Life science
Regenerative medicine, Drug screening, Developmental biology

Generation of functional conjunctival epithelium, including
goblet cells, from human iPSCs
Department of Stem Cells and Applied Medicine, Graduate School of Medicine
Professor Ryuhei Hayashi

Researchmap https://researchmap.jp/stemed0701

SA Researcher Kimihito Nomi

Researchmap https://researchmap.jp/kimi-hitoLife science

Abstract
We have established a new method for generating functional
mucin-producing conjunctival epithelial cell sheets from human
iPSCs (hiPSCs). The conjunctiva, which sits on the sclera (the whiteof the eye) and lines the inside of the eyelids, is essential for mucinsecretion and the establishment of a healthy tear film.
We demonstrated that conjunctival epithelial lineage cells developed in epidermal growth factor (EGF)-treated hiPSC-derived
eye-like organoids and that they could be isolated by using a cellsorter. We then succeeded in reconstituting the conjunctival epithelium, including goblet cells, from the isolated cells under theinfluence of keratinocyte growth factor (KGF).
This study enables us to obtain a large amount of human conjunctival cells, which has been difficult until now, and great progress is expected in drug discovery research and regenerative
therapy for dry eye disease.

▍ Significance of the research and Future perspective
We succeeded in generating human conjunctival cells, whichwere difficult to obtain previously, from iPS cells. This finding has arange of potential applications to help us better understand human
conjunctival development. Furthermore, this study could help withidentifying novel drugs for dry eye syndrome and could further
open new avenues for regenerative therapies.

▍ Background & Results
The conjunctiva, which sits on the sclera and lines the inside ofthe eyelids, is the source of mucins, which are vital for the stabilityof the tear film. Tear film instabilities due to conjunctival disease result in dry eye disease, which is manifested by physical discomfort
and visual impairment
We have previously established a method for inducing "Selfformed Ectoderm Autonomous Multi-zone" (SEAM, Fig. 1), which isan organoid containing various ocular cells using human iPS cells.
And we have succeeded in producing corneal epithelial tissue
from SEAMs. In the present study, we aimed to differentiate, isolate, and mature conjunctival cells from SEAMs by properly using
growth factors under appropriate conditions.
Firstly, we showed that ocular surface epithelial stem/progenitor cells, which express p63 and PAX6, are present in the EGF
or KGF-treated SEAM zone 3, but that differentiation into corneal
epithelial cells does not take place in the EGF-treated SEAM (Fig.
2A–B). Secondly, EGF-treated SEAMs were stained with three cellsurface markers (CD200, SSEA-4, and ITGB4), divided into six
fractions, and analyzed in detail (Fig. 3A). As a result, it was clarified that the cells of the P2 fraction (CD200-, SSEA-4low, ITGB4+
were differentiated into mature conjunctival epithelium including
conjunctival goblet cells by stratified culture. Unexpectedly, expression of the goblet cell marker MUC5AC was upregulated whenKGF, but not EGF, was added to the maturation medium (Fig. 3B–C). The iPSC-derived conjunctival epithelium contained MUC5AC
producing goblet cells (Fig. 3D) and showed characteristic ofnative conjunctival epithelium (Fig. 3E– F). These results suggestthat key complementary roles of EGF and KGF in directing the differentiation and maturation, respectively, of the human conjunctivalepithelium.

Figure 1. Schematic of ocular surface epithelial cell generation
Ocular surface epithelial stem/progenitor cells are present in the zone-3 of multizonal colony (SEAM), which is an organoid containing various ocular cells derived from human iPSCs. In differentiation culture, conjunctival epithelial lineage cells were predominantly induced in the EGF-treated SEAM derivatives, whereas corneal epithelial lineage cells predominated in the KGF-treated
SEAM derivatives. KGF was necessary for maturation of the hiPSC/SEAM-derived conjunctival epithelium, and goblet cells.

