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Establishment of long-term serum-free culture for lacrimal gland stem cells aiming at lacrimal gland repair.

ABSTRACT: BACKGROUND:Aqueous-deficient dry eye disease (ADDED) resulting from dysfunction of the lacrimal gland (LG) is currently incurable. Although LG stem/progenitor cell-based therapy is considered to be a promising strategy for ADDED patients, the lack of a reliable serum-free culture method to obtain enough lacrimal gland stem cells (LGSCs) and the basic standard of LGSC transplantation are obstacles for further research. METHODS:Adult mouse LGSCs were cultured in Matrigel-based 3D culture under serum-free culture condition, which contained EGF, FGF10, Wnt3A, and Y-27632. LGSCs were continuously passaged over 40 times every 7?days, and the morphology and cell numbers were recorded. LGSCs were induced to differentiate to ductal cells by reducing Matrigel rigidity, while fetal bovine serum was used for the induction of acinar cells. RT-PCR or qRT-PCR analysis, RNA-sequence analysis, H&E staining, and immunofluorescence were used for characterization and examining the differentiation of LGSCs. LGSCs were allotransplanted into diseased LGs to examine the ability of repairing the damage. The condition of eye orbits was recorded using a camera, the tear production was measured using phenol red-impregnated cotton threads, and the engraftments of LGSCs were examined by immunohistochemistry. RESULTS:We established an efficient 3D serum-free culture for adult mouse LGSCs, in which LGSCs could be continuously passaged for long-term expansion. LGSCs cultured from both the healthy and ADDED mouse LGs expressed stem/progenitor cell markers Krt14, Krt5, P63, and nestin, had the potential to differentiate into acinar or ductal-like cells in vitro and could engraft into diseased LGs and relieve symptoms of ADDED after orthotopic injection of LGSCs. CONCLUSION:We successfully established an efficient serum-free culture for adult mouse LGSCs aiming at LG repair for the first time. Our approach provides an excellent theoretical and technical reference for future clinical research for ADDED stem cell therapy.

SUBMITTER: Xiao S

PROVIDER: S-EPMC6951017 | biostudies-literature | 2020 Jan

REPOSITORIES: biostudies-literature

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Establishment of long-term serum-free culture for lacrimal gland stem cells aiming at lacrimal gland repair.

Xiao Sa S Zhang Yan Y

Stem cell research & therapy 20200108 1

<h4>Background</h4>Aqueous-deficient dry eye disease (ADDED) resulting from dysfunction of the lacrimal gland (LG) is currently incurable. Although LG stem/progenitor cell-based therapy is considered to be a promising strategy for ADDED patients, the lack of a reliable serum-free culture method to obtain enough lacrimal gland stem cells (LGSCs) and the basic standard of LGSC transplantation are obstacles for further research.<h4>Methods</h4>Adult mouse LGSCs were cultured in Matrigel-based 3D cu ...[more]

PMID: 31915062

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Project description:PurposeTo report the therapeutic efficacy of integrating neoadjuvant chemotherapy with conventional bimodal therapies for lacrimal gland adenoid cystic carcinoma by providing an additional 8 years of follow-up data on the same cohort of patients whose cumulative 10-year disease-free survival outcomes were reported in 2013.DesignNon-randomized, retrospective, interventional case series.MethodsNineteen consecutive patients treated with neoadjuvant intra-arterial cytoreductive chemotherapy (IACC), orbital exenteration, chemoradiotherapy, and adjuvant intravenous chemotherapy at a single institution were included. Analyses were undertaken of locoregional recurrences and distant metastases, disease-free survival time, TNM tumor stage at presentation, response to IACC, and prognostic impact of positive resection margins. The main outcome measures were overall survival, disease-free survival, disease relapse, positive tumor resection margins, and tumor stage at presentation.ResultsEight patients with an intact lacrimal artery (group 1), 7 with AJCC stage T4a-c, had significantly better overall survival (87.5% versus 14.3% at 15 years), disease-specific mortality, and recurrences (all < .001, log-rank test) than prior conventionally treated patients from the Bascom Palmer Eye Institute. Group 1 was superior to group 2, patients lacking an intact lacrimal artery, concerning overall survival (P = .042) and recurrence (P = .017), but with no significant difference in disease-specific mortality (P = .23). Group 2 was associated with a significantly lower cause-specific mortality than the institutional comparator group (P = .039). Prior tumor resection with lateral wall osteotomy and failure to adhere to all protocol elements were adverse prognostic factors for suboptimal outcomes. Positive tumor margins increased the risk of all-cause mortality 4.1 times (P = .036, stratified Cox proportional hazards regression) and disease-specific mortality 8.0 times (P = .043, stratified Cox proportional hazards regression) than a patient with negative margins.ConclusionsExtended follow-up supplemented with AJCC staging data supports neoadjuvant IACC as an integral component of a trimodal treatment strategy in patients with an intact lacrimal artery. Protocol elements implemented as designed appear to have improved overall survival and decreased disease relapse in this cohort. This extended long-term IACC dataset suggests that a critical bar of at least 15 years of follow-up is appropriate for assessing the efficacy of current conventional and future globe-sparing bimodal therapies.

Dataset Information

Establishment of long-term serum-free culture for lacrimal gland stem cells aiming at lacrimal gland repair.

Publications

Establishment of long-term serum-free culture for lacrimal gland stem cells aiming at lacrimal gland repair.

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