Functional characterization of adult human trabecular meshwork stem cells

Sundaresan, Yogapriya; Ramasamy, Krishnadas Subbiah; Muthukkaruppan, Veerappan; Chidambaranathan, Gowri Priya

doi:10.1016/j.yexcr.2021.112709

Cited by 5 publications

(6 citation statements)

References 28 publications

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“…In addition to using the positive expression of SOX2, we also used a negative marker to identify the adult lens epithelial stem cells. The significance of combining two parameters to identify stem cells was established in our previous studies on limbal epithelial stem cells [24], buccal mucosal epithelial stem cells [51], and trabecular meshwork stem cells [23], as well as by other groups [52,53]. In our study, the expression of SOX2 was analysed along with the membrane expression of the differentiated epithelial cell marker GJA1, which is one among the 10 connexins reported to be expressed in the differentiated cells in the human eye.…”

Section: Discussionmentioning

confidence: 92%

“…While there are no exclusive markers for adult stem cells, a combination of two or more markers/parameters is essential to identify stem cells [23,[26][27][28]. Functionally, adult stem cells are defined based on their label-retaining cell property [2,24,29], clonal ability [9,30,31], sphere forming ability [10,24,32], side population property [33][34][35], ability to regenerate lineage-specific cells [8,36,37] and lineage tracing experiments [16,38,39]. With ageing, the adult stem cells decrease in number, as shown in human trabecular meshwork and dental pulp [23,40].…”

Section: Discussionmentioning

confidence: 99%

“…As described by Sundaresan et al (2021) [24], the cultured cells were trypsinized, and 1 × 10 3 viable cells were seeded on ultra low attachment dishes (3471, Corning, Kennebunk, ME, USA). Further, the cells were maintained in sphere medium containing DMEM/F12 (1:1) + Glutamax TM (10565018, Gibco TM , Life Technologies, NY, USA), 2% B27 (17504044, Gibco TM , Life Technologies, NY, USA), 20 ng/mL epidermal growth factor (PHG0311, Thermo Fisher Scientific, MA, USA), 20 ng/mL fibroblast growth factor (13256029, Gibco TM , Thermo Fisher Scientific, MA, USA), 5 µg/mL heparin (375095, EMD Millipore Corp., Danvers, MA, USA), 0.1 mg/mL bovine serum albumin (B8894, Sigma), 2.5 mM L-glutamine (35050061, Gibco TM , Life Technologies, NY, USA), and 250 µg/mL penicillin-streptomycin (15140122, GibcoTM, Life Technologies, NY, USA), and cultured.…”

Section: Establishment Of Lens Sphere Culturesmentioning

confidence: 99%

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Towards the Identification and Characterization of Putative Adult Human Lens Epithelial Stem Cells

Saranya,

Shekhar,

Haripriya

et al. 2023

Cells

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The anterior lens epithelium has the ability to differentiate into lens fibres throughout its life. The present study aims to identify and functionally characterize the adult stem cells in the human lens epithelium. Whole mounts of lens epithelium from donor eyes (normal/cataract) were immunostained for SOX2, gap junction protein alpha 1 (GJA1), PAX6, α, β and γ-crystallins, followed by a confocal analysis. The functional property of adult stem cells was analysed by their sphere forming ability using cultured lens epithelial cells from different zones. Based on marker expression, the lens epithelium was divided into four zones: the central zone, characterized by a small population of PAX6+, GJA1−, β-crystallin− and γ-crystallin− cells; the germinative zone, characterized by PAX6+, GJA1+, β-crystallin− and γ-crystallin−; the transitional zone, characterized by PAX6+, GJA1+, β-crystallin+ and γ-crystallin−; and the equatorial zone, characterized by PAX6+/−, GJA1+, β-crystallin+, and γ-crystallin+ cells. The putative lens epithelial stem cells identified as SOX2+ and GJA1 membrane expression negative cells were located only in the central zone (1.89 ± 0.84%). Compared to the other zones, a significant percentage of spheres were identified in the central zone (1.68 ± 1.04%), consistent with the location of the putative adult lens epithelial stem cells. In the cataractous lens, an absence of SOX2 expression and a significant reduction in sphere forming ability (0.33 ± 0.11%) were observed in the central zone. The above findings confirmed the presence of putative stem cells in the central zone of the adult human lens epithelium and indicated their probable association with cataract development.

