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.

项目总结 肝移植是目前治疗急性和慢性肝功能衰竭的唯一方法。考虑到缺乏 肝细胞移植被认为是一种潜在的肝病治疗方法，也是 等待肝移植的患者，但其应用受到肝细胞供应有限的阻碍。 人诱导多能干细胞(HiPSC)可以提供无限的患者特异性肝细胞。 用于细胞替代疗法。然而，HiPSC来源的肝细胞样细胞(HLC)的产生 重新填充受损的肝脏仍然是一个挑战，也是这项应用所针对的一个主要缺口。障碍 对HLC的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，我们提出了一个多层次的 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， 两者都源于HiPSCs(2)利用新的基因电路技术设计HLC以促进其 植入和成熟；(3)工程内皮细胞支持HLC介导的肝修复。这种方法是 基于我们在将PSC定向分化为HLC方面的长期专业知识开创了这一用途 BMP4诱导肝脏规范，这是目前该领域广泛使用的一种方法。我们最初观察到， 内皮细胞总是与小鼠胚胎干细胞来源的HLC簇联系在一起，并表明内皮细胞作为一个利基发挥作用 抑制WNT和Notch信令以促进HLC规范。最近，我们发现这个EC 支持功能依赖于VEGFR2的激活。与我们的数据一致的是，已经显示出 VEGFA-VEGFR2轴激活受损小鼠肝脏正弦状内皮细胞诱导 作为触发肝细胞增殖的Wnt2和HGF。同样，WNT2和WNT9b由正弦分泌 肝大部切除后，ECS可诱导肝细胞增殖。因此，用内皮细胞进行双细胞治疗 代表了一种克服HLC疗法障碍的潜在策略。此外，我们还将加强EC 通过调节VEGFR2和HGF、WNT2、WNT9b等下游因子发挥支持作用。 同时，我们将改造HLCs激活有丝分裂原受体cMET、EGFR和IL6R并表达转录 对肝脏发育、再生和成熟至关重要的因子(ATF5、PROX1、CEBPA) 对成纤维细胞来源的HLC进行重新编程。我们将测试这些途径和TF加速HLC植入 并在双细胞治疗后成熟。这一建议可能具有重大的临床意义，如VEGFR2， CMET，EGFR和IL6R将在体内被激活，使用临床安全的脂纳米粒-非复合 整合型核苷修饰信使核糖核酸(mRNA-LNP)。我们的初步数据表明，编码mRNA-LNP HGF、EGF和IL6可显著提高HLC移植入肝损伤小鼠模型后的存活率。我们的 因此，中心假设是：HLC和ECs的双细胞疗法旨在促进EC NICE-HLC 串扰与史无前例的mRNA-LNP工具和遗传电路HiPSC相结合 应用将是肝细胞治疗领域的先驱，将导致基于HLC的肝病治疗成功。