Tissue Engineering for Ocular Surface Reconstruction

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Acta Universitatis Tamperensis 2130
Tissue Engineering for Ocular Surface Reconstruction Tissue Engineering for
Ocular Surface Reconstruction
Differentiation of human pluripotent stem cells
towards corneal epithelium
AUT 2130
Tissue Engineering for
Ocular Surface Reconstruction
Differentiation of human pluripotent stem cells
towards corneal epithelium
To be presented, with the permission of
the Board of the BioMediTech of the University of Tampere,
for public discussion in the auditorium Pinni B 1096,
Kanslerinrinne 1, Tampere, on 5 February 2016, at 12 o’clock.
Tissue Engineering for
Ocular Surface Reconstruction
Differentiation of human pluripotent stem cells
towards corneal epithelium
Acta Universitatis Tamperensis 2130
Tampere University Press
Tampere 2016
University of Tampere, BioMediTech
Supervised by
Associate Professor Heli Skottman
University of Tampere
PhD Tanja Ilmarinen
University of Tampere
Reviewed by
Professor Juha Holopainen
University of Helsinki
Docent Frederic Michon
University of Helsinki
The originality of this thesis has been checked using the Turnitin OriginalityCheck service
in accordance with the quality management system of the University of Tampere.
Copyright ©2016 Tampere University Press and the author
Cover design by
Mikko Reinikka
[email protected]
Acta Universitatis Tamperensis 2130
ISBN 978-952-03-0010-4 (print)
ISSN-L 1455-1616
ISSN 1455-1616
Acta Electronica Universitatis Tamperensis 1627
ISBN 978-952-03-0011-1 (pdf )
ISSN 1456-954X
Suomen Yliopistopaino Oy – Juvenes Print
Tampere 2016
441 729
Table of Contents
Introduction ..................................................................................................................... 15
Literature review .............................................................................................................. 17
The human cornea ................................................................................................. 17
Corneal development ........................................................................................ 19
Corneal epithelial renewal ................................................................................ 20
Limbal stem cell deficiency................................................................................... 23
Strategies for ocular surface reconstruction .................................................. 24
Cultivated limbal epithelial transplantation ................................................... 24
Simple limbal epithelial transplantation ......................................................... 26
Alternative cell sources ..................................................................................... 26
Human pluripotent stem cells .............................................................................. 28
Human embryonic stem cells .......................................................................... 28
Human induced pluripotent stem cells .......................................................... 29
Culture and characterization of hPSCs .......................................................... 30
Human PSC-derived corneal epithelium ....................................................... 32
Biomaterials for ocular surface reconstruction ................................................. 35
Aims of the study ............................................................................................................ 39
Materials and methods ................................................................................................... 41
Ethical considerations ........................................................................................... 41
Human tissue collection ........................................................................................ 41
Culture of hPSC lines ............................................................................................ 42
LESC differentiation and culture ........................................................................ 43
Cell characterization methods .............................................................................. 44
Quantitative PCR............................................................................................... 44
Immunofluorescence ........................................................................................ 45
Western blotting ................................................................................................ 46
Flow cytometry .................................................................................................. 46
Cell passaging and colony forming efficiency assay ..................................... 47
Cell proliferation assay ...................................................................................... 48
Comparative proteomics .................................................................................. 48
Fabrication and characterization of bioengineered matrices .......................... 49
Statistical analyses .................................................................................................. 49
Summary of the results ................................................................................................... 51
Differentiation of hPSCs towards LESC-like cells ........................................... 51
Cell morphology during differentiation ......................................................... 51
Gene expression during differentiation ......................................................... 52
Protein expression during differentiation ...................................................... 53
Self-renewal properties of hPSC-LESCs ....................................................... 55
Comparison of hPSC-LESCs with their native counterparts ......................... 56
Bioengineered matrices as carriers for hPSC-LESCs ....................................... 56
Discussion ........................................................................................................................ 59
Directed differentiation of hPSCs towards LESCs .......................................... 59
Characteristics of hPSC-derived LESCs............................................................. 61
Cell surface marker expression ........................................................................ 62
Self-renewal and proliferation ......................................................................... 63
Comparison with native corneal and limbal epithelia .................................. 64
Bioengineered collagen matrix as hPSC-LESC carrier .................................... 65
Future perspectives ................................................................................................ 66
Conclusions ...................................................................................................................... 69
Corneal epithelium, the outermost layer of the transparent and avascular cornea, is
renewed by tissue-specific stem cells, termed limbal epithelial stem cells (LESC).
These stem cells are located in specialized niches of the limbus, a narrow transition
zone between the cornea and sclera, which also serves as a physical barrier between
the clear avascular cornea and the vascularized conjunctiva. Extensive trauma to
the limbus, or LESC dysfunction as a result of certain chronic inflammatory
diseases may lead to limbal stem cell deficiency (LSCD), characterized by the
spread of the vascularized conjunctival tissue over the damaged ocular surface.
This vision-threatening condition varies in its severity, but commonly causes severe
symptoms and is difficult to treat. Conventional corneal transplantation is not a
feasible treatment option, as it only replaces the central corneal epithelium, and not
the damaged limbus. Therefore, various strategies aimed at replacing damaged
LESCs have been explored. Limbal transplantation, with or without ex vivo
expansion of LESCs, has shown great promise. However, patients suffering from
LSCD in both eyes are common and donor corneal tissue is scarce. Alternative
approaches for ocular surface reconstruction are therefore needed.
Human pluripotent stem cells (hPSC) are capable of virtually unlimited selfrenewal and can differentiate into any cell type, including LESCs and corneal
epithelial cells. To date, only a few studies have demonstrated successful
differentiation of corneal epithelial cells from hPSCs, and these methods rely on
the use of chemically undefined and xenogeneic culture components. Such factors
are subject to biologic variation and pathogen transmission, and thereby require a
thorough quality control to ensure the safety of transplantable cell populations.
Nevertheless, hPSC-derived LESCs could provide an alternative cell source for
cell-based therapy of severe ocular surface disorders. In addition, hPSC-derived
LESCs could be applied to studying corneal development and modelling corneal
tissue as a testing platform for in vitro drug development.
This dissertation aimed to investigate differentiation of LESCs and corneal
epithelial cells from hPSCs under conditions that would allow for a smooth
transition to clinical applications. A directed and efficient two-stage differentiation
method was developed, generating LESC-like cells capable of self-renewal and
terminal differentiation towards mature corneal epithelial cells. Differentiation was
carried out in close to chemically-defined and xeno-free conditions, and several
modifications could be explored further. Although there was a certain degree of
variation in differentiation efficiency between the studied cell lines, the method was
consistent and reproducible overall.
Thorough characterization of hPSC-derived cells is crucial to confirm their
authenticity. In this dissertation, several key characteristics of LESCs were
examined. It was demonstrated that hPSC-derived LESCs possess the appropriate
cell morphology, as well as gene and protein expression profiles. They were also
able to self-renew and proliferate in culture, and terminally differentiate towards
mature corneal epithelial cells. Moreover, hPSC-derived LESCs were similar to
native ocular surface epithelial cells, as verified using relative mass spectrometrybased proteomics.
Cell transplantation to the ocular surface would require a transparent,
mechanically durable, yet elastic carrier biomaterial. Currently, human amniotic
membrane is the gold-standard material commonly used to transplant donor
LESCs, despite its limitations. The final aim of this dissertation was to evaluate the
suitability of a bioengineered collagen matrix for the culture and transplantation of
hPSC-derived LESCs. The fabrication of this biomaterial is standardized, it is
suitable for clinical use, and has shown promise in earlier biocompatibility studies
using an animal model. In this study, the bioengineered collagen matrix supported
the attachment and growth of hPSC-derived LESCs in vitro, and could therefore be
applied to clinical use in the future.
In conclusion, this dissertation as a whole describes a novel tissue engineering
approach to ocular surface reconstruction. These results have contributed to
increasing the knowledge of hPSC differentiation towards LESCs, their
characteristics, and potential for use in cell-based therapy.
Sarveiskalvon epiteeli on läpinäkyvän ja verisuonettoman sarveiskalvon uloin
kerros, jonka uusiutumisesta huolehtivat kudosspesifiset limbaaliset kantasolut.
Nämä kantasolut sijaitsevat limbuksessa, sarveiskalvon ja sidekalvon rajapinnassa.
Limbus toimii myös fyysisenä esteenä verisuonettoman sarveiskalvon ja
verisuonitetun sidekalvon välillä. Laajat vauriot limbaalisella alueella tai limbaalisten
kantasolujen vajaatoiminta kroonisen taudin seurauksena saattavat johtaa
kantasolupuutokseen, missä sidekalvo verisuonineen leviää ja korvaa sarveiskalvon
epiteelin. Tämän sairauden oireet ja laajuus vaihtelevat hyvinkin paljon, mutta
siihen yleensä liittyy voimakkaita oireita ja sen hoito on hankala toteuttaa.
Perinteinen sarveiskalvon siirto ei ole käyttökelpoinen hoitomenetelmä näissä
sairauksissa, sillä siirre korvaa vain sarveiskalvon keskiosaa, eikä vaurioitunutta
limbusta. Näin ollen, viime vuosikymmenien aikana on tutkittu useita erilaisia
lähestymistapoja tuhoutuneiden limbaalisten kantasolujen korvaamiseen.
Limbussiirto on osoittautunut lupaavaksi hoitomenetelmäksi: vauriopaikalle
siirretään joko tervettä limbuskudosta tai laboratorio-olosuhteissa viljeltyjä
limbaalisia kantasoluja. Limbaalisten kantasolujen vajaatoiminta esiintyy usein
potilaan molemmassa silmässä, ja näiden potilaiden hoitoa rajoittaa
luovuttajakudosten riittämättömyys. Täten uusia hoitomenetelmiä tarvitaan
sarveiskalvovaurioiden hoitoon.
Ihmisen erittäin monikykyisillä kantasoluilla on lähes rajaton uusiutumiskyky, ja
ne kykenevät erilaistumaan kehon kaikiksi solutyypeiksi, mukaan lukien limbaaliset
kantasolut ja sarveiskalvon epiteelisolut. Tähän mennessä, vain muutamissa
tutkimuksissa on onnistuttu erilaistamaan sarveiskalvon epiteelisoluja ihmisen
erittäin monikykyisistä kantasoluista, ja nämä menetelmät hyödyntävät eläinperäisiä
Tämäntyyppisten tekijöiden käyttö altistaa erilaistusta biologiseen vaihteluun ja
taudinaiheuttajien siirtoon, joten tarkka laadunvalvonta on hyvin tärkeä
turvallisuuden takaamiseksi. Menetelmiä on tärkeä edelleen kehittää, sillä
kantasoluista erilaistetut limbaaliset kantasolut voivat tulevaisuudessa tarjota
vaihtoehtoisia hoitomenetelmiä vakaville sarveiskalvovaurioille. Tämän lisäksi näitä
soluja voidaan hyödyntää ihmisen sarveiskalvon kehityksen tutkimukseen ja
tautimallintamiseen, sekä lääkekehitykseen laboratorio-olosuhteissa.
Tämän väitöskirjatutkimuksen tavoitteena oli kehittää sarveiskalvon
epiteelisolujen erilaistamista olosuhteissa, jotka helpottaisivat menetelmän tuomista
kliinisiin sovelluksiin. Tutkimuksen aikana kehitettiin suunnattu ja tehokas
kaksivaiheinen erilaistusmenetelmä. Tällä menetelmällä erilaistetut limbaaliset
kantasolut osoittautuivat uusiutumiskykyisiksi, ja kykenivät kypsymään
sarveiskalvon epiteelisoluiksi. Menetelmä on lähes täysin vapaa eläinperäisistä tai
määrittelemättömistä aineista, ja sitä on mahdollista edelleen kehittää. Vaikka eri
kokonaisuudessaan osoittautui erittäin toistettavaksi.
Erilaistettujen solujen kattava karakterisointi on erittäin tärkeä niiden laadun
takaamiseen. Tässä väitöskirjatutkimuksessa tarkasteltiin useita solujen
ominaisuuksia. Erilaistetuilla soluilla oli oikeanlainen morfologia, ja ne ilmensivät
limbaalisille kantasoluille tyypillisiä geenejä ja proteiineja. Niillä oli kyky uusiutua ja
jakaantua viljelyssä, sekä kypsyä sarveiskalvon epiteelisoluiksi. Lisäksi,
massaspektrometriaan perustuva proteomiikkatutkimus osoitti, että erilaistetut
limbaaliset kantasolut ovat samanlaisia kuin ihmisen silmän pinnan solut.
Jotta solut olisivat siirrettävissä silmän pinnalle, tarvitaan läpinäkyvä,
mekaanisesti kestävä, mutta elastinen tukimateriaali. Tällä hetkellä käytetyin
biomateriaali luovutettujen limbaalisten kantasolujen siirtoon on ihmisen
amnionkalvo, sen monista puutteista huolimatta. Tämän väitöskirjatutkimuksen
viimeisenä tavoitteena oli tutkia kollageenista valmistetun tukimateriaalin
soveltuvuutta erittäin monikykyisistä kantasoluista erilaistettujen limbaalisten
kantasolujen kasvatukseen ja transplantaatioon. Kyseisen biomateriaalin valmistus
on standardoitu, se soveltuu kliiniseen käyttöön, ja sen on osoitettu olevan
kudosyhteensopiva. Tässä työssä biomateriaali näytti tukevan erittäin
monikykyisistä kantasoluista erilaistettujen limbaalisten kantasolujen kiinnittymistä
sekä kasvua, ja näin ollen voisi soveltua siirtomateriaaliksi soluterapiaa varten.
Kaiken kaikkiaan tässä tutkimuksessa on kehitetty uudenlainen
kudosteknologiaan perustuva menetelmä sarveiskalvovaurioiden hoitoon. Nämä
tulokset ovat edistäneet tietoa erittäin monikykyisten kantasolujen erilaistuksesta
sarveiskalvon epiteelisoluiksi, näiden solujen ominaisuuksista, sekä soveltuvuutta
List of original publications
This dissertation is based on the following original publications, referred to in the
text by their Roman numerals (I-III):
Mikhailova A, Ilmarinen T, Uusitalo H, Skottman H. Small-molecule
induction promotes corneal epithelial cell differentiation from human
induced pluripotent stem cells. Stem Cell Reports, 2014, 2:219-231.
Mikhailova A, Jylhä A, Rieck J, Nättinen J, Ilmarinen T, Veréb Z,
Aapola U, Beuerman R, Petrovski G, Uusitalo H, Skottman H.
Comparative proteomics reveals human pluripotent stem cell-derived
limbal epithelial stem cells are similar to native ocular surface epithelial
cells. Scientific Reports, 2015, 5:14684.
Mikhailova A, Ilmarinen T, Ratnayake A, Petrovski G, Uusitalo H,
Skottman H, Rafat M. Human pluripotent stem cell-derived limbal
epithelial stem cells on bioengineered matrices for corneal
reconstruction. Experimental Eye Research, 2016, 146:26-34.
The original communications included in this dissertation are reproduced with
permission of the copyright holders.
This dissertation contains unpublished data, indicated separately in the text.