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Section: Discussionmentioning

confidence: 92%

Section: Discussionmentioning

confidence: 99%

Section: Establishment Of Lens Sphere Culturesmentioning

confidence: 99%

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Towards the Identification and Characterization of Putative Adult Human Lens Epithelial Stem Cells

Saranya,

Shekhar,

Haripriya

et al. 2023

Cells

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“…We examined single-cell sequence expression data from 10 human anterior segment tissues ( 22 ) and found that the highest THBS1 expression levels were in JCT and the Schwalbe’s cell line, a region that includes TM stem cells ( 23 ) ( Supplemental Figure 1 ; supplemental material available online with this article; https://doi.org/10.1172/JCI156967DS1 ).…”

Section: Resultsmentioning

confidence: 99%

Thrombospondin 1 missense alleles induce extracellular matrix protein aggregation and TM dysfunction in congenital glaucoma

Fu¹,

Siggs²,

Knight³

et al. 2022

Journal of Clinical Investigation

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Glaucoma is a highly heritable disease that is a leading cause of blindness worldwide. Here, we identified heterozygous thrombospondin 1 ( THBS1 ) missense alleles altering p.Arg1034, a highly evolutionarily conserved amino acid, in 3 unrelated and ethnically diverse families affected by congenital glaucoma, a severe form of glaucoma affecting children. Thbs1 R1034C -mutant mice had elevated intraocular pressure (IOP), reduced ocular fluid outflow, and retinal ganglion cell loss. Histology revealed an abundant, abnormal extracellular accumulation of THBS1 with abnormal morphology of juxtacanalicular trabecular meshwork (TM), an ocular tissue critical for aqueous fluid outflow. Functional characterization showed that the THBS1 missense alleles found in affected individuals destabilized the THBS1 C-terminus, causing protein misfolding and extracellular aggregation. Analysis using a range of amino acid substitutions at position R1034 showed that the extent of aggregation was correlated with the change in protein-folding free energy caused by variations in amino acid structure. Extracellular matrix (ECM) proteins, especially fibronectin, which bind to THBS1, also accumulated within THBS1 deposits. These results show that missense variants altering THBS1 p.Arg1034 can cause elevated IOP through a mechanism involving impaired TM fluid outflow in association with accumulation of aggregated THBS1 in the ECM of juxtacanalicular meshwork with altered morphology.

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“… 12 that the cells expressing high ABCG2 and p75 to be located only in the TM insert region and (iii) our recent report Sundaresan et al . 41 , that the native TM-derived neurospheres expressing high ABCG2 and p75 differentiated to TM cells in vitro. Further studies are essential to establish that the cells expressing high ABCG2 and p75 maintain TM tissue homeostasis.…”

Section: Discussionmentioning

confidence: 98%

Reduction in trabecular meshwork stem cell content in donor eyes with primary open angle glaucoma

Sundaresan¹,

Manivannan²,

Radhakrishnan

et al. 2021

Sci Rep

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We previously identified and characterized human trabecular meshwork stem cells (TMSCs) based on high expression of ABCG2/p75 positivity and high nucleus to cytoplasmic ratio. These TMSCs expressing high ABCG2 and p75 were located to the insert region of the human TM. Additionally, we demonstrated an age-related reduction in the TMSC content which was significantly associated with TM cell loss. In continuation, this study was aimed to determine the TMSC content in glaucomatous donor eyes wherein a drastic reduction in TM cellularity has already been reported. Anterior segments from known glaucomatous (n = 6) and age-matched normal (n = 8) donors were dissected into four quadrants. A minimum of three sections from each quadrant were used for histopathological analysis as well as immunostaining. Analysis of hematoxylin and eosin-stained sections from glaucomatous tissues revealed a decrease in total TM cellularity, thickening of trabecular beams, fusion of trabeculae, absence of patent Schlemm’s canal compared to age-matched controls. In addition, the TM thickness at various positions of the meshwork and the coronal as well as the meridional diameters of the Schlemm’s canal were observed to be significantly reduced in glaucomatous eyes. Further, sections from both the groups were immunostained for universal stem cell marker ABCG2 and neural crest derived stem cell marker p75. The images were acquired using Leica SP8 confocal microscope. Quantification of total TM cellularity based on nuclear counterstain (mean ± SD) using ImageJ identified 69.33 ± 12.77 cells/section in control eyes. In glaucomatous donors, the TM cellularity was found to be reduced significantly to 41.83 ± 9.0 (p = 0.0007). In addition, a reduction in the percentage of TMSCs (cells with high ABCG2 expression and p75 positivity) was evident in glaucomatous donors (0.14 ± 0.17%) compared to age-matched controls (4.73 ± 5.46%) (p = 0.064). Thus, the present study confirmed the significant decline in TM cellularity and a reducing trend in the TMSC content, though this reduction was non-significant in glaucomatous donor eyes. Further studies are essential to elucidate the role of TMSCs in the pathogenesis of primary open angle glaucoma.

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Functional characterization of adult human trabecular meshwork stem cells

Cited by 5 publications

References 28 publications

Towards the Identification and Characterization of Putative Adult Human Lens Epithelial Stem Cells

Towards the Identification and Characterization of Putative Adult Human Lens Epithelial Stem Cells

Thrombospondin 1 missense alleles induce extracellular matrix protein aggregation and TM dysfunction in congenital glaucoma

Reduction in trabecular meshwork stem cell content in donor eyes with primary open angle glaucoma

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