Therapeutic Effect of Human Adipose Tissue-Derived Mesenchymal Stem Cells in Experimental Corneal Failure Due to Limbal Stem Cell Niche Damage

Producción CientíficaLimbal stem cells are responsible for the continuous renewal of the corneal epithelium. The destruction or dysfunction of these stem cells or their niche induces limbal stem cell deficiency (LSCD) leading to visual loss, chronic pain, and inflammation of the ocular surface. To restore the ocular surface in cases of bilateral LSCD, an extraocular source of stem cells is needed to avoid dependence on allogeneic limbal stem cells that are difficult to obtain, isolate, and culture. The aim of this work was to test the tolerance and the efficacy of human adipose tissue-derived mesenchymal stem cells (hAT-MSCs) to regenerate the ocular surface in two experimental models of LSCD that closely resemble the different severity grades of the human pathology. hAT-MSCs transplanted to the ocular surface of the partial and total LSCD models developed in rabbits were well tolerated, migrated to inflamed tissues, reduced inflammation, and restrained the evolution of corneal neovascularization and corneal opacity. The expression profile of the corneal epithelial cell markers CK3 and E-cadherin, and the limbal epithelial cell markers CK15 and p63 was lost in the LSCD models, but was partially recovered after hAT-MSC transplantation. For the first time, we demonstrated that hAT-MSCs improves corneal and limbal epithelial phenotypes in animal LSCD models. These results support the potential use of hAT-MSCs as a novel treatment of ocular surface failure due to LSCD. hAT-MSCs represent an available, non-immunogenic source of stem cells that may provide therapeutic benefits in addition to reduce health care expenses.This work was supported by Instituto de Salud Carlos III, CIBER‐BBN, Spain (CB06/01/003 MINECO/FEDER, EU); Regional Center for Regenerative Medicine and Cell Therapy, Castilla y León, Spain; Ministry of Science and Innovation, Spain (SAF2010–14900); Ministry of Economy and Competitiveness and European Regional Development Fund, Spain (SAF2015–63594‐R MINECO/FEDER, EU

Poly-l/dl-lactic acid films functionalized with collagen IV as carrier substrata for corneal epithelial stem cells

Limbal epithelial stem cells (LESCs) are responsible for the renewal of corneal epithelium. Cultivated limbal epithelial transplantation is the current treatment of choice for restoring the loss or dysfunction of LESCs. To perform this procedure, a substratum is necessary for in vitro culturing of limbal epithelial cells and their subsequent transplantation onto the ocular surface. In this work, we evaluated poly-L/DL-lactic acid 70:30 (PLA) films functionalized with type IV collagen (col IV) as potential in vitro carrier substrata for LESCs. We first demonstrated that PLA-col IV films were biocompatible and suitable for the proliferation of human corneal epithelial cells. Subsequently, limbal epithelial cell suspensions, isolated from human limbal rings, were cultivated using culture medium that did not contain animal components. The cells adhered significantly faster to PLA-col IV films than to tissue culture plastic (TCP). The mRNA expression levels for the LESC specific markers, K15, P63α and ABCG2 were similar or greater (significantly in the case of K15) in limbal epithelial cells cultured on PLA-col IV films than limbal epithelial cells cultured on TCP. The percentage of cells expressing the corneal (K3, K12) and the LESC (P63α, ABCG2) specific markers was similar for both substrata. These results suggest that the PLA-col IV films promoted LESC attachment and helped to maintain their undifferentiated stem cell phenotype. Consequently, these substrata offer an alternative for the transplantation of limbal cells onto the ocular surface.This work was supported by the Carlos III National Institute of Health, Spain (CIBER-BBN and Spanish Network on Cell Therapy, (TerCel RD12/0019/0036), MINECO/FEDER, EU), and the Castilla y León Regional Government, Spain (Regional Center for Regenerative Medicine and Cell Therapy, SAN673/VA/28/08 and SAN126/VA11/09)

Age and sex-adjusted reference intervals in tear cytokine levels in healthy subjects

