Multipotent ABCB5-positive cell therapeutics for corneal disease

项目摘要 利姆布斯含有少量稀有的角膜缘干细胞（LSC）亚群，其持续地重新增殖角膜缘干细胞。 角膜上皮角膜缘干细胞缺乏症（LSCD）患者无法再生角膜 上皮，导致角膜基质的"结膜化"，从而触发新血管形成，慢性 炎症，最终由于不可逆的不透明角膜而失明。有几种方法 通过移植角膜缘组织或离体扩增的角膜缘细胞来替代LSC。这些程序 已经取得了一定的成功，主要是使用来自单侧LSCD患者的自体角膜缘组织。 然而，这些治疗在技术上仍然具有挑战性。此外，更大的患者群体 双侧LSCD没有自体LSC来源，同种异体LSC的成功率要低得多。 角膜缘组织移植，表明需要新的方法来成功治疗双侧LSCD 患者我们的合作研究小组现在发现了两种新的纯化供体ABCB 5+来源 用于双侧LSCD患者的干细胞（i）同种异体免疫抑制ABCB 5 + LSC，和（ii） 自体多能真皮皮肤衍生的ABCB 5 + DSC（真皮干细胞）。我们最近发现 ABCB 5基因是ATP结合盒（ABC）超家族的一个新成员， 鉴定了小鼠和人类的利姆布斯（LSC）和皮肤（DSC）中的多能干细胞。重要的是， ABCB 5是一种细胞表面蛋白，我们实验室开发的特异性单克隆抗体， 能够从利姆布斯和皮肤中前瞻性地分离纯的ABCB 5阳性干细胞。最近 已发表的原理验证实验表明，纯化的人ABCB 5+（但不是ABCB 5 阴性）LSC能够长期恢复免疫缺陷小鼠的角膜上皮 LSCD模型，表明这种纯化的LSC群体具有显著改善治疗的潜力， 与LSCD相关的角膜疾病当前拨款申请的目标是将我们的 实验室研究发现，新的ABCB 5+干细胞疗法治疗单侧和双侧 LSCD患者。我们的总体假设是来自利姆布斯或皮肤的ABCB 5+干细胞可以被分离 并在体外扩增作为干细胞的来源，当移植到 患有单侧或双侧LSCD的患者。该项目的三个模块将： 纯化的体外扩增ABCB 5 + LSC（模块1）和纯化的体外扩增ABCB 5 + LSC（模块2）的再生潜力 单侧和双侧LSCD患者的ABCB 5 + DSC（模块2）;和（模块3）创建LSC生物样本库 用于临床移植，并进行导致FDA IND批准的研究。

PROJECT SUMMARY The limbus contains a small subpopulation of rare limbal stem cells (LSC) that continually repopulates the corneal epithelium. Patients with limbal stem cell deficiency (LSCD) are unable to regenerate the corneal epithelium, resulting in "conjunctivalization" of the corneal stroma that triggers neovascularization, chronic inflammation, and ultimately blindness due to an irreversibly opaque cornea. Several approaches have been used to replace LSC by transplanting limbal tissue or ex vivo expanded limbal cells. These procedures have obtained some success, mainly using autologous limbal tissue from patients with unilateral LSCD. However, these treatments remain technically challenging. Moreover, the larger population of patients with bilateral LSCD has no source of autologous LSC and much less success was observed with allogeneic limbal tissue transplants, indicating new approaches are needed for successful treatment of bilateral LSCD patients. Our collaborative research group has now discovered two new sources of purified donor ABCB5+ stem cells for patients with bilateral LSCD (i) allogeneic immunosuppressive ABCB5+ LSC, and (ii) autologous multipotent dermal-skin-derived ABCB5+ DSC (Dermal Stem Cells). We recently discovered that the ABCB5 gene, a new member of the ATP-binding cassette (ABC) superfamily of active transporters, identifies multipotent stem cells in the limbus (LSC) and the skin (DSC) in mice and humans. Importantly, ABCB5 is a cell surface protein and specific monoclonal antibodies developed by our laboratories are capable of prospectively isolating pure ABCB5-positive stem cells from the limbus and skin. Recently published proof-of-principle experiments demonstrated that purified human ABCB5+ (but not ABCB5 negative) LSC were capable of long-term restoration of the corneal epithelium in an immunodeficient mouse model of LSCD, indicating that this purified LSC population has the potential to significantly improve therapy for corneal disease associated with LSCD. The goal of the current grant application is to translate our laboratory research findings into new ABCB5+ stem cell-based therapies to treat unilateral and bilateral LSCD patients. Our overall hypothesis is that ABCB5+ stem cells from the limbus or skin can be isolated and expanded in vitro as a source of stem cells to regenerate the corneal epithelium when transplanted to recipients with either a unilateral or bilateral LSCD. The three Modules of this project will: Validate the regenerative potential of purified in vitro-expanded ABCB5+ LSC (Module 1) and purified in vitro-expanded ABCB5+ DSC (Module 2) for unilateral and bilateral LSCD patients; and (Module 3) create an LSC Biobank for clinical transplantation and conduct studies resulting in FDA IND approval.