accumulate any FM4-64, suggesting that the iHCs possess rudimentary
mechanotransduction machinery. The styryl dye assay was useful in supporting the
findings from the electrophysiology. Taken together, the iHCs demonstrate functionality
reminiscent of primary P1 cochlear hair cells.

58

Chapter 4: Using Induced Hair Cells to Study Ototoxicity

4.1 Induced Hair Cells Selectively Accumulate Gentamicin
Environmental and pharmacological ototoxins that cause selective degeneration
of hair cells are major contributors to hearing loss worldwide (Al-Malky et al., 2015;
Sagwa et al., 2015; Knight et al., 2017). Gentamicin is representative of a large class of
highly effective aminoglycoside antibiotics that result in significant hair cell degeneration
(Alharazneh et al., 2011). Unfortunately, a lack of mammalian models suitable for large
scale screening of ototoxins and otoprotectants has restricted the development of small
molecules to reduce ototoxicity and identification of compounds that can protect against
known ototoxins.
To determine if iHCs are sensitive to ototoxic compounds, we tested their ability
to accumulate gentamicin in a similar manner to primary hair cells. Primary hair cells of
the organ of Corti specifically accumulated Texas-Red conjugated gentamicin (GTTR),
but not Texas Red (TR) alone when treated with 0.5mM of either compound for 3 hours
(Fig. 4.1A,B). MEFs transduced with a GFP-expressing control virus did not accumulate
GTTR (Fig. 4.1D). The iHCs, similarly to primary hair cells, do not accumulate Texas
Red (TR) alone (Fig. 4.1C), but selectively accumulated gentamicin-Texas Red (GTTR)
after a 3-hour treatment at 0.5mM (Fig. 4.1E).
59

Figure 4.1: Gentamicin Accumulates Specifically in Primary and Induced Hair Cells
A. Organ of Corti explants from Atoh1::nGFP transgenic mice accumulate Gentamicin-Texas Red (GTTR)
specifically in the hair cells. Organs were treated with 0.5mM GTTR for 3 hours. Scale bar represents 100
um. B. Organ of Corti explants from Atoh1::nGFP transgenic mice do not accumulate Texas Red (TR)
alone. Organs were treated with 0.5mM TR for 3 hours. Scale bar represents 100 um. C. Induced hair
cells (iHCs) do not accumulate Texas Red alone. iHCs were treated with 0.5mM TR for 3 hours. Merged
image includes Hoechst nuclear stain. Scale bar represents 50 um. D. MEFs infected with eGFP control
virus do not accumulate Gentamicin-Texas Red (GTTR). MEFs were treated with 0.5mM GTTR for 3
hours. Merged image includes Hoechst nuclear stain. Scale bar represents 50 um. E. iHCs can
accumulate GTTR. iHCs were treated with 0.5mM GTTR for 3 hours. iHCs were also labeled for anti-
MyosinVIIa (grey). Merged image includes Hoechst nuclear stain. Scale bar represents 50 um.

4.2 Survival Assay of Ototoxin Treated Induced Hair Cells
To assess whether the gentamicin accumulation seen by GTTR treatment could
cause iHCs to degenerate, we established a longitudinal survival assay using robotic
imaging and automated tracking of iHC survival (Fig. 4.2A). To selectively identify and
track survival of Atoh1::nGFP+ iHCs from daily whole-well images, we customized a
time-lapse nuclei count recipe running on SVCell RS 4.0 (a product of DRVision
Technologies that has been rebranded to Aivia). The software can automatically detect
and count iHCs based on nuclei morphology and the Atoh1::nGFP fluorescence with
60
comparable results to manual counting (p= 0.53). We performed the survival assay with
iHCs, dissociated primary P1 cochlear hair cells (HC) as a positive control, and induced
motor neurons (iMNs) as a negative control. While gentamicin caused primary hair cell
degeneration in a dose-dependent manner, it caused little-to-no toxicity to Hb9::RFP+
induced motor neurons generated from MEFs by transduction with Ngn2, Isl1, Lhx3,
Ascl1, Brn2, and Mty1l (Son et al., 2011) (Fig. 4.2B,C). Similar to primary hair cells,
iHCs treated with gentamicin showed rapid, dose-dependent degeneration (Fig. 4.2B,
C). These data indicate that iHCs possess functional properties of primary hair cells and
display selective vulnerability to the known the ototoxin, gentamicin. Moreover, these
results suggest that iHCs provide a scalable platform for detecting agents that protect
against gentamicin ototoxicity.

Figure 4.2:
Induced Hair
Cells
Recapitulate
Selective
Vulnerability to
Gentamicin
A. Schematic of
experimental
design for
longitudinal
survival of
Atoh1::nGFP+
iHCs. B-C.
Longitudinal
survival tracking
of P1 hair cells
(HC) from
dissociated organ
of Corti preparations, induced hair cells (iHCs) and induced motor neurons (iMNs) treated with gentamicin
at 2 mM and 4 mM respectively. (n=3 replicates each; mean ± SEM; Two-Way ANOVA *p<0.05,
**p<0.01, ***p<0.001, ****p<0.0001)

61
4.3 Large Scale Compound Screen on Ototoxin Treated Induced Hair Cells

Screen against gentamicin ototoxicity
Since iHCs were able to recapitulate a dose dependent decrease in survival we
proceeded to screen 647 compounds for potential otoprotectants. The 647 compounds
came from a combined total of two libraries. The Stem Select Library contained a total
of 320 compounds (EMD Chemicals) and the Protein Kinase Inhibitor Library contained
a total of 327 compounds (EMD Millipore Inhibitor Libraries I-IV). The compounds were
screened at 3uM on iHCs treated with 0.5mM gentamicin. The results of the screen
were quantified using the automated iHC imaging and survival tracking described in
Section 4.2. Unfortunately, due to some image quality issues the automated
quantification only gave reliable cell counts from 143 out of 320 compounds of the Stem
Select Library (SSL) and 208 out of 327 compounds of the Protein Kinase Inhibitor
Library (PKI). The wells were ruled out if the cell counts were not deemed reliable at
either the first day or the last day of the survival assay. This dramatic loss of data was
reason for us to repeat the screen a second time with improved iHC reprogramming
efficiency and imaging parameters. In the second screen we were able to get cell
counts for 293 out of 320 compounds for SSL and 275 out of 327 compounds for PKI.
These much improved numbers helped in the interpretation of the screening results.
The final results for both screens were interpreted based on the survival of iHCs in the
gentamicin treated wells compared to the wells that received gentamicin in addition to
one compound (Fig. 4.3).

62

Figure 4.3: Summary of Gentamicin Screening Results
A. Stem Select Library results from screen 1 showing percent of surviving cells at Day 5 for 143
compounds. B. Stem Select Library results from screen 2 showing percent of surviving cells at Day 5 for
293 compounds. C. Protein Kinase Inhibitor Library results from screen 1 showing percent of surviving
cells at Day 5 for 208 compounds. D. Protein Kinase Inhibitor Library results from screen 2 showing
percent of surviving cells at Day 5 for 275 compounds. A-D) Average survival with gentamicin alone is
marked by red dotted line. The three increasingly opaque boxes represent one, two and three standard
deviations from the average survival with gentamicin alone.

The average survival of iHCs in response to 0.5mM gentamicin in Screen 1 was
69.4% (+/- 6.5) of starting cells surviving at Day 5 of the assay. In Screen 2 the average
survival of iHCs in response to 0.5mM gentamicin increased to 72.4% (+/- 7.8) of
starting cells surviving at Day 5. Due to the differences in average survival across the
screens, and the somewhat large variability of survival with gentamicin alone, the
results of the screens were interpreted separately. Several of the hits identified in the
screen fell in line with the known cellular mechanisms of gentamicin ototoxicity including
accumulation of reactive oxygen species (ROS) (Choung et al., 2009), transcriptional
changes in cell cycle machinery (L. Tao & Segil, 2015), endoplasmic reticulum stress
(Fujinami et al., 2012), and activation of apoptotic signaling cascades such as p38
63
mitogen-activated protein kinase (MAPK) and the Jun-kinase (JNK) pathways (J Wang
et al., 2003; Eshraghi et al., 2007).
Interestingly, we found a publications from 2017 that screened a portion of the
same Protein Kinase Inhibitor Library (EMD Calbiochem Kinase Inhibitor Libraries I and
II) (M. Ryals et al., 2017) on primary hair cells. The study used organs of Corti from
postnatal day 3-5 mice with a transgenic reporter (Pou4f3-GFP) dissected into small
micro explants and plated into 96 well plates. They were imaged daily after treatment
with either 200uM gentamicin for 72 hours or 200uM gentamicin with an experimental
drug for 72 hours. The results were graphed as the percentage of surviving cells over
time and identified 15 protective hits of which 10 were exact compounds also identified
in the iHC screen. The remaining 5 out of 15 compounds targeted pathways that also
came up in the iHC screen. The results of the published screen were a powerful proof of
concept that iHCs, at least to an extent, can be a useful in vitro tool in the search for
otoprotective drugs and can be used at a much higher throughput than the organ of
Corti micro-explants used in the publication.

Figure 4.4: Summary of
Otoprotective Hits from Micro-
Explant Paper
A. The table alphabetically lists the
15 hits from the Protein Kinase
Inhibitor Library identified as
otoprotective in organ of Corti
micro-explants (M. Ryals et al.,
2017). The paper screened 160
compounds in total. Of the 15 hits,
10 matched exactly with hits
identified in our screen on iHCs.
The other 5 hits were not exact
matches, but overlapped in
pathway targets with at least one
other hit from our screen on iHCs.

