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

Abstract

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.

Extracellular Vesicles Isolated from Equine Adipose-Derived Stromal Stem Cells (ASCs) Mitigate Tunicamycin-Induced ER Stress in Equine Corneal Stromal Stem Cells (CSSCs)

“…Flow cytometry analysis showed that equine CSSCs presented a high expression of the surface markers CD44 (98.03%), CD45 (98.73%), and CD90 (97.97%) and low expression of CD200 (4.85%) (Figure 2A), confirming their mesenchymal origin. Then, the effect of EVs on cells' stemness under ER stress conditions was evaluated by measuring by qRT-PCR the expression of the markers SOX2, KLF4, OCT4, and NANOG, known to be expressed in CSSCs [18,76,77]. For RT-qPCR, first, CSSCs were treated with tunicamycin to induce ER stress, increasing the gene expression levels of SOX2, KLF4, and OCT4 (Figure 2B-D).…”

Regenerative Therapy for Corneal Scarring Disorders

“…Our group recently reported that the stromal recovery by CSSCs was further improved by an additional CSK injection treatment, which strengthens the healing stroma with proper collagen types and stromal specific proteoglycans [ 9 ]. Building upon these numerous positive outcomes from pre-clinical studies, we established clinical-grade human CSSC manufacturing with GMP-compliant protocols [ 99 ]. The stem cell phenotypes and the characteristic capability to differentiate into CSKs were confirmed.…”

“…The stem cell phenotypes and the characteristic capability to differentiate into CSKs were confirmed. In addition, we developed an important quality management system using in vitro quality control assays for primary CSSCs screening to identify their in vivo anti-scarring potency and effectiveness [ 99 ]. The calculation of the Scarring Index (SI) in correlation to the in vivo scar inhibitory outcome has demonstrated that CSSCs with SI < 10 had a predicted 50% scar reduction potency while cells with SI > 10 were ineffective to control scarring and should be excluded for patient use (International PCT/US23/27823).…”

“…Anti-inflammatory with TSG-6 expression; anti-fibrosis with TGFβ3 expression; differentiation to keratocytes [98,137] Cell fate and phenotypic variation in response to pH changes and inflammatory response in corneal wound Donor to donor variation in cell characteristics and functions [99] Mesenchymal stem cells from adipose, bone marrow Anti-inflammatory; immuno-modulatory; keratocyte differentiation [76,78,89] Uncertainty in ECM production specific to corneal stroma;risk of angiogenesis [88] Donor to donor variation in cell features…”