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Generation of 3D lacrimal gland organoids from human pluripotent stem cells

Abstract

Lacrimal glands are the main exocrine glands of the eyes. Situated within the orbit, behind the upper eyelid and towards the temporal side of each eye, they secrete lacrimal fluid as a major component of the tear film. Here we identify cells with characteristics of lacrimal gland primordia that emerge in two-dimensional eye-like organoids cultured from human pluripotent stem cells1. When isolated by cell sorting and grown under defined conditions, the cells form a three-dimensional lacrimal-gland-like tissue organoid with ducts and acini, enabled by budding and branching. Clonal colony analyses indicate that the organoids originate from multipotent ocular surface epithelial stem cells. The organoids exhibit notable similarities to native lacrimal glands on the basis of their morphology, immunolabelling characteristics and gene expression patterns, and undergo functional maturation when transplanted adjacent to the eyes of recipient rats, developing lumina and producing tear-film proteins.

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Fig. 1: Development of lacrimal-gland-like clusters in a SEAM.
Fig. 2: Generation and characterization of lacrimal-gland-like organoids.
Fig. 3: Characterization of lacrimal-gland-like primordia.
Fig. 4: Transplantation of lacrimal-gland-like organoids.

Data availability

Bulk RNA-seq and scRNA-seq datasets have been deposited at the NCBI GEO repository under accession numbers GSE157678 and GSE174653Source data are provided with this paper.

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Acknowledgements

We thank K. Nomi, T. Soma, K. Maruyama and J. Mantel for technical support and for providing experimental equipment and supporting research grants; D. Okuzaki and the staff at the NGS Core Facility of the Genome Information Research Center at the Research Institute for Microbial Diseases, Osaka University for support in RNA-seq and data analysis; and Y. Honma of ROHTO Pharmaceutical for scientific discussions. This work was supported in part by the Project for the Realization of Regenerative Medicine from The Japan Agency for Medical Research and Development (AMED, JP19bm0404058h0001, JP20bm0404058h0002, JP21bm0404058h0003), Grant-in-Aid for Scientific Research (17K11480, 20H03842) from Japan Society for the Promotion of Science (JSPS) and AMED-CREST (JP20gm1210004h0002, JP21gm1210004h0004).

Author information

Authors and Affiliations

Authors

Contributions

R.H. and T.O. designed the research plan. T.K. performed the PS cell maintenance culture. T.O., Y.K., Y.I., T.I. and T.K. performed the PS cell differentiation culture. Y.K., S.S. and T.O. performed the flow cytometry experiments and lacrimal gland induction. R.H., T.O., Y.K., T.I. and Y.I. performed immunostaining, western blot and gene expression analyses. Y.K. performed swelling assay. K.S. and T.I. performed the ELISAs and animal experiments. R.H., T.O., S.S. and Y.K. analysed the data. Y.I. and Y.K. performed the siRNA experiments. S.-J.P. and R.H. performed the scRNA-seq analysis. R.D.Y., Y.I. and A.J.Q. performed the transmission electron microscopy experiments. S.S., K.N. and A.J.Q. supervised the research. R.H., T.O. and A.J.Q. wrote the manuscript. R.H. and K.N. obtained financial support.

Corresponding author

Correspondence to Ryuhei Hayashi.

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Competing interests

R.H. is the holder of an Endowed Chair provided by ROHTO Pharmaceutical. The other authors declare no competing interests.

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Nature thanks Karl Koehler and the other, anonymous, reviewer(s) for their contribution to the peer review of this work.

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Extended data figures and tables

Extended Data Fig. 1 Differentiation of multiple ocular cell lineages in the SEAM.

a, Schematic illustration of the method for inducing lacrimal-gland-like clusters in a SEAM. b, Gene expression of three germ layer markers in 2- and 4-week differentiated SEAMs (n = 4 biological replicates). Data are presented as mean ± s.d. c–d, Immunostaining for PAX6 (green) and AQP5 (red) in a 10-week differentiated SEAM. Magnified views of the dashed areas in (c) are shown in (d). n = 3 distinct samples. Nuclei, blue. Scale bars, 100 μm. e, Immunostaining for CD44 (green) and HTN1 (red) in a thin section of lacrimal-gland-like clusters in a 14-week differentiated SEAM. n = 3 biological replicates. BF, bright field; Scale bar, 100 μm. f, Flow cytometric analysis of SSEA-4, ITGB4, and CD200 in isolated lacrimal-gland-like clusters induced in SEAMs after 11 weeks of culture. Four populations in the CD200 cells are categorized as P1–P4. n = 2 biological replicates. Scale bar, 200 μm. OESCs, ocular surface epithelial stem cells. g, 3D culture of the sorted cell populations (P1–P4) to induce lacrimal-gland-like organoids at day 3 (n = 3 biological replicates). Scale bars, 1,000 μm. h, Screening for organoid induction medium using a combination of lacrimal gland-related cytokines and Y-27632. n = 2 biological replicates. Scale bars, 1,000 μm. i, Comparison of lacrimal gland-related gene expression in the organoids cultured in medium containing either Y-27632 and EGF (YE), or Y-27632, EGF, KGF, BMP7 and FGF10 (YEKBF). *p < 0.05 (n = 4 biological replicates; NS, not significant). Data are presented as mean ± s.d. Exact p values are provided in the Source Data.

