Multipotent ABCB5-positive cell therapeutics for corneal disease

项目总结 角膜缘含有一小部分稀有的角膜缘干细胞(LSC)，它们不断地重新填充 角膜上皮细胞。角膜缘干细胞缺乏症患者不能再生角膜 上皮细胞，导致角膜基质的“结膜化”，从而触发新生血管，慢性 炎症，最终由于不可逆转的不透明角膜而致盲。有几种方法 已用于通过移植角膜缘组织或体外扩增的角膜缘细胞来替代LSC。这些程序 已经取得了一些成功，主要是利用单侧LSCD患者的自体角膜缘组织。 然而，这些治疗方法在技术上仍然具有挑战性。此外，更多的患者患有 双侧LSCD没有自体LSC来源，而同种异体LSCD的成功率要低得多 角膜缘组织移植，表明成功治疗双侧LSCD需要新的方法 病人。我们的合作研究小组现在发现了两个新的纯化供体ABCB5+来源 双侧LSCD患者的干细胞(I)同种异体免疫抑制ABCB5+LSC，和(II) 自体多能真皮来源的ABCB5+DSC(真皮干细胞)。我们最近发现 ABCB5基因是ATP结合盒(ABC)活性转运蛋白超家族的新成员， 在小鼠和人类的角膜缘(LSC)和皮肤(DSC)中鉴定多能干细胞。重要的是 ABCB5是一种细胞表面蛋白，我们实验室研制的特异性单抗是 有可能从角膜缘和皮肤中分离出纯净的ABCB5阳性干细胞。最近 已发表的原理证明实验表明，纯化的人ABCB5+(但不是ABCB5 LSC能够长期修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 用于与LSCD相关的角膜疾病。目前拨款申请的目标是将我们的 ABCB5+干细胞治疗单侧和双侧的新疗法的实验室研究结果 LSCD患者。我们的总体假设是可以从角膜缘或皮肤中分离出ABCB5+干细胞 并在体外扩增，作为干细胞的来源，当移植到 单侧或双侧LSCD的受者。本项目的三个模块将：验证 体外扩增纯化的ABCB5+LSC(模块1)和体外扩增纯化的LSC的再生能力 单侧和双侧LSCD患者的ABCB5+DSC(模块2)；和(模块3)创建LSC生物库 用于临床移植，并进行获得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.