ABCB5-positive stem cells for LSCD therapy

摘要-限制为30行 角膜上皮是维持透明角膜所必需的快速自我更新的上皮表面， 正常视力。利姆布斯含有少量角膜缘干细胞（LSC）亚群， 角膜缘干细胞缺乏症（LSCD）患者不能重新填充角膜上皮， 再生角膜上皮，导致“结膜化”，最终导致失明。我们 先前确定ABCB 5基因在小鼠和人类组织中由LSC表达， ABCB 5 + LSC的正常功能是角膜发育、稳态和修复所必需的。此外，委员会认为， ABCB 5是细胞表面蛋白，特异性单克隆抗体能够分离纯ABCB 5 + LSC用于研究和移植。我们之前的赠款支持了第一次临床试验， 用于双侧LSCD患者的纯化的体外扩增的人同种异体ABCB 5 + LSC（ClinicalTrials.gov： 标识符NCT 03549299）。尽管我们取得了成功，但供体同种异体ABCB 5 + LSC移植的使用仍是一个挑战。 因为它需要免疫抑制治疗以防止供体细胞的免疫排斥，所以不是最佳的。 使用同种异体供体ABCB 5 + LSC，因为LSCD患者缺乏健康的自体来源。 ABCB 5 + LSC，因为双眼均受累。为了解决这一问题并治疗双侧LSCD患者， 自体ABCB 5 + LSC，这项资助更新将研究三种新的方法。第一种方法使用 使用完整的ABCB 5 + iLSC（源自皮肤产生的iPSC的诱导的LSC）移植 自体皮肤细胞的重编程，使用Yamanaka基因OSKM（Oct 4，Sox 2，Klf 4，c-Myc）， 产生人类诱导多能干细胞（iPSC），然后在体外分化成2D眼样细胞。 显著含有ABCB 5 + LSC的类器官（Watanabe等人，iScience，2021）。第二种方法 使用不依赖于移植的恢复和扩展残余功能障碍或数量不足 LSC，使用体内部分表观遗传重编程，Co-PI发现的一种新的重编程形式 博士布鲁斯Ksander和合作者（Lu等人，Nature，2020），其使用三个 Yamanaka基因，OSK，以恢复和拯救体内受损的功能失调的细胞。第三种方法使用 小生境产生的LAMA 5配体为基础的BCAM分子途径的信号激活，我们只是 发现的对ABCB 5 + LSC和瞬时扩增细胞（TAC）扩增至关重要（Sasamoto等人，Cell 报告，2022年，在出版），与由此产生的干细胞增殖计划，增强角膜再生。 因此，我们的假设是，有可能从患者中制造和扩增自体ABCB 5 + LSC。 使用（i）体外完全重编程，（ii）体内部分表观遗传重编程，和（iii） 通过特异性BCAM/ABCB 5信号转导的基于小生境产生的LAMA 5配体的LSC活化 途径。这些自体ABCB 5 + LSC将完全再生并维持正常的利姆布斯和角膜基质。 上皮这一假设将在主题相关的三个独立的具体目标中进行检验。

ABSTRACT- limited to 30 lines The corneal epithelium is a rapidly self-renewing epithelial surface essential for maintaining a clear cornea and normal vision. The limbus contains a small subpopulation of limbal stem cells (LSCs) that continually repopulate the corneal epithelium and patients with limbal stem cell deficiency (LSCD) are unable to regenerate the corneal epithelium, resulting in "conjunctivalization" that ultimately leads to blindness. We previously identified that the ABCB5 gene is expressed by LSCs in both mouse and human tissues and that normal function of ABCB5+ LSCs is required for corneal development, homeostasis, and repair. Moreover, ABCB5 is a cell surface protein and specific monoclonal antibodies are capable of isolating pure ABCB5+ LSCs for study and transplantation. Research from our prior grants supported the first clinical trials using purified in vitro-expanded human allogeneic ABCB5+ LSCs for patients with bilateral LSCD (ClinicalTrials.gov: Identifier NCT03549299). Despite our success, the use of donor allogeneic ABCB5+ LSC transplants is suboptimal because it requires immunosuppressive treatment to prevent immune rejection of donor cells. Allogeneic donor ABCB5+ LSCs were used because LSCD patients lack a source of healthy autologous ABCB5+ LSCs since both eyes are involved. To solve this problem and to treat bilateral LSCD patients with autologous ABCB5+ LSCs, this grant renewal will study three novel approaches. The first approach uses transplantation of ABCB5+ iLSCs (induced LSCs derived from iPSCs generated from skin) using full reprogramming of autologous skin cells, using the Yamanaka genes OSKM (Oct4, Sox2, Klf4, c-Myc) to produce human induced pluripotent stem cells (iPSCs) that are then differentiated in vitro into 2D eye-like organoids that remarkably contain ABCB5+ LSCs (Watanabe et al., iScience, 2021). The second approach uses transplantation-independent restoration and expansion of residual dysfunctional or quantity-deficient LSCs, using in vivo partial epigenetic reprogramming, a new form of reprogramming discovered by Co-PI Dr. Bruce Ksander and collaborators (Lu et al., Nature, 2020), which uses transient expression of three Yamanaka genes, OSK, to restore and rescue injured dysfunctional cells in vivo. The third approach uses niche-produced LAMA5 ligand-based signaling activation of the BCAM molecular pathway that we just discovered is critical to ABCB5+ LSC and Transient Amplifying Cell (TAC) expansion (Sasamoto et al. Cell Reports, 2022, in press), with resultant stem cell proliferation programs that enhance corneal regeneration. Thus, our hypothesis is that it is possible to manufacture and expand autologous ABCB5+ LSCs from patients with bilateral LSCD using (i) full reprogramming in vitro, (ii) partial epigenetic reprogramming in vivo, and (iii) niche-produced LAMA5 ligand-based activation of LSCs through specific BCAM/ABCB5 signal transduction pathways. These autologous ABCB5+ LSC will fully regenerate and maintain a normal limbus and corneal epithelium. This hypothesis will be tested in three independent specific aims that are thematically related.