HC-HA/PTX3 Purified From Amniotic Membrane Promotes BMP Signaling in Limbal Niche Cells to Maintain Quiescence of Limbal Epithelial Progenitor/Stem Cells

Abstract

To explore how limbal niche cells (LNCs) may control quiescence, self-renewal, and corneal epithelial lineage commitment/differentiation of limbal epithelial progenitor/stem cells (LEPCs), we have established an in vitro sphere assay by reunion between the two cell types in threedimensional Matrigel. The resultant sphere exhibits inhibition of corneal epithelial lineage commitment/differentiation and marked clonal growth of LEPCs, of which the latter is correlated with activation of canonical Wnt signaling. Herein, we have created a similar reunion assay in immobilized heavy chain-hyaluronic acid/pentraxin 3 (HC-HA/PTX3), which is purified from amniotic membrane (AM) and consists of a complex formed by hyaluronic covalently linked to heavy chain 1 of inter-a-inhibitor and noncovalently linked to pentraxin 3. The resultant spheres exhibited similar suppression of corneal epithelial lineage commitment/differentiation but upregulation of quiescence markers including nuclear translocation of Bmi-1, and negligible clonal growth of LEPCs. This outcome was correlated with the suppression of canonical Wnt but activation of noncanonical (Planar cell polarity) Wnt signaling as well as BMP signaling in both LEPCs and LNCs. The activation of BMP signaling in LNCs was pivotal because nuclear translocation of pSmad1/5/8 was prohibited in hLEPCs when reunioned with mLNCs of conditionally deleted Bmpr1a;Acvr1 DCKO mice. Furthermore, ablation of BMP signaling in LEPCs led to upregulation of cell cycle genes, downregulation of Bmi-1, nuclear exclusion of phosphorylated Bmi-1, and marked promotion of the clonal growth of LEPCs. Hence, HC-HA/PTX3 uniquely upregulates BMP signaling in LNCs which leads to BMP signaling in LEPCs to achieve quiescence, helping explain how AM transplantation is clinically useful to be used as a matrix for ex vivo expansion of LEPCs and to treat corneal blindness caused by limbal stem cells deficiency. STEM CELLS 2015;33:3341-3355 SIGNIFICANCE STATEMENTWe discovered the unique function of HC-HA/PTX3 purified from amniotic membrane to upregulate canonical BMP and non-canonical Wnt (PCP) signalings in limbal niche cells (LNCs). We provide strong evidence supporting that BMP signaling in LNCs plays an important role in maintaining the BMP signaling in limbal epithelial stem/progenitor cells (LEPCs) to achieve quiescence by suppressing canonical Wnt signaling via the activation of non-canonical Wnt (PCP) signaling.

Niche Regulation of Limbal Epithelial Stem Cells: Relationship between Inflammation and Regeneration

“…Expression of CEBPδ, a marker of SC quiescence, 144,145 is significantly higher while expression of CK12 146 is significantly lower on HC-HA/PTX3 than 3D Matrigel. 136 Expression of CEBPδ is higher both in the cytoplasm and nucleus of LEPCs in 3D Matrigel and HC-HA/PTX3. 136 However, phosphorylated Bmi-1 (p-Bmi-1), another marker of SC quiescence, 147 is positive in the cytoplasm of LEPCs in 3D Matrigel, whereas it is positive in the nucleus of LEPCs and LNCs in HC-HA/PTX3 (Figure 1).…”

“…136 Expression of CEBPδ is higher both in the cytoplasm and nucleus of LEPCs in 3D Matrigel and HC-HA/PTX3. 136 However, phosphorylated Bmi-1 (p-Bmi-1), another marker of SC quiescence, 147 is positive in the cytoplasm of LEPCs in 3D Matrigel, whereas it is positive in the nucleus of LEPCs and LNCs in HC-HA/PTX3 (Figure 1). The percentage of BrdU+ cells in total PCK+ cells is significantly reduced in HC-HA/PTX3 compared to 3D Matrigel at both Day 3 and Day 7 (Fig.…”

“…129 Taking advantage of the aforementioned advance of isolation and expansion of LNCs, we have established an in vitro reconstitution of limbal niche by seeding LEPCs over spheres formed by single LNCs 134 or LNC spheres 135 in 3D Matrigel or in HC-HA/PTX3 immobilized on plastic (Figure 4). 136 …”

“…136 Reunion between LEPCs and LNCs in HC-HA/PTX3 also prevents LEPCs from adopting corneal fate decision, as suggested by notable suppression of CK12 more so than 3D Matrigel. When reseeded on 3T3 fibroblast feeders, vivid clonal growth is observed from reunion in 3D Matrigel but nearly nil from reunion in HC-HA/PTX3 (Figure 4).…”

“…136 The activation of BMP signaling in LNCs is pivotal because nuclear translocation of pSmad1/5/8 is prohibited in hLEPCs when reunioned with mLNCs of conditionally deleted Bmpr1a;Acvr1 DCKO mice. 136 Staining for Bmi-1 is all nuclear in both hLEPCs and mLNCs expanded from WT(+) after reunion, but becomes weakly expressed in the nucleus in LEPCs and mLNCs expanded from Bmpr1a;Acvr1 DCKO (+). There was no significant difference in CEBPδ staining.…”