Producción CientíficaAlterations in tear cytokine levels have been associated with various ocular disorders as compared to those in healthy subjects. However, age and sex are not always considered in these comparisons. In this study we aimed to establish age and sex reference intervals (RIs) for tear cytokine levels in healthy people. Tear samples were taken from 75 males and 82 females, aged 18–88 years, and tear cytokine levels were determined. Age- and sex-adjusted RIs for epidermal growth factor (EGF), fractalkine, interleukin (IL)-1 receptor antagonist (RA), IL-7, IL-8, interferon inducible protein (IP)-10, monocyte chemotactic protein (MCP)-1, and vascular endothelial growth factor (VEGF) tear cytokine levels in a healthy sample were established using generalized additive for location, scale and shape (GAMLSS) models. RIs were tested in two external samples: a validation sample of 40 individuals with normal results at four Dry Eye Disease (DED) clinical diagnostic tests (OSDI, T-BUT, corneal staining and Schirmer test); and a utility sample of 13 severe DED cases. IL-1RA, IL-8, IP-10, and MCP-1 levels showed a positive association with age, while EGF was negatively correlated. IL-7 concentration increased up to 40 years and again after 70 years, observing a quasi-linear decrease between them. For VEGF, higher levels were observed in the middle-aged range. Regarding sex-influence, fractalkine tear levels were higher in men, whereas those of IL-7, IL-8, and IP-10 were higher in women. Using the estimated age- and sex-adjusted RIs, more than 92% of the validation sample was correctly classified, and 100% of the severe DED patients in the utility sample had concentrations outside the RIs in at least two of the cytokines evaluated

Biomarkers in ocular chronic graft versus host disease: tear cytokine- and chemokine-based predictive model.

Producción CientíficaPurpose: To develop a tear molecule level-based predictive model based on a panel of tear cytokines and their correlation with clinical features in ocular chronic graft versus host disease (cGVHD). Methods: Twenty-two ocular cGVHD patients and 21 healthy subjects were evaluated in a controlled environmental research laboratory (CERLab). Clinical parameters were recorded, and tears were collected. Levels of 15 molecules (epidermal growth factor [EGF], IL receptor antagonist [IL-1Ra], IL-1β, IL-2, IL-6, IL-8/CXCL8, IL-10, IL-12p70, IL-17A, interferon inducible protein [IP]-10/CXCL10, IFN-γ, VEGF, TNF-α, eotaxin 1, and regulated on activation normal T cell expressed and secreted [RANTES]) were measured by multiplex-bead assay and correlated with clinical parameters. Logistic regression was used to develop a predictive model. Leave-one-out cross-validation was applied. Classification capacity was evaluated in a cohort of individuals with dry eye (DE) of other etiologies different from GVHD. Results: Epidermal growth factor and IP-10/CXCL10 levels were significantly decreased in ocular cGVHD, positively correlating with tear production and stability and negatively correlating with symptoms, hyperemia, and vital staining. Interleukin-1Ra, IL-8/CXCL8, and IL-10 were significantly increased in ocular cGVHD, and the first two correlated positively with symptoms, hyperemia, and ocular surface integrity while negatively correlating with tear production and stability. Predictive models were generated, and the best panel was based on IL-8/CXCL8 and IP-10/CXCL10 tear levels along with age and sex, with an area under the receiving operating curve of 0.9004, sensitivity of 86.36%, and specificity of 95.24%. Conclusions: A predictive model based on tear levels of IL-8/CXCL8 and IP-10/CXCL10 resulted in optimal sensitivity and specificity. These results add further knowledge to the search for potential biomarkers in this devastating ocular inflammatory disease.Ministry of Economy and Competitiveness, Madrid, Spain, SAF-2010 15631 (AES)

Eficacia del trasplante de células madre mesenquimales de médula ósea y de tejido adiposo en un modelo de deficiencia de células madre limbares desarrollado en conejo: estudio comparativo

