Can mesenchymal stromal cells differentiate into corneal cells? A systematic review of published data

The majority of stem cell therapies for corneal repair are based upon the use of progenitor cells isolated from corneal tissue, but a growing body of literature suggests a role for mesenchymal stromal cells (MSC) isolated from non-corneal tissues. While the mechanism of MSC action seems likely to involve their immuno-modulatory properties, claims have emerged of MSC transdifferentiation into corneal cells. Substantial differences in methodology and experimental outcomes, however, have prompted us to perform a systematic review of the published data. Key questions used in our analysis included; the choice of markers used to assess corneal cell phenotype, the techniques employed to detect these markers, adequate reporting of controls, and tracking of MSC when studied in vivo. Our search of the literature revealed 28 papers published since 2006, with half appearing since 2012. MSC cultures established from bone marrow and adipose tissue have been best studied (22 papers). Critically, only 11 studies employed appropriate markers of corneal cell phenotype, along with necessary controls. Ten out of these 11 papers, however, contained positive evidence of corneal cell marker expression by MSC. The clearest evidence is observed with respect to expression of markers for corneal stromal cells by MSC. In comparison, the evidence for MSC conversion into either corneal epithelial cells or corneal endothelial cells is often inconsistent or inconclusive. Our analysis clarifies this emerging body of literature and provides guidance for future studies of MSC differentiation within the cornea as well as other tissues.

Immunosuppressive properties of mesenchymal stromal cell cultures derived from the limbus of human and rabbit corneas

Background aims Mesenchymal stromal cells (MSCs) cultivated from the corneal limbus (L-MSCs) provide a potential source of cells for corneal repair. In the present study, we investigated the immunosuppressive properties of human L-MSCs and putative rabbit L-MSCs to develop an allogeneic therapy and animal model of L-MSC transplantation. Methods MSC-like cultures were established from the limbal stroma of human and rabbit (New Zealand white) corneas using either serum-supplemented medium or a commercial serum-free MSC medium (MesenCult-XF Culture Kit; Stem Cell Technologies, Melbourne, Australia). L-MSC phenotype was examined by flow cytometry. The immunosuppressive properties of L-MSC cultures were assessed using mixed leukocyte reactions. L-MSC cultures were also tested for their ability to support colony formation by primary limbal epithelial (LE) cells. Results Human L-MSC cultures were typically CD34−, CD45− and HLA-DR− and CD73+, CD90+, CD105+ and HLA-ABC+. High levels (>80%) of CD146 expression were observed for L-MSC cultures grown in serum-supplemented medium but not cultures grown in MesenCult-XF (approximately 1%). Rabbit L-MSCs were approximately 95% positive for major histocompatibility complex class I and expressed lower levels of major histocompatibility complex class II (approximately 10%), CD45 (approximately 20%), CD105 (approximately 60%) and CD90 (<10%). Human L-MSCs and rabbit L-MSCs suppressed human T-cell proliferation by up to 75%. Conversely, L-MSCs from either species stimulated a 2-fold to 3-fold increase in LE cell colony formation. Conclusions L-MSCs display immunosuppressive qualities in addition to their established non-immunogenic profile and stimulate LE cell growth in vitro across species boundaries. These results support the potential use of allogeneic L-MSCs in the treatment of corneal disorders and suggest that the rabbit would provide a useful pre-clinical model.

The Use of Mesenchymal Stromal Cells in the Treatment of Diseases of the Cornea

As a key component of the ocular surface required for vision, the cornea has been extensively studied as a site for cell and tissue-based therapies. Historically, these treatments have consisted of donor corneal tissue transplants, but cultivated epithelial autografts have become established over the last 15 years as a routine treatment for ocular surface disease. Ultimately, these treatments are performed with the intention of restoring corneal transparency and a smooth ocular surface. The degree of success, however, is often dependent upon the inherent level of corneal inflammation at time of treatment. In this regard, the anti-inflammatory and immuno-modulatory properties of mesenchymal stromal cells (MSC) have drawn attention to these cells as potential therapeutic agents for corneal repair. The origins for MSC-based therapies are founded in part on observations of the recruitment of endogenous bone marrow-derived cells to injured corneas, however, an increasing quantity of data is emerging for MSC administered following their isolation and ex vivo expansion from a variety of tissues including bone marrow, adipose tissue, umbilical cord and dental pulp. In brief, evidence has emerged of cultured MSC, or their secreted products, having a positive impact on corneal wound healing and retention of corneal allografts in animal models. Optimal dosage, route of administration and timing of treatment, however, all remain active areas of investigation. Intriguingly, amidst these studies, have emerged reports of MSC transdifferentiation into corneal cells. Clearest evidence has been obtained with respect to expression of markers associated with the phenotype of corneal stromal cells. In contrast, the evidence for MSC conversion to corneal epithelial cell types remains inconclusive. In any case, the conversion of MSC into corneal cells seems unlikely to be an essential requirement for their clinical use. This field of research has recently become more complicated by reports of MSC-like properties for cultures established from the peripheral corneal stroma (limbal stroma). The relationship and relative value of corneal-MSC compared to traditional sources of MSC such as bone marrow are at present unclear. This chapter is divided into four main parts. After providing a concise overview of corneal structure and function, we will highlight the types of corneal diseases that are likely to benefit from the anti-inflammatory and immuno-modulatory properties of MSC. We will subsequently summarize the evidence supporting the case for MSC-based therapies in the treatment of corneal diseases. In the third section we will review the literature concerning the keratogenic potential of MSC. Finally, we will review the more recent literature indicating the presence of MSC-like cells derived from corneal tissue.