Source data

Extended Data Fig. 2 Formation of lacrimal-gland-like organoids from human iPS cells and ES cells.

a–c, Development of lacrimal-gland-like organoids from hiPSC lines 201B7 (a) and YZWJs524 (b), and the human ES cell line, KhES-1 (c). For 201B7, n = 413; for YZWJs524, n = 6; for KhES-1, n = 40 organoids. Scale bars, 1,000 μm. d, Ratio of P3 cells in the CD200- fraction of SEAMs derived from pluripotent stem cell lines; 201B7 (n = 91 biological replicates), YZWJs524 (n = 5 biological replicates), and KhES-1 (n = 10 biological replicates). Data are presented as mean ± s.d. e, Gene expression analysis of lacrimal gland markers in lacrimal-gland-like organoids derived from pluripotent stem cell lines, 201B7 (n = 4 biological replicates), YZWJs524 (n = 5 biological replicates), and khES-1 (n = 5 biological replicates). Data are presented as mean ± s.d.

Source data

Extended Data Fig. 3 Characterization of lacrimal-gland-like organoids.

a–c, Light sheet fluorescence microscopy of lacrimal-gland-like organoids stained with CDH1 and PAX6 (a), KRT14 (b) and CD44 (c). Lower panels show magnified views of the upper panels. n = 2 biological replicates. Scale bars, 200 μm. d, H&E staining of hiPSC-derived lacrimal-gland-like organoids. n = 3 biological replicates. Scale bars, 500 μm. e, Immunolocalization of PAX6 (green) and CDH1 (red) in thin sections of hiPSC-derived lacrimal-gland-like organoids. n = 3 biological replicates. Nuclei, blue. Scale bars, 100 μm. f, Immunostaining for SOX9 (green), AQP5 (red) and ACTA2 (magenta) in thin sections of hiPSC-derived lacrimal-gland-like organoids. n = 3 biological replicates. Nuclei, blue. Scale bars, 100 μm. g–i, Immunostaining of KhES-1-derived lacrimal-gland-like organoids for PAX6 (green) and CDH1 (red) (g), CD44 (green), HTN1 (red) and CDH1 (magenta) (h), and SOX9 (green), AQP5 (red) and ACTA2 (magenta) (i). n = 3 biological replicates. Nuclei, blue. Scale bars, 100 μm. j, Gene expression analysis for lacrimal gland-related markers in iPS cells (iPSC), P3 cells, lacrimal-gland-like progenitors (LGP) and organoids (LGO). n = 4 biological replicates. Data are presented as mean ± s.d.

Source data

Extended Data Fig. 4 Characterization of iPS cell-derived lacrimal-gland-like organoids by single-cell RNA sequencing.

a, Uniform manifold approximation and projection (UMAP) plot of all single cells and the summary of enriched GO terms in each cluster; n = 4071 cells (day 0), n = 4566 cells (day 10), n = 2448 cells (day 20). b, Pseudotime trajectory of the single-cell transcriptome showing three branches from the root (cluster 0 of day 0). These represent active epithelial-mesenchymal transition (EMT), cell proliferation, and the development of epithelial secretory cells. c, Cell populations preferentially expressed lacrimal gland-related genes. d, Cell-type annotations of lacrimal-gland-like organoids (day 20) with positive and negative marker genes.

Extended Data Fig. 5 Characterization of domed and flat colonies derived from ocular surface epithelial stem cells.

a–c, Corneal epithelial differentiation from P3-ocular surface epithelial stem cells by 2D culture (a), along with immunostaining for corneal epithelial (b) and lacrimal gland markers (c) in the epithelial cell sheet. Nuclei, blue; n = 3 biological replicates; Scale bars, 100 μm. d, Gene expression analysis for lacrimal gland-related markers in domed and flat colonies. Expression in domed colonies is shown as 1.0. *p < 0.05 (n = 3 distinct colonies from one experiment for each experimental condition; NS, not significant). Data are presented as mean ± s.d. Exact p values are provided in the Source Data. e, A cluster heatmap based on the expression of lacrimal gland, epithelial cell and YAP-related genes. f, Immunostaining for YAP1 (green) in domed (upper panels) and flat (lower panels) colonies derived from sorted P3 cells. n = 3 biological replicates. Nuclei, blue. Scale bars, 100 μm.

