Results of a phase I/II clinical trial: standardized, non-xenogenic, cultivated limbal stem cell transplantation

Abstract

Background: To determine if a standardized, non-xenogenic, reduced manipulation cultivation and surgical transplantation of limbal stem cell grafts is a safe and effective treatment option for patients with total and partial limbal stem cell deficiency.

Methods: In vitro cellular outgrowth and phenotype of the limbal epithelial cell and composite grafts were validated using a new protocol. Patients received either autologous (n = 15) or allogenic (n = 3) explants cultured using a standardized protocol free from xenogenic products. The resulting grafts were transplanted using a reduced manipulation surgical technique.

Results: The majority of cells (>50%) displayed a progenitor phenotype typified by positive immunofluorescence for ∆Np63, CK14 and ABCG2 and low immunofluorescence for CK3/12 and desmoglein 3 proteins. The surgical protocol was designed to minimize manipulation and the graft itself was secured without sutures. The transplant recipients were followed for a mean of 24 months. Twelve of the 18 transplant recipients were graded as anatomically successful (67%), based on the defined success parameters. There was a significant reduction in corneal neovascularization, which was accompanied by an improvement in pain though not photophobia or central corneal opacity post transplant. The transplantation protocol showed no measureable effect on visual acuity.

Conclusion: We conclude that this standardized culture system and surgical approach is safe and effective in reducing corneal neovascularization. The technique is free from animal contaminants and maintains a large proportion of progenitor cells. Although this technique did not improve visual function, restoring a functional epithelial cell layer and reducing corneal neovascularization provides an improved platform for a penetrating keratoplasty to ultimately improve visual function.

Figures

Figure 1

Flow cytometry profiles of limbal epithelial cells grown in CNT-20 medium. LEC cytometry profiles at isolation and at confluence (A), phenotypic profile of LECs at confluence (B) (***p < 0.001).

Figure 2

Limbal epithelial cells cultivated on amniotic membrane in progenitor cell CNT-20 versus SHEM medium. LECs grown in CNT20 and SHEM stained for the expression of Collagen IV and Integin alpha 6 (A), Laminin and Connexin 43 (B) and Collagen II and ∆Np63 (C).

Figure 3

Preoperative images. All three partial deficiency patients are shown (A-C) and three representative total deficiency patients (D-F). Representative images of two patients with total limbal stem cell deficiency prior to (G, J) and following (H, K) limbal stem cell transplantation. The same eyes following penetrating karatoplasty (I, L).

Figure 4

Composite graft generation. Phase contrast microscopy of a limbal biopsy and proliferating cells (A) (magnification × 200) and the outgrowth after the 14-day culture protocol (B) (n = 18).

Figure 5

Immunofluorescence microscopy of composite grafts. Cells were stained for the presence of CK14 (A), CK3/12 (D), ∆Np63 (B, E). (C) and (F) are composites of (A, B) and (D, E) respectively. Negative controls are shown in (G), (H) and (I).

Figure 6

Clinical images. Representative images of patients that achieved anatomical success; pre op (A + D), post LSCT (B + E) and post PK (C + F).

Figure 7

Visual acuity. Post-operative visual acuity outcomes in patients that achieved anatomical success (A) and for the total cohort (B) (* = p < 0.05).

Figure 8

Additional post-operative outcomes. Post-operative outcomes in the anatomically successful groups: pain (A), photophobia (B), vascularization (C) and opacity (D), (* = p < 0.05, **p < 0.01).