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AJAY KUMAR, Yiqin Du; Stemness and regenerative effects of trabecular meshwork stem cells/secretome after long-term storage. Invest. Ophthalmol. Vis. Sci. 2018;59(9):4732. doi: https://doi.org/.
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© ARVO (1962-2015); The Authors (2016-present)
Stem Cell Therapy offers impending avenues for ocular regeneration but long-term stability of stem cells and side effects has been main obstacle in unequivocal clinical translation. We hypothesized that Trabecular Meshwork Stem Cells (TMSCs) can maintain their stemness after long-term storage and secretome derived from TMSCs can enhance the TM regeneration by promoting TM cell proliferation hence increasing TM cellularity which is greatly reduced in glaucoma.
TMSCs were cultivated from four donors and stored in liquid nitrogen at passages 2-3 for 5-7 years before revival. TMSCs were characterized for their stemness by expression of stemness markers by flow cytometry and qPCR. 3D spheroid formation, colony forming efficiency (CFE) and multipotency was also assessed. Secretome was harvested from TMSCs after 24-hr incubation in serum-free medium. Cell viability after secretome harvesting was assessed by MTT assay, Calcein/Hoechst and Annexin/7-AAD. Wound healing assay was performed using live cell time-lapse microscopy and analyzed using ImageJ. Statistical analysis was done using one-way ANOVA.
TMSCs showed good viability post thaw and >90% cells expressed stemness markers CD90, CD73, CD105 and expressed OCT4, KLF4, ABCG2 etc. TMSCs were able to form 3D spheroids and colonies in addition to differentiation into osteogenic. Surprisingly, TMSCs from one donor showed significantly very high osteogenic potential as compared to other three TMSCs. All four TMSCs were able to differentiate into neurons shown by β-III Tubulin and Neurofilament expression. TMSCs also differentiated into TM cells expressing CHI3L1 and responding to dexamethasone treatment, but couldn’t differentiate into adipocytes. Secretome derived from all four TMSCs was able to enhance the CFE significantly. TMSC secretome was also found to speedup TM cell wound healing in vitro by promoting cell proliferation and migration. The expression of fibrotic markers like SPARC and CTGF etc. was reduced after secretome treatment.
This study provides evidence of stemness maintenance of TMSCs after prolonged storage. TMSC secretome induced TM regeneration provides functional evidence for use of TMSCs and their secretome for ocular regeneration (figure attached). This study opens new doors for potential stem cell-free therapy using secretome in Glaucoma.
This is an abstract that was submitted for the 2018 ARVO Annual Meeting, held in Honolulu, Hawaii, April 29 - May 3, 2018.
Regenerative potential of cryo-TMSCs and their secretome
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