Corneal confocal microscopy detects early nerve regeneration in diabetic neuropathy after simultaneous pancreas and kidney transplantation

Diabetic neuropathy is associated with increased morbidity and mortality. To date, limited data in subjects with impaired glucose tolerance and diabetes demonstrate nerve fiber repair after intervention. This may reflect a lack of efficacy of the interventions but may also reflect difficulty of the tests currently deployed to adequately assess nerve fiber repair, particularly in short-term studies. Corneal confocal microscopy (CCM) represents a novel noninvasive means to quantify nerve fiber damage and repair. Fifteen type 1 diabetic patients undergoing simultaneous pancreas-kidney transplantation (SPK) underwent detailed assessment of neurologic deficits, quantitative sensory testing (QST), electrophysiology, skin biopsy, corneal sensitivity, and CCM at baseline and at 6 and 12 months after successful SPK. At baseline, diabetic patients had a significant neuropathy compared with control subjects. After successful SPK there was no significant change in neurologic impairment, neurophysiology, QST, corneal sensitivity, and intraepidermal nerve fiber density (IENFD). However, CCM demonstrated significant improvements in corneal nerve fiber density, branch density, and length at 12 months. Normalization of glycemia after SPK shows no significant improvement in neuropathy assessed by the neurologic deficits, QST, electrophysiology, and IENFD. However, CCM shows a significant improvement in nerve morphology, providing a novel noninvasive means to establish early nerve repair that is missed by currently advocated assessment techniques.

Normative values for corneal nerve morphology assessed using corneal confocal microscopy: A multinational normative data set

OBJECTIVE Corneal confocal microscopy is a novel diagnostic technique for the detection of nerve damage and repair in a range of peripheral neuropathies, in particular diabetic neuropathy. Normative reference values are required to enable clinical translation and wider use of this technique. We have therefore undertaken a multicenter collaboration to provide worldwide age-adjusted normative values of corneal nerve fiber parameters. RESEARCH DESIGN AND METHODS A total of 1,965 corneal nerve images from 343 healthy volunteers were pooled from six clinical academic centers. All subjects underwent examination with the Heidelberg Retina Tomograph corneal confocal microscope. Images of the central corneal subbasal nerve plexus were acquired by each center using a standard protocol and analyzed by three trained examiners using manual tracing and semiautomated software (CCMetrics). Age trends were established using simple linear regression, and normative corneal nerve fiber density (CNFD), corneal nerve fiber branch density (CNBD), corneal nerve fiber length (CNFL), and corneal nerve fiber tortuosity (CNFT) reference values were calculated using quantile regression analysis. RESULTS There was a significant linear age-dependent decrease in CNFD (-0.164 no./mm(2) per year for men, P < 0.01, and -0.161 no./mm(2) per year for women, P < 0.01). There was no change with age in CNBD (0.192 no./mm(2) per year for men, P = 0.26, and -0.050 no./mm(2) per year for women, P = 0.78). CNFL decreased in men (-0.045 mm/mm(2) per year, P = 0.07) and women (-0.060 mm/mm(2) per year, P = 0.02). CNFT increased with age in men (0.044 per year, P < 0.01) and women (0.046 per year, P < 0.01). Height, weight, and BMI did not influence the 5th percentile normative values for any corneal nerve parameter. CONCLUSIONS This study provides robust worldwide normative reference values for corneal nerve parameters to be used in research and clinical practice in the study of diabetic and other peripheral neuropathies.

Strategies for zonal cartilage repair using hydrogels

Articular cartilage is a highly hydrated tissue with depth-dependent cellular and matrix properties that provide low-friction load bearing in joints. However, the structure and function are frequently lost and there is insufficient repair response to regenerate high-quality cartilage. Several hydrogel-based tissue-engineering strategies have recently been developed to form constructs with biomimetic zonal variations to improve cartilage repair. Modular hydrogel systems allow for systematic control over hydrogel properties, and advanced fabrication techniques allow for control over construct organization. These technologies have great potential to address many unanswered questions involved in prescribing zonal properties to tissue-engineered constructs for cartilage repair.

