Limbal Fibroblasts Maintain Normal Phenotype in 3D RAFT Tissue Equivalents Suggesting Potential for Safe Clinical Use in Treatment of Ocular Surface Failure
Limbal Fibroblasts Maintain Normal Phenotype in 3D RAFT Tissue Equivalents Suggesting Potential for Safe Clinical Use in Treatment of Ocular Surface Failure
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DOI：
10.1089/ten.tec.2014.0458
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发表时间：
2015-06-01
影响因子：
3
通讯作者：
Daniels, Julie T.
中科院分区：
文献类型：
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作者：
Massie, Isobel;Dale, Sarah B.;Daniels, Julie T.
角膜缘上皮干细胞缺乏可导致失明，但将这些细胞移植到人类羊膜等载体上可以恢复视力。不幸的是，使用羊膜的临床移植物制造可能不一致。因此，我们开发了一种替代基质，真实的三维组织结构（RAFT），它支持人角膜缘上皮细胞（hLE）扩增。当人角膜缘成纤维细胞（hLF）并入RAFT组织等效物（TE）中时，上皮组织化得到改善。然而，hLF具有转分化成促瘢痕形成细胞类型的潜力，这将与治疗性移植不相容。这项工作的目的是评估hLE+和hLE- RAFT TE以及非空运和空运RAFT TE中hLF的瘢痕形成表型。从眼粘膜类天疱疮（Oc-MMP）患者的纤维化结膜分离的成纤维细胞（dFib）用作促瘢痕形成阳性对照，使用替代瘢痕形成参数：基质金属蛋白酶（MMP）活性、从头胶原蛋白合成、α-平滑肌肌动蛋白（α-SMA）表达和转化生长因子-β（TGF-β）分泌，将hLF与其进行比较。正常hLF和dFib在RAFT TE中保持不同的表型。MMP-2和-9活性、从头胶原合成和α-SMA表达在dFib cf中均增加。正常hLF RAFT TE，尽管TGF-β 1分泌在正常hLF和dFib RAFT TE之间没有差异。正常hLF在RAFT TE中培养期间不会向瘢痕样表型发展，因此，可以安全地包括在治疗性RAFT TE中，其中它们可以支持hLE，尽管需要体内工作来证实这一点。dFib RAFT TE（在本研究中用作阳性对照）可用于开发Oc-MMP的离体疾病模型。
Limbal epithelial stem cell deficiency can cause blindness, but transplantation of these cells on a carrier such as human amniotic membrane can restore vision. Unfortunately, clinical graft manufacture using amnion can be inconsistent. Therefore, we have developed an alternative substrate, Real Architecture for 3D Tissue (RAFT), which supports human limbal epithelial cells (hLE) expansion. Epithelial organization is improved when human limbal fibroblasts (hLF) are incorporated into RAFT tissue equivalent (TE). However, hLF have the potential to transdifferentiate into a pro-scarring cell type, which would be incompatible with therapeutic transplantation. The aim of this work was to assess the scarring phenotype of hLF in RAFT TEs in hLE+ and hLE- RAFT TEs and in nonairlifted and airlifted RAFT TEs. Diseased fibroblasts (dFib) isolated from the fibrotic conjunctivae of ocular mucous membrane pemphigoid (Oc-MMP) patients were used as a pro-scarring positive control against which hLF were compared using surrogate scarring parameters: matrix metalloproteinase (MMP) activity, de novo collagen synthesis, alpha-smooth muscle actin (alpha-SMA) expression, and transforming growth factor-beta (TGF-beta) secretion. Normal hLF and dFib maintained different phenotypes in RAFT TE. MMP-2 and -9 activity, de novo collagen synthesis, and alpha-SMA expression were all increased in dFib cf. normal hLF RAFT TEs, although TGF-beta 1 secretion did not differ between normal hLF and dFib RAFT TEs. Normal hLF do not progress toward a scarring-like phenotype during culture in RAFT TEs and, therefore, may be safe to include in therapeutic RAFT TE, where they can support hLE, although in vivo work is required to confirm this. dFib RAFT TEs (used in this study as a positive control) may be useful toward the development of an ex vivo disease model of Oc-MMP.