64
Interestingly, several of the hits listed from the micro-explant screening
publication as well as hits identified from our iHC screen also came up in our iHC
screen for otoprotectants against cisplatin. The same libraries were screened against
gentamicin and cisplatin. The overlap of hits and reoccurring pathways that arose from
the screens against two separate ototoxins suggests an overlap in the degenerative
mechanisms at play when hair cells and iHCs are treated with gentamicin or cisplatin.

Screen against cisplatin ototoxicity
We decided to focus our screening efforts on the more prevalent cisplatin
ototoxicity because gentamicin already has reduced use in clinics and there are better
alternatives in the antibiotic realm (J. Xie et al., 2011). Cisplatin is widely used for the
treatment of various forms of pediatric and adult solid tumor cancers (Dilruba &
Kalayda, 2016), however, cisplatin causes some degree of hearing loss in as many as
60-80% of patients receiving cancer treatment (Langer et al., 2013; Lanvers-Kaminsky
et al., 2017). This side effect of hearing loss if often the dose limiting factor when
patients are undergoing treatment. The ototoxic side effects of cisplatin have been
studied for several decades and it is well established that it causes dose-dependent
loss of primary hair cells with the outer hair cells being the most susceptible (Schaefer
et al., 1985; Laurell & Bagger-Sjöbäck, 1991). Cisplatin causes DNA adducts, which
block DNA synthesis and transcription in dividing cancer cells, but also has detrimental
effects on the non-dividing hair cells. The accumulation of toxic reactive oxygen species
(ROS) leads to the initiation of cell death through caspase activation (W. Liu et al.,
1998; García-Berrocal et al., 2007).
65
Figure 4.5: Survival Curve of
Induced Hair Cells Treated with
Cisplatin
A. Longitudinal survival tracking of
induced hair cells (iHCs) treated with
cisplatin. A serial dilution of cisplatin
ranging from 1mM to 31.25uM was
used on iHCs. (n=3 replicates each;
mean ± SEM)

Using the longitudinal survival tracking assay, we treated iHCs with a serial
dilution of cisplatin to test for dose-dependent iHC degeneration (Fig. 4.5). Using this
survival curve, we identified that 250uM cisplatin caused ~80% of iHCs to be lost by day
4. We proceeded to screen the Stem Select Library and Protein Kinase Inhibitor Library
on iHCs treated with 250uM cisplatin and 3uM library compound. Using the automated
iHC imaging and survival tracking described in Section 4.2 the results of the screen
were quantified. Due to the image quality issues in Screen 1 the automated
quantification was only able to give reliable cell counts from 138 out of 320 compounds
of the Stem Select Library (SSL) and 184 out of 327 compounds of the Protein Kinase
Inhibitor Library (PKI). Again, the wells were ruled out if the cell counts were not
deemed reliable at either the first day or the last day of the survival assay. In the second
screen we were able to get cell counts for 318 out of 320 compounds for SSL and 315
out of 327 compounds for PKI and the final results for both screens were interpreted
based on the survival of iHCs in the cisplatin treated wells compared to the wells that
received cisplatin and one compound.
66
The average survival of iHCs in response to 250uM cisplatin in Screen 1 was
16.1% (+/- 1.2) of starting cells surviving at Day 5 of the assay (Fig. 4.6A,C). In Screen
2 the average survival of iHCs in response to 250uM cisplatin dropped to 5.7% (+/- 3.9)
of starting cells surviving at Day 5 (Fig. 4.6B,D). Since the average survival with
cisplatin alone was quite variable across both screens the results were graphed
independently in order to normalize the change in survival seen when the library
compounds were present. The results of the screen were ranked for each compound
library in order to identify the targets with the most potent protective effects (Fig. 4.6).

Figure 4.6: Summary of Cisplatin Screening Results
A. Stem Select Library results from screen 1 showing percent of surviving cells at Day 5 for 138
compounds. B. Stem Select Library results from screen 2 showing percent of surviving cells at Day 5 for
318 compounds. C. Protein Kinase Inhibitor Library results from screen 1 showing percent of surviving
cells at Day 5 for 184 compounds. D. Protein Kinase Inhibitor Library results from screen 2 showing
percent of surviving cells at Day 5 for 315 compounds. A-D) Average survival with cisplatin alone is
marked by red dotted line. The three increasingly opaque boxes represent one, two and three standard
deviations from the average survival with cisplatin alone.

From the results of the screen we compiled a list of the compounds that had a
significant rescue of iHC survival. The results of Screen 1 and Screen 2 were kept
67
separate due to the large difference in average survival with cisplatin alone. The list of
significant compounds gave rise to three main categories of hits: 1) Anti-apoptotic hits,
2) Expected mechanistic hits, and 3) Novel hits. The compound hits from the iHC
screen were ranked based on survival increase of >1std, >2std and >3std from the
mean treatment with cisplatin alone. Screen 1 had 46 hits and Screen 2 had 42 hits with
iHC survival increase of at least >1std from cisplatin alone (Fig. 4.7).

Figure 4.7: Table of Identified Potential Otoprotectants Against Cisplatin
Screen 1 (yellow) and Screen 2 (green) hit compounds listed in order of most significant iHC rescue. The
average survival of iHCs treated with 250uM cisplatin alone at Day 5 of the survival assay was 16.1% (+/-
1.2) for Screen 1 and 5.7% (+/- 3.9) for Screen 2. The potential otoprotectant compounds listed are also
ranked based on their survival increase of >1, >2, >3, or >4 standard deviations above the mean survival
with cisplatin alone.

Screen 1 Compounds
% surviving
iHCs
Rank Screen 2 Compounds
% surviving
iHCs
Rank
1 PD 158780 38.46 >4 std VEGF Receptor 2 Kinase Inhibitor II 24.32 >3 std
2 Aurora Kinase Inhibitor III 30.51 Cdk2 Inhibitor III 23.39
3 Roscovitine 29.79 >3 std IKK-2 Inhibitor VI 23.20
4 KN-92 29.17 2,3-Butanedione 2-Monoxime 19.44
5 Flt-3 Inhibitor II 28.89 JNK Inhibitor II 16.67 >2std
6 Pterostilbene, Pterocarpus marsupium 28.57 CR8, (R)-Isomer 16.18
7 STAT3 Inhibitor VII 28.57 5-Iodotubercidin 16.13
8 SU1498 28.21 ZM 336372 15.34
9 PTP LYP Inhibitor 26.67 PD 174265 14.29
10 MK-2 Inhibitor III 25.81 >2std Ras/Rac Transformation Blocker, SCH 51344 14.29
11 SKF-86002 25.58 DMBI 14.14
12 JAK3 Inhibitor II 24.39 Triptolide, Tripterygium wilfordii 13.89
13 Src Inhibitor, PP1 24.00 Meriolin 2 13.71
14 IKK-2 Inhibitor VIII 23.91 AG 879 13.71
15 MNK1 Inhibitor 23.53 Cdk2/9 Inhibitor 13.64
16 JNK Inhibitor, Negative Control 23.26 EGFR Inhibitor III 13.27 >1 std
17 MEK1/2 Inhibitor 22.86 >1 std NF-κB Activation Inhibitor IV 13.24
18 Diacylglycerol Kinase Inhibitor II 22.73 Reversine 13.24
19 ErbB2 Inhibitor II 22.58 DNA-PK Inhibitor II 13.21
20 Daidzein 22.22 PD 158780 13.11
21 GSK-3b Inhibitor VIII 22.22 CR8, (S)-Isomer 12.87
22 NF449 22.22 ERK Inhibitor III 11.49
23 Resveratrol 22.22 Fascaplysin, Synthetic 11.48
24 JAK3 Inhibitor VI 21.95 Tie2 Kinase Inhibitor 11.29
25 5′-Deoxy-5′-methylthioadenosine 21.74 Isogranulatimide 10.96
26 DNA-PK Inhibitor III 21.62 Corticosterone 10.91
27 Casein Kinase I Inhibitor, D4476 21.43 GSK-3b Inhibitor II 10.87
28 PDGF Receptor Tyrosine Kinase Inhibitor III 21.43 Glucagon Receptor Antagonist II 10.81
29 Prionogenesis inhibitor, DAPH-12 21.28 Cdk1/2 Inhibitor III 10.78
30 PD 174265 21.15 Ro-31-8220 10.61
31 Src Kinase Inhibitor I 21.15 GSK-3 Inhibitor IX 10.47
32 Aurora Kinase Inhibitor II 21.05 PKR Inhibitor, Negative Control 10.47
33 GSK-3b Inhibitor II 21.05 BAY 11-7082 10.39
34 PTP Inhibitor V, PHPS1 21.05 IKK-2 Inhibitor VIII 10.38
35 Rho Kinase Inhibitor IV 20.93 PIM1/2 Kinase Inhibitor V 10.24
36 Manumycin A, Streptomyces parvulus 20.90 PI 3-Kg Inhibitor 10.20
37 NF-κB Activation Inhibitor V, 5HPP-33 20.75 p38 MAP Kinase Inhibitor VIII 10.16
38 CFTR-F508del Corrector, KM11060 20.69 Akt Inhibitor V, Triciribine 10.00
39 4-Cyano-3-methylisoquinoline 20.59 Cdk/Crk Inhibitor 10.00
40 Bcr-abl Inhibitor 20.41 FGF Receptor Tyrosine Kinase Inhibitor IV 9.92
41 JAK Inhibitor I 20.37 γ-Secretase Inhibitor VI 9.88
42 17β-Estradiol 20.34 BPIQ-I 9.59
43 BTS 20.31
44 VEGF Inducer, GS4012 20.21
45 IKK-3 Inhibitor IX 20.00
46 Rho Kinase Inhibitor III, Rockout 20.00
68
The anti-apoptotic hits are targets that typically come out of survival
screens as they generally block apoptotic cell signaling pathways. Some of the hits
included inhibitors of p53 and Akt. Previous studies have shown that p53 plays an
important role in the initiation of apoptosis in cisplatin treated hair cells and that
suppression of p53 using pifithrin-alpha conveyed significant protection to hair cells (M.
Zhang et al., 2003). Inhibition of Akt pathway has been shown to be anti-apoptotic in
several cell types, including hair cells treated with gentamicin (Chung et al., 2006; Q. Liu
et al., 2018). We expected to see some anti-apoptotic hits from the results of the iHC
screen, which showed significant iHC rescue by simply blocking apoptosis in a non-
specific way. We also saw compound hits that protected iHC against cisplatin in a more
cisplatin-specific way based on existing literature of the ototoxic mechanisms of
cisplatin. These expected mechanistic hits are compounds that included antioxidants,
calcium metabolism regulators and inhibitors of the Jak/STAT pathway. As mentioned
previously, reactive oxygen species are known to be a main culprit of cisplatin induced
ototoxicity. The accumulation of toxic reactive oxygen species (ROS) leads to depletion
of antioxidant enzymes in the cochlea (Rybak et al., 2000) and the initiation of cell death
through caspase activation (Clerici et al., 1996; W. Liu et al., 1998; García-Berrocal et
al., 2007; Hellberg et al., 2009). Endoplasmic reticulum stress and dysregulation of
calcium and mitochondrial metabolism in hair cells has also been shown to occur in
response to cisplatin treatment (Cheng et al., 2005; Zong et al., 2017; S.-J. Kim et al.,
2018). Lastly, the Jak/STAT pathway has been implicated in cisplatin induced ototoxicity
for several years now. Studies have found that the inhibition of a number of components
of the Jak/STAT pathway block anti-inflammatory cytokine response and enable
69
transcription of anti-apoptotic genes to confer hair cell protection against cisplatin (J.
Huang et al., 2015; Levano & Bodmer, 2015; Rosati et al., 2019). Across both screens
13 of the 88 hits targeted these anti-apoptotic pathways and known cisplatin pathways
of degeneration (Fig. 4.7). The identification of these mechanistic hits in the iHC screen
was reassuring that the iHCs could be recapitulating the known mechanisms of cisplatin
induced ototoxicity in vitro. Although further mechanistic studies remain to be done on
iHCs, these hits prompted us to dig deeper into the novel hits identified in the screen.
The novel hits fell largely in the category of protein kinase inhibitors. The hits
included inhibitors for VEGFR, FGFR, PDGFR, and EGFR. There are currently no
publications implicating VEGFR, FGFR and PDGFR in cisplatin ototoxicity. EGFR has
been shown to be upregulated in hair cells after aminoglycoside treatment, which may
suggest some additional overlap in the hair cell degenerative mechanisms between
aminoglycosides and cisplatin (Zine & de Ribaupierre, 1999). Across both screens, 7
hits were compounds that inhibited EGFR, 4 hits were inhibitors of VEGFR, 2 were
inhibitors of PDGFR and 1 was an inhibitor of FGFR (Fig. 4.7).

