A multi-modular approach for human pluripotent stem cell-based liver regeneration

基于人类多能干细胞的肝再生的多模块方法

• 批准号: 10263323
• 负责人: VALERIE B GOUON-EVANS
• 金额: $ 53.6万
• 依托单位: BOSTON MEDICAL CENTER
• 依托单位国家: 美国
• 财政年份: 2020
• 资助国家: 美国
• 起止时间: 2020-09-15 至 2024-07-31
• 项目状态: 已结题
• 来源: https://reporter.nih.gov/project-details/10263323
• 关键词: Acute; Address; American; BMP4; CEBPA gene; Cell Communication; Cell Line; Cell Survival; Cell Therapy; Cell Transplantation; Cell physiology; Cells; Chronic; Clinical; Clustered Regularly Interspaced Short Palindromic Repeats; Coculture Techniques; Complex; Data; EGF gene; Endothelial Cells; Endothelium; Engineering; Engraftment; Epidermal Growth Factor Receptor; Exhibits; Fibroblasts; Generations; Genetic; Goals; Hepatic; Hepatocyte; Hepatocyte transplantation; Human; Human Engineering; IL6 gene; In Vitro; Individual; Injections; KDR gene; Ligands; Liver; Liver Failure; Liver Regeneration; Liver diseases; Mediating; Messenger RNA; Mitogen Receptors; Mitogens; Modeling; Mus; Natural regeneration; Nucleosides; Partial Hepatectomy; Pathway interactions; Patients; Receptor Activation; Recombinant Proteins; Recombinants; Regenerative capacity; Regenerative pathway; Role; Safety; Signal Transduction; Technology; Testing; Therapeutic; Transplantation; United States; VEGFA gene; WNT2 gene; Waiting Lists; base; beta catenin; cancer clinical trial; cell replacement therapy; cellular engineering; chronic liver injury; clinical translation; clinically relevant; experimental study; human pluripotent stem cell; improved; in vivo; induced pluripotent stem cell; injured; lipid nanoparticle; liver cell proliferation; liver development; liver injury; liver repair; liver transplantation; mouse model; notch protein; novel; overexpression; receptor; regenerative; tool; transcription factor

PROJECT SUMMARY Liver transplantation is currently the only treatment for acute and chronic liver failure. Given the shortage of donor livers, hepatocyte transplantation is considered a potential treatment for liver diseases and a bridge for patients awaiting liver transplantation, but its application has been hampered by a limited supply of hepatocytes. Human induced pluripotent stem cell (hiPSC) could provide an unlimited supply of patient-specific hepatocytes for cell replacement therapy. However, generation of hiPSC-derived hepatocyte like cells (HLCs) that repopulate a damaged liver remains a challenge, and a major gap that this application targets. Obstacles to HLC therapy include poor cell engraftment and insufficient maturation. Thus to fill this gap, we propose a multi- modular approach that includes (1) a bi-cell therapy composed of HLCs and supportive endothelial cells (ECs), both derived from hiPSCs (2) engineering HLCs with novel genetic circuit technology to promote their engraftment and maturation and (3) engineering ECs to support HLC-mediated liver repair. This approach is based on our long-standing expertise in the directed differentiation of PSCs to HLCs having pioneered the use of BMP4 to induce hepatic specification, an approach now utilized widely in the field. We initially observed that ECs were always associated with mouse ESC-derived HLC clusters, and showed that ECs function as a niche to repress Wnt and Notch signaling to promote HLC specification. Recently, we discovered that this EC supportive function is dependent on activation of VEGFR2. Consistent with our data, it has been shown that a VEGFA-VEGFR2 axis activates sinusoidal ECs in injured mouse livers to induce the expression of factors such as WNT2 and HGF that trigger hepatocyte proliferation. Similarly, WNT2 and WNT9b secreted by sinusoidal ECs were shown to induce hepatocyte proliferation following partial hepatectomy. Thus, bi-cell therapy with ECs represents a potential strategy to overcome the obstacles of HLC therapies. In addition, we will enhance EC supportive functions through modulation of VEGFR2 and downstream factors such as HGF, WNT2 and WNT9b. In parallel, we will engineer HLCs to activate mitogen receptors cMET, EGFR and IL6R and express transcription factors (ATF5, PROX1, CEBPA) that are critical for liver development, regeneration and the maturation of reprogrammed fibroblast-derived HLCs. We will test that these pathways and TFs accelerate HLC engraftment and maturation following bi-cell therapy. This proposal may have major clinical implications as VEGFR2, cMET, EGFR and IL6R will be activated in vivo using the clinically safe lipid nanoparticle-complexed non- integrative nucleoside-modified mRNA (mRNA-LNP). Our preliminary data indicate that mRNA-LNP encoding HGF, EGF and IL6 significantly improve HLC survival after transplantation into liver injury mouse models. Our central hypothesis is thus: a bi-cell therapy with HLCs and ECs engineered to promote the EC niche-HLC crosstalk in combination with the unprecedented mRNA-LNP tool and genetic circuit hiPSC lines that this application will pioneer in the liver cell therapy field will lead to successful HLC-based treatment for liver disease.