List of abbreviations
Adenosine triphosphate binding cassette sub-family B
member 5
Adenosine triphosphate binding cassette sub-family G
member 2
Array comparative genomic hybridization
Age-related macular degeneration
Acellular porcine corneal matrix
Advanced therapy medicinal product
Polycomb complex protein BMI-1
Bone morphogenetic protein
Bovine pituitary extract
Bovine serum albumin
Myc proto-oncogene protein
CCAAT/Enhancer binding protein delta
Cluster of differentiation
Corneal epithelial cells
Colony forming efficiency
Cultivated limbal epithelial transplantation
Cultivated oral mucosal epithelial transplantation
European Bank for Induced Pluripotent Stem Cells
Extracellular matrix
1-[3-(dimethylamino)propyl]-3-ethylcarbodiimide methiodide
Epidermal growth factor
European Medicines Agency
Fetal bovine serum
Fibroblast growth factor
Fluorescein isothiocyanate
Forkhead box 1
Glyceraldehyde 3-phosphate dehydrogenase
Good manufacturing practice
Human amniotic membrane
Human dermal fibroblasts
Human embryonic stem cells
Human foreskin fibroblasts
Human induced pluripotent stem cells
Human leukocyte antigen
Human limbal fibroblasts
Human pluripotent stem cells
Intercellular adhesion molecule 1
Inner cell mass
Immortalized human corneal epithelial cell line
Isobaric tag for relative and absolute quantitation
In vitro fertilization
Krüppel-like factor 4
KnockOut Dulbecco’s Modified Eagle Medium
KnockOut Serum Replacement
Leukocyte common antigen
Limbal epithelial cells
Limbal epithelial stem cells
Limbal stem cell deficiency
Melanoma cell adhesion molecule
Mouse embryonic fibroblasts
Micro ribonucleic acid
Mesenchymal stem cells
Mucin 18
Non-essential amino acids
Octamer-binding transcription factor 3/4
Paired box 6
Phosphate buffered saline
Platelet endothelial cell adhesion molecule 1
Paired-like homeodomain 2
Quantitative polymerase chain reaction
Real architecture for 3D tissue
Reduced expression protein 1
Recombinant human collagen
Retinal pigment epithelium
Scanning electron microscopy
Simple limbal epithelial transplantation
Superoxide dismutase 2
(Sex-determining region Y)-box 2
Stage-specific embryonic antigen
Transient amplifying cells
Transcription factor 4
Terminally-differentiated cells
Transforming growth factor β
Tumor-related antigen
United Kingdom Stem Cell Bank
Universal protein knowledge base
Vascular endothelial cadherin
Corneal epithelium is the outermost layer of the transparent and avascular cornea.
It is constantly renewed by limbal epithelial stem cells (LESC), tissue-specific stem
cells located in specialized niches at the corneo-scleral junction, which also serve to
maintain a physical barrier between the clear cornea and the vascularized
conjunctiva (Dua & Azuara-Blanco, 2000; Ordonez & Di Girolamo, 2012).
Significant loss or dysfunction of LESCs due to acute trauma or various chronic
disorders can result in limbal stem cell deficiency (LSCD) – a painful and visionthreatening condition, which is difficult to treat using conventional methods
(Ahmad, 2012). Various tissue engineering approaches aiming at restoring the
ocular surface with the help of tissue-specific stem cells have been investigated
within the past two decades. Most importantly, cultivated limbal epithelial
transplantation (CLET) offers an advantage over a more traditional sector limbal
transplantation – autologous or allogeneic LESCs are obtained from a small biopsy
and expanded ex vivo prior transplantation (Baylis et al., 2011; Pellegrini et al.,
1997). Several other adult stem cell types have also been studied, but the main
disadvantage of such techniques is their limited capacity for self-renewal.
Human pluripotent stem cells (hPSC) can be used to generate LESCs or mature
corneal epithelial cells, providing a virtually unlimited supply of transplantable cell
populations (Ahmad et al., 2007; Hayashi et al., 2012). With the advance of human
leukocyte antigen (HLA) haplotype-matched hPSC banking, it may soon be
possible to generate readily-available hPSC lines with a minimal risk of immune
response for each individual patient (Zimmermann et al., 2012). However,
differentiation of LESCs often requires the use of biologically variable components
such as human amniotic membrane (hAM) or cell culture medium conditioned by
limbal or corneal fibroblasts. Ideally, the differentiation method needs to be robust,
reproducible, and free from xenogeneic and chemically undefined components.
Furthermore, differentiated hPSC-derived LESCs need to be extensively
characterized, preferably using high-throughput methods, in order to ensure they
possess the appropriate characteristics and behave in a way similar to that of their
native counterparts. In the future, hPSC-LESCs could be applied to cell-based
therapy of severe ocular surface disorders such as LSCD, as well as studying and
modelling the human cornea, or in vitro drug development.
In order to transplant hPSC-LESCs to the ocular surface, a carrier biomaterial is
needed to provide support for the cells. Optimally, the biomaterial needs to be
transparent, biocompatible and mechanically durable, yet sufficiently elastic so as to
follow the natural curvature of the cornea (Menzel-Severing et al., 2013). Collagenbased bioengineered matrices, typically reinforced using cross-linking or
compression, are currently widely researched for corneal reconstruction, mainly
due to the fact that collagen is the main component of the corneal stroma. Several
preclinical studies have demonstrated that collagen-based carriers are
biocompatible in vivo, despite being fabricated using collagen of animal origin (Chae
et al., 2015; Koulikovska et al., 2015; Petsch et al., 2014). The main benefit of using
fabricated bioengineered carriers, rather than readily-available scaffolds such as
hAM, is the possibility of standardized and reproducible production.
The main objectives of this dissertation were to develop an efficient and
reproducible method for differentiation of hPSCs towards LESCs and mature
corneal epithelial cells, to study the characteristics and functionality of hPSCderived LESCs and compare them with native ocular surface epithelial cells, and to
find a potential carrier for clinical applications of these cells.
Literature review
The human cornea
The cornea is located at the outer surface of the eye (Figure 1), surrounded by the
limbus, conjunctiva, and sclera (DelMonte & Kim, 2011). Its main functions are to
protect the eye from external environmental factors, including mechanical damage,
pathogens and ultraviolet (UV) light, while allowing accurate focusing of light to
produce a sharp image on the retina (Ghezzi et al., 2015). The human cornea is
approximately 0.5 mm thick and composed of three cellular layers (Figure 1).
Corneal epithelium is the topmost layer, consisting of four to six layers of nonkeratinized squamous epithelial cells. The epithelium is covered by a tear film,
which is secreted by the various types of lacrimal glands (e.g. major and minor
lacrimal glands and Meibomian glands) and conjunctival goblet cells (BolanosJimenez et al., 2015). Tear film provides protection from external noxious stimuli
and pathogens, while supplying various growth factors and cytokines important for
epithelial health, proliferation and repair (DelMonte & Kim, 2011). The epithelial
basement membrane is composed primarily of type IV collagen and several laminin
isoforms, with regional heterogeneity from central cornea to limbus to conjunctiva
(Torricelli et al., 2013). Bowman’s layer is described as an acellular condensation of
the anterior stroma, composed primarily of collagen types I, III, and V, and lacking
an apparent critical function in corneal physiology (Wilson & Hong, 2000). Corneal
stroma comprises roughly 80-90% of the entire corneal thickness. It consists
primarily of densely and regularly packed collagen fibrils (predominantly type I,
with smaller amounts of type V), interspersed with four proteoglycans (decorin,
lumican, keratocan and mimecan), arranged in a highly organized network to
maintain the optical transparency and mechanical strength of the tissue (Ghezzi et
al., 2015; Hassell & Birk, 2010). The normally quiescent stromal keratocytes reside
in the anterior stroma and maintain homeostasis by synthesizing collagen
molecules and glycosaminoglycans, as well as matrix metalloproteinases (DelMonte
& Kim, 2011; Meek & Knupp, 2015). The Descemet’s membrane is a thin acellular
layer composed of collagen fibrils, separating the stroma from the endothelium
(Bolanos-Jimenez et al., 2015). Corneal endothelium is a monolayer of hexagonal
cells, the main function of which is to provide the upper layers with nutrients from
the anterior chamber, and to keep the water content of the stroma at around 78%,
thereby maintaining its transparency (Bolanos-Jimenez et al., 2015; DelMonte &
Kim, 2011). The in vivo wound healing capacity of the corneal endothelium is
limited, although several studies have demonstrated that slow-cycling progenitor
cells exist at the endothelial periphery (Espana et al., 2015; He et al., 2012;
Whikehart et al., 2005).
Figure 1. Structure of the human eye, cornea and the limbus.
The limbus is a specialized narrow transitional zone, located circumferentially along
the periphery of the cornea, at its junction with the sclera and the conjunctiva
(Figure 1). It is generally accepted that the stem cells of the corneal epithelium,
known as limbal epithelial stem cells (LESC), and their immediate progeny reside
within limbal crypts in the palisades of Vogt (Dua et al., 2005; Grieve et al., 2015;
Shanmuganathan et al., 2007). Moreover, the limbus acts as a physical barrier to the
conjunctiva and its blood vessels (Osei-Bempong et al., 2013). LESCs will be
described in greater detail in later chapters.
Transparency, lack of vascularization and dense innervation are the main
structural features of the cornea. In the healthy eye, blood and lymphatic vessels do
not enter the corneal stroma, but rather surround it underneath the peripheral
limbal epithelium (Dhouailly et al., 2014). This unique property of the cornea is
referred to as angiogenic privilege.
Corneal development
In humans, the primitive bi-layered corneal epithelium first becomes apparent
between five and six weeks of gestation (DelMonte & Kim, 2011; Zieske, 2004). It
derives from the ocular surface ectoderm, a multipotent region of head ectoderm,
shortly after lens vesicle detachment (Collomb et al., 2013; Zhang et al., 2015). The
corneal stroma and endothelium, on the other hand, derive from migrating neural
crest cells (Dhouailly et al., 2014). By the seventh week of gestation, two waves of
neural crest migration into the space between the primitive epithelium and the lens
take place: the primitive endothelium forms during the first wave and the corneal
stroma forms during the second wave (Collomb et al., 2013; DelMonte & Kim,
2011). Upon opening of the eyelids, around 24 weeks of gestation, the primitive
epithelium starts to stratify and undergoes morphological changes, finally forming
the mature corneal epithelium with four to six distinct cell layers (Zieske, 2004).
Corneal development involves inductive interactions between the ocular surface
ectoderm and the underlying mesenchyme. In addition to corneal epithelium,
ocular surface ectoderm gives rise to lens and conjunctival epithelia, as well as the
epidermis of the eyelids (Gage et al., 2014; Zhang et al., 2015). The specification of
corneal epithelium development is guided by the inhibition of bone morphogenetic
protein (BMP) signaling, while lens epithelium formation is dependent on active
BMP signaling (Collomb et al., 2013). Shortly after lens vesicle detachment,
fibroblast growth factor (FGF) signaling is required for cell proliferation in the
ocular surface ectoderm, which then differentiates into corneal epithelium (Zhang
et al., 2015). Furthermore, inhibition of the canonical Wnt signaling pathway is
crucial for corneal epithelial commitment, and establishing its angiogenic privilege
(Dhouailly et al., 2014; Gage et al., 2014). Meanwhile, primitive corneal stroma
appears to be involved in stabilizing paired box 6 (PAX6) expression in the corneal
epithelium (Collomb et al., 2013). Overall, corneal epithelium is finally committed
during stroma formation in the seventh week of gestation, as determined by the
persistence of intrinsic PAX6 signaling (Collomb et al., 2013; Dhouailly et al.,
2014). Loss of PAX6, on the other hand, leads to skin-like differentiation,
indicating that PAX6 expression is a central event in corneal cell fate control (Li et
al., 2015). Although the main features of corneal development are relatively well
established, most studies are done using animal models, and the precise signaling
mechanisms behind human corneal morphogenesis remain largely unknown.
Corneal epithelial renewal
In the fully-developed human eye, corneal epithelial cells have an average lifespan
of seven to ten days (DelMonte & Kim, 2011). This continuous renewal is
explained by the XYZ hypothesis of corneal epithelial homeostasis: the
proliferation and movement of cells from the basal layers (X) and centripetal
movement from the periphery of the cornea (Y) replace cells lost from the central
corneal surface (Z), giving the equation X+Y=Z (Ahmad, 2012; Osei-Bempong et
al., 2013). The process of corneal epithelial renewal is schematically presented in
Figure 2. As mentioned earlier, the unipotent LESCs are primarily responsible for
corneal epithelial renewal, under both normal and wound healing conditions. They
reside in the specialized niches in the limbal region of the eye, providing a
protective environment for the LESCs, and helping maintain their undifferentiated
state (Ordonez & Di Girolamo, 2012). LESCs are normally slow-cycling, but have
a high proliferative potential (Dua & Azuara-Blanco, 2000). Only about 5% of all
cells at the human limbus are considered to be true LESCs, while the rest are
transient amplifying cells (TAC) at varying levels of maturity (Pellegrini et al.,
2013). Corneal TACs have a more limited proliferative potential, and are
considered to be committed on the pathway to replace the terminally-differentiated
cells (TDC) constantly shed from the corneal surface (Dua & Azuara-Blanco, 2000;
Pellegrini et al., 1999).
Although it is widely accepted that stem cells responsible for corneal epithelial
cell renewal reside exclusively in the limbus, several studies have recently
challenged this view. Serial transplantation of the mouse corneal epithelium was
shown to be possible, implying on existence of progenitor cells in the central
cornea as well as the limbus (Majo et al., 2008). Additionally, an observational study
of five LSCD patients revealed clear central corneal islands despite the lack of
functional LESCs (Dua et al., 2009). Nevertheless, these findings remain
outnumbered by the extensive evidence in support of the limbus as the principal
source of corneal epithelial stem cells in humans. More specifically, centripetal
migration of corneal epithelial cells has been documented through lineage tracing
in mice (Amitai-Lange et al., 2015; Di Girolamo et al., 2015), LESCs have been
shown to possess slow-cycling characteristics and superior in vitro proliferative
capacity (Figueira et al., 2007; Pellegrini et al., 1999), and most importantly, there
are numerous clinical reports of LESCs restoring the corneal epithelium once
transplanted to the damaged ocular surface (Baylis et al., 2011; Zhao & Ma, 2015).
Overall, although it is likely that some early-stage progenitor cells are in fact
distributed throughout the basal layers of the corneal epithelium, the limbus
appears to be crucial for long-term corneal epithelial renewal.
Figure 2. Corneal epithelial renewal. LESCs and their immediate progeny reside within limbal
crypts, where they self-renew and give rise to TACs, which move towards the central cornea
and differentiate to replace TDCs lost from the ocular surface.
Despite the ongoing efforts to find a molecular marker specific for LESCs,
distinguishing between true LESCs and their immediate progeny remains a
challenge. Therefore, it is currently common practice to assess a wider molecular
signature of cell populations, selected from a growing set of positive and negative
makers (Table 1). One of the most widely used LESC markers is the nuclear
transcription factor p63, which appears to regulate corneal epithelial renewal
through control of cell proliferation, and was demonstrated to be functionally
significant and important for graft survival in a clinical trial (Pellegrini et al., 2001;
Rama et al., 2010). More than six isoforms of p63 have been described, and
ΔNp63α is predominantly expressed within the ocular surface epithelia (Kawasaki
et al., 2006; Robertson et al., 2008). Unlike nuclear transcription factors, cell
surface proteins such as adenosine triphosphate (ATP)-binding cassette sub-family
B member 5 (ABCB5) or ATP-binding cassette sub-family G member 2 (ABCG2)
enable antibody-based sorting and enrichment of heterogeneous cell populations
for clinical use (Ksander et al., 2014).