4.4 Kinase Inhibitor Compounds and FDA Approved Analogs
Following the large scale screen, we proceeded to order 8 compounds to validate
in more detail. The eight compounds were chosen due to their reproducibility across
both Screen 1 and Screen 2, as well as the relative potency of rescue. In the large
screen all of the compounds were tested at 3uM in combination with cisplatin at 250uM.
We proceeded to repeat the survival assay using the 8 compounds for validation and
performed an 8 point dose response curve to see which concentration was the most
70
effective at rescuing the iHC survival. The results of the 8 point dose response are from
only one experiment and need to be repeated in order to draw concrete conclusions.
Only one compound, JNK inhibitor II, reproduced a protective effect at 3uM
concentration. This JNK inhibitor showed the largest protection at 10uM and only had
marginal protective effects at 3, 0.3, 0.1 and 0.03uM concentrations (Fig. 4.8A).
Activation of the JNK pathway has been seen in hair cells in response to
aminoglycoside treatment (Francis et al., 2013) and cisplatin treatment (Nicholas et al.,
2017). The inhibition of JNK signaling in hair cells has previously been shown to be
protective against ototoxicity (J Wang et al., 2003; Levano & Bodmer, 2015).
Two of the other compounds, PD158780 and PD174265, are EGFR inhibitors.
Both of these inhibitors showed significant iHC survival rescue in Screen 1 and Screen
2. In the 8 point dose response validation PD158780 showed protection at a range of
concentrations from 0.3uM to 0.03uM with each concentration showing a comparable
level of rescue (Fig. 4.8B). PD174265 also showed protection at a similar range of
concentrations from 0.3, 0.03 and 0.01uM (Fig. 4.8C). Interestingly, PD174265 showed
a protective effect at the highest concentration of 30uM, but did not show a protective
effect at 0.1uM.
The Cdk2 Inhibitor III showed protection between a range of concentrations from
1uM to 0.03uM (Fig. 4.8D). Cdk2 Inhibitor III was chosen for validation due to its high
survival rescue in Screen 2. Screen 2 also had seven other hits that target cyclin
dependent kinases, Cdks. Cdks have been implicated in aminoglycoside ototoxicity (L.
Tao & Segil, 2015; M. Ryals et al., 2017) and more recently other groups had identified
a role for Cdk2 inhibitors in cisplatin ototoxicity (Hazlitt et al., 2018; Teitz et al., 2018).
71
The two IKK2 inhibitors showed protection at different concentrations. IKK2
Inhibitor VI showed a toxic effect at the higher concentrations of 30uM and 10uM, while
also showing protection at much lower concentrations ranging between 0.3uM and
0.03uM (Fig. 4.8E). IKK2 Inhibitor VIII showed protection at 30, 1 and 0.1uM, which did
not provide any obvious concentration dependence (Fig. 4.8F).
BDM is an abbreviation for 2,3-Butanedione 2-Monoxime. This compound is a
non-selective, reversible inhibitor of myosin ATPase and has also been demonstrated to
inhibit calcium channels. BDM showed iHC protection at 30, 1, 0.3 and 0.03uM (Fig.
4.8G). A potential hypothesis for how BDM may confer protection is by blocking entry of
cisplatin into the iHCs. Several forms of myosin are required for proper tip link
organization on the stereocilia and for opening of the mechanically gated ion channels.
The results for the GSK3b Inhibitor II were the least compelling because it
showed a protective effect at the highest concentration of 30uM and a much lower
concentrations of 0.3uM. These inhibitors were all chosen for further validation based
on their level of rescue and reproducibility of rescue. The GSK3b Inhibitor II showed a
significant rescue of iHCs in both Screen 1 and Screen 2, however the dose response
validation did not turn out as expected. Again, the results of the 8 point dose response
are from only one experiment with three wells per condition. Further validation needs to
be repeated in order to draw definitive conclusions about the compounds’ potential
protective effects in iHCs treated with cisplatin.

72

Figure 4.8: Protein Kinase Inhibitor Compound Validation
Survival of iHCs in the eight point dose response curve for 8 compounds. A. JNK Inhibitor II reproduced a
protective effect at 3uM, as well as 10, 0.3, 0.1 and 0.03uM. B. PD158780 showed protection at 0.3-
0.03uM. C. PD174265 showed protection at 30, 0.3, 0.03 and 0.01uM. D. Cdk2 Inhibitor III showed
protection between 1-0.03uM. E. IKK2 Inhibitor VI showed protection between 0.3-0.03uM. F. IKK2
Inhibitor VIII showed protection at 30, 1 and 0.1uM. G. BDM showed protection at 30, 1, 0.3 and 0.03uM.
H. GSK3b Inhibitor II showed protection at 30 and 0.3uM. (n=3 replicates each; mean ± SEM; Student’s
T-test *p<0.05, **p<0.01, ***p<0.001)

73
Since many of the drugs identified as novel hits from the large scale screen were
kinase inhibitors, we also ordered FDA approved kinase inhibitor analogs to test on
iHCs. A total of 20 FDA approved compounds were order for further testing (Fig. 4.9A).
The FDA approved kinase inhibitors were used in a survival assay on iHCs treated with
cisplatin at 250uM. The results of the survival assay were graphed as a percent of
surviving iHCs at day 5 and compared to the cisplatin treated control. The FDA
approved compounds were resuspended in DMSO at stock concentrations of 30mM
and used in the survival assay at 3uM. The respective amount of DMSO was added into
the well that received only cisplatin. Nine of the 20 compounds showed a protective
effect with variability in significance. Two of the compounds, Nintedanib and Sunitinib,
cause autofluorescent precipitation in the wells so no cell counts could be calculated.
Afatinib and Vandetanib cause significant decrease in iHC survival compared to
cisplatin alone.

