Niche factors are important in the maintenance and regulation of stem cells. Limbal stromal cells are potentially a component of limbal stem cell (LSC) niche. We investigated the role of the limbal stromal cells in the ex vivo expansion of limbal stem/progenitor cells. Limbal epithelial cells were cultured as single-cell suspension and cell clusters from dispase II or collagenase A (ColA), or tissue explant. ColA isolated limbal stromal cells along with limbal epithelial cells. In the presence of limbal stromal cells, a higher absolute number of p63α(bright) cells (p < 0.05) and a higher proportion of K14 positive epithelial cells were obtained from both ColA and explant tissue cultures. Expansion of the stem/progenitor population from dispase isolation was more efficient in the form of cell clusters than single cell suspension based on the absolute number of p63α(bright) cells. Expansion of the stem cell population is similar in the single cell and cell cluster cultures that are derived from ColA isolation. Our finding suggests that limbal stromal cells and an intact cell-cell contact help to maintain LSCs in an undifferentiated state in vitro during expansion.

The application of human amniotic membrane (AM) has a wide spectrum of indications in the treatment of ocular surface disorders. Transplantation of AM has been incorporated routinely as a component of ocular surface reconstruction in a variety of ocular pathologies. The application of human AM can be combined with nearly all types of limbal transplantation in treating limbal stem cell deficiency (LSCD). AM provides support and possible protection to the transplanted limbal tissues and limbal stem cells owing to its mechanical and biological properties, and these properties are thought to enhance the success rate of LSC transplantation. This paper reviews the current literature on the applications of AM in the surgical management of LSCD and summarizes the outcome of different surgical approaches. The current literature contains mostly low-level evidences in supporting the role of AM. The efficacy of AM in LSC transplantation needs to be confirmed by randomized controlled clinical trials.

Notch signaling has been shown to regulate the homeostasis and wound healing of the corneal epithelium. We investigated the effect of Notch inhibition in the human limbal stem/progenitor cells (LSCs) in vitro by using small molecules. Treatment of the LSCs with DAPT and SAHM1 reduced the proliferation rate and maintained the undifferentiated state of the LSCs in a concentration dependent manner. Stratification and differentiation of the corneal epithelium were not reduced after Notch inhibition, indicating that the function of the corneal basal cells is retained. Our findings suggest that Notch signaling plays a role in the proliferation and maintenance of LSCs.

The culture of human limbal epithelial stem/progenitor cells (LSCs) in the presence of animal components poses the risk of cross-species contamination in clinical applications. We quantitatively compared different xenobiotic-free culture media for the cultivation of human LSCs. LSCs were cultured from 2 × 2 mm limbal tissue explants on denuded human amniotic membrane with different xenobiotic-free culture media: CnT-Prime (CnT-PR) supplemented with 0%, 1%, 5%, and 10% human serum (HS), embryonic stem cell medium (ESCM) alone or in combination with the standard supplemented hormonal epithelium medium (SHEM, control) at a 1:1 dilution ratio, and modified SHEM (mSHEM), in which cholera toxin and dimethyl sulfoxide (DMSO) were removed, isoproterenol was added, and the epidermal growth factor concentration was reduced. Several parameters were quantified to assess the LSC phenotype: cell morphology, cell growth, cell size, outgrowth size, and expression of the undifferentiated LSC markers cytokeratin (K) 14, and p63α high-expressing (p63α^bright) cells, a mature keratinocyte marker K12, epithelial marker pancytokeratin (PanK), and stromal cell marker vimentin (Vim). Compared with the standard SHEM control, CnT-PR base medium was associated with a lower cell growth and reduction in the proportion of stem cells generated regardless of the amount of HS supplemented (p < 0.05); ESCM resulted in an increased proportion of PanK^-/Vim⁺ stromal cells (p < 0.05) and a decreased proportion of p63α^bright cells (p < 0.05); mSHEM supported a similar cell growth (p > 0.05), increased the number of small cells (diameter ≤12 μm; p < 0.05), and provided a similar proportion of p63α^bright cells (p > 0.05). Among all the conditions tested, mSHEM was the most efficient and consistent in supporting the LSC phenotype and growth.

Purpose

To evaluate outcomes of Descemet membrane endothelial keratoplasty (DMEK) in eyes with previous trabeculectomy or a drainage device.

Methods

This is a retrospective study of 108 consecutive DMEK performed between October 2013 and December 2015. All eyes were divided into 3 groups: surgical treatment (ST) group, medical treatment (MT) group, and control group. Visual improvement, endothelial cell loss, and postoperative complications, including rejection, graft failure, and intraocular pressure elevation (≥25 mm Hg) were evaluated.

Results

The length of follow-up was 9.7 ± 7.3 months. Best-corrected visual acuity (BCVA) improved postoperatively in 85.3% of the ST group, 100% of the MT group, and 93% of the control (P = 0.24). Significantly more lines of BCVA were gained in the ST and MT groups (8.1 ± 8.1 and 9.2 ± 6.3 lines, respectively) than in the control (4.8 ± 5.6 lines, P < 0.05). The mean time to BCVA was 2.9 ± 2.8 months for the ST group, 4.7 ± 5.3 months for the MT group, and 3.0 ± 3.3 months for the control (P = 0.75). Endothelial cell loss was greater in the ST group (44.6 ± 17.8%) than in the MT group (29.9 ± 12.0%) and the control group (32.7 ± 11.3%, P = 0.001). There was one primary failure and no secondary graft failures. The overall rejection rate was 0.9%. Postoperative intraocular pressure elevation was less common in the ST group (14.7%) and control (23.3%) than in the MT group (50.0%, P = 0.04). There was no difference in the air injection rate among all groups (P = 1.0).

