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Lacrimal Gland Repair Using Progenitor Cells.

ABSTRACT: In humans, the lacrimal gland (LG) is the primary contributor to the aqueous layer of the tear film. Production of tears in insufficient quantity or of inadequate quality may lead to aqueous-deficiency dry eye (ADDE). Currently there is no cure for ADDE. The development of strategies to reliably isolate LG stem/progenitor cells from the LG tissue brings great promise for the design of cell replacement therapies for patients with ADDE. We analyzed the therapeutic potential of epithelial progenitor cells (EPCPs) isolated from adult wild-type mouse LGs by transplanting them into the LGs of TSP -1^-/- mice, which represent a novel mouse model for ADDE. TSP-1^-/- mice are normal at birth but progressively develop a chronic form of ocular surface disease, characterized by deterioration, inflammation, and secretory dysfunction of the lacrimal gland. Our study shows that, among c-kit-positive epithelial cell adhesion molecule (EpCAM⁺ ) populations sorted from mouse LGs, the c-kit⁺ dim/EpCAM⁺ /Sca1 ^- /CD34 ^- /CD45 ^- cells have the hallmarks of an epithelial cell progenitor population. Isolated EPCPs express pluripotency factors and markers of the epithelial cell lineage Runx1 and EpCAM, and they form acini and ducts when grown in reaggregated three-dimensional cultures. Moreover, when transplanted into injured or "diseased" LGs, they engraft into acinar and ductal compartments. EPCP-injected TSP-1^-/- LGs showed reduction of cell infiltration, differentiation of the donor EPCPs within secretory acini, and substantial improvement in LG structural integrity and function. This study provides the first evidence for the effective use of adult EPCP cell transplantation to rescue LG dysfunction in a model system. Stem Cells Translational Medicine 2017;6:88-98.

SUBMITTER: Gromova A

PROVIDER: S-EPMC5442743 | biostudies-literature | 2017 Jan

REPOSITORIES: biostudies-literature

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Lacrimal Gland Repair Using Progenitor Cells.

Gromova Anastasia A Voronov Dmitry A DA Yoshida Miya M Thotakura Suharika S Meech Robyn R Dartt Darlene A DA Makarenkova Helen P HP

Stem cells translational medicine 20160815 1

In humans, the lacrimal gland (LG) is the primary contributor to the aqueous layer of the tear film. Production of tears in insufficient quantity or of inadequate quality may lead to aqueous-deficiency dry eye (ADDE). Currently there is no cure for ADDE. The development of strategies to reliably isolate LG stem/progenitor cells from the LG tissue brings great promise for the design of cell replacement therapies for patients with ADDE. We analyzed the therapeutic potential of epithelial progenito ...[more]

PMID: 28170196

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Project description:BackgroundAqueous-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.MethodsAdult 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.ResultsWe 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.ConclusionWe 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.

Project description:In the lacrimal gland, myoepithelial cells (MEC) express muscle contractile proteins such as alpha smooth muscle actin (SMA) and calponin and therefore can contract to help expel lacrimal fluid. In a previous study, we demonstrated that lacrimal gland MEC express the oxytocin receptor (OXTR) and they contract under oxytocin (OXT) stimulation. Using NOD and MRL/lpr mice (animal models of Sjogren's syndrome), we reported a decrease in SMA and calponin protein levels plus a decline in acini contraction after stimulation with OXT. It is known that proinflammatory cytokines, such as interleukin-1β (IL-1β), tumor necrosis factor alpha (TNF-α) or interferon gamma (IFN-γ), can affect OXTR expression and signaling capacity and inhibit MEC contraction. The aim of the current study was to investigate if proinflammatory cytokines are implicated in the loss of MEC contractile ability. Thus, lacrimal gland MEC from a SMA-GFP transgenic mouse were treated with IL-1β (10 ng/ml) for a total of 7 days. At days 0, 2, 4 and 7, GFP intensity, cell size/area, contractile proteins amounts and MEC contraction were assessed. At day 0, control and treated cells showed no differences in GFP intensity and cell size. GFP intensity started to decrease in treated MEC at day 2 (20%; p=0.02), continuing after day 4 (25%; p=0.007) and 7 (30%; p=0.0001). Mean cell area was also reduced at day 2 (34%; p=0.0005), and after 4 (51%; p<0.0001) and 7 days (30%; p=0.0015). The contraction assay at day 2 showed a 70% decrease of contraction in treated MEC (p<0.0001), 73% (p<0.0001) at day 4 and 82% (p=0.0015) at day 7 when compared to control. Levels of contractile proteins were measured on day 7 showing a decrease in SMA and calponin amount in treated MEC compared with the control group (around 30%; p=0.0016 and p=0.0206; respectively). Similar results were observed when TNF-α and IFN-γ were added along with IL-1β. Taken together the present data and those from our previous studies with Sjogren's syndrome mouse models, they strongly suggest that proinflammatory cytokines affect lacrimal gland MEC contractile ability that may account for the reduced tear secretion associated with Sjogren's syndrome dry eye disease.

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Lacrimal Gland Repair Using Progenitor Cells.

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Lacrimal Gland Repair Using Progenitor Cells.

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