Conjunctival reconstruction with progenitor cell-derived autologous epidermal sheets in rhesus monkey

Abstract

Severe ocular surface diseases are some of the most challenging problems that the clinician faces today. Conventional management is generally unsatisfactory, and the long-term ocular consequences of these conditions are devastating. It is significantly important to find a substitute for conjunctival epithelial cells. This study was to explore the possibility of progenitor cell-derived epidermal sheets on denuded amniotic membrane to reconstruct ocular surface of conjunctiva damaged monkeys. We isolated epidermal progenitor cells of rhesus monkeys by type IV collagen adhesion, and then expanded progenitor cell-derived epidermal sheets on denuded amniotic membrane ex vivo. At 3 weeks after the conjunctiva injury, the damaged ocular surface of four monkeys was surgically reconstructed by transplanting the autologous cultivated epidermal progenitor cells. At 2 weeks after surgery, transplants were removed and examined with Hematoxylin-eosin staining, Periodic acid Schiff staining, immunofluorescent staining, scanning and transmission electron microscopy. Histological examination of transplanted sheets revealed that the cell sheets were healthy alive, adhered well to the denuded amniotic membrane, and had several layers of epithelial cells. Electron microscopy showed that the epithelial cells were very similar in appearance to those of normal conjunctival epithelium, even without goblet cell detected. Epithelial cells of transplants had numerous desmosomal junctions and were attached to the amniotic membrane with hemidesmosomes. Immunohistochemistry confirmed the presence of the conjunctival specific markers, mucin 4 and keratin 4, in the transplanted epidermal progenitor cells. In conclusion, our present study successfully reconstructed conjunctiva with autologous transplantation of progenitor cell-derived epidermal sheets on denuded AM in conjunctival damaged monkeys, which is the first step toward assessing the use of autologous transplantation of progenitor cells of nonocular surface origin. Epidermal progenitor cells could be provided as a new substitute for conjunctival epithelial cells to overcome the problems of autologous conjunctiva shortage.

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.