Figure 2. EGF inhibits the differentiation of hiPSC-derived SEAMs into corneal epithelial cells

ure 2. EGF inhib
itsImmunostaining
the differfor
entEGFiatio
nof hiPSC-SEAMs
derive(+EGFd SEand
AM+KGF).
s intoK12co(corneal
rneal epithelicellcmarker)
ells(A)andKGF-treatedepithelial
FigureFig2.
EGF inhibits
the differentiationof hiPSC-derivedSEAMs
intoalcorneal
epithelial cellsexpression was not detected in the EGF-treated SEAM derivatives. (B) Gene expression analysis for EGF and(A) Immunostaining
and KGF-treatedSEAMsand+KGF).
K12(cornealcellepithelialmarker) was not(A) Immunostainingfor EGFforandEGF
KGF-treatedSEAMs (+EGF
and(+EGF+KGF).
K12(cornealepithelialmarker)cellexpression
KGF-treated SEAMs (+EGF and +KGF). K12, and K3 (corneal epithelial differentiation cell markers) weredetected in the EGF-treatedSEAM derivatives. (B) Gene expression analysis for EGF and KGF-treated SEAMs (+EGF and +KGF).
expression
wasnotdetectedin the inEGF-treated
SEAM(B) Gene expression
analysis forEGF(pandsignificantlydownregulatedthe markers)
EGF-treated wereSEAM(p<derivatives.
0.01).
K12, andK3 (cornealepithelial
differentiation
cellsignificantlydownregulated
in the EGF-treated
SEAM< 0.01).

KGF-treated SEAMs (+EGF and +KGF). K12, and K3 (corneal epithelial differentiation cell markers) weresignificantly downregulated in the EGF-treated SEAM (p < 0.01).

Figure 3. Isolation of Conjunctival epithelial progenitors from EGF-treated SEAM derivatives and generation ofFigure 3. Isolation ofConjunctival epithelial progenitors from EGF-treated SEAM derivatives andconjunctival epithelial tissue by maturation culture in KGF containing medium.
generation of conjunctivalepithelial tissue by maturation culture in KGF containing medium.
(A) FACS analysis of the EGF-treated SEAM derivatives (10‒12 weeks) and isolation of each cell fraction. (B)(A) FACS analysis of the EGF-treated SEAM derivatives (10–12 weeks) and isolation of each cell fraction. (B) MUC5AC immunostaining for P2 and P3cells after cultivation. (C) The density of MUC5AC+ cells from the P2 and P3 fraction cells after maturation culture. (D) ELISA for MUC5AC in culture
supernatants of the P2 and P3 fraction cells after maturation culture. (E) Immunostaining for conjunctival functional proteins and ocular surface epithelium-related markers (green) in the hiPSC/SEAM-derived conjunctival epithelium. (F) PAS-staining of the hiPSC/SEAM-derived conjunctival epithelium.

P a t e n t Japanese Patent Application No. 2018-98954, PCT/JP2019/20364
igure 3. Isolation oepithelium,
f Conjunctival epitincluding
helial progenitogoblet
rs from EGcells,
F-treatefrom
d SEAMhumanderivativeiPSCs.
s and geneCellration oRep.
fNomi, Kimihito; Hayashi, Ryuhei; Ishikawa, Yuki et al. Generation of functional Fconjunctival
conjunctival epithelial tissue by maturation culture in KGF containing medium.
2021 Feb 2; 34(5): 108715. doi: 10.1016/j.celrep.2021.108715.
Tr e a t i s e
Hayashi, Ryuhei; Ishikawa, Yuki; Sasamoto, Yuzuru et al. Co-ordinated ocular development
human SEAM
iPS derivatives
cells andrecoveryofisolation
cornealfunction.
Nature.
(A) FACS analysis offrom
the EGF-treated(10‒12weeks) andof eachcell fraction.
(B)
2016 Mar 17; 531(7594): 376-80. doi: 10.1038/nature17000. Epub 2016 Mar 9.
https://resou.osaka-u.ac.jp/ja/research/2021/20210203_1
URL
https://www.amed.go.jp/news/release_20210203-02.html
K e y w o r d conjunctival epithelium, goblet cells, EGF, KGF, SEAM