Figure 1.
Growth pattern of monkey keratinocytes and Rapid Adherent Cells (RAC).
A. Representative phase images of primary monkey keratinocytes (P0) and RAC. a. keratinocytes (P0) growing around hair follicle and almost reaching confluence (×200); b. keratinocytes growing from explants (×200); c. RAC adhered on Type IV collagen coated culture flask within 20 minutes (×200); d. RAC reaching confluence (×200); B. Scanning electron microscopy photography of RAC (×3500); C. Representative phase image of conjunctival epithelial cells growing from explant (×200).
Figure 2.
Growth capacity and cell phenotype of RAC isolated with Type IV collagen adhesion.
A. Representative images of immunofluorescent staining of epidermal stem cell associated markers, β1 Integrin (red) and Keratin 15 (green) with Hoechst (blue) counterstaining; B. Flow cytometry analysis of these markers by keratinocytes before and after isolation; C. RT-PCR image showing the expression levels (mRNA) of these markers by keratinocytes before and after isolation; D. Representative cultures stained with 1% rhodamine on day 8 showing growth capacity of RAC; E. Representative phase images of RAC on day 3 showing single colonies.
Figure 3.
Cultivation of epidermal progenitor cell-derived sheets on denuded AM and autologous transplanted to ocular surface.
A and B. Images of Denuded AM. A. Representative phase image (×100); B. Hematoxylin and eosin staining image (×100); C and D. Representative images of epidermal progenitor cells cultured on denuded AM at 2 days. C. Representative phase image showing epidermal progenitor cells form colonies on AM within 48 hours (×400); D. Scanning electron microscopy photography showing epidermal progenitor cells adhered tightly with AM (×2000); E to H. Electron microscopy photography of progenitor cell-derived epidermal sheets at 2 week after transplantation. E. TEM photography showed hemidesmosomes between basal cells and AM (×15000); F. Adjacent cells were joined with numerous desmosomal junctions (×13000); G and H. SEM photography showed the cells appeared to be in good condition and were closely attached to each other with tightly fitting cell junctions (×3500); I. HE staining image of transplanted preserved AM in controls, which showed discontinuous monolayler epithelium, and no nuclei found in the epithelial cells (×200); J. HE staining image of autologous transplantation of progenitor cell-derived epidermal sheets, which showed several layers of healthy epithelial cells on AM (×200); K and L. Surgical images of monkey eyes.
Figure 4.
Representative images showing immunofluorescent staining of MUC4, keratin4 or β1 Integrin (green) with Propidium Iodide (PI) (red) counterstaining of cell sheets before transplantion and 2 weeks after transplantation, normal conjunctival epithelium and epidermal epithelium.