Long-Term Results of Cultured Limbal Stem Cell Versus Limbal Tissue Transplantation in Stage III Limbal Deficiency.

We aimed to evaluate efficiency and safety of transplantation of limbal stem cells (LSC) cultured on human amniotic membrane with no feeders and to compare cultured LSC with limbal tissue transplantation. Thirty eyes with stage III LSC deficiency were treated with autologous (autoLSC) or allogeneic (alloLSC) cultured LSC transplantation (prospective phase II clinical trial; average follow-up time, 72 months) or autologous (autoLT) or allogeneic (alloLT) limbal tissue transplantation (retrospective control group; average follow-up time, 132 months) between 1993 and 2014. The 5-year graft survival defined by absence of recurrence of the clinical signs of limbal deficiency was 71% for autoLSC, 0% for alloLSC, 75% for autoLT, and 33% for alloLT. Visual acuity improved by 9.2 lines for autoLSC and 3.3 lines for autoLT. It decreased by 0.7 lines for alloLSC and 1.9 lines for alloLT. Adverse events were recorded in 1/7 autoLSC, 7/7 alloLSC, 6/8 autoLT, and 8/8 alloLT patients. Corneal epithelial defect was the only adverse event recorded after autoLSC, whereas severe sight-threatening adverse events were recorded in the remaining three groups. Compared with failed grafts, successful grafts featured greater decrease in fluorescein staining, greater superficial vascularization-free corneal area, lower variability of the corneal epithelial thickness, and higher corneal epithelial basal cell density. Autologous cultured LSC transplantation was associated with high long-term survival and dramatic improvement in vision and was very safe. Autologous limbal tissue transplantation resulted in similar efficiency but was less safe. Cadaver allogeneic grafts resulted in low long-term success rate and high prevalence of serious adverse events. Stem Cells Translational Medicine 2019;8:1230&1241.

Easy xeno-free and feeder-free method for isolating and growing limbal stromal and epithelial stem cells of the human cornea.

Epithelial and stromal stem cells are required to maintain corneal transparency. The aim of the study was to develop a new method to isolate and grow both corneal stromal (SSC) and epithelial limbal (LSC) stem cells from small human limbal biopsies under culture conditions in accordance with safety requirements mandatory for clinical use in humans. Superficial limbal explants were retrieved from human donor corneo-scleral rims. Human limbal cells were dissociated by digestion with collagenase A, either after epithelial scraping or with no scraping. Isolated cells were cultured with Essential 8 medium (E8), E8 supplemented with EGF (E8+) or Green's medium with 3T3 feeder-layers. Cells were characterized by immunostaining, RT-qPCR, colony forming efficiency, sphere formation, population doubling, second harmonic generation microscopy and differentiation potentials. LSC were obtained from unscraped explants in E8, E8+ and Green's media and were characterized by colony formation and expression of PAX6, ΔNP63α, Bmi1, ABCG2, SOX9, CK14, CK15 and vimentin, with a few cells positive for CK3. LSC underwent 28 population doublings still forming colonies. SSC were obtained from both scraped and unscraped explants in E8 and E8+ media and were characterized by sphere formation, expression of PAX6, SOX2, BMI1, NESTIN, ABCG2, KERATOCAN, VIMENTIN, SOX9, SOX10 and HNK1, production of collagen fibrils and differentiation into keratocytes, fibroblasts, myofibroblasts, neurons, adipocytes, chondrocytes and osteocytes. SSC underwent 48 population doublings still forming spheres, Thus, this new method allows both SSC and LSC to be isolated from small superficial limbal biopsies and to be primary cultured in feeder-free and xeno-free conditions, which will be useful for clinical purposes.

New parameters in assessment of human donor corneal stroma.

PURPOSE: To provide quantitative parameters for assessment of human donor corneal stroma by imaging stromal features of diseased and normal human corneas with full-field optical coherence microscopy (FFOCM), using confocal microscopy (CM) and histology as reference techniques. METHODS: Bowman's layer (BL) thickness and keratocyte density were assessed ex vivo in 23 human donor corneas and 27 human pathological corneas (keratoconus and other corneal disorders) with FFOCM, CM and histology. Stromal backscattering was assessed with FFOCM. Additionally, 10 normal human corneas were assessed in vivo with CM. RESULTS: In FFOCM, the logarithm of the normalized stromal reflectivity was a linear function of stromal depth (R2  = 0.95) in human donor corneas. Compared with keratoconus corneas, human donor corneas featured higher BL thickness (p = 0.0014) with lower coefficient of variation (BL-COV; p = 0.0002), and linear logarithmic stromal reflectivity with depth (higher R2 , p = 0.0001). Compared with other corneal disorders, human donor corneas featured lower BL-COV (p = 0.012) and higher R2 (p = 0.0001). Using the 95% confidence limits of the human donor cornea group, BL thickness < 6.5 µm (sensitivity, 57%; specificity, 100%), BL-COV > 18.6% (79%; 100%) and R2  < 0.94 (93%; 71%) were revealed as indictors of abnormal cornea. In CM, keratocyte density decreased with stromal depth (r = -0.56). The mean overall keratocyte density (cells/mm2 ) was 205 in human donor corneas, 244 in keratoconus, 176 in other corneal disorders and 386 in normal corneas. CONCLUSION: Full-field optical coherence microscopy (FFOCM) provides precise and reliable parameters for non-invasive assessment of human donor corneal stroma during storage, enabling detection of stromal disorders that could impair the results of keratoplasty.