Table 1. Expression and localization of widely used corneal and limbal epithelial markers.
The following references were used: Bian et al., 2010; Figueira et al., 2007; Joe & Yeung, 2014;
Ksander et al., 2014; Lu et al., 2012; Lyngholm et al., 2008; Nieto-Miguel et al., 2011; Notara et
al., 2010; Qu et al., 2015; Schlotzer-Schrehardt & Kruse, 2005.
Corneal cell type
Limbal epithelium
Cell membrane
Limbal epithelium
Cell membrane
Limbal epithelium, basal corneal epithelium
Limbal epithelium
Limbal epithelium
Connexin 43
Basal corneal epithelium
Gap junctions
Cytokeratin 3
Mature corneal epithelium
Cytokeratin 12
Mature corneal epithelium
Cytokeratin 14
Limbal epithelium
Cytokeratin 15
Limbal epithelium
Cytokeratin 19
Limbal epithelium
Desmoglein 3
Limbal epithelium
Limbal epithelium
Integrin α9
Limbal epithelium
Cell surface
Integrin β1
Limbal epithelium
Cell surface
Mature corneal epithelium
Limbal epithelium
Mature corneal epithelium
Limbal epithelium
Limbal and corneal epithelia
Limbal epithelium
Cell membrane
Limbal epithelium
Limbal epithelium
Limbal epithelium
Limbal epithelium
Limbal epithelium
Cell membrane, secreted
Abbreviations: ABCB5, ATP-binding cassette sub-family B member 5; ABCG2, ATP-binding cassette
sub-family G member 2; BMI-1, polycomb complex protein BMI-1; C/EBPδ, CCAAT/Enhancer binding
protein delta; PAX6, paired box 6; SOD2, superoxide dismutase 2; TCF4, transcription factor 4
Recently, several microRNAs (miRNA), a type of non-coding regulatory RNA
involved in modulating post-transcriptional gene expression, have been identified
in the corneal and limbal epithelia. Specifically, miR-184 was detected during early
eye development in the mouse, and in a pluripotent stem cell model, demonstrating
its importance in corneal lineage specification (Shalom-Feuerstein et al., 2012). In
addition, miR-103 and miR-107 were found to be preferentially expressed in the
basal limbal epithelium of mice, regulating the stem cell characteristics of LESCs
and contributing to their slow-cycling phenotype (Peng et al., 2015). Finally,
miR-143 and miR-145 were found to be expressed predominantly in the limbal
epithelium of the human donor corneas, and miR-145 was shown to be involved in
regulating corneal epithelial formation and maintenance (Lee et al., 2011).
Nevertheless, more research is needed to fully understand the complex signaling
cascades involved in LESC maintenance and self-renewal.
Limbal stem cell deficiency
Corneal diseases are major causes of blindness especially in developing countries,
where they are second only to cataract (Whitcher et al., 2001). Ocular surface
diseases where there is either a significant loss or dysfunction of LESCs are
collectively referred to as limbal stem cell deficiency (LSCD). These disorders are
characterized by disruption of corneal epithelial renewal and loss of barrier
function of the limbus, leading to the invasion of conjunctival epithelium onto the
cornea (Osei-Bempong et al., 2013). Consequently, patients with LSCD suffer
from recurrent epithelial defects, persistent pain and inflammation, loss of corneal
clarity, decreased visual acuity, and in severe cases blindness (Ahmad, 2012). Each
year, corneal vascularization and opacity have been estimated to cause blindness in
eight million people worldwide – roughly 10% of total cases (Whitcher et al., 2001).
The severity of LSCD varies depending on the extent of the injury – it can be
partial or total, and either unilateral or bilateral (Dua & Azuara-Blanco, 2000). In
addition to the damaged epithelium, the stroma is often involved in LSCD cases.
Complete wound healing of a stromal injury can take months or even years, and
thus affects corneal clarity long after primary wound healing has occurred
(DelMonte & Kim, 2011).
There are many known causes of LSCD: hereditary diseases such as aniridia,
inflammatory disorders including ocular cicatricial pemphigoid and StevensJohnson syndrome, prolonged contact lens wear, extensive cryotherapy or surgery,
chemical burns and other acute trauma of the ocular surface (Ahmad, 2012; OseiBempong et al., 2013). There are also idiopathic cases of LSCD, meaning that there
is no known cause for the disorder. For each patient, the etiology of LSCD and its
degree of severity need to be taken into consideration when deciding on a
treatment plan.
Strategies for ocular surface reconstruction
For partial LSCD not affecting the patient’s vision, topical lubricant drops and
anti-inflammatory drugs could help sufficiently reduce ocular discomfort and
nurture the remaining viable LESCs, and no surgical intervention may be necessary
(Dua et al., 2010). However, in more severe cases, surgery is required to first clear
the ocular surface from the vascularized conjunctival epithelium, and then
transplant healthy tissue. Patients with LSCD are generally poor candidates for
conventional corneal transplantation, as it does not permanently reconstitute the
function of the limbus (Dua & Azuara-Blanco, 2000; Pellegrini et al., 2013). Sector
limbal transplantation of autologous or allogeneic tissue has been reported, where a
limbal biopsy is taken from a healthy donor eye (patient’s own, living related
donor, or cadaveric) and transplanted to the injured eye (Dua & Azuara-Blanco,
2000; Dua et al., 2010; Kenyon & Tseng, 1989). Autologous grafts generally give
the best results, but are only available in cases of unilateral LSCD. Moreover, the
technique is limited by the need for fairly large amounts of donor tissue, putting
the healthy donor eye at risk (Osei-Bempong et al., 2013). Therefore, other
strategies have been developed and are beginning to gain popularity. The most
commonly implemented and widely studied techniques for treatment of partial and
total LSCD are summarized in a flow diagram at the end of this chapter (Figure 3).
Cultivated limbal epithelial transplantation
About 18 years ago, the first successful clinical use of cultivated limbal epithelial
transplantation (CLET) was reported, using autologous limbal tissue obtained from
a 1-2 mm2 biopsy expanded ex vivo to treat two patients (Pellegrini et al., 1997).
Subsequently, the technique has become fairly widely used worldwide, and the
overall success rate of the procedure is around 76%, based on the data gathered
over a period of 13 years from a total of 583 patients (Baylis et al., 2011). LSCD is
a highly heterogeneous disease with respect to cause and severity, and the methods
for the isolation and cultivation of LESCs vary greatly among laboratories, making
it difficult to compare the efficacy of each technique (Baylis et al., 2011; Zhao &
Ma, 2015). Typically, limbal biopsies are subjected to either mechanical disruption
(explant culture) or enzymatic digestion (single-cell suspension), followed by 2-3
weeks of culture on mitotically-inactivated mouse fibroblast feeder cells or a
transplantable carrier, such as human amniotic membrane (hAM), fibrin, or contact
lens (Joe & Yeung, 2014). Most of the available culture protocols rely on the use of
xenogeneic and undefined culture components, although efforts are being made to
eliminate such potential contaminants and standardize the techniques (Kolli et al.,
2010; Shortt et al., 2008; Zakaria et al., 2014). On the other hand, some laboratories
continue using fetal calf serum and mouse feeder cells, provided they are clinicalgrade and good manufacturing practice (GMP) certified (Pellegrini et al., 2013). In
early 2015, the first advanced-therapy medicinal product (ATMP) containing stem
cells was granted conditional approval by the European Medicines Agency (EMA).
This product, Holoclar (Holostem Terapie Avanzate, Modena, Italy), consists of
autologous LESCs expanded ex vivo and transplanted to treat severe LSCD caused
by chemical or thermal burn (Dolgin, 2015; http://www.ema.europa.eu). Notably,
expansion of LESCs is carried out on mouse feeder cells in the presence of animal
serum, thereby requiring thorough quality assessment to guarantee the safety of the
product prior transplantation. Most importantly, Holoclar is not applicable to
treating bilateral LSCD, as it requires a biopsy of healthy autologous limbus.
Although CLET alone may stabilize the ocular surface, the patient’s vision
often remains poor due to corneal neovascularization and stromal scarring, making
it necessary to perform corneal transplantation later on (Joe & Yeung, 2014;
O'Callaghan & Daniels, 2011; Zakaria et al., 2014). Additionally, localized
conjunctival invasion of the cornea is sometimes observed within the first year
after transplantation (Kolli et al., 2010). Surprisingly, there appears to be no
significant difference in success rates between autologous and allogeneic CLET,
while the cause of LSCD does have an effect on graft survival (Baylis et al., 2011).
There are several factors that may contribute to CLET outcome, regardless of
whether autologous or allogeneic LESCs are used. Firstly, LESC cultures used for
CLET are transplanted as heterogeneous cell populations, and therefore likely
contain a mixture of LESCs, TACs, as well as mature corneal and conjunctival
epithelial cells. This may contribute to graft failure, as it is believed that only true
LESCs are capable of long-term tissue homeostasis (Joe & Yeung, 2014). Secondly,
clinical evidence shows a correlation between p63 expression and CLET success –
grafts containing less than 3% of p63-positive cells were shown to have a
significantly higher risk for failure (Rama et al., 2010). Finally, the mechanisms
through which CLET functions remain unclear – the transplanted cell populations
either replace the patient’s damaged or lost LESCs, or stimulate them to reactivate
(O'Callaghan & Daniels, 2011). Contradictory evidence exists, with a clinical study
showing that donor LESCs are observed for up to 3.5 years after allogeneic limbal
tissue transplantation (Djalilian et al., 2005), yet a different study demonstrating an
absence of donor cells nine months after allogeneic CLET (Daya et al., 2005).
Overall, it may be that the mode of action depends on LSCD etiology, the extent
of damage and whether the LESC niche environment is compromised.
Simple limbal epithelial transplantation
In 2012, a novel surgical technique for the treatment of unilateral LSCD was
described. Similarly to CLET, a small limbal biopsy (2 mm2) is first obtained from
the healthy eye. However, rather than expanding LESCs ex vivo, the tissue is
divided into 8-10 small pieces, which are then distributed evenly over a sheet of
hAM placed on the corneal surface, secured with fibrin glue (Sangwan et al., 2012).
The technique, termed simple limbal epithelial transplantation (SLET), is therefore
a relatively affordable single-stage procedure, not requiring a specialized GMP
certified facility for cell culture. So far, only a small number of patients have been
treated with SLET, although the clinical outcomes show promise (Amescua et al.,
2014; Bhalekar et al., 2013; Sangwan et al., 2012; Vazirani et al., 2013). The longterm efficacy of the technique is yet to be demonstrated.
Alternative cell sources
LSCD more commonly presents bilaterally, rather than unilaterally (Utheim, 2015).
In cases of bilateral and total LSCD, there is not enough autologous limbal tissue
for CLET, and alternative autologous cell sources may be considered. In 2004,
cultivated oral mucosal epithelial transplantation (COMET) was first introduced in
two separate studies, each demonstrating successful treatment of four LSCD
patients (Nakamura et al., 2004; Nishida et al., 2004). Subsequently, many other
clinical studies have been reported (Hirayama et al., 2012; Kolli et al., 2014;
Utheim, 2015). Although the definition of clinical success varies among different
studies, the overall success rate of COMET is around 72%, similar to that of
CLET (Utheim, 2015). Complications include peripheral corneal
neovascularization, corneal epithelial defects, increased ocular pressure and
infections (O'Callaghan & Daniels, 2011; Utheim, 2015).
In addition to oral mucosal epithelium, several other autologous sources of
epithelial stem cells have been explored in hopes of finding an alternative to
CLET. Conjunctival epithelium is the only other tissue besides oral mucosal
epithelium which has been tested in human patients (Ricardo et al., 2013). Finally,
many pre-clinical studies in search of a novel cell therapy for LSCD are in progress
using mesenchymal stem cells (MSC) of various origin (Holan et al., 2015;
Katikireddy et al., 2014; Nieto-Miguel et al., 2013; Tsai et al., 2015), corneal stromal
stem cells (Hashmani et al., 2013), hair follicle stem cells (Blazejewska et al., 2009),
umbilical cord lining stem cells (Reza et al., 2011) and nasal mucosal epithelium
(Kobayashi et al., 2015a). The main disadvantage of using somatic cells or adult
stem cells is their limited capacity for self-renewal, resulting in limited yields of
transplantable cells. Therefore, more extensive studies are needed to determine
whether these cell types are capable of self-sustaining corneal reconstruction. It
could also be beneficial to investigate alternative cell sources with a higher potential
for proliferation and self-renewal, such as human pluripotent stem cells (hPSC).
Figure 3. Strategies for ocular surface reconstruction, depending on the severity of the
disorder. Representative images of partial and total LSCD modified from Zakaria et al., 2014.
Human pluripotent stem cells
Stem cells are identified by two characteristics: they have the capacity to self-renew,
and differentiate into specific cell lineages (Fortier, 2005). Human stem cells are
typically classified by their differentiation potential. Unipotent and multipotent
stem cells are found in adult tissues: unipotent stem cells are only capable of
differentiating into one other cell type, while multipotent stem cells can
differentiate into several cell types. LESCs are an example of unipotent stem cells,
as described earlier in Chapter 2.1.2. Hematopoietic stem cells, on the other hand,
are an example of multipotent stem cells, giving rise to all types of blood cells
(Fortier, 2005). Pluripotent and totipotent cells are only found in the fertilized
embryo, and have a much wider differentiation potential. Totipotent cells have the
ability to give rise to an entire individual, and exist until the eight-cell stage of the
morula. Later, once the morula develops into a blastocyst, the cells within this
structure become segregated into two distinct populations. The inner cell mass
(ICM) cells have the capacity to form the embryo, while the outer trophoblast cells
form extraembryonic tissues such as the placenta. Although the pluripotent cells of
the ICM are capable of forming all tissues and cell types of the human body, they
cannot give rise to an entire individual due to their inability to form
extraembryonic tissues. Human PSCs offer insights into human development, drug
discovery, toxicology and personalized medicine, as well as cell-based therapy (Pera
et al., 2000). There are two sources of hPSCs – human embryonic stem cells
(hESC) are derived from early-stage embryos, while human induced pluripotent
stem cells (hiPSC) are obtained by reprogramming somatic cells to a pluripotent
Human embryonic stem cells
The first hESC lines were established in 1998, by culturing embryos to the
blastocyst stage, isolating ICM cells, and plating them onto mouse embryonic
fibroblast (MEF) feeder cell layers to give rise to hESC colonies (Thomson et al.,
1998). The preimplantation embryos used for hESC derivation are produced by in
vitro fertilization (IVF), donated for research by couples undergoing IVF treatment
(Hasegawa et al., 2010). In Finland, both partners are required to sign an informed
consent form after receiving both an oral and written description of the research,
and no financial compensation is provided to the donors (Skottman, 2010). During
the last two decades, rapid progress in the field of hESC research has been made
worldwide, and currently 1304 hESC lines are registered at the International Stem
Cell Registry (http://www.iscr-admin.com) and 683 hESC lines at the European
Human Pluripotent Stem Cell Registry (http://hpscreg.eu/). In addition to the
conventional hESC derivation on feeder cell layers, feeder-independent derivation
of hESC lines on surfaces coated with recombinant laminin-521 and E-cadherin
has recently been described (Rodin et al., 2014). Elimination of feeder cells,
regardless of whether or not they are of animal origin, improves the reproducibility
of hESC culture. Currently, Phase I/II clinical trials using hESC-derived retinal
pigment epithelial (RPE) cells to treat advanced age-related macular degeneration
(AMD) and Stargardt’s macular dystrophy are ongoing, showing promising results
regarding the safety of treatments (Schwartz et al., 2015; Song et al., 2015).
