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Jana Dietrich, Mathias Roth, Gerd Geerling, Sonja Mertsch, Stefan Schrader; Evaluation of the regenerative potential of the secretome of lacrimal gland derived mesenchymal stem cells isolated by explant technique or cell sorting. Invest. Ophthalmol. Vis. Sci. 2018;59(9):4929.
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© ARVO (1962-2015); The Authors (2016-present)
Regeneration of lacrimal gland (LG) tissue in situ would be a promising approach to curatively treat dry eye disease (DED). Mesenchymal stem cells (MSC) have emerged as a potential source for cell therapy and were found in the native and regenerating LG. Thus, the application of LG-MSC or MSC-secreted proteins is promising to induce/enhance LG regeneration. This study aims to optimize the isolation of murine LG-MSC and analyze their secretome.
Murine LG-MSC were isolated by explant technique or cell sorting using PDGFRα and Sca-1 (PαS) as positive markers. Characterization was performed by immunophenotyping, differentiation assays and assessing the cumulative population doublings (cpd) and colony-forming units (CFU). The secretome of two MSC-conditioned medium (MSCCM) types (basal (αMEM) and inflammatory (50ng/ml each IL-1α and IFN-γ)) was identified by LC-MS/MS. The effects of MSCCM on LG epithelial cells viability after ethanol injury were assessed.
MSC from both isolation methods exhibit a spindle-shaped, fibroblastic morphology. Growth behavior was linear with 12.5±0.2 and 11.7±2.0 cpd for explant and sorted (PαS) MSC respectively after 6 passages (P). CFU efficiency was stable with 32.2±5.0 and 25.8±8.8 colonies in P2 and P6 for explant and 31.6±7.8 and 32.0±13.2 for PαS. Immunophenotyping revealed >95% positive cells for nestin, Sca-1, PDGFRα, CD29, CD90, >70% for CD105 and <5% for CD34, c-Kit, CD45, Ter119. Both MSC populations were able to differentiate into osteocytes and adipocytes. Epithelial cell viability significantly increased when cultured with basal MSCCM over 48h from explant (113.4±72.8%) and PαS (94.5±44.8%) compared to control (63.4±4.8%), whereas inflammatory MSCCM is only beneficial at 24h. A total of 227 and 232 proteins were upregulated in the inflammatory MSCCM for explant and PαS respectively.
The results of the study show, that both methods are useful to isolate a specific and functional MSC population from murine LG as only minor differences were detected during characterization. While cell sorting might be beneficial to prepare cells on demand, explant technique is more economical in effort and cost. Analysis on MSCCM revealed that the secretome of MSC exhibit regenerative effects on LG epithelial cells and could be useful to induce/enhance LG regeneration in patients with DED.
This is an abstract that was submitted for the 2018 ARVO Annual Meeting, held in Honolulu, Hawaii, April 29 - May 3, 2018.
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