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

被引:22
作者
Xiao, Sa [1 ]
Zhang, Yan [1 ]
机构
[1] Sun Yat Sen Univ, MOE Key Lab Gene Funct & Regulat, Sch Life Sci, Guangzhou 510006, Guangdong, Peoples R China
基金
中国国家自然科学基金;
关键词
Lacrimal gland stem cell; Dry eye disease; 3D culture; Serum-free; Stem cell therapy; BRANCHING-MORPHOGENESIS; PROGENITOR CELLS; DRY EYE; EXPANSION; ADULT; ORGANOIDS; LUNG; DIFFERENTIATION; INFLAMMATION; HOMEOSTASIS;
D O I
10.1186/s13287-019-1541-1
中图分类号
Q813 [细胞工程];
学科分类号
摘要
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.
引用
收藏
页数:13
相关论文
共 49 条
  • [11] Lacrimal Gland Repair Using Progenitor Cells
    Gromova, Anastasia
    Voronov, Dmitry A.
    Yoshida, Miya
    Thotakura, Suharika
    Meech, Robyn
    Dartt, Darlene A.
    Makarenkova, Helen P.
    [J]. STEM CELLS TRANSLATIONAL MEDICINE, 2017, 6 (01) : 88 - 98
  • [12] HANN LE, 1991, INVEST OPHTH VIS SCI, V32, P2610
  • [13] Cytokeratin expression in mouse lacrimal gland germ epithelium
    Hirayama, Masatoshi
    Liu, Ying
    Kawakita, Tetsuya
    Shimmura, Shigeto
    Tsubota, Kazuo
    [J]. EXPERIMENTAL EYE RESEARCH, 2016, 146 : 54 - 59
  • [14] Challenges and Strategies for Regenerating the Lacrimal Gland
    Hirayama, Masatoshi
    Kawakita, Tetsuya
    Tsubota, Kazuo
    Shimmura, Shigeto
    [J]. OCULAR SURFACE, 2016, 14 (02) : 135 - 143
  • [15] Unlimited in vitro expansion of adult bi-potent pancreas progenitors through the Lgr5/R-spondin axis
    Huch, Meritxell
    Bonfanti, Paola
    Boj, Sylvia F.
    Sato, Toshiro
    Loomans, Cindy J. M.
    van de Wetering, Marc
    Sojoodi, Mozhdeh
    Li, Vivian S. W.
    Schuijers, Jurian
    Gracanin, Ana
    Ringnalda, Femke
    Begthel, Harry
    Hamer, Karien
    Mulder, Joyce
    van Es, Johan H.
    de Koning, Eelco
    Vries, Robert G. J.
    Heimberg, Harry
    Clevers, Hans
    [J]. EMBO JOURNAL, 2013, 32 (20) : 2708 - 2721
  • [16] Differentiation of human embryonic stem cells into embryoid bodies comprising the three embryonic germ layers
    Itskovitz-Eldor, J
    Schuldiner, M
    Karsenti, D
    Eden, A
    Yanuka, O
    Amit, M
    Soreq, H
    Benvenisty, N
    [J]. MOLECULAR MEDICINE, 2000, 6 (02) : 88 - 95
  • [17] Organoids: Modeling Development and the Stem Cell Niche in a Dish
    Kretzschmar, Kai
    Clevers, Hans
    [J]. DEVELOPMENTAL CELL, 2016, 38 (06) : 590 - 600
  • [18] Epithelial Markers aSMA, Krt14, and Krt19 Unveil Elements of Murine Lacrimal Gland Morphogenesis and Maturation
    Kuony, Alison
    Michon, Frederic
    [J]. FRONTIERS IN PHYSIOLOGY, 2017, 8
  • [19] Y-27632, a ROCK inhibitor, delays senescence of putative murine salivary gland stem cells in culture
    Lee, Jingu
    Park, Sangkyu
    Roh, Sangho
    [J]. ARCHIVES OF ORAL BIOLOGY, 2015, 60 (06) : 875 - 882
  • [20] Mesenchymal Stem/Stromal Cells Protect the Ocular Surface by Suppressing Inflammation in an Experimental Dry Eye
    Lee, Min Joung
    Ko, Ah Young
    Ko, Jung Hwa
    Lee, Hyun Ju
    Kim, Mee Kum
    Wee, Won Ryang
    Khwarg, Sang In
    Oh, Joo Youn
    [J]. MOLECULAR THERAPY, 2015, 23 (01) : 139 - 146