Primer sequences used for RT-PCR.

<p>Primer sequences used for RT-PCR.</p

The functional properties of FAK/Wnt signaling pathways in the maintenance of the biological characteristics in the LSC cultured in ESC-CM system.

<p>(A) The expression levels of pFAK, FAK, pAkt, Akt, pGSK3β, GSK3β and p21 in the ESC-CM, ESC-CM+FAK inhibitor14 ESC-CM+GSK3β inhibitor or ESC-CM+SiRNA-TERT group. (B) The expression level of β-catenin of LSC cultured in ESC-CM after treatment with Wnt3a in the absence or presence of Dkk1. (C) MTT proliferation assay of LSC cultured in ESC-CM after treatment with Wnt3a in the absence or presence of Dkk1. Data are expressed as the mean±SD (n = 3;*,p<0.05;**, p<0.01; ***, p<0.001).</p

Rates of apoptosis in LSC grown in CnT-20 and ESC-CM.

<p>(A) Representative flow cytometry images showing apoptotic LSC (P0, P1 …P6) in ESC-CM and CnT-20 media. (B) Rates of apoptosis of LSC (P0, P1…P6) in ESC-CM and CnT-20 media.</p

Expression percentages of telomerase activity in LSC in each group.

<p>Expression percentages of telomerase activity in LSC in each group.</p

The LSC cultured in different media.

<p>(A) Before reaching 100% confluence, the cells cultured in ESC-CM had small cell size with nucleus/cytoplasm ratio, while the cells in CnT-20 exhibited larger morphology. And the TDC cells were tightly packed and highly elongated. (B) Clonal growth of LSC at day 7 in ESC-CM,CnT-20, or TDCwithout a feeder layer. (C) Representative immunofluorescence staining images of CK3 in LSCcultured in ESC-CM, CnT-20, and ESC-CM withdrawn group. (D) Percentage of colony-forming efficiency (CFE) of LSC at day 7 in ESC-CM, CnT-20, or TDC. (E) Percentages of positive cells stained for CK3 in ESC-CM, CnT-20, and ESC-CM withdrawn group. Data are expressed as the mean±SD (n = 3;**, p<0.01; ***, p<0.001).</p

Model graph for the functional role of the ES micro-environment in LSC.

<p>The telomerase-p21-mitochondrial axis, telomerase activity and the activation of the FAK/Wnt signaling pathways enhance the maintenance of undifferentiated status and the inhibition of apoptosis in human limbal stem cells cultured in ESC-CM.</p

Changes of apoptosis in LSC grown in ESC-CM and CnT-20 or after RNA interference.

<p>(A) Representative immunofluorescence staining images showing the reactive oxygen species (ROS) of the LSC in the ESC-CM, CnT-20, siRNA-F or siRNA-TERT groups. (B) Flow cytometry images showing the levels of ROS of the LSC in the ESC-CM, CnT-20, siRNA-F or siRNA-TERT groups. (C) Percentages of the levels of ROS in each group. (D) Rates of apoptosis of LSC in ESC-CM after RNA interference, with siRNA-F used as a negative control group. (E) Representative flow cytometry images showing the levels of Δψm of the LSC in the ESC-CM, CnT-20, siRNA-F or siRNA-TERT groups. (F) Percentages of the levels of Δψm in each group. Data are expressed as the mean±SD (n = 3;**, p<0.01).</p

Changes in the stem cell-associated markers in LSC after RNA interference.

<p>(A) Representative images of immunofluorescence staining of stem cell associated markers, p63, ABCG2, and integrinβ1, as well as differentiation marker, CK3 in LSC in siRNA-F or siRNA-TERT group. (B) Percentages of positive cells stained for each marker in LSC in siRNA-F or siRNA-TERT group. (C) Quantitative real-time PCR data showing the expression levels (relative fold of mRNA) of these markers by LSC in siRNA-F or siRNA-TERT group. (D)Western blot results showing the protein levels of P63 and ABCG2 in the ESC-CM, CnT-20, siRNA-F or siRNA-TERT groups. Data are expressed as the mean±SD (n = 3;**, p<0.01).</p

Expression of TERT in LSC in each group.

<p>(A) Representative immunofluorescence staining images of TERT expression in LSC from P0 and P2 in the ESC-CM, CnT-20, siRNA-F or siRNA-TERT group. (B) Representative flow cytometry images of TERT expression in LSC from P0 and P2 in the ESC-CM, CnT-20, siRNA-F or siRNA-TERT group. (C) Percentages of TERT expression in each group. Data are expressed as the mean±SD (n = 3;**, p<0.01).</p