Conclusions

DMEK in eyes with previous trabeculectomy and drainage device can result in very good short-term outcomes.

Limbal stem cells (LSCs) maintain the normal homeostasis and wound healing of corneal epithelium. Limbal stem cell deficiency (LSCD) is a pathologic condition that results from the dysfunction and/or an insufficient quantity of LSCs. The diagnosis of LSCD has been made mainly based on medical history and clinical signs, which often are not specific to LSCD. Methods to stage the severity of LSCD have been lacking. With the application of newly developed ocular imaging modalities and molecular methods as diagnostic tools, standardized quantitative criteria for the staging of LSCD can be established. Because of these recent advancements, effective patient-specific therapy for different stages of LSCD may be feasible.

An accurate diagnosis of limbal stem cell deficiency (LSCD) is the premise of an appropriate treatment; however, there is no consensus about the diagnostic criteria for LSCD. We performed a systematic literature search of the peer-reviewed articles on PubMed, Medline, and Ovid to investigate how LSCD was diagnosed before surgical intervention. The methods used to diagnose LSCD included clinical presentation, impression cytology, and in vivo confocal microscopy. Among 131 eligible studies (4054 eyes), 26 studies (459 eyes, 11.3%) did not mention the diagnostic criteria. In the remaining 105 studies, the diagnosis of LSCD was made on the basis of clinical examination alone in 2398 eyes (62.9%), and additional diagnostic tests were used in 1047 (25.8%) eyes. Impression cytology was used in 981 eyes (24.2%), in vivo confocal microscopy was used in 29 eyes (0.7%), and both impression cytology and in vivo confocal microscopy were used in 37 eyes (0.9%). Our findings suggest that only a small portion of patients underwent a diagnostic test to confirm the diagnosis of LSCD. Treating physicians should be aware of the limitations of clinical examination in diagnosing LSCD and perform a diagnostic test whenever possible before surgical intervention.

Purpose

We tested the viability of human limbal mesenchymal cells (LMCs) to support the expansion of human corneal epithelial stem/progenitor cells (LSCs).

Methods

Human LMCs were isolated from sclerocorneal tissue using collagenase A. Primary limbal epithelial cells (LECs) in the form of single cell suspension or cell clusters were cocultured on a monolayer of either 3T3 cells (control) or LMCs (SC-LMC culture). The LEC clusters also were grown directly on LMCs (CC-LMC culture) and in an optimized 3-dimensional culture method (3D CC-LMC culture). Colony-forming efficiency (CFE) and LEC proliferation were analyzed. The phenotype of the cultured LECs was assessed by their expression level of putative stem cell markers and a differentiation marker by qRT-PCR and immunocytochemistry.

Results

The LECs in the SC-LMC culture had a very limited growth and the stem/progenitor phenotype was lost compared to the control. Growth and cell morphology improved using the CC-LMC culture. The 3D CC-LMC culture method was the best to support the growth of the LSC population. Expression of ATP-binding cassette family G2 and ΔNp63 at the mRNA level was maintained or increased in CC-LMCs and 3D CC-LMC cultures compared to the control. The percentage of the K14(+) and K12(+) cells was comparable in these three cultures. There was no significant difference in the percentage of p63α high expressing cells in the control (21%) and 3D CC-LMC culture (17%, P > 0.05).

Conclusions

Human LMCs can substitute 3T3 cells in the expansion of LSCs using the 3-dimensional culture system.

Wnt signaling pathway plays an important role in the regulation of human limbal stem/progenitor cells (LSCs). To examine the possible function of Frizzled (Fz) receptors in LSCs, the expression of 10 Fz receptors was profiled in the limbus and cornea. Only Fz7 had preferential expression in the basal limbal epithelium which contains the LSCs. The expression of Fz7 was colocalized with the putative LSC markers including p63α, N-cadherin and keratin (K) 14, and was minimum in cells expressing the corneal maturation marker K12. The expression of Fz7 was higher in the enriched LSCs population and decreased in cultured LSCs when there was a loss of progenitor phenotype. When the Fz7 was knocked down (Fz(KD)) using shRNA in primary LSCs, the expression of putative LSC markers ABCG2, ΔNp63α, and K14 was decreased significantly. The colony forming efficiency of the Fz7(KD) LSCs was significantly decreased in the subsequent passage 1 and 2 compared to the control. Our finding suggests that Wnt signaling is one of the factors of LSC niche, and Fz7 helps to maintain the undifferentiated state of LSCs.

The function of limbal stem/progenitor cells (LSCs) is critical to maintain corneal epithelial homeostasis. Many external insults and intrinsic defects can be deleterious to LSCs and their niche microenvironment, resulting in limbal stem cell dysfunction or deficiency (LSCD). Ocular comorbidities, frequent in eyes with LSCD, can exacerbate the dysfunction of residual LSCs, and limit the survival of transplanted LSCs. Clinical presentation and disease evolution vary among different etiologies of LSCD. New ocular imaging modalities and molecular markers are now available to standardize the diagnosis criteria and stage the severity of the disease. Medical therapies may be sufficient to reverse the disease if residual LSCs are present. A stepwise approach should be followed to optimize the ocular surface, eliminate the causative factors and treat comorbid conditions, before considering surgical interventions. Furthermore, surgical options are selected depending on the severity and laterality of the disease. The standardized diagnostic criteria to stage the disease is necessary to objectively evaluate and compare the efficacy of the emerging customized therapies.