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Effect of Isolation Technique and Location on the Phenotype of Human Corneal Stroma-Derived Cells.

ABSTRACT: Purpose:To determine the effect of the isolation technique and location upon the phenotype of human corneal stroma-derived cells (CSCs). Methods:CSCs were isolated from the corneal stroma center and periphery using the explant or enzymatic digestion technique. The native tissue was stained for functional markers, while cultured cells were analysed by FACS. PCR was used to determine gene expression in the cultured versus native cells. Results:The native stroma was positive for ?-actinin, ALDH1A1, CD31, CD34, Collagen I, and Vimentin. Cultured cells expressed CD73, CD90, CD105, CD51, Nestin, CD49a, CD49d, ABCG2, and CD47. PCR demonstrated a significant upregulation of ALDH1A1, AQP1, ITGB4, KLF4, CD31, CD34, and CXCR4 in the native tissue, while the expression of ABCG2, ITGAV, Nestin, CD73, CD90, CD105, and Vimentin were significantly higher in the cultured cells. GPC did not change. Conclusion:The study finds no significant difference between the phenotype of CSCs generated by the explant or enzymatic digestion technique from the center or periphery of the stroma. Isolation of the cells can be performed without regard to the location and isolation technique used for research. Cultivated CSCs undergo a complete surface marker and genotype profile change compared to the state in situ.

SUBMITTER: Nagymihaly R

PROVIDER: S-EPMC5682086 | biostudies-literature | 2017

REPOSITORIES: biostudies-literature

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Effect of Isolation Technique and Location on the Phenotype of Human Corneal Stroma-Derived Cells.

Nagymihály Richárd R Veréb Zoltán Z Facskó Andrea A Moe Morten C MC Petrovski Goran G

Stem cells international 20171029

<h4>Purpose</h4>To determine the effect of the isolation technique and location upon the phenotype of human corneal stroma-derived cells (CSCs).<h4>Methods</h4>CSCs were isolated from the corneal stroma center and periphery using the explant or enzymatic digestion technique. The native tissue was stained for functional markers, while cultured cells were analysed by FACS. PCR was used to determine gene expression in the cultured versus native cells.<h4>Results</h4>The native stroma was positive f ...[more]

PMID: 29213290

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Project description:The unwounded, normal corneal stroma is a relatively simple, avascular tissue populated with quiescent keratocytes, along with corneal nerves and a few resident dendritic and monocyte/macrophage cells. In the past, the resting keratocytes were thought of as a homogenous cellular population, but recent work has shown local variations in vimentin and nestin expression, and responsiveness to transforming growth factor (TGF)-β1. Studies have also supported there being "stromal stem cells" in localized areas. After corneal wounding, depending on the site and severity of injury, profound changes in stromal cellularity occur. Anterior or posterior injuries to the epithelium or endothelium, respectively, trigger apoptosis of adjacent keratocytes. Many contiguous keratocytes transition to keratocan-negative corneal fibroblasts that are proliferative and produce limited amounts of disorganized extracellular matrix components. Simultaneously, large numbers of bone marrow-derived cells, including monocytes, neutrophils, fibrocytes and lymphocytes, invade the stroma from the limbal blood vessels. Ongoing adequate levels of TGFβ1, TGFβ2 and platelet-derived growth factor (PDGF) from epithelium, tears, endothelium and aqueous humor that penetrate defective or absent epithelial barrier function (EBF) and epithelial basement membrane (EBM) and/or Descemet's basement membrane (DBM) drive corneal fibroblasts and fibrocytes to differentiate into alpha-smooth muscle actin (SMA)-positive myofibroblasts. If the EBF, EBM and/or DBM are repaired or replaced in a timely manner, typically measured in weeks, then corneal fibroblast and fibrocyte progeny, deprived of requisite levels of TGFβ1 and TGFβ2, undergo apoptosis or revert to their precursor cell-types. If the EBF, EBM and/or DBM are not repaired or replaced, stromal levels of TGFβ1 and TGFβ2 remain elevated, and mature myofibroblasts are generated from corneal fibroblasts and fibrocyte precursors that produce prodigious amounts of disordered extracellular matrix materials associated with scarring fibrosis. This fibrotic stromal matrix persists, at least until the EBF, EBM and/or DBM are regenerated or replaced, and keratocytes remove and reorganize the affected stromal matrix.

Project description:PurposeTo compare the effects of the Ahmed glaucoma valve (AGV; New World Medical, Rancho Cucamonga, CA) with sulcus versus anterior chamber (AC) tube placement on the corneal endothelial density and morphology over time.DesignNonrandomized, interventional study.ParticipantsThis study included 106 eyes from 101 pseudophakic patients who had the AGV tube placed in the AC (acAGV) and 105 eyes from 94 pseudophakic patients who had the AGV tube placed in the ciliary sulcus (sAGV).MethodsAll patients underwent preoperative specular microscopy, which was repeated postoperatively in 2019. The patients' demographic information, glaucoma diagnoses, and basic ocular information were obtained on chart review. Anterior segment OCT was conducted for patients who underwent sAGV to evaluate the sulcus tube position. Gonioscopy was performed to document peripheral anterior synechiae (PAS). Linear mixed-effects models were used to compare the different ocular and endothelial measurements between the 2 groups and to identify risk factors for endothelial cell density (ECD) loss over time.Main outcome measuresMonthly change in corneal endothelial measurements, including ECD and coefficient of variation (CV), calculated as the difference between preoperative and postoperative measurements divided by the number of months from the time of surgery to postoperative specular microscopy.ResultsThe acAGV and sAGV groups were comparable in all baseline characteristics except that the acAGV group had longer follow-up (37.6 vs. 20.1 months, respectively, P < 0.001). Mean monthly loss in central ECD was significantly more in the acAGV group (mean ± standard deviation: 29.3±29.7 cells/mm2) than in the sAGV group (15.3±20.7 cells/mm2, P < 0.0001). Mean monthly change in CV was similar between the 2 groups (P = 0.28). Multivariate analyses revealed that younger age and tube location in the AC were associated with faster central ECD loss (P = 0.02, P < 0.0001, respectively). For patients with sAGV, while PAS was associated with faster central ECD loss (P = 0.002), a more forward tube position tenting the iris was not (P > 0.05).ConclusionsCompared with anterior segment placement, ciliary sulcus tube implantation may be a preferred surgery approach to reduce endothelial cell loss in pseudophakic patients.

Dataset Information

Effect of Isolation Technique and Location on the Phenotype of Human Corneal Stroma-Derived Cells.

Publications

Effect of Isolation Technique and Location on the Phenotype of Human Corneal Stroma-Derived Cells.

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