Figure 4.9: FDA Approved Kinase Inhibitor Compound Testing
A. Full list of FDA approved kinase inhibitors tested. B. Results of the iHC survival assay. 250uM cisplatin
treatment was paired with 3uM kinase inhibitor treatment. P-values are listed for the nine compounds that
showed a protective effect on the survival of iHCs (n=3 replicates each; mean ± SEM; Student’s T-test
*p<0.05, **p<0.01, ***p<0.001, ***p<0.0001)
74

4.5 Antisense Oligonucleotides and Neutralizing Antibodies for Target Validation
The compounds we were able to validate in our iHC model still represented a
wide variety of kinase targets. All together the compounds listed above had the
following overlapping targets: VEGFR, EGFR, FGFR, and PDGFR. We decided to try
and narrow down the targets that may be conferring otoprotection in vitro by using both
antisense oligonucleotides (ASOs) and neutralizing antibodies (nABs). Both ASOs and
nABs bind to specific intracellular targets.
ASOs work by binding to mRNA in the cell and promoting its degradation, leading
to knocked down protein expression (Crooke & Bennett, 1996). ASOs can be designed
to target any specific mRNA nucleotide of interest, which has made them an easy tool to
study mRNA and protein knock down while also becoming a useful in potential
therapeutics (Agrawal, 1996; Geary, 2009). We designed ASOs to the various
overlapping targets of the FDA approved kinase inhibitors hoping to tease apart which
target was playing a protective role (Fig. 4.10A). The iHC survival assay used cisplatin
in conjunction with one of three different ASOs for each of the following targets: FGFR1-
3, VEGFR1-3, and PDGFR alpha and beta (Fig. 4.10A,B). The iHCs were pre-treated
with ASO at 9uM for 24 hours and then the survival assay began with the addition of
100uM cisplatin and re-addition of the respective ASO. The cisplatin and ASOs were
reapplied to the cells with each media change. Cisplatin was used at 100uM instead of
the previously described 250uM concentration because several experiments showed
increasingly drastic loss of iHCs at 250uM. We decided to lower the concentration such
any potential rescue was not outweighed by the higher cisplatin concentration. The
75
results of the assay show that for the most part the ASOs alone at 9uM do not have any
effect on iHC survival, however some of the ASOs did have a detrimental effect of iHC
survival (Fig. 4.10B). In the set of iHCs that received 100uM cisplatin as well as ASOs
at 9uM there was only very marginal improvement in iHC survival.
Since the results of the ASO survival assay did not show any compelling rescue
of iHCs, we decided to approach the various targets using neutralizing antibodies.
Neutralizing antibodies (nABs) have been recently developed for the treatment of
Human Immunodeficiency Virus (HIV), various forms of cancer, and to act as vaccines
(K.-H. Kim & Kim, 2017; Koyanagi et al., 2017; Marcandalli et al., 2019). They work by
recognizing and binding the proteins of interest to inhibit their activity and downstream
signaling. There were nABs available for VEGFR1, VEGFR2, FGFR2, FGFR3, and
PDGFRa (R&D Systems). Similar to the ASO experimental design, the iHCs were pre-
treated with nABs for 24 hours and then the survival assay began with addition of
100uM cisplatin and re-addition of the respective nAB. The cisplatin and nABs were
reapplied to the cells with each media change. The results were graphed as the percent
of surviving iHCs at day 5 of the survival assay (Fig. 4.10). The nAB survival assay was
performed twice and the average survival with 100uM cisplatin alone was 66.9% (+/-
4.8) (Fig. 4.10D,E). The results of the study showed that nABs had little to no protective
effects on the survival of iHCs. Due to the relatively high survival seen in iHCs treated
with cisplatin alone it is unclear how effective the nABs were in rescuing the iHC
survival. It remains to be repeated for more definitive results with reliable statistical
testing.
76
Figure 4.10: Induced
Hari Cell Survival
with ASOs and nABs
A. Table summarizing
FDA approved kinase
inhibitors that showed
significant iHC
survival rescue
against cisplatin.
Inhibitors are listed
alongside their
respecting kinase
targets. B. ASOs are
abbreviated by target.
Examples: F1-1 is
FGFR1 paired with
ASO number 1. V2-2
is VEGFR2 paired
with ASO number 2.
Pa-3 is PDGFRalpha
paired with ASO
number 3.
Quantification of iHC
survival with ASOs at
9uM and no cisplatin.
Bars represent mean
(+/- std) C. The same
abbreviations are
used as in Panel C.
Quantification of iHC
survival with ASOs at
9uM in combination
with 100uM cisplatin.
Bars represent mean
(+/- std) D.
Quantification of first
iHC survival assay
with nABs at various
concentrations (ng/ul)
in combination with
100uM cisplatin. E.
Quantification of
second iHC survival
assay with nABs at
various
concentrations (ng/ul)
in combination with
100uM cisplatin. D-E.
Red dotted line
represents the
average survival with
cisplatin alone. Bars
represent mean (+/-
std)

77
4.6 Drug Treatment of Organ of Corti Explant Cultures
In order to look at the effects of cisplatin and our identified potential protective
compounds in an intact cochlea we used an organ of Corti explant drug treatment
protocol previously established (Doetzlhofer et al., 2009; Rainey et al., 2016). Organ of
Corti explant cultures were dissected from postnatal day 1 (P1) mice. Once the intact
cochlea was isolated from each ear they were placed onto porous membranes (22um
pore size) floating on the surface of the culture medium. After 24 hours the organ
explants became attached to the membranes and media changes could be performed
to the culture medium below the membrane in order to introduce various treatment
conditions.
The treatment protocol consisted of 2 hours treatment with 80uM cisplatin
followed by a wash out and then kept in culture for 48 more hours. In the organs that
received the experimental FDA approved kinase inhibitors there was a 24 hour pre-
treatment with the inhibitor at either 3uM or 10uM, then the cisplatin was added for 2
hours at 80uM, lastly both drugs were washed out and the organs were kept in culture
for 48 more hours. Using this in vitro treatment system, we also tested a couple of
combinations of kinase inhibitors to test if there was an additive effect. Following
treatment, the organs were fixed and immunolabeled with anti-Pou4f3 antibody for
imaging and quantification (Fig. 4.11A). The organs were imaged as a whole and then
divided into three sections corresponding to apex, middle, and base. The images were
then sectioned into 150um segments and the number of Pou4f3 positive hair cells was
counted in each segment. The negative control with cisplatin treatment alone lost a
large number of hair cells compared to the untreated positive control (Fig. 4.11B).
78

Figure 4.11: Primary Hair Cell Protection in Organ of Corti Explants
A. Whole organ of Corti explants images after drug treatment. Explants were dissected from P1 mice and
cultured for 24 hours before treatment. The drug treatment protocol consisted of 24hrs pre-treatment with
kinase inhibitor (3uM or 10uM) or DMSO vehicle control. This was followed by 2hrs co-treatment with
80uM cisplatin. After treatment the drugs were washed off and the explants were cultured for an
additional 48 hours before fixation and imaging. B. Quantification of the number of hair cells per 150um
section of the organ of Corti explants. The cisplatin treated organs showed a base to apex loss of hair
cells compared to the untreated (UT) control. C. Quantification of the number of hair cells per 150um
section in the organ of Corti explants that were untreated (UT), treated with cisplatin alone, or treated with
cisplatin in addition to a kinase inhibitor at 3uM. D. Quantification of the number of hair cells per 150um
section in the organ of Corti explants that were untreated (UT), treated with cisplatin alone, or treated with
cisplatin in addition to a kinase inhibitor at 3uM. E. Summary of the quantification for the drug treatment
conditions with significantly increased hair cell numbers per 150um section. Several of the conditions
include kinase inhibitors at 3uM or 10uM, as well as a couple of combinations of kinase inhibitors at 3uM
each. (B-E. All quantification consists of n=3 organs per condition.)

79
The results of the organ of Corti explant treatments showed promising rescue of
hair cell survival in several conditions. The most significant rescue from single
compounds came from the combinations of 80uM cisplatin with either Gefitinib at 3uM
or Pazopanib at 10uM (Fig. 4.11C-E). The treatment group that received cisplatin at
80uM in combination with Ruxolitinib at 3uM showed a marginal rescue in hair cells
numbers, however when Ruxolitinib was paired with either Imatinib at 3uM or
Pazopanib at 3uM then the amount of hair cell rescue was dramatically improved (Fig.
4.11E). These results suggest that some of the compounds and pathways originally
identified in iHCs can also convey protection in primary hair cells of organ of Corti
explants.

4.7 Preliminary Modeling of In Vivo Cisplatin Ototoxicity
Following the organ of Corti explant experiments, we decided to begin testing our
compounds in vivo. There are existing techniques have been established to cause
cisplatin induced ototoxicity in laboratory models. Using the mouse model, a protocol
has been established in the literature that uses a 63 day treatment regimen (DeBacker
et al., 2020). The regimen consists of a cumulative dose of 48 mg/kg delivered as
4mg/kg/day for four consecutive days followed by 17 days of recovery and then
repeated two more times. In order to test the mice for cisplatin induced ototoxicity
several hearing tests can be performed followed by postmortem immunohistochemistry
to quantify the number of hair cells. The two hearing tests we decided to us in our model
are Auditory Brainstem Recordings (ABRs) and Distortion Product Otoacoustic
Emissions (DPOAEs). ABR is a technique based on measuring the electrically
80
generated response to an acoustic stimulus. DPOAE is the physiological measure
based upon detection of sounds generated by the functioning cochlear hair cells. Both
ABR and DPOAE testing are mandatory test in newborn screening at hospitals and can
also be performed on mice in a laboratory setting (Choo, 2002).
We started our in vivo cisplatin treatments using single bolus intraperitoneal
injections of cisplatin. Cisplatin given once at 15mg/kg cause no hearing loss, no hair
cell loss and the mice remained healthy. A higher dose of 20mg/kg also caused no
hearing loss, but the mice became increasingly unhealthy. At the highest bolus dose of
30mg/kg the mice were so unhealthy that they were sacrificed. The bolus intraperitoneal
delivery of cisplatin caused systemic toxicity in the mice prior to any signs of ototoxicity.
After testing bolus delivery, we tested the published protocol with a cumulative dose of
48mg/kg delivered over the course of 63 days. The results were not reproducible in our
hands as the mice were incredibly unhealthy showed no detectable hearing loss by
ABR and DPOAE testing.
The difficulty in causing cisplatin induced ototoxicity by intraperitoneal injection
led us to test a more localized delivery of the ototoxin. We began to use intratympanic
injections to deliver cisplatin. The procedure consists of puncturing the tympanic
membrane with a small syringe and delivering the cisplatin solution into the middle ear
allowing for passive diffusion of the drug to the inner ear. Some preliminary testing
allowed us to detect noticeable ABR threshold changes in mice treated with cisplatin via
intratympanic injection. In summary we found that a treatment with 30ug of cisplatin
delivered intratympanically caused detectable ABR threshold changes (Fig. 4.12). The
results of our finding suggest that 30ug was the optimal dose for intratympanic cisplatin
81
because we could still see surviving hair cells in the immunohistochemistry. The 60ug
treatment group had a complete loss of outer hair cells and was excessively toxic for a
study to test ototprotection. In recent data not shown we found that 20ug of cisplatin
delivered intratympanically created reproducible ABR threshold changes.

