Life science
Regenerative therapy, Drug development

Generation of 3D lacrimal gland organoids from humanpluripotent stem cells
Department of Stem Cells and Applied Medicine, Graduate School of Medicine
Professor Ryuhei Hayashi
https://researchmap.jp/stemed0701?lang=en

The surface of the eye (ocular surface) consists of the cornea
and conjunctiva, and the presence of a lacrimal fluid layer is essential for the homeostasis of the ocular surface. The lacrimal fluidlayer is mainly maintained by the secretion of tear fluid and mucinfrom the lacrimal gland, but when the lacrimal gland is damagedby autoimmune diseases such as Sjögren’s syndrome, the ocularsurface becomes dry, resulting in severe dry eye. Our researchgroup had previously succeeded in producing the cornea andconjunctiva from human iPS cells, but the generation of the lacrimal gland had not yet been reported. Noting that the cornea, conjunctiva and lacrimal gland have the same developmental origin,
we applied the two-dimensional ocular organoids (named SEAM)
previously used to induce the cornea and conjunctiva and newly
attempted to induce lacrimal gland primordia. First, we found thatlacrimal gland-like cell clusters appeared in the iPS cell-derived
SEAM, and successfully produced 3D lacrimal gland organoids byconducting 3D culture in Matrigel.

Fig.1 Summary of the study
Ocular surface ectoderm, which is thought to be the common primordium of thelacrimal gland and cornea, is induced in zone-3 of eye-like organoids derived
from human iPS cells. After further differentiation culture, we induced and isolated
lacrimal gland progenitor cells by cell sorting and successfully made 3D lacrimal
gland organoids by 3D culture in Matrigel.

▍ Background & Results
We found that in the region of the ocular surface ectoderm (common developmental primordium of the lacrimal gland and cornea)
within the induced SEAM (zone-3), there were widely distributed ascell clusters with PAX6/SOX9-positive gland-like structures. Whenthese gland-like cell clusters were isolated and cultured in Matrigel
in 3D culture, budding and elongation of ducts were observed, andfurther branching occurred, resulting in a lacrimal gland-like structure. Furthermore, lacrimal gland progenitor cells were successfully isolated using a cell sorter. Immunostaining of the generated
lacrimal gland-like organoids confirmed the expression of lacrimal
gland markers such as PAX6, AQP5 and SOX9. In addition, aswelling assay was performed to verify the lacrimal gland organoid
lacrimal fluid secretion capacity, and significant swelling of the terminal end of the organoid was observed upon forskolin stimulation.
Next, transplantation experiments were performed using nuderats to investigate the viability and functional maturation of thelacrimal gland organoids in vivo. When lacrimal gland organoids
were examined 4 weeks after transplantation, lumen formation andcell polarity, which were insufficient in vitro, became remarkable.
Furthermore, the expression of lactoferrin and lysozyme, defence
proteins of the lacrimal fluid, which were hardly expressed in vitro,
was observed and significantly increased after transplantation
evaluated by ELISA. These results indicate that human iPS cell-derived lacrimal gland organoids are functional in vivo.

▍ Significance of the research and Future perspective
The results of this research have made it possible to produce 3D lacrimal gland tissues from human iPS cells and will enable the developmentof regenerative Therapy for severe dry eye. In addition, the ability to produce large quantities of human lacrimal gland organoids from human
iPS cells is expected to make significant progress in drug discovery andresearch for the pathology of dry eye and other ocular diseases.

Fig.2 Schematic illustration of SEAMSEAM typically forms a concentric four zones, and the ocularsurface ectoderm, which is the primordium of the lacrimal
gland and corneal epithelium, develops in zone 3.

Proportion of area increased (%)
Proportionof area increased (%)

Life science

Abstract

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1100 mµmo rs k o l i n
M fforskolin

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Fig.3
Characterization
lacrimalFig.3 Characterizationof oflacrimal
gland
glandorganoids
organoids(A)
Lacrimalglandorganoids
were(A) Lacrimal gland organoids were fabrifabricatedby3D
theculture
3D cultureof lacrimal
cated by theof lacrimal
gland
gland
progenitor
cellsisolated
by cellprogenitor cells isolated by cell sorting.
(B)sorting.
(B) 3D lacrimal gland organoids
3D lacrimal gland organoids expressed
expressed
lacrimalglandmarkers.
(C)lacrimal gland markers. (C) swelling assay
swelling assay to determine water/ion
to determine water/ion exchange capacity
exchange capacity showed a significant
showed a significant increase in swellingincrease in swelling of the organoids byof the organoids by forskolin stimulation.
forskolin stimulation.

P a t e n t Japanese Patent Application No.2020-511023, WO2019189640
Hayashi, Ryuhei; Okubo, Toru; Kudo, Yuji et al. Generation of 3D lacrimal gland organoids from human pluripotent stem cells. Nature 2022, 605,126-131.
doi: 10.1038/s41586-022-04613-4
Nomi, Kimihito; Hayashi, Ryuhei; Ishikawa, Yuki et al. Generation of functional conjunctival epithelium, including goblet cells, from human iPSCs. Cell Rep.
Tr e a t i s e
2021, 34, 108715. doi: 10.1016/j.celrep.2021.108715
Hayashi, Ryuhei; Ishikawa, Yuki; Sasamoto, Yuzuru et al. Co-ordinated ocular development from human iPS cells and recovery of corneal function. Nature2016, 531, 376-380. doi: 10.1038/nature17000
URL
K e y w o r d pluripotent stem cells, lacrimal gland, iPS cells, ocular surface, regenerative medicine