Additional Supporting Information may be found in the online version of this article.

sc-12-0870_sm_SupplFigure1.tif910KSupplemental online Figure 1. Effect of SHH stimulation on LGR5. (A) Real-time PCR showed that the expression of LGR5 in the human cultured CECs with SHH stimulation was elevated as compared to those without SHH stimulation. Mean±SEM. **P<0.01. n = 3. (B) Immunohistochemistry showed that after SHH stimulation, the expression of LGR5 in the cultured CECs was elevated in some of the cells (arrows), yet not in all of the cells. Bar = 200μm.
sc-12-0870_sm_SupplFigure2.tif2116KSupplemental online Figure 2. Expression of β-catenin in CECs. (A) We found that the expression of β-catenin shifted from the cell membrane to the cytoplasm and nucleus in most of non-target (NT) transfected CECs. (B) In contrast, we could observe the expression of β-catenin in cell membrane of LGR5-transfected CECs. Bar = 100μm.
sc-12-0870_sm_SupplFigure3.pdf562KSupplemental online Figure 3. Expression and function of RSPOs in human CECs. (A) Real-time PCR for RSPO1, RSPO2, RSPO3, and RSPO4 in human corneal epithelial cells (Epi), stroma, and endothelial cells (Endo). (B) Phase contrast microscopy image of cultivated human CECs (7 days, 49 year-old-donor) with or without RSPO1, RSPO2, RSPO3, and RSPO4 (50ng/ml). Bar = 100μm. (C) Immunostaining of Ki67 in human donor CECs (56 year-old-donor) with or without RSPO1, RSPO2, RSPO3, and RSPO4 (50ng/ml). Bar = 100μm.
sc-12-0870_sm_SupplFigure4.tif1738KSupplemental online Figure 4. Schema of the molecular mechanism of CEC maintenance. Human CECs exhibit regional diversity with respect to LGR5 expression. LGR5 was uniquely expressed in the peripheral region of the CECs, where HH signaling was clearly activated. LGR5 was the target molecule of the HH pathway, and in in vitro conditions, the HH pathway was able to induce the CEC proliferation. However, in donor situations, stimulation of the HH pathway alone was not sufficient enough to induce the CEC Corneal endothelial cell is maintained by LGR5 2 proliferation. Persistent LGR5 expression maintained the normal CEC phenotypes through the inhibition of the Wnt pathway. The LGR5 ligand RSPO1 accelerated the CEC proliferation and inhibited the MT through the Wnt pathway. However, the exact mechanism of RSPO1 on CEC maintenance (via LGR5 or not) has yet to be elucidated.

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