项目摘要 目前，肝移植是急性和慢性肝衰竭的唯一治疗方法。考虑到短缺 供体肝脏，肝细胞移植被认为是肝病的潜在治疗方法和桥梁 等待肝移植的患者，但其应用受到肝细胞供应有限的阻碍。 人类诱导的多能干细胞（HIPSC）可以提供无限的患者特异性肝细胞的供应 用于替代细胞疗法。但是，hipsc衍生的肝细胞类似细胞（HLC）的产生 重新填充受损的肝脏仍然是一个挑战，这是该应用程序针对的主要差距。障碍 HLC治疗包括细胞植入不良和成熟不足。因此，为了填补这一空白，我们提出了一个多 模块化方法包括（1）由HLC和支持性内皮细胞（ECS）组成的双细胞疗法， 两者都源自HIPSC（2）具有新型遗传回路技术的工程HLC，以促进其 植入和成熟以及（3）ECS支持HLC介导的肝脏修复。这种方法是 基于我们在PSC对HLC的定向差异化方面的长期专业知识，这是我们开创使用的HLC BMP4诱导肝规范，这是一种现场广泛使用的方法。我们最初观察到 EC始终与鼠标ESC衍生的HLC簇相关，并表明EC可以充当利基市场 抑制Wnt和Notch信号传导以促进HLC规范。最近，我们发现这个EC 支持功能取决于VEGFR2的激活。与我们的数据一致，已经表明 VEGFA-VEGFR2轴激活受伤的小鼠肝脏中的正弦EC，以诱导因素的表达 如触发肝细胞增殖的Wnt2和HGF。同样，由正弦分泌的Wnt2和Wnt9b 显示EC在部分肝切除术后诱导肝细胞增殖。因此，使用EC的双细胞疗法 代表了克服HLC疗法障碍的潜在策略。此外，我们将增强EC 通过调节VEGFR2和下游因素（例如HGF，WNT2和WNT9B）的支持功能。 同时，我们将设计HLC激活有丝分裂原受体CMET，EGFR和IL6R和Express转录 因素（ATF5，Prox1，cebpa）对于肝脏发育，再生和成熟至关重要 重编程的成纤维细胞衍生的HLC。我们将测试这些途径和TFS加速HLC植入 双胞胎疗法后的成熟。该建议可能具有vegfr2的主要临床意义， CMET，EGFR和IL6R将使用临床安全的脂质纳米粒子复合非 - 整合核苷修饰的mRNA（mRNA-LNP）。我们的初步数据表明mRNA-LNP编码 HGF，EGF和IL6在移植到肝损伤小鼠模型中显着提高了HLC的存活。我们的 因此，中央假设是：一种使用HLC和ECS进行的双细胞疗法，旨在促进EC Niche-HLC 串扰与前所未有的mRNA-LNP工具和遗传回路hipsc线相结合 应用将在肝细胞疗法领域进行开拓性，将导致成功基于HLC的肝病治疗。