Figure 4.12: Summary of Results for
Intratympanic Cisplatin Treatment
A. ABR threshold measurements were made at 4,
8, 12, 16, 24, and 32 kHz. The graph shows the
threshold in dB at which and ABR response was
detected. The right ear (R) was treated with
cisplatin intratympanically while the left ear (L) was
treated with a vehicle control. A dose of 15ug of
cisplatin did not cause significant threshold changes
between pre and post-treatment recordings. B. A
does of 30ug of cisplatin did cause a significant
threshold change in the range of 12-24 kHz. The
points that are missing represent data that was not
able to be collected. C. A dose of 60ug of cisplatin
caused significant threshold changes in the range of
8-24 kHZ. (n=2 mice per treatment condition;
missing points or error bars were due to technical
difficulties acquiring a full data set from both mice
per treatment group).

4.8 Discussion
The discovery of treatments for
hearing loss, as well as screening for drugs
that can protect the sensory hair cells of
the inner ear from environmental stress,
such as chemotherapy, have been
hampered by the small number and
inaccessibility of the sensory cells of the
inner ear. The current study is an effort to alleviate this problem through the use of
direct lineage reprogramming of somatic cells to generate the large numbers of induced
82
hair cells needed for high-throughput screening. Taken together, we believe that iHCs
are a valuable source of in vitro cells to screen for otoprotectants.
We have shown that iHCs recapitulate sensitivity to known ototoxins gentamicin
and cisplatin. We demonstrate a method for high-throughput screening, that in the
future will allow the discovery of new otoprotectants, as well as gene-
therapy/regenerative medicine approaches to treat hearing loss. A large scale screen
was performed on iHCs treated with either gentamicin or cisplatin. The screen against
gentamicin resulted in numerous hits that fall into the known mechanisms of gentamicin
ototoxicity and they largely overlapped with hits identified in a study using organ of Corti
micro-explants. The screen against cisplatin also yielded numerous results that fit in
well with the established mechanisms of hair cell ototoxicity. Preliminary data of novel
targeted inhibitors suggest a role for either VEGFR, EGFR, FGFR and/or PDGFR in the
protection of iHCs in vitro. We tested these novel targets by using FDA approved kinase
inhibitors on iHCs and organ of Corti explants. Several of the kinase inhibitors showed a
protective effect on the hair cells which led to a more specifically targeted survival assay
using ASOs and nABs. Overall we have not found a clear indication of which target
could be most beneficial for in vivo testing. However, we have begun to establish an in
vivo model of cisplatin ototoxicity in mice. The modification of cisplatin delivery from
intraperitoneal to intratympanic has allowed us to bypass the systemic toxicity seen with
IP delivery as well as provided an internal contralateral control. The data summarized
above is preliminary and will require much more optimization.

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Chapter 5: Conclusions

5.1 Discussion

Reprogramming efficiency and the level of maturity of iHCs
As confirmation of the importance of the four transcription factors identified in our
unbiased reprogramming investigation, three of the four transcription factors, Atoh1,
Pou4f3, and Gfi1, were previously used to induce a hair cell like-fate from mouse ES
cells that had been partially differentiated into ectodermal organoids (Costa et al.,
2015). In our hands, these factors are able to activate reporter expression in MEFs from
Atoh1::nGFP mice, but yield a mixed population in which many GFP-positive cells failed
to upregulate the hair cell markers MyosinVIIa and Parvalbumin. We hypothesize that
the addition of Six1 increases reprogramming efficiency by pushing cells towards the
sensory ectodermal lineage. Six1 has been reported previously to promote competency
and progenitor-like state, as well as being an essential determinant of early sensory
inner ear lineage (W. Zheng et al., 2003; Ozaki et al., 2004; T. Zhang et al., 2017). Six1
directly targets the Atoh1 autoregulatory enhancer, as well as other essential hair cell
enhancers for Pou4f3 and Gfi1, with an increase in Six1 occupancy as hair cells
differentiate with in the sensory epithelium (J. Li et al., 2020). Additionally, Six1 has
been shown to play a role in the maturation of hair cells by regulating key genes
involved in the establishment of planar cell polarity and hair-bundle orientation (J. Li et
al., 2020). These studies support our findings that, in addition to Atoh1, Pou4f3 and
84
Gfi1, Six1 is an important upstream transcription factor in establishing a hair cell-specific
gene expression program in our direct reprogramming.
At perinatal times, only modest differences in gene expression are able to
distinguish inner and outer hair cells of the cochlea (Liu et al., 2014b), as well as
vestibular vs. cochlear hair cells (Burns et al., 2015). As a result, we are unable to
statistically classify iHCs as being more similar to one or another hair cell type based on
the bulk RNA sequencing. While many of the specialized gene characteristics of the
cochlear hair cells are clearly upregulated during reprogramming (Fig. 2), the iHCs fail
to activate other important genes essential for the functional maturation of the sensory
receptors in the cochlea, such as Prestin and Gata3 (Liberman et al., 2002; Bardhan et
al., 2019).
The imperfections of iHC reprogramming could have several causes. First, as
noted, we could be lacking additional, hair cell-type transcription factors to drive cells to
a more mature state. For example, the transcription factor Zfp503 identified in the initial
set of 16 factors for reprogramming does not get activated in response to the SAPG
reprogramming cocktail. We saw that the addition of Zfp503 to the core group SAPG
negatively impacted the activation of the Atoh1::nGFP reporter (Fig. S1), however it
remains to be investigated whether the addition of Zfp503 can confer a more mature
induced hair cell. Zfp503 is highly expressed in postnatal day 1 cochlear hair cells but
not in utricular hair cells suggesting it could play an important role in conferring subtype
specificity in the iHC reprogramming. Another bioinformatic analysis showed that some
of the relatively small group of distal elements that are present in P1 hair cells, but fail to
open in iHCs, associate with hair cell-specific genes that also fail to be robustly
85
upregulated during reprogramming (data not shown). This further suggests additional
factors, such as Gata3, that may improve the quality and maturity of the iHCs. In
addition, transcriptional characterization of older hair cells will allow for identification of
additional TFs, which may improve the reprogramming strategy. Second, our current
strategy relies on constitutive expression of the reprogramming factors, while
continuous expression of Atoh1 is known to halt hair cell maturation (Liu et al., 2012b;
Liu et al., 2014a). We plan to overcome this limitation by using inducible gene
expression constructs to drive reprogramming in the future. Finally, lack of organ-
specific context in vitro may not provide additional signals for maturation. In fact, we
demonstrate that iHCs co-cultured with dissociated organ of Corti cells, promoted
morphological and electrophysiological maturation of iHCs (Fig. 6). This functional
maturation may be mediated, at least in part, by improved trafficking and assembly of
ion channel subunits conferred by the co-cultures. We plan to examine the
transcriptional profile changes that occur in iHCs after co-culture in order to understand
what genes may be contributing to the morphological and functional maturation.
As has been documented in other reprogramming experiments (Wapinski et al.,
2013, 2017; Rhee et al., 2017), the chromatin landscape was also drastically remodeled
during reprogramming of MEFs to a hair cell-like state, readily opening de novo distal
elements to change their chromatin to resemble P1 cochlear hair cells. Similar to the
RNA sequencing results, there is a residual MEF signature of the chromatin landscape,
and the failure to close down these chromatin regions may be acting as a barrier for
more efficient and/or faithful reprogramming. In addition, the opening of a large number
of peaks inappropriately (peaks that occur in neither MEFs of primary P1 hair cells)
86
suggests that our cocktail may lack a transcriptional repressor, and that these
inappropriately open distal elements may provide some explanation for the low level of
inappropriate gene expression (Fig. 2,4).
Despite these pitfalls, common to most if not all reprogramming strategies
published to date (Ieda et al., 2010; Son et al., 2011; Treutlein et al., 2016; Kaminski et
al., 2016; Van Pham et al., 2017), induced hair cells are highly similar to the primary
counterparts on transcriptional and epigenetic levels, as well as functional levels,
making this the first report of direct reprogramming of the somatic cells into the highly
specialized sensory hair cells of the inner ear.

5.2 Significance

Direct reprogramming as a strategy for gene therapy and identification of genetic
causes of hearing loss
Recently the use of Anc80-based adeno associated vectors made inner ear gene
delivery feasible (Suzuki et al., 2017; Y. Tao et al., 2018; Tan et al., 2019). The proof of
concept studies have demonstrated functional recovery after administration of TMC1
gene therapy in animals carrying a mutation in the gene (Yoshimura et al., 2019). Yet,
the genetic causes of deafness are often unknown in patients and loss of hair cells
remains the leading contributor to hearing loss worldwide. We found that the SAPG four
transcription factor combination is significantly more effective at activating the
expression of hair cell genes MyosinVIIa and Parvalbumin in adult tail tip fibroblasts,
and postnatal (P8) supporting cells, when compared to Atoh1 alone (Figure 5). This
87
later demonstration highlights the potential of SAPG for gene therapy to treat hearing
loss, since residual supporting cells have been shown to be maintained in long
deafened mice (Oesterle & Campbell, 2009) and humans (Johnsson et al., 1981).
Additionally, primary human fibroblasts can be taken from patients for reprogramming
and patient specific studies opening the opportunity to study hearing loss mutations,
ototoxicity and regenerative medicine (Koch et al., 2011; S. M. Lim, Choi, Oh, Choi, et
al., 2016; S. M. Lim, Choi, Oh, Xue, et al., 2016; Shi et al., 2018; P. Huang et al., 2019;
Villanueva-Paz et al., 2019; M. Lee et al., 2019).

