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Manipulation of Panx1 Activity Increases the Engraftment of Transplanted Lacrimal Gland Epithelial Progenitor Cells.

ABSTRACT: Purpose:Sjögren's syndrome is a systemic chronic autoimmune inflammatory disease that primarily targets the salivary and lacrimal glands (LGs). Currently there is no cure; therefore, cell-based regenerative therapy may be a viable option. LG inflammation is facilitated by extracellular ATP and mediated by the Pannexin-1 (Panx1) membrane channel glycoprotein. We propose that suppression of inflammation through manipulation of Panx1 activity can stimulate epithelial cell progenitor (EPCP) engraftment. Methods:The expression of pannexins in the mouse and human LG was assayed by qRT-PCR and immunostaining. Acute LG inflammation was induced by interleukin-1? (IL1?) injection. Prior to EPCP transplantation, IL1?-injured or chronically inflamed LGs of thrombospondin-1-null mice (TSP-1-/-) were treated with the Panx1-specific blocking peptide (10panx) or the self-deliverable RNAi (sdRNAi). The efficacy of cell engraftment and the area of inflammation were analyzed by microscopy. Results:Panx1 and Panx2 were detected in the mouse and human LGs. Panx1 and proinflammatory factors were upregulated during acute inflammation at days 1 to 3 after the IL1? injection. The analysis of EPCP engraftment demonstrated a significant and reproducible positive correlation between the 10panx peptide or Panx1 sdRNAi treatment and the number of engrafted cells. Similarly, treatment of the LG of the TSP-1-/- mouse (mouse model of chronic LG inflammation) by either Panx1 or Caspase-4 (also known as Casp11) sdRNAi showed a significant decrease in expression of proinflammatory markers and the lymphocyte infiltration. Conclusions:Our results suggest that blocking Panx1 and/or Casp4 activities is a beneficial strategy to enhance donor cell engraftment and LG regeneration through the reduction of inflammation.

SUBMITTER: Basova LV

PROVIDER: S-EPMC5678547 | biostudies-literature | 2017 Nov

REPOSITORIES: biostudies-literature

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Manipulation of Panx1 Activity Increases the Engraftment of Transplanted Lacrimal Gland Epithelial Progenitor Cells.

Basova Liana V LV Tang Xin X Umasume Takeshi T Gromova Anastasia A Zyrianova Tatiana T Shmushkovich Taisia T Wolfson Alexey A Hawley Dillon D Zoukhri Driss D Shestopalov Valery I VI Makarenkova Helen P HP

Investigative ophthalmology & visual science 20171101 13

<h4>Purpose</h4>Sjögren's syndrome is a systemic chronic autoimmune inflammatory disease that primarily targets the salivary and lacrimal glands (LGs). Currently there is no cure; therefore, cell-based regenerative therapy may be a viable option. LG inflammation is facilitated by extracellular ATP and mediated by the Pannexin-1 (Panx1) membrane channel glycoprotein. We propose that suppression of inflammation through manipulation of Panx1 activity can stimulate epithelial cell progenitor (EPCP) ...[more]

PMID: 29098296

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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.

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Manipulation of Panx1 Activity Increases the Engraftment of Transplanted Lacrimal Gland Epithelial Progenitor Cells.

Publications

Manipulation of Panx1 Activity Increases the Engraftment of Transplanted Lacrimal Gland Epithelial Progenitor Cells.

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