Figure 1.
Primary culture and identification of rat neural crest cells.
(a) Spindle-like cells migrated from the neural tube of a rat embryo 24 hours after culture. (b) The cells reached confluence 7 days after culture. Spindle-shaped cells simultaneously expressed P75 (c) and HNK-1(d). (e) DAPI staining of cell nuclei. (f) Overlay image of c, d and e. NT: neural tube; NCC: neural crest cell. Scale bar: 100 µm.
Figure 2.
Morphological change of NCC after induction.
(a) A small part of NCC changed to polygonal shape (arrow) 7 days after induction. (b) On day 14, about half of the cells in one field became polygonal (arrow). (c) Normal rat CEC control. Insets in a and b are higher magnifications of polygonal cells. Scale bars: 100 µm.
Figure 3.
Immunofluorescence detection of N-cadherin.
(a) Cells were stained with N-cadherin antibodies after induction, green fluorescence mainly distributed in the plasma and cell membrane. (b) No positive signal was detected among non-induced NCC. (c) No positive signal was detected in negative control. (d) Normal rat CEC were stained with N-cadherin antibodies, green fluorescence was mainly detected at cell boundaries. Scale bars: 50 µm.
Figure 4.
RT-PCR detection of transcription factor genes FoxC1 and Pitx2.
Both of these two genes were detected as early as 5 days after induction. The expression level of FoxC1 peaked on day 7, and quickly fell afterwards. Expression of Pitx2 was steady but lower than that of FoxC1, and the peak value appeared around day 9. Error bars = SD.
Figure 5.
Cell inductivity determined by flow cytometery analysis.
(a) The mean proportion of N-cadherin positive cells was about 49.75% 14 days after induction. (b) The mean proportion of N-cadherin positive cells was about 49.01% 20 days after induction. There was no increase in cell inductivity by prolonging the inducing time from 14 days to 20 days. M1: N-cadherin negative cells; M2: N-cadherin positive cells.
Figure 6.
Expression of function-related proteins of the CEC-like cell monolayer on APCM scaffolds.
Positive signals for ZO-1 (a) and Na+/K+ ATPase (b) were detected between cell boundaries of the CEC-like cell monolayer. Expression patterns were similar to normal rat corneal endothelium (c, d), except that fluorescence was weaker for reconstructed corneal endothelium. Primary antibodies were omitted as negative controls (e, f). Scale bars: 50 µm.
Figure 7.
Contact-inhibition analysis of CEC-like cells.
(a) HE staining of the reconstructed corneal endothelium showed that CEC-like cells kept a monolayer after 30 days cultivation on APCM in cross section view. (b) Alizarin red S and trypan blue double-staining of the reconstructed endothelium showed the “lake reaction” of cell borders and no multiple layers from plane view. Scale bars: 50 um.
Figure 8.
Representative corneal photos after the surgery.
Corneas in the experimental group were clear, and the anterior chamber and the iris were clearly visible at 28 days (a). In contrast, corneas in the control group were opaque (b). Corneas in the experimental group were much thinner than that in the control group and the endothelium surface was also smoother under slit observation at 28 days (c and d). Confocal microscopy confirmed the polygonal cell coverage of the Descemet’s membrane in the experimental group at 28 days and 2 months (e and g), while there was only denuded Descemet’s membrane in the control group at 28 days (f), and there was some newborn CEC at the marginal region at 2 months in the control group (h).
Figure 9.
Changes of corneal thickness during clinical observation.
Mean corneal thickness of normal rats was about 116±5.27 um. 1 day after cryo-injury, it sharply increased in both experimental and control groups to about two times the normal value. Then it rapidly decreased in the experimental group and was significantly less than the control group on 7, 14, 21 and 28 days after the surgery. Error bars = SD(*P<0.05).
Figure 10.
Histological examination of the corneas after the surgery.
(a) Cells covering the Descemet’s membrane in the experimental group were CFDA SE-positive under the fluorescence microscope. (b and c) No signal was detected from the corneas of negative control and normal control groups. (d and e) HE-stained cross-section showed similar results that the Descemet’s membrane was covered by a cell monolayer in the experimental group, and no cells were present on Descemet’s membrane in the control group. (f) HE staining of normal rat cornea. (g, h and i) High magnification images of corneal endothelia in d, e and f respectively. Scale bars: 50 µm.