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Szu-Yu Chen, Bo Han, Yingting Zhu, Megha Mahabole, Huang Jie, David C Beebe, Scheffer C G Tseng; HC-HA/PTX3 from Amniotic Membrane Promotes BMP Signaling in Limbal Niche Cells to Maintain Quiescence and Self Renewal of Limbal Epithelial Progenitor/Stem Cells. Invest. Ophthalmol. Vis. Sci. 2015;56(7 ):3472.
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© ARVO (1962-2015); The Authors (2016-present)
We have previously reported that clonal expansion of LEPCs after reunion with aggregates of limbal niche cells (LNCs) in 3D Matrigel (MG) is governed by activation of WNT signaling that is integrated with BMP signaling. We would like to determine whether HC-HA/PTX3, a novel matrix purified from amniotic membrane, might change the above signaling to modulate functions of LEPCs.
As reported, human LEPCs and LNCs were isolated from donor corneoscleral rings by dispase and collagenase, respectively. Mouse LNCs were also similarly isolated from wild type and Bbmpr1a ;Acvr1 DCKO mice. For reunion, 1x105 cells/cm2 human and mouse LNCs were pre-seeded on immobilized HC-HA/PTX3, MG or plastic for 24 h and added with 1x105 cells/cm2 of LEPCs for 4 days before cells were assayed for RT2 profiler PCR array, qPCR, immunostaining, and 3T3 clonal assay.
Compared to MG, human LNCs also formed aggregates, which expressed more ESC markers, such as Oct4, Sox2, Nanog and Rex1 when seeded on immobilized HC-HA/PTX3. Furthermore, upon reunion with LNC aggregates, LEPCs expressed more quiescence markers, CEBPd and BMI-1, less corneal differentiation marker, CK12, and significantly lower clonal growth on 3T3 feeder layers. RT2 Profiler PCR array revealed that HC-HA/PTX3 downregulated canonical WNT signaling but promoted the "counteractive" non-canonical planar cell polarity (PCP) signaling, and promoted BMP signaling in both LNCs and LEPCs. On immobilized HC-HA/PTX3, human LEPCs generate significantly higher holoclones when reunioned with mouse LNCs from Bmpr1a ;Acvr1 DCKO (i.e., with null BMP signaling) than those from the wild type. Such a promotion effect on HC-HA/PTX3 was significantly greater than MG and plastic. The resultant holoclones from 3T3 feeder layers were more, larger and lack of CK12 expression when reunion with mouse LNCs occurred on HC-HA/PTX3 than on MG.
For the first time, our data disclose a novel function of HC-HA/PTX3 derived from amniotic membrane that uniquely upregulates BMP signaling but downregulates WNT signaling in LNCs so as to maintain quiescence and self-renewal of LEPCs.
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