5.3 Future Directions

Reprogramming strategy vs other approaches
The evaluation of ototoxic compounds, and the identification of new
otoprotectants has been severely limited by the lack of sufficient numbers of
mammalian hair cells available for study. Although directed differentiation of ESCs
towards the sensory hair cells have been described (H. Li et al., 2003; Oshima et al.,
2010; K R Koehler et al., 2013; Ronaghi et al., 2014), these three-dimensional protocols
are time consuming and the outcomes are ESC-line dependent with variable efficiency
across protocols (Hiler et al., 2015; Mellough et al., 2019; Yoon et al., 2019). Direct
reprogramming allows for a much faster and more reliable approach to produce induced
hair cells and overcomes many of the short-comings of directed differentiation. We
demonstrate that iHCs are selectively vulnerable to gentamicin, and can be
reprogrammed and cultured in microtiter plate format, providing a robotic-imaging
88
platform for scalable monitoring of iHC survival and small-molecule screening.
Importantly, reprogramming does not require an iPSC intermediate, thus generating
iHCs from human patients with known or novel genetic mutations associated with
hearing loss will enable screening for new therapeutic targets and agents for the
treatment of genetic causes of deafness.
Overall, iHC technology can accelerate research studies investigating
mechanisms underlying hair cell hypersensitivity to environmental stress, as well as
contributing to the identification of otoprotective and regenerative strategies for reducing
the burden of hearing loss.
We hypothesize that the iHC model can provide the means to study important
biological questions, such as how hair cells in the cochlea become inner vs, outer hair
cells, mechanisms of degeneration in the many mutants that cause hearing loss, and
most importantly, why hair cells are so susceptible to degeneration and lack
regenerative capacity.

89
Appendix I: Materials and Methods

Mice
All experiments were performed at the University of Southern California. All animal
experiments were conducted according to the National Institutes of Health Guide for
Care and Use of Laboratory Animals. Protocols and experiments using animals were
approved by the Institutional Animal Care and Use Committee at the University of
Southern California.
Mice were housed with free access to chow and water and a 12hr day/night cycle.
Breeding and genotyping of the mice was performed according to USC standard
procedures.
Atoh1::nGFP (previously known as Math1-GFP) transgenic line was obtained from Jane
Johnson. Atoh1::nGFP transgenic mice were mated with wild type CD1 mice to obtain
litters for mouse embryonic fibroblast isolations and tail tip fibroblast isolations. Wild
type CD1 mice were used for harvesting wild type organs of Corti in co-culture
experiments. Lfng-CreERt2::tdTomato transgenic mice were used for harvesting organs
of Corti with lineage traced supporting cells.

Molecular cloning of viral plasmids and virus production
Complimentary DNAs for the 16 candidate transcription factors were each cloned into
viral expression vectors using the Gateway cloning (Invitrogen). Retroviral and lentiviral
plasmids were constructed into the entry vector pDONR221. Entry clones were
recombined into destination vectors via LR reaction into the pMXS-DEST (retro) FUWO-
tetO-DEST (lenti).
Plat-E cells and HEK293 cells were cultured in MEF medium (DMEM containing 10%
fetal bovine serum) and used to produce retroviruses and lentiviruses respectively. Cells
were transfected at 90% confluency with viral vectors containing genes of interest and
viral packaging plasmids (PIK-MLV-gp and pHDM for retrovirus; pPAX2 and VSVG for
lentivirus) using linear polyethylenimine (PEI) (Sigma-Aldrich). After 24 hours of
incubation with the plasmid DNA and PEI, the medium was replaced with fresh MEF
medium and the culture was continued. Supernatants from the transfected cells were
collected at 24 hrs and 48 hrs after medium replacement, filtered through 0.45um filters
and used immediately if generated from Plat-E or concentrated using Lenti-X
concentrator (Clontech) and stored at -80°C if generated from HEK293T.
MEFs were transduced by mixing virus with MEF media. The virus containing media
was removed from the MEFs after 24hrs and replaced with MEF media. The next day,
medium was switched to hair cell medium (HCM: DMEM/F-12 (supplier), N2 and B27
supplements (supplier), EGF (2.5 ng/ml), and FGF (5 ng/ml)).

MEF isolation
Mouse embryonic fibroblasts (MEFs) were obtained from E13-14 embryos taking care
to exclude contamination with other Atoh1 expressing tissues (kidney, brain, spinal cord
and webbing between digits). Tissue was minced with a razor blade and enzymatically
dissociated with 0.25% trypsin-EDTA for 30 minutes at 37°C. Trypsinization was
quenched by addition of MEF media (previously described). The isolated cells were
centrifuged (800g for 10min) and the pellet was resuspended in MEF media before
90
plating onto gelatin coated T75 tissue culture flasks. We found that plating 2 embryos
per T75 gave optimal survival post-dissection. The MEFs were cultured until confluency
was reached and then cryopreserved in liquid nitrogen using freezing media (1:1
mixture of MEF media and Freezing media (FM; 80% fetal bovine serum and 20%
DMSO)). MEFs were used without further passaging for reprogramming experiments.
All cells were tested for mycoplasma contamination and came back negative.

TTF isolation
Tail tip fibroblasts (TTFs) were obtained from 6 month old adult Atoh1::nGFP transgenic
mice. The tail was harvested from the sacrificed mouse by removing the skin and
dissecting the remaining tail tissue into small segments. After plating on gelatin coated
dishes, the tissue adhered to the dish and the expanding cells eventually covered the
dish. The TTFs were harvested for reprogramming by simply moving the segments to a
new dish and then collecting the remaining adherent cells. TTFs were cultured in DMEM
with 40% fetal bovine serum. TTFs were frozen in liquid nitrogen and used without
further passaging for reprogramming experiments. All cells were tested for mycoplasma
contamination and came back negative.

Merkel Cell Isolation
Skin was obtained from postnatal day 1 (P1) mice. Skin was incubated overnight at 4
degrees Celsius in Accutase. The epithelium (epidermis and hair follicles) was
separated from the underlying dermis with forceps and the epidermal cells were
dissociated with trypsin for 10 min at 37 degrees Celsius then dissociated to single cell
suspension. The freshly isolated epidermal cell suspension was then FACS purified to
sort for Atoh1::nGFP positive cells Merkel cells.

Gut Secretory Cell Isolation
Small intestines were obtained from adult mice. Intestinal villi were scraped away, crypt
epithelium was collected by shaking in 5mM EDTA for 50 minutes at 4 degrees Celsius,
and single cell suspensions were prepared by digestion in 4x TrypLE (Invitrogen) for 50
minutes at 37 degrees Celsius. The freshly isolated cell suspension was then FACS
purified to sort for Atoh1::nGFP positive cells gut secretory cells.

Cerebellar Granule Precursor Isolation
Cerebellums were obtained from postnatal day 1 (P1) mice. Tissue was minced and
enzymatically digested using 0.25% trypsin for 10 minutes at 37 degrees Celsius then
dissociated to single cell suspension. The freshly isolated cerebellar cell suspension
was then FACS purified to sort for Atoh1::nGFP positive cells cerebellar granule
precursors.

Primary hair cell culture
The primary hair cell culture was established by dissecting the organs of Corti from P1
transgenic Atoh1::nGFP mice. The cells were dissociated to a single cell suspension
and plated onto laminin coated tissue culture plates or cover slips.
The primary cultures from Lfng-CreERt2::tdTomato transgenic mice were done at
postnatal day 8 (P8). Lfng-CreERt2::tdTomato transgenic mice were injected with
tamoxifen at postnatal day 3 for lineage tracing of the Lfng+ supporting cell population.
91
The organs of Corti were harvested at P8, dissociated to single cell suspension in HCM
and the reprogramming factors were added to the cell suspension. The cells were then
plated onto laminin coated tissue culture treated cover slips. The virus containing media
was replaced after 24 hours with fresh HCM.
All primary cultures were plated using ROCK Inhibitor (Y-27632) (Sigma-Aldrich) for the
first 24 hours to help promote survival.

Co-culture of iHCs
Induced hair cells were FACS purified to obtain the Atoh1::nGFP positive cells and
collected in HCM. The primary organ of Corti was dissected from wild type mice at
E13.5 and enzymatically dissociated to a single cell suspension. The iHCs were then
mixed with the dissociated organs of Corti. The ratio of iHC to cells of the organ of Corti
was kept at about 1:33. This ratio was determined from the fact that the primary organ
of Corti contains approximately 3000 hair cells and upon dissociation gives
approximately 100,000 total cells. Co-cultures were grown on tissue culture treated
coverslips in wells of 24 well plates that had been coated with a 20ul drop of matrigel
(10% in HCM) at the center of the coverslip. The co-culture cell suspension was plated
as 30ul drops (2,500-3,000 cells per ul) in the center of the matrigel coated drop on the
cover slip. 12-24 hours after plating the drops the well was flooded with 500ul of HCM.
All cocultures were maintained in HCM.

Immunostaining
Cells for staining were washed with PBS and fixed using 4% paraformaldehyde (PFA) in
phosphate-buffered saline (PBS) for 15 minutes at room temperature. For
permeabilization and blocking the cells were incubated in PBST (0.1% Triton-X 100 in
PBS) with 10% fetal bovine serum for 2 hours at room temperature or overnight at 4°C.
After blocking, the cells were washed 3 times for 5 minutes with PBS. Cells were then
incubated with the primary antibody overnight at 4°C. Then the cells were washed with
PBS again before incubation with the secondary antibody for one hour at room
temperature or overnight at 4°C. Primary and secondary antibodies were diluted in
PBST with 10% serum. The DNA was stained with Hoechst diluted 1:1000 in PBS for 10
minutes at room temperature.