Furthermore, clinical trials aiming to treat spinal cord injury, post-infarction heart
failure and type 1 diabetes mellitus using hESC-derived progenitor cells are
currently recruiting participants (https://clinicaltrials.gov/).
Human induced pluripotent stem cells
In 2007, the first hiPSC line was generated from adult human dermal fibroblasts
(hDF) by retroviral transduction of four transcription factors (octamer-binding
transcription factor 3/4 (OCT3/4), (sex-determining region Y)-box 2 (SOX2),
Krüppel-like factor 4 (KLF4) and myc proto-oncogene protein (c-MYC)), and
shown to behave similarly to hESCs (Takahashi et al., 2007). After successful
reprogramming, these transcription factors become silenced, indicating that hiPSCs
do not depend on continuous expression of the transgenes for self-renewal
(Takahashi et al., 2007). Although the technique was introduced less than a decade
ago, it has been widely used and modified. Various somatic tissues besides
fibroblasts and different combinations of reprogramming factors have been used
to successfully generate hiPSC lines (Hu, 2014; Trokovic et al., 2014; Zhou et al.,
2012). As recognition for his ground-breaking work in reprogramming adult cells
to hPSCs, Professor Yamanaka received the Millennium Technology Prize and the
Nobel Prize for Physiology or Medicine in 2012.
The discovery of hiPSCs has opened new opportunities in the field of
regenerative medicine. For instance, hiPSC technology enables the generation of
disease-specific cell lines, useful for understanding disease mechanisms, drug
screening, and toxicology (Park et al., 2008; Takahashi et al., 2007). Regenerative
medicine is the most widely studied application of both types of hPSCs. A unique
property of hiPSCs is that it is possible to generate patient-specific cell lines to be
used for autologous cell therapy. In fact, the first clinical trial using cells
differentiated from autologous hiPSCs was initiated in late 2014 in Japan, aiming to
treat AMD with hiPSC-derived RPE cells (Kamao et al., 2014). Unfortunately, the
trial was suspended in March 2015, after several mutations were detected in hiPSCs
of a prospective second patient, likely caused by the reprogramming procedure
(Garber, 2015). In light of the setbacks concerning hiPSC-derived cells, there are
still some issues that need to be addressed before wider clinical implementation. In
attempts to minimize the risk of harmful mutations and transgene reactivation,
several non-integrating methods for reprogramming have been developed, utilizing
the non-integrating Sendai virus, recombinant proteins, or synthetic modified
mRNA (Fusaki et al., 2009; Warren et al., 2010; Zhou et al., 2009). Moreover,
reprogramming methods in feeder-independent and chemically-defined conditions
have been described (Beers et al., 2015; Chen et al., 2011). It has been noted that
genomic stability of hiPSCs is mainly affected by the reprogramming methods and
culture conditions, highlighting the importance of protocol optimization (Bai et al.,
2013). Interestingly, an extensive karyotype analysis on >1700 hiPSC and hESC
cultures collected from 97 investigators revealed no notable difference in the
incidence of chromosomal aberrations between the two hPSC types (Taapken et
al., 2011). On the other hand, it appears that hiPSCs partially retain the DNA
methylation patterns of parental somatic cells, suggesting that transcription-factor
based reprogramming is associated with incomplete epigenetic reprogramming
(Lister et al., 2011; Ma et al., 2014). However, the biological consequence of these
aberrations remains unclear and requires further studies (Lund et al., 2012a).
Ultimately, there is substantial molecular and functional evidence showing
similarity between hiPSCs and hESCs, and the choice of cell type for
differentiation studies is largely dictated by the end application. In addition,
guidelines defining acceptable levels of genomic and epigenetic stability need to be
established for both hPSC types.
Culture and characterization of hPSCs
Human PSCs are conventionally cultured as colonies on feeder cell layers (MEF or
human foreskin fibroblasts (hFF)), or on Matrigel, a complex mixture of matrix
proteins derived from Engelbreth-Holm-Swarm mouse sarcomas, in a medium
containing xenogeneic serum or serum albumin (Hasegawa et al., 2010; Hoffman &
Carpenter, 2005; Skottman & Hovatta, 2006). Ideally, culture conditions will
include a defined matrix and a defined medium supplemented with recombinant
proteins to allow the establishment of more reproducible hPSC cultures (Hoffman
& Carpenter, 2005; Villa-Diaz et al., 2013). Recently, various methods of culturing
hPSCs in chemically-defined and xeno-free conditions, on surfaces coated with
recombinant extracellular matrix (ECM) proteins, or synthetic coatings, have been
described (Chen et al., 2011; Rodin et al., 2014; Villa-Diaz et al., 2013). Passaging
of hPSCs is done either mechanically, by manually selecting and transferring
undifferentiated colonies onto fresh substrates, or enzymatically, where hPSC
colonies are passaged as single cell suspensions or clusters (Hasegawa et al., 2010;
Hoffman & Carpenter, 2005). Mechanical passaging is generally regarded as the
gentler technique, yet it is fairly laborious and subject to variation. Enzymatic
passaging, on the other hand, allows for more uniform plating of hPSCs, but has
been shown to increase the risk of genetic and epigenetic instability (Bai et al.,
2015; Garitaonandia et al., 2015).
The quality of hPSCs in culture needs to be routinely verified. Human PSCs
have a high nucleus to cytoplasm ratio, prominent nucleoli, and grow in flat
colonies with distinct borders. Undifferentiated hPSCs are commonly characterized
by their gene and protein expression of cell surface markers and transcription
factors associated with an undifferentiated state, such as stage-specific embryonic
antigen (SSEA)-3, SSEA-4, tumor-related antigen (TRA)-1-60, TRA-1-81,
OCT3/4, reduced expression protein 1 (REX1), SOX2 and NANOG (Hovatta et
al., 2014; Pera et al., 2000; Takahashi et al., 2007). Telomerase and alkaline
phosphatase activities are also linked with pluripotency, and often assessed in
hPSCs. Additionally, hPSCs are required to maintain the potential to form
derivatives of all three embryonic germ layers (mesoderm, endoderm and
ectoderm). This may be evaluated in vitro, using spontaneous differentiation in
embryoid body culture, or in vivo, by injecting undifferentiated cells into nude mice
and following teratoma formation (Hasegawa et al., 2010; Hoffman & Carpenter,
2005; Itskovitz-Eldor et al., 2000). Furthermore, a normal euploid karyotype
despite continuous passaging is essential in hPSCs. Karyotyping can be done by
chromosomal G-band analysis or higher resolution techniques such as array
comparative genomic hybridization (aCGH), which enable the detection of
unbalanced genomic changes at the kilobase level (Hovatta et al., 2014; Lund et al.,
2012a). In addition to evaluation of genomic stability, it is becoming increasingly
evident that epigenetic stability of hPSCs also needs to be studied. For instance, X31
chromosome inactivation and variation in DNA methylation of a subset of
imprinted and developmental genes is fairly common in both hESC and hiPSC
lines (Lund et al., 2012a).
Human PSC-derived corneal epithelium
There are several reasons why the ocular surface is a good target for therapy using
hPSCs. Firstly, the eye is easily accessible, making the surgery and follow-up
procedures less invasive than for internal organs. Secondly, it is possible to treat
only one eye and use the fellow eye as the control, whereby assessing efficacy is
more reliable. Finally, the eye is an immune-privileged organ, and the cornea lacks
blood vessels, meaning that there is a lower risk of immune rejection than in other
tissues. This, however, may be altered in the case of severe LSCD, making regular
follow-up very important in order to recognize possible signs of rejection or graft
dysfunction early on. Efficient production of hPSC-derived LESCs or stratified
corneal epithelium (Figure 4) would potentially solve the issues related to CLET,
most importantly donor tissue shortage. In addition, hPSC-LESCs could be
applied to studying and modelling the human cornea, as well as drug development
in vitro (Figure 4).
Although corneal transplantation and CLET are often successfully performed
without matching for human leukocyte antigen (HLA), it may be beneficial
especially for high risk cases with extensive vascularization (Van Essen et al., 2015).
This is an important aspect to take into consideration also when dealing with
hPSC-LESCs, which are likely to be allogeneic. In order to make cell-based therapy
more readily available and minimize the risk of immune rejection posed by
allogeneic hPSCs, HLA haplotype-based banking of hPSC lines could be
considered (Wilmut et al., 2015; Zimmermann et al., 2012). A haplotype is a set of
alleles encoded by a group of closely linked genes, and is usually inherited as a unit,
making HLA haplotype-matching feasible. Selection of donors with homozygous
HLA haplotypes for hiPSC production and banking would provide cell lines
matching large groups of patients (Wilmut et al., 2015; Zimmermann et al., 2012).
For instance, it was estimated that as few as 30 HLA-homozygous hiPSC lines
would provide a match for 82% of the Japanese population (Nakatsuji et al., 2008).
Similarly to Japan, there is a limited amount of HLA allele diversity and fewer
haplotypes in Finland than in countries with a more mixed population (Haimila et
al., 2013). The more diverse the HLA haplotypes in a certain population, the more
homozygous hiPSC lines would be needed to provide a possible match for a
patient. There are currently two hPSC banks in Europe – The European Bank for
Induced Pluripotent Stem Cells (EBiSC), and The UK Stem Cell Bank (UKSCB).
Nevertheless, the idea of collecting homozygous hiPSC lines has not yet been
Figure 4. Establishment of hPSC lines and generation of hPSC-LESCs and corneal
epithelium. Human ESC lines are derived from the ICM of the early-stage surplus embryos,
while hiPSC lines are obtained by reprogramming somatic cells with a combination of
transcription factors. Collecting hPSC lines in specialized banks can allow generation of HLA
haplotype-matched cell populations. Differentiation of hPSCs can be directed towards corneal
epithelium, first producing monolayers of LESC-like cells, which stratify upon further
maturation. Finally, hPSC-derived LESC-like cells or corneal epithelium can be used to treat
ocular surface disorders, or as an in vitro model system.
The first study describing differentiation of corneal epithelial cells from hPSCs was
published in 2007, where corneal epithelial-like cells were obtained from hESCs by
in vitro replication of the corneal epithelial stem cell niche with the help of a
differentiation medium conditioned by human limbal fibroblasts (hLF) and
collagen IV-coated substrate (Ahmad et al., 2007). Since then, hESC and hiPSC
differentiation towards mature or progenitor corneal epithelial cells has been
reported by several groups (Table 2).
Table 2. Published corneal epithelial differentiation methods using hPSCs.
Collagen IV
Fixed MEF
PA6 feeder
Key medium components
Limbal fibroblast CM, contains FCS,
hydrocortisone, insulin, adenine, triiodothyronine, cholera toxin, EGF
FCII, adenine, HEPES, hydrocortisone,
cholera toxin, insulin, EGF
up to 21
Ahmad et al.,
~30 days
KO-SR, NaPyr, NEAA, 2-mE
21 days
Collagen IV
Corneal fibroblast CM, contains FCII,
adenine, hydrocortisone, cholera toxin,
insulin, EGF, BMP-4
12 days
Collagen IV
and APCM
LESC CM, contains FBS, EGF,
hydrocortisone, insulin, transferrin, BPE
23 days
Hewitt et al.,
Hanson et al.,
Hayashi et al.,
ShalomFeuerstein et al.,
2012 and 2013
Zhu et al., 2013
et al., 2014
FCL, hAM, or
Sareen et al.,
15 days
human cornea
EpiLife®, B27, N2, HKGS, EGF
Abbreviations: 2-mE, 2-mercaptoethanol; APCM, acellular porcine corneal matrix; B27, growth
supplement (contains BSA); CM, conditioned medium; Epilife®, chemically-defined medium for
keratinocyte culture (Invitrogen); FCII, FetalClone II™ serum (HyClone™); FCL, mixture of fibronectin,
collagen type IV and laminin; FCS, fetal calf serum; HKGS, human keratinocyte growth supplement
(contains BPE, human insulin-like growth factor 1, hydrocortisone, bovine transferrin, and EGF); N2,
chemically-defined and xeno-free growth supplement; NaPyr, sodium pyruvate; PA6, mouse stromal cells.
Other abbreviations are listed on pages 12-14.
Limbal fibroblast CM
21 days
Generally, differentiation is driven by a combination of two factors – the culture
substrate and an appropriate cell culture medium, commonly relying on undefined
or xenogeneic components. A favorable culture substrate is usually obtained with
the help of ECM coatings, feeder cells or biological scaffolds, while cell culture
medium is often conditioned by limbal or corneal fibroblasts, so as to offer the
necessary differentiation cues. Characterization of hPSC-derived cells is largely
based on verification of LESC and corneal epithelial marker expression using RTPCR, immunocytochemistry, or flow cytometry. The duration required for
adequate differentiation varies from 12 days to 16 weeks, and efficiency is rarely
quantified. Moreover, two of the studies have shown that limbal epithelial-derived
hiPSCs differentiate into corneal epithelium more efficiently than dermal
fibroblast-derived hiPSCs, likely due to their epigenetic memory (Hayashi et al.,
2012; Sareen et al., 2014).
Ideally, the differentiation method should be robust and reproducible, carried
out in chemically-defined conditions free from xenogeneic components. Most
importantly, hPSC-derived corneal epithelial cells should be similar to native
human corneal epithelium with regard to cell morphology, gene and protein
expression and functionality.
Biomaterials for ocular surface reconstruction
Transplantation of LESC-like cells to the ocular surface requires a carrier that is
biocompatible, mechanically stable, transparent, and capable of supporting cell
attachment and proliferation both in culture and after transplantation (MenzelSevering et al., 2013). Currently, the gold standard for culture and transplantation
of LESCs is hAM, the inner of the two fetal membranes obtained following
elective Cesarean section, with a long-standing history in corneal applications
(Gomes et al., 2005; de Rotth, 1940; Sorsby & Symons, 1946). The main
advantages of hAM are its anti-inflammatory, anti-angiogenic and anti-microbial
properties, combined with the ability to promote epithelialization and inhibit
fibrosis (Gomes et al., 2005; Zhao & Ma, 2015). The disadvantages of hAM include
lack of standardization, biological variability in morphological, chemical, and
optical properties, poor mechanical strength and difficulty of handling, as well as
limited availability (Connon et al., 2010; Ghezzi et al., 2015; Menzel-Severing et al.,
Various biomaterials are being investigated in the hopes of finding an
alternative superior to hAM. While some approaches aim at replacing the corneal
epithelium alone, others attempt using a thicker construct to simultaneously
reconstruct part of the stroma. Some of the most recent studies investigating
transplantable carriers for human LESC-like cells are summarized in Table 3.
Biomaterials of natural origin have been more widely studied than synthetic
scaffolds. Some of the most widely studied biomaterials include various hydrogels
(such as alginate, collagen and fibrin), silk fibroin or keratin films, and electrospun
membranes (Table 3). Hydrogels are a particularly promising type of transplantable
carrier, owing to their highly hydrated network structure and the possibility of coculturing two types of cells – supporting stromal cells incorporated within the
hydrogel, and epithelial cells cultured on the surface (Wright et al., 2013a). This
type of construct could potentially replace part of the damaged corneal stroma
along with the epithelium.
