Transcriptomic Profiling of Human Limbus-Derived Stromal/Mesenchymal Stem Cells—Novel Mechanistic Insights into the Pathways Involved in Corneal Wound Healing

Tavakkoli, Fatemeh; Damala, Mukesh; Koduri, Madhuri Amulya; Gangadharan, Abhilash; Amit, K.; Dash, Debasis; Basu, Sayan; Singh, Vivek

doi:10.3390/ijms23158226

Cited by 6 publications

(4 citation statements)

References 109 publications

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“…Hence, a culture medium with correct glucose level could favor CSSC growth in vitro. Supplementation with a low level of serum (2%) stimulates CSSC proliferation [ 27 ]. However, the poorly defined composition and batch-to-batch variability of sera have complicated the optimization step.…”

Section: Discussionmentioning

confidence: 99%

Good manufacturing practice production of human corneal limbus-derived stromal stem cells and in vitro quality screening for therapeutic inhibition of corneal scarring

Santra,

Geary,

Rubin

et al. 2024

Stem Cell Res Ther

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Background Mesenchymal stem cells in the adult corneal stroma (named corneal stromal stem cells, CSSCs) inhibit corneal inflammation and scarring and restore corneal clarity in pre-clinical corneal injury models. This cell therapy could alleviate the heavy reliance on donor materials for corneal transplantation to treat corneal opacities. Herein, we established Good Manufacturing Practice (GMP) protocols for CSSC isolation, propagation, and cryostorage, and developed in vitro quality control (QC) metric for in vivo anti-scarring potency of CSSCs in treating corneal opacities. Methods A total of 24 donor corneal rims with informed consent were used—18 were processed for the GMP optimization of CSSC culture and QC assay development, while CSSCs from the remaining 6 were raised under GMP-optimized conditions and used for QC validation. The cell viability, growth, substrate adhesion, stem cell phenotypes, and differentiation into stromal keratocytes were assayed by monitoring the electric impedance changes using xCELLigence real-time cell analyzer, quantitative PCR, and immunofluorescence. CSSC’s conditioned media were tested for the anti-inflammatory activity using an osteoclastogenesis assay with mouse macrophage RAW264.7 cells. In vivo scar inhibitory outcomes were verified using a mouse model of anterior stromal injury caused by mechanical ablation using an Algerbrush burring. Results By comparatively assessing various GMP-compliant reagents with the corresponding non-GMP research-grade chemicals used in the laboratory-based protocols, we finalized GMP protocols covering donor limbal stromal tissue processing, enzymatic digestion, primary CSSC culture, and cryopreservation. In establishing the in vitro QC metric, two parameters—stemness stability of ABCG2 and nestin and anti-inflammatory ability (rate of inflammation)—were factored into a novel formula to calculate a Scarring Index (SI) for each CSSC batch. Correlating with the in vivo scar inhibitory outcomes, the CSSC batches with SI < 10 had a predicted 50% scar reduction potency, whereas cells with SI > 10 were ineffective to inhibit scarring. Conclusions We established a full GMP-compliant protocol for donor CSSC cultivation, which is essential toward clinical-grade cell manufacturing. A novel in vitro QC–in vivo potency correlation was developed to predict the anti-scarring efficacy of donor CSSCs in treating corneal opacities. This method is applicable to other cell-based therapies and pharmacological treatments.

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Section: Discussionmentioning

confidence: 99%

Good manufacturing practice production of human corneal limbus-derived stromal stem cells and in vitro quality screening for therapeutic inhibition of corneal scarring

Santra,

Geary,

Rubin

et al. 2024

Stem Cell Res Ther

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“…As collagen IV is mainly present in Descemet’s membrane, the expression is supposed to be higher in native tissue [ 85 , 86 ]. Similarly, concerning PAX6, which is omnipresent in both corneal and limbal epithelia relative to the stroma, its decreased expression in hLMSCs is self-explanatory [ 87 ].…”

