当前位置： SCI文献检索 > EXPERIMENTAL EYE RESEARCH期刊下所有文献 > Expansion and maintenance of primary corneal epithelial stem/progenitor cells by inhibition of TGFβ receptor I-mediated signaling.

Expansion and maintenance of primary corneal epithelial stem/progenitor cells by inhibition of TGFβ receptor I-mediated signaling.

Abstract：

:Transforming growth factor β (TGFβ) signaling is one of the most important signaling pathways regulating cell behavior in ocular tissues. Its functions are mainly linked to tissue fibrosis and inflammatory responses in ophthalmology. In epithelial cells, however, the growth inhibitory activity of TGFβ was reported in both non-ocular and ocular tissues. Since TGFβ is a bifunctional regulator that either inhibits or stimulates cell proliferation according to the specific context, we examined the effect of inhibition of TGFβ receptor (TβR) I-mediated signaling on primary corneal epithelial cells (CECs) in serum- and feeder-free conditions. The mouse CECs were isolated from the eyeballs of 6-8 weeks old female C57BL/6 mice using dispase and trypsin separately, cultivated in defined Keratinocyte serum-free medium (KSFM) with supplements (the complete medium) without feeder layer. Cells were divided into three groups, those cultured in complete medium additionally supplemented with 10 μM SB-431542, a specific inhibitor of TβR-I, were SB-CECs; those cultured in complete medium additionally supplemented with 10 ng/ml SRI-011381, a TGF-beta signaling agonist, were SRI-CECs; those cultured in complete medium without SB-431542 or SRI-011381 were control CECs. The growth rate and morphology were analyzed by light microscopy. The identity and stemness of cells was investigated through marker staining of p63, inhibitor of differentiation 1 (ID1), cytokeratin 12 (K12), cytokeratin 14 (K14), PAX6, pSmad3, alpha smooth muscle Actin (αSMA) and E-cadherin (E-cad); Real-time quantitative (RT-PCR) analysis of p63; Western blot analysis of ID1; as well as colony forming assay, sphere forming assay, healing wound in vitro assay and air-lifting interface assay. The results showed SB-CECs subcultured steadily, achieved sustained expansion, and expanded almost thrice faster than control CECs. Expanded SB-CECs exhibited smaller and more compact morphology, up-regulated p63 and ID1, as well as better performed colony-forming capacity, sphere-forming capacity, in vitro wound healing capacity, and the capacity to stratify and differentiate on air-lifting interface. Preliminary tests on human limbal epithelial cells (HLECs) showed the same results as mouse CECs. Interestingly, the ID1 expression pattern was almost identical to p63, the typical marker for corneal epithelial stem/progenitor cell (CESC/CEPC), in cultured CECs and normal corneal sections. Since ID1 has been proven to be regulated negatively by TGFβ signaling in epithelial cells and plays a role in blocking cell differentiation, its derepression by TβR-I inhibitor could be, at least in part, the underlying cause of CESC/CEPC expansion and the synchronously up-regulated expression of p63 in SB-CECs. In conclusion, inhibition of TβR-I-mediated signaling, CESCs/CEPCs achieved efficient long-term expansion in a feeder- and serum-free condition in vitro. And derepression of ID1 could be the underlying cause. Meanwhile, ID1 could serve as a marker for CESC/CEPC. These results may advance the basic and clinical CESC/CEPC research.

journal_name

Exp Eye Res

journal_title

Experimental eye research

authors

Hu L,Pu Q,Zhang Y,Ma Q,Li G,Li X

doi

10.1016/j.exer.2019.03.014

subject

Has Abstract

pub_date

2019-05-01 00:00:00

pages

44-56

eissn

0014-4835

issn

1096-0007

pii

S0014-4835(18)30802-9

journal_volume

182

pub_type

杂志文章

在线工具

Expansion and maintenance of primary corneal epithelial stem/progenitor cells by inhibition of TGFβ receptor I-mediated signaling.