Construction safety in the repair and maintenance sector

Statistics indicate that the percentage of fatal industrial accidents arising from repair, maintenance, minor alteration and addition (RMAA) works in Hong Kong was disturbingly high and was over 56% in 2006. This paper provides an initial report of a research project funded by the Research Grants Council (RGC) of the HKSAR to address this safety issue. The aim of this study is to scrutinize the causal relationship between safety climate and safety performance in the RMAA sector. It aims to evaluate the safety climate in the RMAA sector; examine its impacts on safety performance, and recommend measures to improve safety performance in the RMAA sector. This paper firstly reports on the statistics of construction accidents arising from RMAA works. Qualitative and quantitative research methods applied in conducting the research are dis-cussed. The study will critically review these related problems and provide recommendations for improving safety performance in the RMAA sector.

Establishment of a preclinical ovine model for tibial segmental bone defect repair by applying bone tissue engineering strategies

Currently, well-established clinical therapeutic approaches for bone reconstruction are restricted to the transplantation of autografts and allografts, and the implantation of metal devices or ceramic-based implants to assist bone regeneration. Bone grafts possess osteoconductive and osteoinductive properties, however they are limited in access and availability and associated with donor site morbidity, haemorrhage, risk of infection, insufficient transplant integration, graft devitalisation, and subsequent resorption resulting in decreased mechanical stability. As a result, recent research focuses on the development of alternative therapeutic concepts. The field of tissue engineering has emerged as an important approach to bone regeneration. However, bench to bedside translations are still infrequent as the process towards approval by regulatory bodies is protracted and costly, requiring both comprehensive in vitro and in vivo studies. The subsequent gap between research and clinical translation, hence commercialization, is referred to as the ‘Valley of Death’ and describes a large number of projects and/or ventures that are ceased due to a lack of funding during the transition from product/technology development to regulatory approval and subsequently commercialization. One of the greatest difficulties in bridging the Valley of Death is to develop good manufacturing processes (GMP) and scalable designs and to apply these in pre-clinical studies. In this article, we describe part of the rationale and road map of how our multidisciplinary research team has approached the first steps to translate orthopaedic bone engineering from bench to bedside byestablishing a pre-clinical ovine critical-sized tibial segmental bone defect model and discuss our preliminary data relating to this decisive step.

QoS-Based Web Service Scheduling Accommodating Inter-Service Dependencies Using Minimal-Conflict Hill-Climbing Repair Genetic Algorithm

In the filed of semantic grid, QoS-based Web service scheduling for workflow optimization is an important problem.However, in semantic and service rich environment like semantic grid, the emergence of context constraints on Web services is very common making the scheduling consider not only quality properties of Web services, but also inter service dependencies which are formed due to the context constraints imposed on Web services. In this paper, we present a repair genetic algorithm, namely minimal-conflict hill-climbing repair genetic algorithm, to address scheduling optimization problems in workflow applications in the presence of domain constraints and inter service dependencies. Experimental results demonstrate the scalability and effectiveness of the genetic algorithm.

Corneal sensitivity and slit scanning in vivo confocal microscopy of the subbasal nerve plexus of the normal central and peripheral human cornea

Purpose: To determine the subbasal nerve density and tortuosity at 5 corneal locations and to investigate whether these microstructural observations correlate with corneal sensitivity. Method: Sixty eyes of 60 normal human subjects were recruited into 1 of 3 age groups, group 1: aged ,35 years, group 2: aged 35–50 years, and group 3: aged .50 years. All eyes were examined using slit-lamp biomicroscopy, noncontact corneal esthesiometry, and slit scanning in vivo confocal microscopy. Results: The mean subbasal nerve density and the mean corneal sensitivity were greatest centrally (14,731 6 6056 mm/mm2 and 0.38 6 0.21 millibars, respectively) and lowest in the nasal mid periphery (7850 6 4947 mm/mm2 and 0.49 6 0.25 millibars, respectively). The mean subbasal nerve tortuosity coefficient was greatest in the temporal mid periphery (27.3 6 6.4) and lowest in the superior mid periphery (19.3 6 14.1). There was no significant difference in mean total subbasal nerve density between age groups. However, corneal sensation (P = 0.001) and subbasal nerve tortuosity (P = 0.004) demonstrated significant differences between age groups. Subbasal nerve density only showed significant correlations with corneal sensitivity threshold in the temporal cornea and with subbasal nerve tortuosity in the inferior and nasal cornea. However, these correlations were weak. Conclusions: This study quantitatively analyzes living human corneal nerve structure and an aspect of nerve function. There is no strong correlation between subbasal nerve density and corneal sensation. This study provides useful baseline data for the normal living human cornea at central and mid-peripheral locations