Source data

Extended Data Fig. 6 The involvement of BARX2 in lacrimal-gland-like organoid formation.

a, Immunostaining for BARX2 (green) and KRT14 (red) in domed (upper panels) and flat (lower panels) colonies derived from sorted P3 cells. n = 3 biological replicates. Nuclei, blue. Scale bars, 100 μm. b, Colony-forming efficiency and the proportion (at day 21) of domed colonies of P3 cells sorted from SEAMs after 8 and 14 weeks of differentiation culture. n = 3 biological replicates. Data are presented as mean ± s.d. Exact p values are provided in the Source Data. c–d, Immunostaining for PAX6 (green) and KRT12 (red) in 8-week (c) and 14-week (d) differentiated SEAMs. n = 3 biological replicates. Nuclei, blue. Scale bars, 100 μm. e, Immunostaining for BARX2 (green) and PAX6 (red) in lacrimal-gland-like progenitors (left panels) and organoids (right panels). n = 3 biological replicates. Nuclei, blue. Scale bars, 100 μm. f, Effect of BARX2 knockdown using siRNA on domed-colony formation. *p < 0.01 (n = 8 biological replicates; NS, not significant). Data are presented as mean ± s.d. Exact p values are provided in the Source Data. g, Effect of BARX2 knockdown using siRNA on corneal epithelial differentiation. n = 1 sample for each experimental condition. Nuclei, blue, Scale bars, 100 μm.

Source data

Extended Data Fig. 7 Differentiation capability of SEAM-derived lacrimal-gland-like organoids and corneal epithelial cell sheets.

a, 3D organoid culture using cells from SEAM-derived corneal epithelial cells. n = 8 biological replicates. Scale bars, 1,000 μm. b, 2D corneal epithelial culture using cells from lacrimal-gland-like organoids. n = 19 biological replicates. Scale bars, 1,000 μm. c–f, Immunostaining of cells derived from lacrimal-gland-like organoids after 2D cultivation as shown in (b): (c) CD44 (green) and KRT12 (orange); (d) PAX6 (green) and p63 (orange); (e) AQP5 (green), KRT13 (orange), and KRT14 (magenta); (f) HTN1 (green), SOX9 (orange), and KRT3 (magenta). n = 3 biological replicates in (c) - (f). Nuclei, blue. Scale bars, 100 μm. g, Prolonged 3D cultivation of lacrimal-gland-like organoids up to 41 days. n = 2 biological replicates. Scale bars, 1,000 μm. h, Cell passaging of lacrimal-gland-like organoids. n = 2 technical replicates. Scale bars, 1,000 μm.

Extended Data Fig. 8 Transplanted lacrimal-gland-like organoids.

a–b, Images depicting (a) partial and (b) whole removal of rat lacrimal gland and transplantation of lacrimal-gland-like organoids. Scale bar (inset images), 1,000 μm. c–e, Immunostaining for (c) PAX6 (green) and KRT14 (red), (d) SOX9 (green) and CDH1 (red), and (e) CD44 and CD31 (green) in serial sections of grafted lacrimal-gland-like organoids (5×) four weeks after transplantation into rat lacrimal glands. Lower panels in (c-d) show magnified views of the upper panels. Dotted lines in (e) indicate CD44+ lacrimal gland cells. n = 3 biological replicates. Nuclei, blue. Scale bars, 100 μm. f, H&E staining of 2 week-transplanted tissue. n = 3 biological replicates. Scale bar, 500 μm. g–k, Immunostaining of grafted lacrimal-gland-like organoids (5×) two weeks following transplantation to rat lacrimal glands: (g) human nuclei (green), AQP5 (red) and ACTA2 (magenta); (h) CD44 (green) and HTN1 (red); (i) SOX9 (green) and CDH1 (red); (j) human nuclei (green) and KRT14 (red); (k) LTF (green) and CDH1 (red), n = 3 biological replicates. Nuclei, blue; Scale bars, 100 μm. l, Immunostaining of human nuclei (green), ACTA2 (red) and CD31 (magenta) in grafted lacrimal-gland-like organoids four weeks after transplantation into rat lacrimal glands. Arrowheads indicate stromal cells, asterisks indicate vascular endothelial cells, and short arrows indicate blood vessels. n = 3 biological replicates. Nuclei, blue. Scale bars, 100 μm.

Extended Data Fig. 9 Transmission electron microscopy and ELISA of transplanted lacrimal-gland-like organoids.