INTRODUCCIÓN: La deficiencia de células madre limbares (LSCD, por sus siglas en inglés) se produce como resultado de la destrucción y/o disfunción de las células madre del epitelio limbar, y tiene entre sus consecuencias la pérdida de visión y la inflamación crónica de la superficie ocular. Nuestro grupo de investigación ha demostrado recientemente que el trasplante de células madre mesenquimales (MSC) derivadas de la médula ósea (BM-MSC) en la superficie ocular de pacientes que padecen LSCD es seguro y facilita de manera eficaz la reparación del epitelio corneal. Sin embargo, las MSC derivadas del tejido adiposo (AT-MSC) se ha demostrado que son más accesibles, más económicas y una fuente de células madre más segura que las BM-MSC. OBJETIVO: Comparar la eficacia del trasplante de BM-MSC versus AT-MSC en un modelo de LSCD desarrollado en conejo. MÉTODOS: Dieciocho conejos fueron tratados con 250.000 MSC cultivadas sobre una membrana amniótica (9 con BM-MSC y 9 con AT-MSC) y trasplantadas tres semanas después de inducirles un LSCD. Mediante desepitelización completa de la córnea con n-heptanol, seguida de una peritomía limbar quirúrgica de 360º, se indujo un modelo de LSCD en 27 conejos. Transcurridas 3 semanas de evolución, 18 conejos fueron trasplantados con 250.000 MSC cultivadas sobre una membrana amniótica (9 con BM-MSC y 9 con AT-MSC). Nueve conejos no trasplantados formaron el grupo de control. Semanalmente, se evaluó clínicamente la invasión conjuntival, la neovascularización, la opacidad y el defecto epitelial corneal con una lámpara de hendidura. Mediante un análisis histopatológico realizado al final del periodo del seguimiento (11 semanas), se evaluó el nivel de daño tisular y la presencia de linfocitos (como signo de inflamación) y de células caliciformes (como signo de conjuntivalización) en el limbo y en la córnea. RESULTADOS: El grupo trasplantado con BM-MSC mostró menos neovascularización y menos opacidad corneal en las semanas 6-8 y 6-9, respectivamente, que el grupo no tratado. El grupo tratado con AT-MSC tuvo menos invasión conjuntival y opacidad corneal en las semanas 6-8 y 6-10, respectivamente, que el grupo de control. No hubo diferencias en los defectos epiteliales entre los 3 grupos. Los grupos trasplantados con BM-MSC y AT-MSC mostraron un mayor número de capas epiteliales en la córnea y en el limbo, menos desorganización del estroma, menor cantidad de células inflamatorias en el estroma de la córnea y menos células caliciformes en el epitelio del limbo y/o de la córnea que el grupo no tratado. CONCLUSIONES: El trasplante de BM-MSC y AT-MSC en un modelo de LSCD desarrollado en conejo redujo el desarrollo de la opacidad corneal y restauró parcialmente la estructura dañada del tejido limbar y corneal. Las BM-MSC fueron mejores en la reducción del desarrollo de la neovascularización corneal, mientras que las AT-MSC evitaron mejor la invasión conjuntival. Ambos tipos de MSC parecen alternativas válidas para el tratamiento de la LSCD.Ministerios de Economía y Competitividad (SAF2015-63594-R MINECO/FEDER, UE) y de Ciencia e Innovación, España (SAF2010-14900); Instituto de Salud Carlos III, España (CIBER-BBN, CB06/01/003 MINECO/FEDER). Centro en Red de Medicina Regenerativa y Terapia Celular de Castilla y León, España

A proof-of-concept clinical trial using mesenchymal stem cells for the treatment of corneal epithelial stem cell deficiency