Table 3. Some of the most recently developed transplantable carriers for LSCD treatment.
Alginate hydrogel
Oxidation, ColIV
Cell type
In vitro
Wright et al., 2014
iHCE or
In vitro
de la Mata et al.,
In vitro
Tidu et al., 2015
In vitro
Levis et al., 2013
In vivo (rabbit)
Collagen I (bovine)
(up to 90 mg/ml)
Plastic compression
Collagen I (porcine)
In vivo (rabbit)
Collagen I (equine)
Cross- linking
In vivo (rabbit)
In vivo (pig)
Liu et al., 2008
In vivo (pig)
Rafat et al., 2008
Human corneal
Clinical trial
Rama et al., 2010
Sections, 200 µm
In vitro
Human lens capsule
In vitro
Keratin film
In vitro
Collagen I (rat tail)
Collagen I (bovine)
Lin et al., 2012
Albert et al., 2012;
Galal et al., 2007
Feng et al., 2014;
Reichl et al., 2011
iHCE and
In vitro
Sharma et al., 2014
LESC (rabbit
Deshpande et al.,
In vitro
or human)
Silk fibroin film
In vitro
Liu et al., 2012
LESC and
Silk fibroin dual layer Film and fibrous mat
In vitro
Bray et al., 2012
Silicone contact lens
Clinical trial
Bobba et al., 2015
Abbreviations: ColIV, collagen type IV; CjE, conjunctival epithelium; hOME, human oral mucosal
epithelium; iHCE, immortalized human corneal epithelial cell line; L-MSC, limbal mesenchymal stromal
cells; PCL, poly-ε-caprolactone; PLGA, poly-lactide-co-glycolide; RAFT, real architecture for 3D tissue;
RHCI or III, recombinant human collagen type I or III.
plasma treament
Chae et al., 2015
Koulikovska et al.,
Petsch et al., 2014
Collagen hydrogels have gained exceptional popularity as a potential LESC carrier,
mainly due to the fact that collagen is the main structural component of the corneal
stroma (Hassell & Birk, 2010). Collagen forms highly hydrated and inherently weak
hydrogels, and is therefore often modified either by plastic compression (Levis et
al., 2010; Mi et al., 2010) or chemical cross-linking (Koulikovska et al., 2015; Liu et
al., 2008; Petsch et al., 2014). Recently, commercial kits designed to simplify and
standardize plastic compression of collagen hydrogels such as the RAFT™ 3D Cell
Culture System (Lonza Group Ltd., Basel, Switzerland) have become available.
Another interesting approach to corneal reconstruction was proposed using a
thermoresponsive polymer Mebiol Gel: autologous rabbit LESCs were cultured on
the surface of a solid hydrogel, but transplanted as a liquid gel, the transition being
triggered by a temperature change (Sitalakshmi et al., 2009). Additionally, several
biomaterials are being studied for their compatibility with corneal stromal cells (Mi
et al., 2010; Wu et al., 2014), or as cell-free scaffolds aimed at corneal stromal
reconstruction (Fagerholm et al., 2014; Van Essen et al., 2013).
With regard to transplantable carriers for hPSC-LESCs, so far only two in vitro
studies have been published – one using hAM (Sareen et al., 2014) and the other
using acellular porcine corneal matrix (Zhu et al., 2013). Neither of these scaffolds
is optimal, mainly due to their biological variability and potential for pathogen
transmission. More research is needed before a suitable carrier for hPSC-LESCs is
thoroughly characterized in vitro, the construct’s safety verified in an animal model,
and eventually tested in human patients.
Aims of the study
The aim of this dissertation was to study the differentiation of hPSC lines towards
functional LESC-like cells capable of terminal differentiation towards mature
corneal epithelial cells. Moreover, it was important to minimize the use of
undefined and animal-derived components, aiming for clinical applications in the
long run. The specific aims of the dissertation were:
1. To develop a directed and efficient differentiation method for hPSCLESCs in serum-free and feeder-free conditions (Study I).
2. To characterize the molecular properties and functionality of hPSC-LESCs
(Studies I and II).
3. To compare hPSC-LESCs with their in vivo counterparts (Study II).
4. To evaluate in vitro the suitability of a bioengineered collagen matrix for
clinical applications using hPSC-LESCs (Study III).
Materials and methods
Ethical considerations
The use of human embryos for research purposes at BioMediTech has been
approved by the National Authority for Medicolegal Affairs in Finland (Dnro
1426/32/300/05). The institute also has supportive statements of the Ethical
Committee of the Pirkanmaa Hospital District to derive, culture, and differentiate
hESC lines (Skottman/R05116), and use hiPSC lines derived in other laboratories
for ophthalmic research (Skottman/R14023). The research groups of Prof. Timo
Otonkoski at the University of Helsinki and Prof. Katriina Aalto-Setälä at the
University of Tampere have the appropriate permissions of the Ethics Committee
for generation of hiPSC lines. No new cell lines were established for the studies
conducted as part of this dissertation.
Use of human corneal donor tissue unsuitable for corneal transplantation for
research purposes was approved by the local ethics research committee (Valvira,
Dnro 7797/
Collection of human corneal tissue was carried out in Debrecen, Hungary, with
approval by the National Medical Ethics Committee of Hungary
(14415/2013/EKU-183/2013 and DEOEC RKEB/IKEB 3094/2010), and in
compliance with the guidelines of the Helsinki Declaration. Hungary follows the
EU Member States’ Directive 2004/23/EC on presumed consent practice for
tissue collection.
Human tissue collection
Donor corneas unsuitable for transplantation were obtained from Regea Cell and
Tissue Center, to be used as positive control in Study I. The epithelium was
collected from the surface of the corneas by mechanical scraping, washed with
phosphate buffered saline (PBS) and pelleted by centrifugation. Cell pellets were
lysed and stored at -80 °C until total RNA extraction. Additionally, corneal tissue
was fixed with 4% paraformaldehyde (PFA; Sigma-Aldrich, St. Louis, MO),
embedded in paraffin and sectioned for immunofluorescence antibody verification.
Limbal epithelial cell (LEC) and corneal epithelial cell (CEC) samples were
obtained within 12 hours post-mortem by gently scraping the surface of the limbus
or the central cornea, respectively. All samples were collected in sterile PBS,
pelleted by centrifugation and stored as dry pellets at -80 °C until protein
extraction for comparative proteomics (Study II) or Western blotting (Study III).
Culture of hPSC lines
Three hESC lines and three hiPSC lines were used in the original publications. The
hESC lines Regea08/017 (Studies I, II and III), Regea08/023 and Regea11/013
(Study I) were previously derived at the University of Tampere (Skottman, 2010).
The hiPSC lines FiPS5-7, A116 and HEL24.3 (Study I) were generated from
neonatal hFFs (FiPS5-7 and HEL24.3) or adult hDFs (A116) by Professor Timo
Otonkoski’s research group at the University of Helsinki (Hussein et al., 2011;
Toivonen et al., 2013; Trokovic et al., 2015). The hiPSC line UTA.04511.WT
(Studies II and III) was generated from adult hDFs by Professor Katriina AaltoSetälä’s research group at the University of Tampere (Ojala et al., 2015).
All hPSC lines were routinely cultured on mitotically inactivated hFF feeder
cells (CRL-2429, ATCC, Manassas, VA) in a basic hPSC culture medium consisting
of KnockOut Dulbecco’s Modified Eagle Medium (KO-DMEM) supplemented
with 20% KnockOut Serum Replacement (KO-SR), 2 mM Glutamax, 0.1 mM 2mercaptoethanol (all from Invitrogen, Carlsbad, CA), 1% Non-Essential Amino
Acids (NEAA), 50 U/ml penicillin/streptomycin (both from Lonza Group Ltd.),
and 8 ng/ml human basic FGF (bFGF; PeproTech, Rocky Hill, NJ).
Undifferentiated colonies were enzymatically passaged onto fresh feeder layers at
ten-day intervals.
All hPSC lines were regularly characterized for their pluripotency and ability to
generate derivatives of all three embryonic germ layers as previously described
(Skottman, 2010). Karyotype analysis was performed either using conventional Gbanding at United Medix Laboratories Ltd in Helsinki (Study I), or using a highthroughput bead-based KaryoLite™ BoBs™ assay (Lund et al., 2012b) at the
Finnish Microarray and Sequencing Center in Turku (Studies II and III).
LESC differentiation and culture
Differentiation was initiated by manually dissecting the hPSC colonies, transferring
them to suspension culture, and directing differentiation towards surface ectoderm
using an induction medium supplemented with 10 µM of transforming growth
factor β (TGF-β) inhibitor SB-505124, 10 µM of Wnt inhibitor IWP-2 (both from
Sigma-Aldrich) and 50 ng/ml bFGF (PeproTech). During this induction stage,
three-dimensional cell aggregates would form, and they were maintained for 4-7
days, changing the induction medium daily. The cell aggregates were then plated
onto well-plates (Corning CellBIND; Corning, NY) coated with human placental
collagen IV (Sigma-Aldrich), and differentiation was continued as adherent culture
in the commercial serum-free and defined corneal epithelium medium CnT-30
(CELLnTEC Advanced Cell Systems, Bern, Switzerland). Differentiated cells were
characterized at several time-points, as described in the following chapters, and
considered to have reached the LESC-like state after a total of 30 ±5 days of
differentiation. Further maturation until day 44 resulted in corneal epithelial cells.
In Study I, xeno-free RegES medium (Rajala et al., 2010) modified by omitting
retinol and activin A was used as the base for surface ectoderm induction medium.
The effect of the small molecules was assessed by comparing differentiation
efficiency in supplemented and unsupplemented RegES media. In addition,
differentiation in only RegES medium (i.e. spontaneous differentiation) or only
CnT-30 medium throughout the duration of the study was evaluated. In Studies II
and III, instead of RegES medium, hPSC medium modified by lowering KO-SR
concentration to 15% was used as the base for the induction medium.
In Study III, bioengineered matrices fabricated using porcine atelo-collagen I
as transparent membranes of uniform 100 µm thickness (LinkoCare Life Sciences
AB, Linköping, Sweden), were tested as carriers for hPSC-LESCs. For culture on
the bioengineered collagen matrices LESC-like cells (28-33 days in differentiation
culture) were enzymatically detached from their substrate, and plated onto the
bioengineered matrices or well-plates coated with human placental collagen IV at a
density of 20 000 cells/cm2. After re-plating, hPSC-LESCs were maintained in the
progenitor cell targeted serum-free and defined medium CnT-20 (CELLnTEC
Advanced Cell Systems).
Cell characterization methods
During hPSC-LESC differentiation, cell growth and morphology were regularly
monitored using Nikon Eclipse TE2000-S phase contrast microscope (Nikon
Instruments, The Netherlands). In Study I, three days after plating cell aggregates
onto collagen IV-coated substrate, the adhesion ratios were quantified and
compared between the three studied induction media. In Study III, cell
attachment and proliferation on bioengineered collagen matrices were regularly
monitored using Zeiss Axio Vert A1 inverted brightfield microscope (Carl Zeiss,
Jena, Germany).
Quantitative PCR
Quantitative polymerase chain reaction (qPCR) was used to evaluate differentiation
efficiency in different culture conditions (Study I). Total RNA extraction, cDNA
synthesis and qPCR protocols are described in the original publication. The qPCR
reactions were run in triplicates using the 7300 Real-time PCR system (Applied
Biosystems, Foster City, CA). The studied genes and respective TaqMan primers
(Applied Biosystems) are presented in Table 4.
Table 4. Gene expression studied using qPCR.
Gene symbol
TaqMan assay
Cell type
Pluripotent stem cells
d0, d4, d44
Pluripotent stem cells
d0, d4, d44
Pluripotent stem cells
d0, d4, d44
Pluripotent stem cells
d0, d4, d44
Surface ectoderm
d0, d4, d44
Surface ectoderm
d0, d4, d44
Surface ectoderm
d0, d4, d44
Eye precursors, cornea
d0, d4, d44
d0, d44
d0, d44
Corneal epithelium
d0, d44
Corneal epithelium
d0, d44
Abbreviations: OCT3/4, octamer-binding transcription factor 3/4; SOX2, (sex determining
region Y)-box 2; PITX2, paired-like homeodomain 2; BMP4, bone morphogenetic protein 4;
FOX1, forkhead box 1; PAX6, paired box 6; KRT, keratin.
Relative expression analyses were performed using the 2∆∆Ct method (Livak &
Schmittgen, 2001), with Glyceraldehyde 3-phosphate dehydrogenase (GAPDH,
Hs99999905_m1) as the endogenous control gene, and undifferentiated hPSCs
(day 0) as the calibrator. Corneal epithelium obtained from human donor corneas
was used as a positive control of LESC and corneal epithelial gene expression.
Protein expression of the putative LESC markers and proteins specific to the
mature corneal epithelium, as well as their subcellular localization, were evaluated
using immunofluorescence. The primary antibodies are presented in Table 5. Their
detection was carried out with the following secondary antibodies, diluted 1:800:
donkey anti-goat Alexa Fluor 568, donkey anti-mouse Alexa Fluor 488 or 568,
donkey anti-rabbit Alexa Fluor 488 or 568 (all from Molecular Probes®, Thermo
Fisher Scientific, Waltham, MA). The staining protocol is described in detail in the
original publications (Studies I, II and III).
Table 5. Protein expression studied using immunofluorescence
Qual, quant
Santa Cruz Biotech.
Santa Cruz Biotech.
Qual, quant
Thermo Fisher Scientific
Qual, quant
US Biological
Qual, quant
Biocare Medical
Santa Cruz Biotech.
Qual, quant
Cell Signaling Tech.
R&D Systems
Santa Cruz Biotech.
Abbreviations: ABCG2, ATP-binding cassette subfamily G member 2; CK, cytokeratin; DG-3,
desmoglein 3; OCT3/4, octamer-binding protein 3/4; PAX6, paired box 6; TCF4, transcription
factor 4; qual, qualitative; quant, quantitative.
Qualitative evaluation was performed by staining LESC-like cells directly on their
culture substrate (Studies I, II and III). Quantitative immunofluorescence was
carried out by preparing and staining cytospin samples, and counting the positively
stained cells in relation to the total nuclei (Studies I and III). In addition, p63
expression was quantified at ten-day intervals in Study I, from cells cultured on
collagen IV-coated hanging cell culture inserts. Images of stained cells were
captured using Olympus IX51 fluorescence microscope (Olympus, Hamburg,
Germany), or Zeiss LSM700 confocal microscope (Carl Zeiss).
Western blotting
Protein expression of CK3 and CK12 in hiPSC-LESCs cultured on bioengineered
collagen matrices and human native LECs was analyzed using Western blotting
(Study III). The following primary antibodies were used: mouse anti-CK3 (diluted
1:1000, Abcam), goat anti-CK12 (diluted 1:500, Santa Cruz Biotechnology), and
mouse anti-β-actin (diluted 1:2000, Santa Cruz Biotechnology) as loading control.
Their detection was carried out using horseradish peroxidase-conjugated goat antimouse and rabbit anti-goat secondary antibodies (both diluted 1:3000, Santa Cruz
Biotechnology). The detailed protocol is described in the original publication.