Antibodies
Anti-Parvalbumin (Sigma-Aldrich, catalog# P3088-100UL)
Anti-Espin (Gift from Hudspeth Lab)
Anti-MyosinVIIa (Proteus Bioscience, catalog# 25-6790)
Molecular Probes Phalloidin Rhodamine (Thermo Fisher Scientific, catalog# R415)
Phalloidin-iFluor 647 Reagent (Abcam Biochemicals, catalog# ab176759)
Anti-acetylated Tubulin (Sigma Aldrich, catalog# T7451-100UL)
Anti-Sox2 (Abcam, catalog# ab97959)

Imaging
Immunostaining images of adherent cell cultures were acquired on an LSM780 confocal
microscope using Carl Zeiss Zen blue/black software and processed using Adobe
Illustrator CS6 software. For quantification of reprogramming efficiency in adherent
cultures, images were acquired at 10x using the Molecular Devices ImageExpress. The
92
images were either processed manually using ImageJ software and the Cell Counter
plug in or automatically using SVCell RS (described below). Counts are represented as
reprogramming efficiency (percent of Atoh1::nGFP+ cells per well of 5000 MEFs
infected).

iHC detection and counting method
For each time frame, the customized time-lapse nuclei count recipe of SVCell RS is
applied to first reduce noise with image smoothing. Objects are detected by performing
background removal followed by adaptive thresholding. A size filtering is then applied to
remove objects that are either too large or too small. The count of remaining objects is
measured for the time point. Batch processing is available for applying the recipe to
multiple time-lapse images and saving results.

Flow cytometry preparations
Primary hair cells were harvested from Atoh1::nGFP transgenic mice. The cochleas
were incubated in 0.25% trypsin for 8 minutes and gently triturated to single cell
suspension. Media (DMEM with 10% FBS) was added to the dissociated cells and then
spun down at 1000 rpm for 5 minutes, resuspended in Hair Cell Media, passed through
a 70um cell strainer and then FACS-purified). The same procedure was used to FACS-
purify dsRED MEFs and Atoh1::nGFP+ cells from the reprogramming cultures.

RNA Sequencing
Total RNA was extracted from primary mouse hair cells (at postnatal day 1), mouse
iHCs (at day 14 post infection with reprogramming factors) and MEFs (at 14 days post
transduction with dsRed). For each replicate 20,000 FACS-sorted cells were used as
input for RNA-seq. Total RNA was extracted with either Quick-RNA Microprep kit (Zymo
Research), quantified by bioanalyzer and then processed for libraries with either QIAseq
FX Single Cell RNA Library Kit (Qiagen) or TruSeq RNA Library Prep Kit v2 (Illumina).
Specific sequencing parameters and instrument models were submitted with GEO
datasets. At least 3 replicates were collected for each condition and sequenced to a
depth of at least 20 million reads.
Reads were mapped to the mouse reference genome (Gencode Mm10v11) using
STAR. Read counts were quantified by RSEM. Only protein coding polyA tail transcripts
and autosomal genes were kept. Transcript counts were collapsed to gene counts.
Differentially expressed genes were identified using the DESeq2 package. Genes with a
log fold change threshold greater than 1 and adjusted P-value of less than 0.1 were
considered significant. Principle component analysis and unsupervised hierarchical
clustering of RNA-seq was performed using counts transformed by DESeq2’s
regularized logarithm (Rlog).

GO Analysis
Gene ontology analysis was performed on categorized gene sets using R clusterProfiler
package. GO results were visualized using the R enrichplot package.

GSEA Analysis
Gene Set Enrichment Analysis was performed using the R package fgsea. The Wald
statistic from the differential comparison of reprogrammed cells versus MEFs was used
93
to pre-rank genes for subsequent GSEA analysis. Gene sets representing unique
signatures for each Atoh1 positive cell-type were tested for enrichment in SAPG. To
determine signature gene sets for each Atoh1 cell type, only genes with a log2
foldchange greater than or equal to 2 with adjusted P-value less than 0.01 compared
between each profiled Atoh1 positive cell-type were used. Utricle and cochlear hair cells
were treated as a single cell type due to small number of unique genes at the postnatal
day 1 developmental stage used.

ATAC-seq
Cells were collected by FACS purification into cold PBS, and centrifuged 500 xg for 15
min. Cell pellet were resuspended with 50 ul transposition buffer consisting of 10mM
Tris-HCL pH8.0, 5 mM MgCl2, 10% DMF, 0.2% NP40, and home-made transposase
Tn5. Transposition was performed at 37 degree for 20 min. DNA was collected
immediately after transposition using Qiagen Mini-elute kit.
Encode pipeline was adapted for alignment and QC for ATAC-seq and ChIP-seq data.
Paired-end reads were quality trimmed with cutadapt (v1.18) and aligned to mouse
reference genome (Gencode Mm10v11) with bowtie2 (v2.2.6) using parameters -X2000
-mm –local. PCR duplicates were removed based on genomic coordinates. Only
autosomal chromosomes were selected and used for downstream analysis.
Specific sequencing parameters and instrument models were submitted with GEO
datasets.

ChIP-seq
Histone ChIP-seq protocol was developed by us based on μChIP-PCR protocol
published previously (Zlatka P Stojanova et al., 2015) with additional Tn5 tagmentation
step. Briefly, chromatin was cross-linked with 1% formaldehyde (Thermo Fisher) for 8
min, quenched with 125mM Glycine (Sigma) for 5 min at room temperature, sonicated
using the microtip of a High Intensity Ultrasonic Processor (Sonics & Materials,
Newtown, CT; amplitude 50, power 50) for 8 x 30 sec with 30 sec pause, tagmentated
with Tn5 transposase for 30 min at 37°C, incubated with antibody complexed with
Dynabeads Protein A (Thermo Fisher) overnight at 4°C, precipitated and washed three
times on magnetic rack, and finally PCR amplified with primers matching Tn5 adapters.
Encode pipeline was adapted for alignment and QC for ChIP-seq data. Paired-end
reads were quality trimmed to 36 bp with cutadapt (v1.18) and aligned to mm10
reference genome (Gencode Mm10v11) with STAR aligner using parameters end-to-
end and alignIntronMax=1 for DNA alignment. PCR duplicates were removed with
STAR. Only autosomal chromosomes were selected and used for downstream analysis.
Specific sequencing parameters and instrument models were submitted with GEO
datasets.

Chromatin analysis
ATAC peaks and H3K27ac peaks were identified using the R package chromstaR
(parameters: binsize=500bp, stepsize=250bp, mode=full). An equal number of reads
were randomly sampled for H3K27ac replicates (17.5 million) and ATAC replicates (15
million reads) as input for subsequent chromatin analysis. For peak calling, a false
discovery rate (FDR) cutoff of 0.01 and 0.001 was used for ATAC and H3K27ac
respectively and an RPKM cut off >2. Promoter regions were defined by 2 kb upstream
94
of 500 bp downstream of protein coding transcription start sites; all remaining regions
were considered distal. Enhancers were defined by cooccurrence of an ATAC peak and
H3K27ac peak at distal regions.
Differential ATAC peak analysis was performed between P1HC, SAPG iHCs, and MEFs
using chromstaR. Regions with a differential score of at least 0.999999 were considered
differentially accessible. Regions with differential score less than 1E-06 were
considered non-differentially accessible.
Deeptools was used to average replicates and calculate coverage tracks and for ATAC-
seq and ChIP-seq data for visualization on IGV and heatmaps.

Electrophysiology
Whole cell patch clamping was performed on three different preparations of cells. The
first preparation was iHCs in the monolayer culture of MEFs at D14-15 post infection
with SAPG. The second preparation was iHCs FACS purified and replated with
dissociated wild type organ of Corti. The third preparation was postnatal day 1 primary
hair cells from the dissociated transgenic Atoh1::nGFP organ of Corti. Preparations
were viewed at X630 using a Zeiss Axios Examiner D1 microscope fitted with Zeiss W
Plan-Aprochromat optics. Signals were driven, recorded, and amplified with an
Multiclamp 700B amplifier, Digidata 1440 board and pClamp 10.7 software (pClamp,
RRID:SCR_011323). Recording and cleaning pipettes were fabricated using filamented
borosilicate glass. Pipettes were fired polished to yield an access resistance between 4-
8MΩ. Each recording pipette was covered in a layer of parafilm to reduce pipette
capacitance. Recording pipettes were filled with standard internal solution. The contents
of the standard internal solution are (in mM): 135 KCl, 3.5 MgCl2, 3 Na2ATP, 5 HEPES,
5 EGTA, 0.1 CaCl2, 0.1 Li-GTP, and titrated with 1M KOH to a pH of 7.35 and an
osmolarity of about 300 mmol/kg. The voltage clamp protocol was performed by holding
the cell at -60mV followed by a stimulus of voltage steps (-120 to +70mV, by intervals of
10mV). The current response of the cell was recorded along with measures of ionic
current peak amplitudes, channel conductance values, and current activation kinetics.
Analysis of the data was performed using a combination of pClamp (pClamp,
RRID:SCR_011323), Matlab (MATLAB, RRID:SCR_001622), JMP (JMP,
RRID:SCR_014242), Origin Pro (OriginPro, RRID:SCR_015636), and Imaris (Imaris,
RRID:SCR_007370). pClamp software was be used to gather and quantify raw data
from electrophysiological recordings.