Producción CientíficaOcular stem cell transplantation derived from either autologous or allogeneic donor corneoscleral junction is a functional cell therapy to manage extensive and/or severe limbal stem cell deficiencies that lead to corneal epithelial failure. Mesenchymal stem cells have been properly tested in animal models of this ophthalmic pathology, but never in human eyes despite their potential advantages. We conducted a 6- to 12-month proof-of-concept, randomized, and double-masked pilot trial to test whether allogeneic bone marrow-derived mesenchymal stem cell transplantation (MSCT], n = 17) was as safe and as equally efficient as allogeneic cultivated limbal epithelial transplantation (CLET), (n = 11) to improve corneal epithelial damage due to limbal stem cell deficiency. Primary endpoints demanded combination of symptoms, signs, and the objective improvement of the epithelial phenotype in central cornea by in vivo confocal microscopy. This proof-of-concept trial showed that MSCT was as safe and efficacious as CLET. Global success at 6–12 months was 72.7%–77.8% for CLET cases and 76.5%–85.7% for MSCT cases (not significant differences). Central corneal epithelial phenotype improved in 71.4% and 66.7% of MSCT and CLET cases, respectively at 12 months (P = 1.000). There were no adverse events related to cell products. This trial suggests first evidence that MSCT facilitated improvement of a diseased corneal epithelium due to lack of its stem cells as efficiently as CLET. Consequently, not only CLET but also MSCT deserves more preclinical investigational resources before the favorable results of this proof-of-concept trial could be transformed into the larger numbers of the multicenter trials that would provide stronger evidence. (ClinicalTrials.gov number, NCT01562002.)Ministerio de Sanidad, Consumo y Bienestar Social (project SAS/2481/2009)Centro en Red de Medicina Regenerativa y Terapia Celular de Castilla y León (grant SAN 1178/200)Red de Terapia Celular TerCel (project RD12/0019/0036

Clinical and Molecular Inflammatory Response in Sjögren Syndrome–Associated Dry Eye Patients Under Desiccating Stress

Producción CientíficaTo evaluate the response of the lacrimal function unit in Sjogren syndrome (SS)-associated dry eye patients exposed to 2 simulated daily life environ-mental conditions. Fourteen female SS dry eye patients were exposed for 2 hours to a controlled normal condition (23 C, 45% relative humidity, and air flow 0.10 m/s) and a controlled adverse condition that simulates desic- cating stress (23 C, 5% relative humidity, and air flow 0.10 m/s). The following dry eye tests were performed before and after the exposure: tear osmolarity, phenol red thread test, conjunctival hyperemia, fluorescein tear break-up time, corneal fluorescein staining, conjunctival lissamine green staining, and Schirmer test. Levels of 16 molecules were analyzed in tears by multiplex immu- nobead analysis.Junta de Castilla y León (programa de apoyo a proyectos de investigación – Ref. VA174U14

Inflammation-related molecules in tears of patients with chronic ocular pain and dry eye disease

Producción CientíficaThe purpose of this study was to analyze inflammation- and pain-related molecules in tears of patients suffering from chronic ocular pain associated with dry eye (DE) and/or a previous corneal refractive surgery (RS). Based on history, symptomatology, and clinical signs, the subjects (n = 180, 51.0 ± 14.7 years, 118 females, 62 males) in this cross-sectional study were assigned to one of five groups: DE and chronic ocular pain after RS (P/DE-RS, n = 52); asymptomatic subjects, i.e., without DE and chronic ocular pain, after RS (A-RS, n = 30); DE and chronic ocular pain without previous RS (P/DE-nonRS, n = 31); DE, no pain, and no previous RS (DE-nonRS, n = 35); and asymptomatic subjects with no previous RS (controls, n = 32). The tear concentrations of 20 cytokines and substance P (SP) were analyzed by immunobead-based assay and enzyme-linked immunosorbent assay, respectively. We found that tear levels of interleukin (IL)-10 and SP were increased in the RS groups. There were significant differences in IL-8/CXCL8 among the five groups. Nerve growth factor (NGF) tear levels were significantly higher in P/DE-RS than in DE-nonRS and controls. IL-9 had the highest percentage of detection in the P/DE-RS and P/DE-nonRS groups, while macrophage inflammatory protein (MIP)-1α, IL-2, and interferon (IFN)-γ were higher in the P/DE-RS, A-RS, and P/DE-nonRS groups. IL-17A was detected only in the A-RS group. Moderate correlations were observed in the A-RS, P/DE-nonRS, DE-nonRS and controls groups. A positive correlation was obtained between growth related oncogene concentration and tear break-up time (rho = 0.550; p = 0.012), while negative correlation was found between monocyte chemoattractant protein-3/CCL7 and conjunctival staining (rho = −0.560; p = 0.001), both in the A-RS group. IL-10 correlated positively with ocular pain intensity (rho = 0.513; p = 0.003) in the P/DE-nonRS group. Regulated on Activation Normal T Cell Expressed and Secreted/CCL5 correlated negatively with conjunctival staining (rho = −0.545; p = 0.001) in the DE-nonRS group. SP correlated negatively with corneal staining (rho = −0.559; p = 0.001) in the controls. In conclusion, chronic ocular pain was associated with higher IL-9 tear levels. IL-10, SP, MIP-1α/CCL3, IL-2, and IFN-γ were associated with previous RS. Higher levels of IL-8/CXCL8, MIP-1α/CCL3, IL-2, and IFN-γ were associated with DE-related inflammation, while NGF levels were related to chronic ocular pain and DE in RS patients. These findings suggest that improved knowledge of tear cytokines and neuromodulators will lead to a more nuanced understanding of how these molecules can serve as biomarkers of chronic ocular pain, leading to better therapeutic and disease management decisions.Ministerio de Ciencia, Innovación y Universidades (grants SAF-2016-77080-P, FPU17/02715 and FPU15/01443

