Fidelity of long-term cryopreserved adipose-derived stem cells for differentiation into cells of ocular and other lineages

Abstract Adipose-Derived Stem Cells (ADSCs) have an important contribution in regenerative medicine ranging from testing stem cell therapy for disease treatment in pre-clinical models to clinical trials. For immediate use of stem cells for therapy, there is a requirement of the high dose of stem cells at different time points which can be met by cryopreservation. In this study, we evaluated the characteristics of long-term cryopreserved ADSCs and their regenerative potential after an average of twelve-year cryopreservation. Revived ADSCs were examined for cell viability and proliferation by trypan blue, Calcein/Hoechst and MTT assay. Expression of stem cell markers was examined by flow cytometry, immunostaining and qPCR. Colony forming efficiency and spheroid formation ability were also assessed. Multilineage differentiation potential was evaluated by induction into osteocytes, adipocytes, neural cells, corneal keratocytes and trabecular meshwork (TM) cells. Post-thaw, ADSCs maintained expression of stem cell markers CD90, CD73, CD105, CD166, NOTCH1, STRO-1, ABCG2, OCT4, KLF4. ADSCs retained colony and spheroid forming potential. These cells were able to differentiate into osteocytes, confirmed by Alizarin Red S staining and elevated expression of osteocalcin and osteopontin; into adipocytes by Oil Red O staining and elevated expression of PPARγ2. ADSCs could differentiate into neural cells, stained positive to β-III tubulin, neurofilament, GFAP as well as elevated expression of nestin and neurofilament mRNAs. ADSCs could also give rise to corneal keratocytes expressing keratocan, keratan sulfate, ALDH and collagen V, and to TM cells expressing CHI3L1 and AQP1. Differentiated TM cells responded to dexamethasone treatment with increased Myocilin expression, which could be used as in vitro glaucoma model for further studies. Conditioned medium from ADSCs was found to impart a regenerative effect on primary TM cells. In conclusion, ADSCs maintained their stemness and multipotency after long-term cryopreservation with variability between different donors. This study can have great repercussions in regenerative medicine and pave the way for future clinical trials using cryopreserved ADSCs.