ABSTRACT
To understand the response of oral epithelial cells, transplanted on corneal surface to the ocular cues in vivo. The corneal bu on obtained after penetrating keratoplasty (PK) of an eye of a patient with total limbal stem cell defi ciency (LSCD), previously treated with cultured oral mucosal epithelial transplantation (COMET) was examined by immunohistochemistry for the expression of keratins, p63, p75, PAX6, Ki-67, CD31, and CD34. COMET followed by optical-PK has improved visual acuity to 20/40 and rendered a stable ocular surface. The excised corneal tissue showed the presence of stratifi ed epithelium with vasculatures. The epithelial cells of the corneal bu on expressed K3, K19, Ki- 67, p63, p75 and the cornea-specifi c PAX6 and K12. This study confi rms that the oral cells, transplanted to corneal surface, survive and stably reconstruct the ocular surface. They maintain their stemness at the ectopic site and acquire some of the corneal epithelial-like characters.
ABSTRACT
Background: Cultivated limbal epithelium for reconstruction of corneal surface is a well-established procedure; however, it is not adequate for damage which also extensively involves the conjunctiva. In severe cases of ocular surface damage that warrant additional conjunctival transplantation apart from cultivated limbal stem cell transplantation, we describe the long-term survival of a novel method of cocultivating autologous limbal and conjunctival epithelium on a single substrate. Materials and Methods: Forty eyes of 39 patients with severe limbal stem cell deficiency and conjunctival scarring or symblepharon underwent transplantation of autologous cocultivated epithelium on human amniotic membrane. A ring barrier was used to segregate the central limbal and peripheral conjunctival epithelia in vitro. Patients were followed up at regular intervals to assess stability of the ocular surface, defined by absence of conjunctivalization into the central 4 mm of the cornea and absence of diffuse fluorescein staining. Penetrating keratoplasty (PKP) was subsequently performed, where indicated, in patients with surface stability. Results: The cumulative survival probability was 60% at 1 year and 45% at 4 years by Kaplan–Meier analysis (mean follow-up duration: 33 ± 29 months, range: 1–87 months). Best-corrected visual acuity improved to greater than 20/200 in 38% eyes at the last follow-up, compared with 5% eyes before surgery. Immunohistochemistry in five of the corneal buttons excised for PKP showed an epithelial phenotype similar to cornea in all five. Conclusions: Synchronous use of cultured limbal and conjunctival epithelium offers a feasible alternative and a simpler one-step surgical approach to treat severe ocular surface disorders involving limbus and conjunctiva.
ABSTRACT
Millions of people world over suffer visual disability due to retinal dystrophies which can be age-related or a genetic disorder resulting in gradual degeneration of the retinal pigmented epithelial (RPE) cells and photoreceptors. Therefore, cell replacement therapy offers a great promise in treating such diseases. Since the adult retina does not harbour any stem cells, alternative stem cell sources like the embryonic stem cells (ESCs) and induced pluripotent stem cells (iPSCs) offer a great promise for generating different cell types of the retina. Here, we report the derivation of four iPSC lines from mouse embryonic fibroblasts (MEFs) using a cocktail of recombinant retroviruses carrying the genes for Oct4, Sox2, Klf4 and cMyc. The iPS clone MEF-4F3 was further characterized for stemness marker expression and stable reprogramming by immunocytochemistry, FACS and RT-PCR analysis. Methylation analysis of the nanog promoter confirmed the reprogrammed epigenetic state. Pluripotency was confirmed by embryoid body (EB) formation and lineage-specific marker expression. Also, upon retinal differentiation, patches of pigmented cells with typical cobble-stone phenotype similar to RPE cells are generated within 6 weeks and they expressed ZO-1 (tight junction protein), RPE65 and bestrophin (mature RPE markers) and showed phagocytic activity by the uptake of fluorescent latex beads.