Flow cytometry
Protein expression of the LESC marker BMI-1 in hPSC-LESCs was analyzed using
flow cytometry (Study II). The detailed protocol is described in the original
publication. Briefly, single-cell suspensions were treated either with the fluorescein
isothiocyanate (FITC)-conjugated mouse anti-human BMI-1 antibody or the FITCconjugated mouse anti-human IgG2A isotype control (both from R&D Systems,
Minneapolis, MN), and analyzed using the BD Accuri C6 Flow Cytometer (BD
Biosciences, Franklin Lakes, NJ).
In addition, hiPSC-LESCs (HEL24.3 and UTA.04511.WT cell lines), and their
undifferentiated counterparts were analyzed for protein expression of the cell
surface markers presented in Table 6. Single-cell suspensions were incubated with
the appropriate fluorochrome-conjugated antibody for 30 min, protected from
light. Before and after the staining, samples were washed with a buffer containing
0.5% bovine serum albumin (BSA) and 0.01% NaN3, and collected by
centrifugation. Matched isotype controls were used to account for unspecific
binding. Samples were analyzed using the BD Accuri C6 Flow Cytometer.
Table 6. Additional cell surface markers analyzed by flow cytometry
CD18 / Integrin β2
Mouse IgG1
CD29 / Integrin β1
Mouse IgG1
BD Pharmingen
CD31 / PECAM-1
Mouse IgG1
Mouse IgG1
4H11[APG] Immunotools
CD45 / LCA
Mouse IgG1
BD Pharmingen
CD49a / Integrin α1
Mouse IgG1
CD49d / Integrin α4
Mouse IgG1
BD Pharmingen
CD51 / Integrin αV
Mouse IgG2A
CD54 / ICAM-1
Mouse IgG1
R&D Systems
CD73 / NT5E
Mouse IgG1
BD Pharmingen
CD117 / c-kit
Mouse IgG1
Miltenyi Biotec
CD144 / VE-cadherin Mouse IgG2B
R&D Systems
CD146 / MCAM /
Mouse IgG1
BD Pharmingen
Mouse IgM
BD Pharmingen
Abbreviations: APC, allophycocyanin; CD, cluster of differentiation; FITC, fluorescein
isothiocyanate; ICAM-1, intercellular adhesion molecule 1; LCA, leukocyte common antigen;
MCAM, melanoma cell adhesion molecule; MUC18, mucin 18; NT5E, ecto-5’-nucleotidase;
PE, phycoerythrin; PECAM-1, platelet endothelial cell adhesion molecule 1; VE-cadherin,
vascular endothelial cadherin
Cell passaging and colony forming efficiency assay
Like other types of tissue-specific stem cells, LESCs are known to be capable of
self-replication. To test this function in hPSC-LESCs, differentiated cells were
enzymatically detached from their substrate, collected by centrifugation, and replated onto human placental collagen IV-coated well-plates. Plating densities of
20 000 cells/cm2 and 5 000 cells/cm2 were used for cell passaging and colony
forming efficiency (CFE) assays, respectively. Re-plated hPSC-LESCs were
maintained in the progenitor cell targeted CnT-20 medium. Cells were serially
passaged upon reaching sub-confluency, roughly at two-week intervals, until
cultures no longer proliferated.
In order to assess their CFE, hPSC-LESCs were cultured in CnT-20 medium
for 14 days, after which they were fixed with 4% PFA for 15 min and stained with
0.1% Rhodamine B for 15 min, with PBS washes in between each step. The
visibly-stained colonies were counted and CFE calculated as follows:
× 100%.
Cell proliferation assay
In Study III, cell proliferation on bioengineered collagen matrices and human
placental collagen IV-coated well-plates was evaluated using the WST-1 Cell
Proliferation Assay (Takara Bio Inc., Shiga, Japan). This method is based on the
cleavage of the tetrazolium salt WST-1 to formazan by cellular mitochondrial
dehydrogenases in living cells. The formazan produced by viable cells is quantified
after 4 h incubation at +37 °C by measuring the absorbance at 450 nm. The
protocol is described in more detail in the original publication.
Comparative proteomics
In Study II, hESC-LESCs and hiPSC-LESCs were compared with native human
CECs and LECs using isobaric tag for relative and absolute quantitation (iTRAQ)
proteomics. The detailed protocols for all the following steps are available in the
original publication. Briefly, human corneal and limbal epithelial cells were
collected from three cadaveric donors as described in Chapter 4.2. Both hPSC lines
were differentiated towards LESC-like cells as described in Chapter 4.4, and
enzymatically detached from their substrate after 30-35 days in differentiation
culture. Protein was extracted from all samples, and equal amounts (25 µg/sample)
were digested with Trypsin (AB Sciex, Concord, Canada). Digested peptides were
labeled with iTRAQ reagents and analyzed in duplicate by Nano-RPLC-TripleTOF
instrumentation using Eksigent 425 NanoLC coupled to high speed TripleTOF™
mass spectrometer (AB Sciex). Raw data processing was carried out in ProteinPilot
software (AB Sciex), and all identified proteins were converted to the Universal
Protein Knowledgebase (UniProtKB) accession numbers. Data normalization was
performed using log transformation and central tendency normalization. Finally,
only proteins that were detected in at least two of the biological replicates were
used for further biological interpretation.
Fabrication and characterization of bioengineered
In Study III, bioengineered collagen matrices were evaluated as potential carriers
for hPSC-LESCs. These matrices were fabricated from medical-grade, high-purity
porcine atelo-collagen type I. Briefly, the 18% collagen solution was cross-linked
with 1-[3-(dimethylamino)propyl]-3-ethylcarbodiimide methiodide (EDCM; SigmaAldrich) and dicyclohexyl-carbodiimide (DCC; Thermo Fisher Scientific), and
molded between glass plates with a 100 µm thick spacer. The resulting matrices
were cured at room temperature in 100% humidity chambers for 25 h, and demolded by immersion in PBS for 1 h. Prior to cell culture experiments, the sheet of
bioengineered collagen matrix was cut into round pieces (1 cm in diameter) using a
sterile trephine, and the pieces were sterilized by soaking in antibiotic solution
(150 U/ml penicillin/streptomycin in PBS), with thorough PBS washes after each
step. Finally, each piece of the matrix was placed in a separate well of a 48-well
plate, and incubated in CnT-20 cell culture medium overnight at +37 °C, 5% CO2.
Water content and swelling capacity of bioengineered matrices and research
grade human donor corneas (obtained from the Eye Bank of Canada) were
calculated by comparing dry and hydrated masses of five replicate samples as
follows:   = (
ℎ −

ℎ −
) × 100% and   =
) × 100%. To evaluate the transparency of the matrices, light
transmission and scatter measurements were performed for white light and for
narrow spectral regions. Finally, the microstructure of the matrix surface and crosssection was visualized using scanning electron microscopy (SEM). The detailed
protocols describing the fabrication and characterization methods are available in
the original publication (Study III).
Statistical analyses
Mann-Whitney U test was used to assess the statistical significance of differences
between culture conditions in gene expression and p63 protein expression data in
Study I, as well as differences in cell proliferation and Ki67 and p63 protein
expression in Study III. Analyses were carried out in IBM SPSS Statistics
Software, and results were considered significant if p < 0.05, and highly significant
if p < 0.01.
Summary of the results
Differentiation of hPSCs towards LESC-like cells
The aim of Study I was to develop an efficient differentiation method for
production of corneal epithelial cells and their progenitors from hPSCs. Four
different conditions were tested: 1) spontaneous differentiation, 2) commercial
medium for corneal epithelial cell culture (CnT-30), 3) small-molecule induction,
and 4) induction in unsupplemented medium, both followed by maturation in
CnT-30 medium. All four conditions were free from serum and feeder cells.
During the first four days (i.e. induction stage), differentiation was carried out in
suspension culture as three-dimensional cell aggregates, followed by adherent
culture on human placental collagen IV-coated substrate (schematic outline in
Study I/Figure 1).
Spontaneous differentiation proved to be very inefficient and did not yield
detectable amounts of LESCs or CECs. In contrast, pigmented cells were often
observed in spontaneous differentiation cultures (Study I/Figure 3).
Differentiation in the commercial CnT-30 medium was adequate, yet subject to a
high degree of variation. Supplementing the induction medium with two small
molecule inhibitors (SB-505124 and IWP-2) and bFGF was seen to promote
corneal epithelial differentiation. Of all the studied differentiation conditions,
small-molecule induction followed by maturation in CnT-30 was deemed superior.
This method was therefore utilized in Studies II and III, modified by replacing
RegES medium with 15% KO-SR hPSC medium as the induction medium base.
Cell morphology during differentiation
During the induction stage in suspension culture, three-dimensional cell aggregates
would form, and they would then be plated onto cell culture substrate coated with
human placental collagen IV. Degree of adhesion to collagen IV was substantially
higher after small-molecule induction, than in the other studied conditions
(Study I/Figure 2). Under these differentiation conditions, cell outgrowths from
the attached aggregates possessed predominantly fibroblast-like cell morphology at
first, gradually changing to polygonal morphology typical to epithelial cells, and
spontaneously stratifying after prolonged culture (Study I/Figure 3). LESC-like
cells were typically obtained after approximately 30 days of differentiation. They
possessed polygonal morphology and were small in size (Study III/Figure 3).
Representative phase contrast microscopy images of an undifferentiated hPSC
colony, three-dimensional cell aggregates, and differentiated LESC-like cells are
presented in Figure 5.
Figure 5. Differentiation of hPSCs towards LESC-like cells. Scale bars 200 µm.
Gene expression during differentiation
Differentiation efficiency was assessed using qPCR, relative to undifferentiated
hPSCs (Study I/Figures 2 and 6). Undifferentiated hiPSCs (HEL24.3 cell line)
expressed endogenous pluripotency markers OCT3/4, NANOG, SOX2 and
c-MYC. Expression of these genes decreased after the four-day induction towards
surface ectoderm, and even more so by the end-point of the study at day 44. Gene
expression of the eye progenitor and corneal marker PAX6 increased to about
100-fold after the four-day induction stage, and remained upregulated until the
end-point of the study. Gene expression of OCT3/4 and PAX6 was analyzed for
four additional hPSC lines (A116, Regea08/017, Regea08/023 and Regea11/013),
showing similar trends after the four-day induction stage (Study I/Figure S1).
Transcription factors involved in early eye development (PITX2, BMP4 and FOX1)
were slightly upregulated after the four-day induction stage, but their expression
decreased by the end-point of the study. Finally, LESC-markers TP63 and KRT15,
and corneal epithelial markers KRT3 and KRT12 were highly upregulated after 44
days of differentiation. At this time-point, gene expression of TP63 and KRT15
was at levels comparable to native human cornea, while KRT3 and KRT12 were
expressed at substantially lower levels (Study I/Figure 6). Small-molecule
induction promoted corneal epithelial differentiation, with the most pronounced
differences in gene expression compared to undifferentiated hPSCs.
Protein expression during differentiation
Differentiating hPSC-LESCs were analyzed for their protein expression using
immunofluorescence (Studies I, II and III) and flow cytometry (Study II and
unpublished results). Several of the key antibodies were verified by staining paraffin
sections of native human corneal tissue. Putative LESC markers p63, CK15 and
ABCG2 were localized to the limbus, while CK3 and CK12 were expressed
exclusively in central corneal epithelium (Figure 6).
Figure 6. Localization of LESC and mature corneal epithelial markers in native human cornea.
CK3 was not detected at the limbus (A), but was strongly expressed in the central corneal
epithelium (B), while the opposite was true for p63. CK15 and ABCG2 were expressed at the
limbus and CK12 in central corneal epithelium (C). Scale bars 20 µm.
Already after 20 days of differentiation, hiPSC-LESCs (HEL24.3 cell line) were
shown to express several putative LESC markers, namely ABCG2, CK15, DG-3,
Ki67, p63, and PAX6 (Study I/Figure 4). Moreover, the pluripotency marker
OCT3/4 was no longer expressed, and CK3 and 12 were faintly visible already at
this time-point (Study I/Figure 4). On the other hand, LESC-like cells
differentiated from UTA.04511.WT and Regea08/017 cell lines did not express
CK3 or 12 after 28 days of differentiation, but did express several putative LESC
markers (Study III/Figure 3). In addition, after 28-35 days of differentiation, colocalization of p63 and TCF4 and a uniform expression of ABCG2
(Study II/Figure 6) were observed in hPSC-LESCs (UTA.04511.WT and
Regea08/017 cell lines). Protein expression of the ΔNp63α isoform was indirectly
verified via co-localization of p40 and p63α proteins (Study III/Figure 3).
Importantly, protein expression of CK10/13, a marker of epidermal
differentiation, was not detected in hPSC-LESCs (Study III/Figure 3).
Efficiency of LESC differentiation was assessed by counting cells positively
stained for the clinically-relevant p63 protein at ten-day intervals
(Study I/Figure 5). Small-molecule induction promoted differentiation towards
p63-positive LESC-like cells. For HEL24.3 cell line, an average of 50% of cells
expressed this protein at day 10, and by day 30 up to 95% of cells were p63positive. The studied cell lines showed variation in overall differentiation efficiency.
For instance, A116 and UTA.04511.WT hiPSC lines yielded an average of 64%
p63-positive cells by day 30 (Study I/Figure S1 and Study III/Figure 3), while
only 50% of analyzed cells of Regea08/017 hESC line were clearly positive at this
time-point (Study III/Figure 4). Additionally, the proliferation marker Ki67 was
expressed in approximately 50% of hPSC-LESCs (UTA.04511.WT and
Regea08/017 cell lines) after 28-35 days of differentiation (Study III/Figure 4).
Additional quantitative measurements of protein expression were obtained
using flow cytometry. The putative LESC marker BMI-1 showed an average of
80% and 84% positivity for Regea08/017 and UTA.04511.WT cell lines,
respectively (Study II/Figure 6). Additionally, protein expression of several cell
surface markers was analyzed in hiPSC-LESCs (UTA.04511.WT and HEL24.3 cell
lines) and their undifferentiated counterparts (Unpublished results, Figure 7). Most
importantly, the pluripotency marker TRA-1-81 was highly expressed in
undifferentiated hiPSCs, but virtually undetected in hiPSC-LESCs.
Figure 7. Cell surface marker expression in undifferentiated hiPSCs (undiff) and hiPSC-LESCs
Self-renewal properties of hPSC-LESCs
In order to evaluate whether hPSC-LESCs are capable of self-renewal, the
differentiated cells (UTA.04511.WT and Regea08/017 cell lines) were serially
passaged in progenitor cell targeted CnT-20 medium. Passaging hPSC-LESCs three
times at two-week intervals did not seem to affect their overall cell morphology
(Figure 8), and p63 protein expression was maintained in more than 50% of cells.
Additionally, CFE assay was carried out for two hiPSC lines (UTA.04511.WT
and HEL24.3). Differentiated hiPSC-LESCs were seeded onto human placental
collagen IV-coated well-plates at clonal density (5000 cells/cm2) in CnT-20
medium. During the two week culture period, colonies of LESC-like cells would
appear (Figure 8). The colonies were visualized and counted following
Rhodamine B staining. The average CFE of hiPSC-LESCs was 0.029% (±0.02%)
and 0.03% (±0.02%) for UTA.04511.WT and HEL24.3 cell lines, respectively.
Figure 8. Self-renewal properties of hPSC-LESCs. Representative light microscopy images of
hiPSC-LESC morphology before (A) and after (B) passaging. When seeded at clonal density,
colony formation was observed (C). Scale bar 100 µm.