Corneal confocal microscopy : a novel noninvasive means to diagnose neuropathy in patients with Fabry disease

ABSTRACT: Neuropathy is a cause of significant disability in patients with Fabry disease, yet its diagnosis is difficult. In this study we compared the novel noninvasive techniques of corneal confocal microscopy (CCM) to quantify small-fiber pathology, and non-contact corneal esthesiometry (NCCA) to quantify loss of corneal sensation, with established tests of neuropathy in patients with Fabry disease. Ten heterozygous females with Fabry disease not on enzyme replacement therapy (ERT), 6 heterozygous females, 6 hemizygous males on ERT, and 14 age-matched, healthy volunteers underwent detailed quantification of neuropathic symptoms, neurological deficits, neurophysiology, quantitative sensory testing (QST), NCCA, and CCM. All patients with Fabry disease had significant neuropathic symptoms and an elevated Mainz score. Peroneal nerve amplitude was reduced in all patients and vibration perception threshold was elevated in both male and female patients on ERT. Cold sensation (CS) threshold was significantly reduced in both male and female patients on ERT (P < 0.02), but warm sensation (WS)and heat-induced pain (HIP) were only significantly increased in males onERT (P<0.01). However, corneal sensation assessed withNCCAwas significantly reduced in female (P < 0.02) and male (P < 0.04) patients on ERT compared with control subjects. According to CCM, corneal nerve fiber and branch density was significantly reduced in female (P < 0.03) and male (P < 0.02) patients on ERT compared with control subjects. Furthermore, the severity of neuropathic symptoms and the neurological component of the Mainz Severity Score Index correlated significantly with QSTand CCM. This study shows that CCM and NCCA provide a novel means to detect early nerve fiber damage and dysfunction, respectively, in patients with Fabry disease.

Aberrations and pupil location under corneal topography and Hartmann-Shack illumination conditions

PURPOSE. This study was conducted to determine the magnitude of pupil center shift between the illumination conditions provided by corneal topography measurement (photopic illuminance) and by Hartmann-Shack aberrometry (mesopic illuminance) and to investigate the importance of this shift when calculating corneal aberrations and for the success of wavefront-guided surgical procedures. METHODS. Sixty-two subjects with emmetropia underwent corneal topography and Hartmann-Shack aberrometry. Corneal limbus and pupil edges were detected, and the differences between their respective centers were determined for both procedures. Corneal aberrations were calculated using the pupil centers for corneal topography and for Hartmann-Shack aberrometry. Bland-Altmann plots and paired t-tests were used to analyze the differences between corneal aberrations referenced to the two pupil centers. RESULTS. The mean magnitude (modulus) of the displacement of the pupil with the change of the illumination conditions was 0.21 ± 0.11 mm. The effect of this pupillary shift was manifest for coma corneal aberrations for 5-mm pupils, but the two sets of aberrations calculated with the two pupil positions were not significantly different. Sixty-eight percent of the population had differences in coma smaller than 0.05 µm, and only 4% had differences larger than 0.1 µm. Pupil displacement was not large enough to significantly affect other higher-order Zernike modes. CONCLUSIONS. Estimated corneal aberrations changed slightly between photopic and mesopic illumination conditions given by corneal topography and Hartmann-Shack aberrometry. However, this systematic pupil shift, according to the published tolerances ranges, is enough to deteriorate the optical quality below the theoretically predicted diffraction limit of wavefront-guided corneal surgery.

Enhancement of DNA repair using topical T4 endonuclease V does not inhibit melanoma formation in Cdk4R24C/R24C/Tyr-NrasQ61K mice following neonatal UVR

To further investigate the use of DNA repair-enhancing agents for skin cancer prevention, we treated Cdk4R24C/R24C/NrasQ61K mice topically with the T4 endonuclease V DNA repair enzyme (known as Dimericine) immediately prior to neonatal ultraviolet radiation (UVR) exposure, which has a powerful effect in exacerbating melanoma development in the mouse model. Dimericine has been shown to reduce the incidence of basal-cell and squamous cell carcinoma. Unexpectedly, we saw no difference in penetrance or age of onset of melanoma after neonatal UVR between Dimericine-treated and control animals, although the drug reduced DNA damage and cellular proliferation in the skin. Interestingly, epidermal melanocytes removed cyclobutane pyrimidine dimers (CPDs) more efficiently than surrounding keratinocytes. Our study indicates that neonatal UVR-initiated melanomas may be driven by mechanisms other than solely that of a large CPD load and/or their inefficient repair. This is further suggestive of different mechanisms by which UVR may enhance the transformation of keratinocytes and melanocytes.