Corneal regeneration using adipose-derived mesenchymal stem cells

Producción CientíficaAdipose-derived stem cells are a subtype of mesenchymal stem cell that offers the important advantage of being easily obtained (in an autologous manner) from low invasive procedures, rendering a high number of multipotent stem cells with the potential to differentiate into several cellular lineages, to show immunomodulatory properties, and to promote tissue regeneration by a paracrine action through the secretion of extracellular vesicles containing trophic factors. This secretome is currently being investigated as a potential source for a cell-free based regenerative therapy for human tissues, which would significantly reduce the involved costs, risks and law regulations, allowing for a broader application in real clinical practice. In the current article, we will review the existing preclinical and human clinical evidence regarding the use of such adipose-derived mesenchymal stem cells for the regeneration of the three main layers of the human cornea: the epithelium (derived from the surface ectoderm), the stroma (derived from the neural crest mesenchyme), and the endothelium (derived from the neural crest cells)

Clinical and tear cytokine profiles after advanced surface ablation refractive surgery: A six-month follow-up

Producción CientíficaNeuropathic dry eye is one of the most frequently seen complications after corneal refractive surgery, however, its incidence decreases in a significant manner along the first six months postoperative, reaching between 10 and 45% incidence. However, little is known on the inflammatory status of the ocular surface during this recovery process. We aim to analyze the clinical and tear molecule concentration changes along six months after advanced surface ablation for myopia correction, in a prospective study including 18 eyes of 18 subjects who bilaterally underwent advanced surface ablation corneal refractive surgery. Clinical variables (uncorrected distance visual acuity, symptoms, conjunctival hyperemia, tear osmolarity, tear stability, corneal fluorescein staining, conjunctival lissamine staining, Schirmer test, and corneal esthesiometry) and a panel of 23 pro and anti-inflammatory cytokines/chemokines concentration in tears preoperatively and at 1, 3 and 6 months postoperatively were evaluated. We found that uncorrected distance visual acuity improved significantly from baseline at 1-month visit, symptoms improved and tear osmolarity decreased significantly from baseline at 3-month visit and there was a decrease in mechanical corneal threshold between 1-month and 3- and 6-month visits. Regarding tear molecules, IL-4, IL-5, IL-6, IL-13, IL-17A, and IFN-γ tear levels were significantly increased at all the three visits, compared to preoperative levels at V0; IL-2 and VEGF were also significantly increased at 1-month and 6-month visits, but not at 3-month visit, whereas IL-9 IL-10 and IL-12 were only significantly increased at 6-month visit. Although we found that there is a recovery in clinical variables at 6 months postoperatively (i.e. neuropathic dry eye was not developed in the sample), ocular surface homeostasis is not completely restored, as it can be seen by the changes in concentration of some pro and anti-inflammatory molecules measured in tears.Partially supported by national grant (AES) from the Ministry of 465 Economy and Competitiveness, Madrid, Spain, SAF2016-77080 AEI/FEDER, U