Comparison of hPSC-LESCs with their native
In Study II, high-throughput mass spectrometry-based proteomics were used to
compare hPSC-LESCs (UTA.04511.WT and Regea08/017 cell lines) with CECs
and LECs obtained from human cadaveric donors (schematic outline in
Study II/Figure 1). Using this approach, a total of 860 unique proteins expressed
in all four samples were identified (Study II/Figure 2). Approximately 57% of
these proteins were present in at least two of the three biological replicates, and
were selected for further analyses. Roughly two thirds of these proteins were
similarly expressed in hPSC-derived LESCs and their native counterparts
(Study II/Figure 3). Identified proteins were grouped according to their function:
proteins involved in maintaining stem cell or TAC behavior (i.e. cell cycling,
proliferation, differentiation and apoptosis), as well as various niche components
of the ocular surface, and corneal and limbal markers (Study II/Figures 4-6).
Relatively few proteins involved in cell adhesion, immune response or angiogenesis
were identified, likely due to the fact that hPSC-LESCs are maintained in far
poorer conditions than what native cells are exposed to at the ocular surface. Most
importantly, protein expression of CK3 and 12 in hPSC-LESCs was higher than in
LECs, but lower than in CECs. The opposite was true for the putative LESC
markers CK19, S100A8 and S100A9. Together, these observations indicate that
hPSC-LESCs are perhaps more mature than LECs, but less mature than CECs.
The overall protein expression profiles of LESC-like cells obtained from the
two hPSC lines were very similar, demonstrating that the differentiation method is
highly reproducible and yields homogeneous cell populations. Surprisingly, protein
expression profiles of CECs and LECs were also very similar to each other, likely
attributable to the innate heterogeneity of limbal cell populations, biological
variation between human donors and tissue collection method used in this study.
Nevertheless, hPSC-LESCs were clearly similar to the native ocular surface
epithelial cells, and possessed LESC-like characteristics.
Bioengineered matrices as carriers for hPSC-LESCs
In Study III, bioengineered matrices fabricated using medical-grade porcine
collagen type I were evaluated as carriers for hPSC-LESCs (UTA.04511.WT and
Regea08/017 cell lines). These 100 µm thick bioengineered matrices had a parallel
lamellar microstructure and were fully transparent, transmitting over 92% of light
at visible wavelengths, and scattering less than 4% (Study III/Figures 1 and 2).
They also exhibited water content of 91% (±0.2%), and were capable of absorbing
water 9.6 times of their dry weight – both measurements slightly higher than those
of the native human cornea (Study III/Figure 1).
Growth of hPSC-LESCs was supported by the bioengineered matrices in
serum-free conditions. Proliferative activity of hPSC-LESCs cultured on the
matrices was approximately four times higher than on well-plates coated with
human placental collagen IV (Study III/Figure 5). Moreover, protein expression
of LESC markers p63 and CK15, along with the proliferation marker Ki67 was
maintained for at least 30 days in culture on bioengineered matrices
(Study III/Figure 5). Finally, upon stimulation by the corneal epithelium medium
CnT-30, hPSC-LESCs were induced to differentiate, as demonstrated by colocalization of proteins CK3 and 12, yet the construct remained fully transparent
(Study III/Figure 6). Protein expression of CK3 and 12 was also analyzed using
Western blotting, and these proteins were found to be expressed at lower levels in
hiPSC-LESCs than in native human LECs (Study III/Figure 6).
Directed differentiation of hPSCs towards LESCs
The first aim of this dissertation was to optimize a directed and efficient
differentiation method, minimizing the use of undefined and xenogeneic
components. Differentiation efficiency of several hPSC lines towards corneal
epithelial cell lineage was evaluated by following gene and protein expression of
several putative LESC markers, as well as CK3 and 12 – both specific to mature
corneal epithelium.
Spontaneous differentiation in a cell culture medium lacking inductive
molecules did not yield detectable amounts of CECs or their progenitors, but
rather resulted in heterogeneous cell populations. It is known that the default
pathway of hPSC differentiation is towards neuroectoderm (Vallier et al., 2004),
and even that is subject to a high degree of variation between cell lines (Osafune et
al., 2008; Toivonen et al., 2013). Being a derivative of the surface ectoderm, a
directed differentiation method is likely needed in order to obtain LESC-like cells.
To date, most of the available corneal epithelial differentiation methods rely on the
use of niche components, such as conditioned medium (Ahmad et al., 2007;
Brzeszczynska et al., 2014; Shalom-Feuerstein et al., 2012; Zhu et al., 2013), hAM
(Sareen et al., 2014), or Bowman’s membrane (Hanson et al., 2013). The reasoning
behind using these culture components is to mimic the ocular surface
environment, thereby providing appropriate signals to drive hPSC differentiation
towards corneal epithelial cell fate. Although these methods are relatively
successful in producing CECs, their reproducibility and scalability suffer due to the
biological variability of such undefined components. Primary LESCs and hLFs
secrete proteins that affect cell growth and proliferation (Shimmura et al., 2006;
Wright et al., 2013b), which may vary depending on the culture conditions. This
makes the differentiation methods using conditioned medium subject to batch-tobatch variation. Similarly, there is variation among hAM obtained from different
donors in respect to growth factor secretion (Hopkinson et al., 2006).
Furthermore, the handling and processing of hAM prior cell seeding affects cell
viability and proliferation (Shortt et al., 2009). Finally, culture components of
animal origin, such as fetal bovine serum (FBS), pose a risk of infection by
nonhuman pathogens, and incorporation of immunogenic nonhuman sialic acids
(Hoffman & Carpenter, 2005; Martin et al., 2005). Overall, standardized conditions
for LESC differentiation and culture are needed in order to obtain cell populations
applicable to the clinical setting.
In this dissertation, hPSC differentiation was induced towards surface ectoderm
by mimicking the early eye development. As described in Chapter 2.1.1, corneal
epithelial development involves activation of FGF signaling, as well as inhibition of
the canonical Wnt signaling pathway (Dhouailly et al., 2014; Zhang et al., 2015).
These mechanisms were replicated in vitro using two small molecule inhibitors and
the recombinant growth factor bFGF. This induction was shown to promote earlystage differentiation by down-regulating pluripotency markers and up-regulating
PAX6 and several surface ectodermal transcription factors. Moreover, expression
of LESC markers at later stages of differentiation was also enhanced by the small
molecule induction. Plating the three-dimensional cell aggregates onto human
placental collagen IV coupled with the transition to corneal epithelium medium
CnT-30 was aimed at further directing differentiation towards LESC-like cells.
Collagen type IV was chosen for two reasons: it is one of the main components of
the corneal epithelial basement membrane (Torricelli et al., 2013), and there is
evidence of LESCs preferentially adhering to type IV collagen (Bian et al., 2010; Li
et al., 2005). The commercial cell culture medium was used in order to provide a
corneal epithelial environment to the differentiating cells without the use of
conditioned medium. Successful differentiation of hPSC-LESCs was verified
through gene and protein expression of several markers, as well as appropriate cell
morphology and spontaneous stratification upon prolonged culture.
Ideally, LESC-like cells would be differentiated from hPSCs in entirely xenofree and chemically-defined conditions. In this work, although differentiation is
carried out in the absence of a biological substrate or serum, there are several issues
which could still be addressed. First of all, hPSCs used in this study were
maintained on hFF feeder cells, in undefined culture conditions. It remains to be
seen whether or not hPSCs maintained in feeder-independent and chemicallydefined conditions behave differently and require a modified differentiation
method. Secondly, the recombinant bFGF added to the induction medium is a
growth factor, which could be replaced with a small molecular compound. The use
of chemically-defined small molecules rather than growth factors is generally more
affordable and reliable as they tend to be more specific in their mode of action.
Thirdly, human placental collagen IV is used as a coating for adherent culture, and
it is subject to batch-to-batch variation. Various synthetic coatings incorporating
specific binding sequences are currently on the market, and they may provide a
more optimal culture substrate than human placental collagen (Villa-Diaz et al.,
2013). Finally, although the corneal epithelium medium CnT-30 is chemicallydefined, it does contain unspecified animal-derived components. The novel
simplified and xeno-free media CnT-Prime and CnT-Prime-2D, manufactured by
the same company, could be tested to see if they could replace CnT-30. Overall,
further studies are needed to refine the differentiation method in fully-defined and
xeno-free conditions.
Characteristics of hPSC-derived LESCs
After adhering to the human placental collagen IV coating, cell migration and
outgrowth from three-dimensional cell aggregates was primarily fibroblast-like.
Upon reaching confluence, cells would obtain the compact epithelial morphology
similar to that of primary LESCs. In this dissertation, LESC-like cells were
obtained from hPSCs within approximately 30 days of differentiation. At this timepoint, protein expression of the clinically significant marker p63 was at its highest,
decreasing slightly by day 44 (Study I). Furthermore, the putative LESC marker
BMI-1 was expressed in over 80% of cells at this time-point (Study II), and
positive expression of ABCG2, CK15, DG-3, p40, p63α, PAX6, and TCF4 was
assessed qualitatively (Studies I, II and III). Meanwhile, protein expression of an
epidermal differentiation marker CK10/13 was not detected. Gene and protein
expression of CK3 and 12, markers specific to mature corneal epithelium, was
observed by day 44 in differentiation, and faint protein expression was detected
already at day 20 (Study I). Confirming positive expression of several putative
LESC markers is essential, because there is currently no known marker capable of
distinguishing between LESCs and early-stage TACs. Importantly, co-localization
of p40 (i.e. ΔNp63) and p63α proteins indirectly verifies expression of the ΔNp63α
isoform. Judging by their gene and protein expression profiles, hPSC-derived cells
obtained in this work do in fact possess LESC-like characteristics, although it
cannot be said with certainty whether they are closer to true LESCs or early-stage
progenitor cells.
To compare, the first study demonstrating successful differentiation of CECs
from hESCs reported p63 expression to peak after six days of differentiation,
yielding 15-25% positive cells (Ahmad et al., 2007). However, close to 60% of cells
were also expressing CK3/12 at the same time-point, and gene and protein
expression of CK10 was also detected in the same cultures, suggesting that skinlike epithelial cells are also obtained using this differentiation method. Similarly,
other differentiation studies carried out in the presence of conditioned medium
also show that hPSC-derived cells bypass the LESC-like state fairly early on and
express primarily CK3 and 12 after roughly two weeks of differentiation
(Brzeszczynska et al., 2014; Shalom-Feuerstein et al., 2012). In addition, two
separate studies found that hiPSCs derived from limbal epithelium have a much
higher propensity for corneal epithelial differentiation than hiPSCs derived from
hDFs (Hayashi et al., 2012; Sareen et al., 2014). In particular, using mouse feeder
cells to differentiate hiPSCs derived from hDFs for 12-16 weeks yielded RPE and
lens epithelial cells in addition to low amounts of CK12 and 14 positive cell
colonies (Hayashi et al., 2012). Using a mixture of fibronectin, type IV collagen and
laminin as an ECM coating was also fairly inefficient at inducing corneal
differentiation of hiPSCs derived from hDFs: after two weeks of culture only 2030% of cells expressed CK14 and 15, and close to 10% expressed ΔNp63 (Sareen
et al., 2014). For comparison, hiPSCs derived from limbal epithelium generated
about 60% CK14 and 15 positive cells, and close to 20% ΔNp63 positive cells
under identical culture conditions. Lastly, differentiation on hAM or denuded
human cornea was more effective, supporting the premise that a LESC-like niche
microenvironment plays an important role in guiding hPSC differentiation (Sareen
et al., 2014). Nevertheless, validation of LESCs is challenging because their identity
has not been clearly defined. This is a hindrance especially when considering
LESC-like cells derived from another cell type, such as somatic cells, adult stem
cells or hPSCs. Thus, there is a need for a consensus regarding the basic
characteristics and qualities that are sufficient for identification of LESC-like cells.
Cell surface marker expression
Protein expression of several cell surface markers was analyzed using flow
cytometry in LESC-like cells differentiated from two hiPSC lines, and compared to
their undifferentiated counterparts (unpublished results). Most importantly, the
pluripotency marker TRA-1-81 was expressed in 73–97% (seven biological
replicates) of undifferentiated hiPSCs, and in 0.6–3.6% (six biological replicates) of
hiPSC-LESCs after 30-35 days of differentiation (Figure 7). In order to ensure
safety of cell-based therapy, it is important that hPSC-LESCs do not contain
potentially tumorigenic pluripotent cells. Recently, a novel strategy to eliminate
pluripotent cells from potentially heterogeneous cell populations has been
introduced, where a small molecular compound selectively eliminates
undifferentiated hPSCs by inhibiting oleic acid biosynthesis (Ben-David et al.,
2013). Alternatively, enrichment of LESC-like cell populations can be implemented
via cell sorting, utilizing a LESC-specific cell surface marker such as ABCB5 or
ABCG2. Tumorigenic potential of hPSC-derived cells, whether or not they have
been enriched or purified, could be assessed in vivo using immune-deficient rodent
models (Kanemura et al., 2014).
Integrins are cell adhesion molecules essential for cell attachment to various
ECM proteins. Different types of cells express different integrins, and primary
LESCs have previously been shown to express integrins α1, α2, α6, β1 and β4
(Albert et al., 2012; Vereb et al., 2013). To compare, hiPSC-LESCs expressed high
levels of integrin β1, and moderate levels of integrin αV, while protein expression
of integrins α1 and α4 was low and variable between replicates (Figure 7).
Moreover, the MSC markers CD31, CD34 and CD45, as well as the leukocyte cell
adhesion molecule integrin β2 and VE-cadherin were not expressed in hiPSCLESCs. Finally, protein expression of ICAM-1, CD73, c-kit and MCAM was quite
low, and varied between replicates. MCAM and c-kit were previously detected in
primary LESCs, but not in mature CECs, while the opposite was true for ICAM-1
expression (Vereb et al., 2013). The expression differences between hPSC-LESCs
derived and analyzed in this dissertation and primary LESCs reported by other
laboratories may be explained by the poor serum-free culture conditions that
hPSC-LESCs are differentiated and maintained in, coupled with the lack of
interactions with other cell types and ocular niche factors. Further research using
animal models or ex vivo organotypic culture is needed to see whether or not this
changes if hPSC-LESCs are transplanted onto the ocular surface.
Self-renewal and proliferation
LESCs are tissue-specific stem cells, and therefore have a capacity for producing
cell generations, and a potential for self-renewal. To test their proliferative capacity
in vitro, hPSC-LESCs were serially passaged at roughly two-week intervals in a
serum-free progenitor cell targeted medium CnT-20. The LESC-like cells retained
their morphology for at least three passages, and p63 expression was maintained in
over 50% of cells. Further passaging of hPSC-LESCs was not attempted, and could
be tested in the future. Primary LESCs cultured on mouse feeder cells in a medium
containing FBS have been shown to maintain their phenotype for up to 14
passages before reaching senescence (Pellegrini et al., 1999). However, frequent
enzymatic dissociation subjects cells to stress and may induce chromosomal
aberrations (Bai et al., 2015; Hoffman & Carpenter, 2005). Therefore, extensive cell
passaging may not be desirable in practice, especially if cell therapy is the target.