FM Lipophilic Styryl Dye uptake assay
Cells were incubated with 1uM FM 4-64FX, the fixable analog of FM4-64 (Life
Technologies, catalog# F34653). Prior to incubation the FM 4-64FX was resuspended
in ice cold HBSS at a 1mM concentration. The cells were incubated with a final
concentration of 1uM FM 4-64FX in ice cold HBSS for 30 seconds. After incubation, the
cells were rinsed in HBSS 3 times and then immediately fixed using 4% PFA in PBS for
15 minutes at room temperature. Using the software ImageJ, the images were filtered
on minimum background intensity in order to reduce the amount of background signal.
The filter measures the minimum signal intensity found in the image and applies the
filter to remove the minimum signal across the entire image. This image enhancement
was used uniformly on all images and all channels for each cell type.

95
GTTR uptake assay
Gentamicin sulfate salt (Sigma Aldrich catalog# G3632-5G, 50mg/ml in K2CO3, pH0)
and Texas-Red (Thermo Fisher Scientific catalog# T20175, 2mg/ml in dimethyl
formamide) were agitated together overnight to produce gentamicin-Texas Red
conjugate (GTTR). The mixture contained 4.4mls of 50mg/ml gentamicin (GT) with
0.6mls of 2mg/ml Texas Red (TR) to produce approximately 300:1 molar ratio of
GT:GTTR. A high ratio of gentamicin ensures a minimum of unbound Texas Red
molecules. The molecular weight of GT is 477.6 g/mol and the molecular weight of TR is
816.94 g/mol. The GTTR was made at a stock concentration of 100mM. The cells were
incubated with HCM containing 0.5mM or 1mM GTTR for three hours. After incubation
the cells were washed three times with PBS and then immediately fixed using 4% PFA
in PBS for 15 minutes at room temperature.

Ototoxicity assay
The cells were cultured (for primary hair cells) or reprogrammed (for iHCs and iMNs) in
a 96 well tissue culture plate. The primary hair cells were used 24hrs post dissociation
of the organ of Corti and plating. The iHCs and iMNs were reprogrammed for 14 days
prior to starting the survival assay. The gentamicin was dissolved in HCM at a
concentration of 100mM and subsequently diluted to 8mM in HCM. The stock at 8mM in
HCM was used for serial dilution to get the desired range of concentrations (8mM, 4mM,
2mM, 1mM, 0.5mM, 0.25mM, and 0.125mM). The control wells received only HCM.
The assay was performed over a period of 5 days. The HCM containing with or without
gentamicin was added to the cells on Day 1 and the cells were imaged every 24hrs after
the initial treatment. Subsequent media changes were performed every other day (Day
3). The HCM for gentamicin treated wells and control wells was made fresh for each
media change. The assay ended on Day 5. The Molecular Devices ImageExpress was
used for imaging the plate robotically every 24 hours. The images were taken at 10x.

Statistics
Sample numbers, experimental repeats and statistical test used are indicated in figure
legends. Unless otherwise stated, data presented as mean + SEM of at least three
biological replicates. Significance summary: p > 0.05 (ns), ∗p ≤ 0.05, ∗∗p ≤ 0.01, ∗∗∗p ≤
0.001, and ∗∗∗∗p ≤ 0.0001.

Data availability
RNA sequencing data sets:
P1 organ Atoh1::nGFP+ Hair cells (n=3)
P1 organ Atoh1::nGFP- populations (n=3)
dsRed transduced MEFs (n=3)
SAPG iHCs (n=3)
Merkel cells (n=3)
Cerebellar granule precursor cells (n=3)
Gut secretory cells (n=2)

ATAC sequencing data sets:
P1 organ Atoh1::nGFP+ Hair cells (n=2)
dsRed transduced MEFs (n=2)
96
SAPG iHCs (n=2)

ChIP sequencing data sets:
H3K27ac on P1 organ Atoh1::nGFP+ Hair cells (n=2)

Software Analysis PMID DOI IDENTIFIER
R version 3.6.1 (2019-07-05) General https://www.r-project.org
Bioconductor 3.9 General https://www.bioconductor.org/
GenomicRanges (v1.34.0) General 23950696 10.1371/journal.pcbi.1003118 https://www.bioconductor.org/packages/release/bioc/html/GenomicRanges.html
DEseq2 (v1.22.2) Differential 25516281 10.1186/s13059-014-0550-8 https://www.bioconductor.org/packages/release/bioc/html/DESeq2.html
biomaRt Package (v2.38.0) General 19617889 10.1038/nprot.2009.97 https://www.bioconductor.org/packages/release/bioc/html/biomaRt.html
STAR (v2.5.4b) Alignment 23104886 10.1093/bioinformatics/bts635 https://github.com/alexdobin/STAR
cutadapt (v1.18) Preprocessing https://doi.org/10.14806/ej.17.1.200. https://cutadapt.readthedocs.io/
FastQC(v0.11.6) Preprocessing https://www.bioinformatics.babraham.ac.uk/projects/fastqc/
ggpubr (v0.2) Visualization https://github.com/kassambara/ggpubr
ggplots2 (v3.1.0) Visualization https://github.com/tidyverse/ggplot2
fgsea GSEA https://doi.org/10.1101/060012 https://bioconductor.org/packages/release/bioc/html/fgsea.html
clusterProfiler GO Analysis 22455463 10.1089/omi.2011.0118 https://bioconductor.org/packages/release/bioc/html/clusterProfiler.html
org.Mm.eg.db Annotation http://bioconductor.org/packages/release/data/annotation/html/org.Mm.eg.db.html
ReactomePA Database 26661513 10.1039/c5mb00663e https://bioconductor.org/packages/release/bioc/html/ReactomePA.html
DOSE GO Analysis 25677125 10.1093/bioinformatics/btu684 https://bioconductor.org/packages/release/bioc/html/DOSE.html
chromstaR Chromatin state calls https://doi.org/10.1101/038612 https://bioconductor.org/packages/release/bioc/html/chromstaR.html
deeptools 3.0 Visualization 27079975 10.1093/nar/gkw257 https://deeptools.readthedocs.io/
ComplexHeatmap Visualization 27207943 10.1093/bioinformatics/btw313 https://bioconductor.org/packages/release/bioc/html/ComplexHeatmap.html
GenomicFeatures Annotation 23950696 10.1371/journal.pcbi.1003118 https://bioconductor.org/packages/release/bioc/html/GenomicFeatures.html
AnnotationDbi Annotation https://bioconductor.org/packages/release/bioc/html/AnnotationDbi.html
ChIPpeakAnno Annotation 20459804 10.1186/1471-2105-11-237 https://bioconductor.org/packages/release/bioc/html/ChIPpeakAnno.html
bedtools (v2.25.0) General 20110278 10.1093/bioinformatics/btq033 https://github.com/arq5x/bedtools2
SAMtools (v1.9) General 19505943 10.1093/bioinformatics/btp352 http://www.htslib.org/
Picard General https://github.com/broadinstitute/picard
RSEM Transcript quantification 21816040 10.1186/1471-2105-12-323 https://github.com/deweylab/RSEM
ENCODE-DCC/atac-seq-pipeline General https://github.com/ENCODE-DCC/atac-seq-pipeline
ENCODe-DCC/chip-seq-pipeline https://github.com/ENCODE-DCC/chip-seq-pipeline
97
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Abstract (if available)

Abstract The specialized mechanoreceptive hair cells in the mammalian inner ear are selectively vulnerable to numerous genetic and environmental insults. The lack of regenerative capacity leads to permanent hearing loss and vestibular dysfunction when they degenerate. The small numbers and difficult accessibility of primary hair cells limits the search for otoprotectants and regenerative initiatives. Direct cellular reprogramming could offer a robust route to the generation of sensory hair cells. Here we report a combination of four transcription factors (Atoh1, Pou4f3, Gfi1 and Six1) can induce cell fate conversion to a hair cell-like state. Induced hair cells (iHCs) exhibit robust similarities to primary hair cells at the transcriptome and epigenome level, as well as rudimentary stereocilia, uptake of styryl dyes, and hypersensitivity to gentamicin. Our demonstration of direct reprogramming to iHCs provides a platform to study causes of acquired and genetic hearing loss, high throughput drug screens to identify otoprotectants, and regenerative initiatives.

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Asset Metadata

Creator Menendez, Louise Madeleine (author)

Core Title Generation and characterization of an in vitro model of inner ear sensory hair cells using transcription factor mediated cellular reprogramming

School College of Letters, Arts and Sciences

Degree Doctor of Philosophy

Degree Program Neuroscience

Publication Date 07/23/2020

Defense Date 04/17/2020

Publisher University of Southern California (original), University of Southern California. Libraries (digital)

Tag cellular reprogramming,hearing loss,inner ear,Neuroscience,OAI-PMH Harvest,sensory hair cells,stem cells

Language English

Contributor Electronically uploaded by the author (provenance)

Advisor Segil, Neil (committee chair), Ichida, Justin (committee member), Kalluri, Radha (committee member), Ying, Qilong (committee member)

Creator Email lmenende@usc.edu,louise.menendez@gmail.com

Permanent Link (DOI) https://doi.org/10.25549/usctheses-c89-340116

Unique identifier UC11663280

Identifier etd-MenendezLo-8736.pdf (filename),usctheses-c89-340116 (legacy record id)

Legacy Identifier etd-MenendezLo-8736.pdf

Dmrecord 340116

Document Type Dissertation

Rights Menendez, Louise Madeleine

Type texts

Source University of Southern California (contributing entity), University of Southern California Dissertations and Theses (collection)

Access Conditions The author retains rights to his/her dissertation, thesis or other graduate work according to U.S. copyright law. Electronic access is being provided by the USC Libraries in agreement with the a...

Repository Name University of Southern California Digital Library

Repository Location USC Digital Library, University of Southern California, University Park Campus MC 2810, 3434 South Grand Avenue, 2nd Floor, Los Angeles, California 90089-2810, USA