Section: Discussionmentioning

confidence: 99%

Expansion and characterization of human limbus-derived stromal/mesenchymal stem cells in xeno-free medium for therapeutic applications

et al. 2023

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Background Mesenchymal stem cells (MSCs) have been proven to prevent and clear corneal scarring and limbal stem cell deficiency. However, using animal-derived serum in a culture medium raises the ethical and regulatory bar. This study aims to expand and characterize human limbus-derived stromal/mesenchymal stem cells (hLMSCs) for the first time in vitro in the xeno-free medium. Methods Limbal tissue was obtained from therapeutic grade corneoscleral rims and subjected to explant culture till tertiary passage in media with and without serum (STEM MACS XF; SM), to obtain pure hLMSCs. Population doubling time, cell proliferation, expression of phenotypic markers, tri-lineage differentiation, colony-forming potential and gene expression analysis were carried out to assess the retention of phenotypic and genotypic characteristics of hLMSCs. Results The serum-free medium supported the growth of hLMSCs, retaining similar morphology but a significantly lower doubling time of 23 h (*p < 0.01) compared to the control medium. FACS analysis demonstrated ≥ 90% hLMSCs were positive for CD90+, CD73+, CD105+, and ≤ 6% were positive for CD45−, CD34−andHLA-DR−. Immunofluorescence analysis confirmed similar expression of Pax6+, COLIV+, ABCG2+, ABCB5+, VIM+, CD90+, CD105+, CD73+, HLA-DR−andCD45−, αSMA− in both the media. Tri-lineage differentiation potential and gene expression of hLMSCs were retained similarly to that of the control medium. Conclusion The findings of this study demonstrate successful isolation, characterization and culture optimization of hLMSCs for the first time in vitro in a serum-free environment. This will help in the future pre-clinical and clinical applications of MSCs in translational research.

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“…Both groups that received ADASC demonstrated improvement in paracentral scars. Meanwhile, other candidates for cell replacement are being explored, such as corneal stromal SC which have been shown to control inflammation and fibrosis [28,29]; stromal keratocytes which demonstrated ability to recover the native stromal matrix [30]; or a combination of both [31]. Similarly, additional tissue engineering approaches are exploring different scaffolds [32][33][34].…”

Section: Stromamentioning

confidence: 99%

Recent advances in cell-based regenerative therapies for corneal disease

Kaufman

Jun

2023

Current Opinion in Ophthalmology

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Purpose of reviewWith limited access of more than half the world's population to corneal transplantation, regenerative medicine may represent a promising alternative. This review explores the main advancements achieved in cell-based therapies for corneal epithelium, stroma, and endothelium during 2021--2022. Recent findingsMultiple surgical techniques have been developed for epithelial limbal stem cell replacement. Recent studies aimed to gain greater understanding and characterization of these techniques. Though no clear superiority could be demonstrated, simple limbal epithelial transplantation seems to have the most clinical and cost effectiveness. For stromal disease, autologous adipose-derived stem cells have shown favorable results. For endothelial dysfunction, the validity of intracameral cultivated allogeneic endothelial cell injection and Descemetorrhexis without endothelial keratoplasty, as well as the benefits of adjunctive rho-associated kinase inhibitors, were emphasized. SummaryA plethora of innovative cell-based regenerative therapies for corneal diseases have been developed in past years. While recent literature solidifies our knowledge, most studies are still in preliminary or preclinical stages. Though showing great promise, these approaches will require larger studies with betterdefined endpoints to establish their benefits over currently available treatments.

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Transcriptomic Profiling of Human Limbus-Derived Stromal/Mesenchymal Stem Cells—Novel Mechanistic Insights into the Pathways Involved in Corneal Wound Healing

Cited by 6 publications

References 109 publications

Good manufacturing practice production of human corneal limbus-derived stromal stem cells and in vitro quality screening for therapeutic inhibition of corneal scarring

Good manufacturing practice production of human corneal limbus-derived stromal stem cells and in vitro quality screening for therapeutic inhibition of corneal scarring

Expansion and characterization of human limbus-derived stromal/mesenchymal stem cells in xeno-free medium for therapeutic applications

Recent advances in cell-based regenerative therapies for corneal disease

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