The CFE assay is an in vitro functionality test commonly used for evaluating the
self-renewal properties of a cell population. Generally, single-cell suspensions are
plated onto mitotically inactivated feeder cells at clonal densities, although the assay
has also been carried out in feeder-independent conditions, on ECM coatings
(Albert et al., 2012). There is a high degree of variation in the average CFE values
for primary LESCs among different laboratories, which is likely caused by
discrepancies in isolation and culture methods (Albert et al., 2012; Kolli et al., 2010;
Li et al., 2005; Pellegrini et al., 1999). Also, only a small amount of cells at the
limbus are authentic LESCs, while most are considered to be TACs of varying
maturity levels (Pellegrini et al., 1999). In this work, because hPSC-LESCs were
differentiated and cultured in the absence of feeder cells, the CFE assay was
performed using human placental collagen IV coating. The values obtained for
LESCs differentiated from two hiPSC lines in serum-free conditions are low, and
would require a more careful testing and validation. It remains to be seen whether
purifying hPSC-LESC populations or enriching the culture conditions for the
duration of the assay would enhance their CFE. For instance, using feeder cells or
adding serum to the culture medium may in fact create a more favorable
microenvironment and promote the clonal growth of hPSC-LESCs.
Comparison with native corneal and limbal epithelia
High-throughput characterization methods are generally more informative than
conventional characterization techniques, allowing a broader analysis of target cell
populations. Therefore, mass spectrometry-based iTRAQ proteomics was used to
compare hPSC-LESCs with their native counterparts obtained directly from the
ocular surface of cadaveric human donors. Study II was the first study to utilize a
high-throughput proteomics approach for hPSC-LESC characterization. A total of
860 unique proteins present in all samples were identified, including various LESC
niche components, proteins involved in cell cycling, proliferation, differentiation
and apoptosis, and most importantly corneal and limbal markers. Judging by their
overall protein expression profiles, it appears that hPSC-LESCs fall in between
LESCs and terminally-differentiated CECs. To compare, 2737 proteins have been
previously identified in the corneal epithelium, yet only a fraction of them was
quantified (Dyrlund et al., 2012). The limitation of the iTRAQ method is that it is
only capable of detecting proteins present in all analyzed samples. Therefore,
proteins expressed exclusively in the native ocular surface epithelial cells, or
exclusively in hPSC-LESCs, were not detected. This explains why only a few
proteins involved in angiogenesis or immune response were identified in this study
– hPSC-LESCs lack interactions with other cell types or blood vessels, and are not
exposed to pathogens in the same way as the ocular surface in vivo. Mechanisms
such as angiogenesis and immune response are therefore not necessarily needed,
and this is reflected in the protein expression profile. In the future, it would be
interesting and important to characterize entire proteomes of hPSC-LESCs and
native ocular surface epithelial cells, to better assess the differences between these
cell populations. Nevertheless, iTRAQ proteomics did reveal clear similarities
between hPSC-LESCs and their native counterparts, providing valuable
information for further studies.
Bioengineered collagen matrix as hPSC-LESC carrier
Transplantation of an epithelial cell sheet to the ocular surface requires a
supportive carrier. Collagen is the most abundant structural component of the
corneal stroma, and therefore has been widely researched in attempts to provide a
better alternative to hAM. Collagen is also biodegradable, possesses low
immunogenicity and has shown promising results in vitro and in vivo, as a cell-free
scaffold, or in combination with primary LESCs (Chae et al., 2015; Fagerholm et
al., 2014; Levis et al., 2013). Conventional collagen hydrogels are fairly soft due to
high water content, requiring plastic compression or chemical cross-linking to
enhance their mechanical strength (Ahn et al., 2013; Levis et al., 2010; Mi et al.,
The aim of Study III was to evaluate the suitability of a cross-linked collagen
hydrogel fabricated as thin membranes to act as a carrier for hPSC-derived LESCs.
The bioengineered matrix possessed excellent optical properties, had high water
content and was mechanically stable yet elastic. The cross-linking agents used
during fabrication do not become incorporated into the hydrogel, eliminating the
risk of toxic degradation products being released into the tissue (Ahn et al., 2013).
The biocompatibility of the similarly-fabricated, yet thicker bioengineered matrices
was previously verified in vivo, by implanting cell-free matrices into the corneal
stroma of rabbits (Koulikovska et al., 2015). Although the collagen used for
fabrication of these matrices is of porcine origin, and immunosuppressive
medication was not used post-operatively, no adverse immune reaction was
observed. This offers hope for possible clinical applications, as long as the matrix
production is standardized and carefully monitored according to GMP standards.
The in vitro study presented as part of this dissertation demonstrated that the
bioengineered matrix supports the adhesion and proliferation of hPSC-LESCs in
serum-free conditions. Further studies are needed to assess the performance of this
tissue engineered construct in vivo, using animal models. In addition, immunogenic
properties of hPSC-LESCs and bioengineered matrix could be studied using
various ex vivo assays, such as mixed lymphocyte culture, lymphocyte
transformation tests or the enzyme-linked immunospot assay, which is able to
detect cytokine production on a single-cell level (Lindemann, 2014). Finding a
suitable surgical technique for transplantation onto the ocular surface will also
require attention. Ideally, this type of tissue engineered construct combining a
sufficiently thick carrier with LESC-like cells could serve as a replacement for
damaged corneal stroma, while providing a self-renewing source of LESCs and
corneal epithelium.
Future perspectives
The field of regenerative medicine is relatively young – the first hESC lines were
derived in 1998 (Thomson et al., 1998) and the first hiPSC lines were generated in
2007 (Takahashi et al., 2007). However, both cell types have rapidly progressed
towards cell-based therapies in the recent years. Transplantation of hPSC-derived
LESC-like cells could be possible in the future, yet several issues need to be
addressed to ensure the high quality and safety of the approach.
Although the native corneal epithelium is composed of four to six cell layers,
evidence supporting transplantation of LESC-like cell monolayers is rapidly
accumulating. First of all, clinical studies show that a monolayer of primary LESCs
transplanted on hAM is capable of differentiating and stratifying on the ocular
surface post-transplantation (Kolli et al., 2010; Shortt et al., 2008). Secondly, a
recent in vitro study investigating the effects of air-lifting on cell functionality
revealed that primary LESCs lose their ability to re-epithelialize a wounded area
upon stratification (Massie et al., 2014). And finally, culturing human limbal
explants at an air-liquid interface was shown to induce squamous metaplasia –
abnormal epidermal differentiation confirmed by co-localization of the corneal
CK12 and epidermal CK10 (Li et al., 2008). Taken together, these results suggest
that in vitro stratification of LESC-like cells prior transplantation may in fact be
disadvantageous. Nevertheless, the ability to differentiate and give rise to the
stratified corneal epithelium is a key characteristic of LESCs, and it may therefore
be a requirement to demonstrate the functionality of hPSC-derived LESCs preclinically. However, the eye likely provides a more optimal environment to induce
cell differentiation and stratification than an in vitro culture system, due to the
various niche components and growth factors present at the ocular surface
(Bolanos-Jimenez et al., 2015; Ordonez & Di Girolamo, 2012).
The interactions between the corneal epithelium and the underlying corneal
stroma may play an important role in maintaining corneal integrity. For instance,
incorporating hLFs into plastically-compressed collagen hydrogels was shown to
enhance the production of basement membrane components by LESCs (Levis et
al., 2010). However, the ability of LESCs to re-epithelialize a wounded area was not
affected by hLF incorporation (Massie et al., 2014). Interestingly, an in vitro study
conducted in a different laboratory demonstrated that human corneal epithelial
cells only stratified if human corneal fibroblasts were incorporated in the culture
system (Kobayashi et al., 2015b). The conflicting results are possibly due to the
differences in cell isolation and culture methods. Furthermore, the recently
identified limbal niche cells, also known as limbal mesenchymal cells, were shown
to support LESCs in co-culture, emphasizing the importance of a niche
microenvironment for LESC function (Li et al., 2014; Nakatsu et al., 2014).
Alternatively, co-culture and co-transfer of mouse LESCs with bone marrowderived MSCs was shown to inhibit local inflammatory reactions and support the
healing process in a mouse model (Zajicova et al., 2010). MSCs may be beneficial
even when administered systemically. Studies using mouse models have shown that
MSCs possess the ability to migrate to the inflamed ocular surface and suppress
inflammation, thereby improving allograft survival (Lan et al., 2012; Oh et al.,
2012; Omoto et al., 2014). It remains to be seen whether similar effects can be
achieved in human patients. More research is needed to determine the optimal
strategy for hPSC-LESC transplantation and whether stromal cells are needed
either as part of the graft, or administered systemically.
In addition to regenerative medicine, hPSCs offer novel opportunities for tissue
modeling and drug development. More specifically, hPSC-derived LESCs or fully
stratified corneal epithelial constructs could be used as an in vitro model to study
drug absorption, permeability and transport (Vellonen et al., 2014). Traditionally,
animal models, most commonly rabbits, are used to evaluate ocular drug
absorption and chemical irritation. For instance, the Draize eye irritation test
performed on rabbits has been widely criticized due to its lack of reproducibility,
overestimation of human responses, and animal cruelty (Bartok et al., 2015).
Alternative ex vivo models using porcine corneas have been developed to study
transcorneal drug permeation and predict eye irritation of cosmetic ingredients
(Pescina et al., 2015; Van den Berghe et al., 2005). Moreover, various in vitro cell
culture models have been established, and there are currently two commerciallyavailable tissue models for ocular toxicity and irritation studies: SkinEthic™
Reconstructed Human Corneal Epithelium (EpiSkin, Lyon, France) and
EpiOcular™ (MatTek Corporation, Ashland, MA). Utilizing primary and
immortalized cell lines have shown promise as a possible alternative to the Draize
test (Bartok et al., 2015; Reichl, 2008), and hPSC-derived corneal epithelium could
be evaluated in a similar way. Ideally, an in vitro model for permeation and toxicity
studies should exhibit a multilayered structure with tight junctions and barrier
properties similar to that of the native corneal epithelium. For this purpose,
differentiation and culture does not necessarily need to be carried out in
chemically-defined and xeno-free conditions, as long as a functional corneal
epithelium-like structure is obtained.
To conclude, the novel tissue engineering approach described in this
dissertation provides a valuable and clinically relevant treatment strategy for ocular
surface reconstruction. The method could be translated to the clinic after further
optimization and testing in animal models in accordance with the regulatory
guidelines for ATMPs defined by EMA.
The aim of this dissertation was to examine the ability of several hPSC lines to
differentiate towards LESC-like cells capable of self-renewal and terminal
differentiation. LESC differentiation was carried out in the absence of feeder cells
and serum, in order to minimize biological variation and improve reproducibility.
The resulting hPSC-derived LESCs were characterized and compared with native
ocular surface epithelial cells using high-throughput proteomics. Finally, a
bioengineered collagen matrix was evaluated as a possible carrier for
transplantation of hPSC-derived LESCs, cultured in serum-free conditions. Based
on the results of these studies, the following conclusions can be drawn:
1. Several hPSC lines were successfully differentiated towards LESCs.
Spontaneous differentiation did not yield detectable amounts of
Mimicking in vivo corneal development using two smallmolecule inhibitors along with bFGF promoted corneal
epithelial differentiation.
Small-molecule induction followed by maturation in a
commercial corneal epithelium medium CnT-30 resulted in
efficient and reproducible differentiation of LESC-like cells.
2. Human PSC-derived LESCs possessed appropriate cell morphology,
gene and protein expression, and were capable of both self-renewal and
terminal differentiation – features typical to authentic LESCs.
3. Comparative proteomics revealed a total of 860 unique proteins that
hPSC-derived LESCs have in common with their native counterparts.
Their overall protein expression profile demonstrated a similarity
between the cell types, and strengthened the evidence for LESC-like
properties of hPSC-derived LESCs.
4. Bioengineered collagen matrices supported the growth of hPSC-derived
LESCs in serum-free conditions in vitro, showing potential for use as a
transplantable carrier of these cells in clinical applications. However,
further in vivo testing using animal models will be necessary to
definitively test the functionality of hPSC-derived LESCs.
The research for this dissertation was carried out at the Institute of Biosciences and
Medical Technology (BioMediTech), University of Tampere, during the years
2011-2015. I am grateful to the Dean of the institute Hannu Hanhijärvi for
maintaining excellent research facilities and a collaborative working environment
during my studies.
I would like to thank the Doctoral Program in Biomedicine and Biotechnology
at the University of Tampere, the University of Tampere Foundation, the Finnish
Funding Agency for Technology and Innovation (TEKES), the Finnish Eye and
Tissue Bank Foundation, the Emil Aaltonen Foundation, the Foundation
Supporting Research in Tampere, and the Finnish Concordia Fund for financially
enabling my research and journeys to international conferences.
Most importantly, I am deeply grateful to my supervisors Associate Professor
Heli Skottman and Tanja Ilmarinen, PhD. Both of you are an inspiration and I
greatly admire your expertise, enthusiasm, and overall passion for science. Heli, you
introduced me to the fascinating world of stem cells already during my
undergraduate studies, and afterwards gave me the opportunity to work with the
corneal differentiation project. Thank you for granting me scientific freedom, yet
always finding the time to provide guidance. Tanja, thank you for all the help,
reassurance and optimism during these years – they have been invaluable for this
dissertation and my sanity. Also, your multitasking skills are incredible, and deserve
special recognition.
The members of my thesis committee Professor Hannu Uusitalo and Docent
Susanna Miettinen are warmly thanked for the encouraging annual meetings and
thought-provoking discussions. I also owe my gratitude to Professor Juha
Holopainen and Docent Frederic Michon for finding the time to review this
dissertation and providing constructive feedback to improve its quality. I am
thankful to my co-authors Ulla Aapola, Roger Beuerman, Antti Jylhä, Janika
Nättinen, Goran Petrovski, Mehrdad Rafat, Anjula Ratnayake, Jochen Rieck, and
Zoltán Veréb – without your contributions to the publications, this dissertation
would not have been possible.
The entire staff of BioMediTech, especially the regenerative medicine research
groups, is thanked for such a friendly working environment. Regea Cell and Tissue
Center personnel, particularly Annika Hakamäki and Minna Sjöblom, are thanked
for providing human donor corneas for research during this dissertation, and
Marja-Leena Koskinen is acknowledged for skillful paraffin embedding and
sectioning of these corneas.
I wish to thank the past and present members of the Eye Group – it’s been a
pleasure to work with all of you! Outi Heikkilä, Outi Melin and Hanna Pekkanen,
thank you for technical assistance and more importantly for outstanding company
in the cell culture lab. Kati Juuti-Uusitalo, thank you for scientific advice during
these years. Hanna Hiidenmaa and Heidi Hongisto, thank you both for supervising
me back in the days of my undergraduate studies and showing me that research
(especially in the right company) can be fun. Also special thanks to Heidi for all the
help and support while I was writing this thesis, and sorry for all the distractions I
caused you! Anni Sorkio – the other half of the A-team – thank you for sharing the
fun times, existential crises, and cheesecake. You are a wonderful person and an
amazing friend.
Thanks to my friends from the university world for the annual picnics, sangria
evenings and other traditions, and for sharing the ups and downs of life as a
researcher. Thanks to “the gang” for your friendship and silliness, and for
reminding me that there’s more to life than science. And last but not least, I am
eternally grateful to my family for encouraging me in all my endeavors, and to
Johan for the much needed moral support and understanding during these stressful
times. I am lucky to have you in my life.
Tampere, December 2015
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