Transmission electron microscopy of human iPS cell-derived lacrimal-gland-like organoids after 4 weeks of transplantation. a, lumen (l) and lining cells with numerous microvilli (arrows). b, variable basophilia of secretory acinar cells peripheral to lumen. c, multiple desmosomes (arrows) are located along membranes between adjacent secretory cells. d, lateral membranes of secretory cells express interdigitating finger-like processes (p) and tonofilaments (t) associated with desmosomes (arrows). e, lipid vesicle inclusions (lv) are present within basal secretory cells, which rest on a basement membrane (arrow). f, clusters of mitochondria (m) and a lipid droplet (v) in the cytoplasm of a basal acinar cell, adjacent to multi-layered basal lamina (arrow). g, acinar cells towards lumen exhibit secretory granules (sg) with variable size and density, lipid droplets (v) and extensive Golgi bodies (g). h, regions of rough-surfaced endoplasmic reticulum (r) are also present. n = 2 biological replicates in (a) - (h). i, ELISA for human lysozyme (hLYZ) in lacrimal-gland-like organoids (LGOs). (w) and (p) indicate whole and partial lacrimal gland-removal surgeries, respectively. *p < 0.05, **p < 0.01 (LGO×1, LGO×5, LGO×1 (w) and LGO×5 (w), n = 8. Others, n = 4 biological replicates). Data are presented as mean ± s.d. Exact p values are provided in the Source Data. j, ELISA for rat lactoferrin in lacrimal gland tissues and tear fluid. n = 4 biological replicates. Data are presented as mean ± s.d.k, Expression of lacrimal gland-related genes in lacrimal-gland-like organoids (LGOs), transplanted organoids (tLGO), and rat lacrimal gland (Rat LG). The expression level in the LGO is shown to be 1.0. *p < 0.05 (n = 4 biological replicates; NS, not significant). Data are presented as mean ± s.d. Exact p values are provided in the Source Data. ND, not detected.

Source data

Extended Data Fig. 10 Comparison of exocrine gland cells.

a, Immunostaining for CD44 (green), AQP5 (red) and PAX6 (magenta) in lacrimal gland (rat), salivary gland, mammary gland and pancreas (all humans). Lower panels show magnified views of the upper panels. Dotted lines in pancreas tissue indicate the presumptive region of islets. n = 3 biological replicates (rat lacrimal gland) and n = 1 (human gland tissue). Nuclei, blue. Scale bars, 100 μm. b, Expression of PAX6, AQP5 and CD44 in human exocrine gland tissues, lacrimal glands (n = 8), salivary glands (n = 5), and mammary glands (n = 8) based on existing microarray datasets. c, Expression of lacrimal gland and ocular surface epithelium-related genes in transplanted lacrimal-gland-like organoids (tLGO) and in human corneal and conjunctival epithelial cells. CE, corneal epithelial cell; CjE, conjunctival epithelial cell. tLGO, n = 4; CE, n = 6; CjE, n = 3 biological replicates. Data are presented as mean ± s.d. d, Summary of expression of lacrimal gland-related markers in glandular tissues and ocular surface epithelium. Data indicate that PAX6, AQP5, LTF and LYZ-expressing cells are lacrimal gland type cells. SG, salivary gland; MG, mammary gland; PC, pancreas; ND, not determined.

Source data

Supplementary information

Supplementary Information

Supplementary Discussion and additional references.

Reporting Summary

Supplementary Figure 1

Uncropped western blots used for the preparation of Fig. 3j. The bands corresponding to BARX2 and ACTB were electrophoresed at about 40 kDa and 42 kDa, respectively.

Supplementary Table 1

Gene expression for each single-cell cluster. Log-normalized read counts were averaged for individual clusters and profiled in log-space.

Supplementary Table 2

Gene expression for each single-cell cluster in day-20 lacrimal-gland-like organoids. Log-normalized read counts were averaged for individual clusters and profiled in log-space.

Supplementary Table 3

List of the TaqMan probe sets.

Supplementary Table 4

Composition of differentiation cultivation media.

Supplementary Table 5

Details of antibodies used for immunostaining, western blotting and flow cytometry.

Supplementary Video 1

Video of lacrimal-gland-like organoid formation, in which hiPS-cell-derived lacrimal-gland-like progenitor cells form a 3D lacrimal-gland-like organoid in Matrigel.

Supplementary Video 2

Video in 3D of AQP5 expression in a hiPS-cell-derived lacrimal-gland-like organoid. AQP5 is located mainly on acinar-like cells, where it exhibits a punctate staining pattern.

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Hayashi, R., Okubo, T., Kudo, Y. et al. Generation of 3D lacrimal gland organoids from human pluripotent stem cells. Nature 605, 126–131 (2022). https://doi.org/10.1038/s41586-022-04613-4

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  • DOI: https://doi.org/10.1038/s41586-022-04613-4

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