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: 10459609
• 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/10459609
• 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; directed differentiation; end stage liver disease; experimental study; human pluripotent stem cell; improved; in vivo; in vivo engraftment; 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.

项目概要 肝移植是目前治疗急慢性肝功能衰竭的唯一方法。鉴于短缺 供体肝脏，肝细胞移植被认为是肝脏疾病的潜在治疗方法和桥梁 等待肝移植的患者，但由于肝细胞供应有限，其应用受到阻碍。 人类诱导多能干细胞（hiPSC）可以无限供应患者特异性肝细胞 用于细胞替代疗法。然而，hiPSC 衍生的肝细胞样细胞 (HLC) 的产生 重新填充受损的肝脏仍然是一个挑战，也是该应用所针对的主要差距。障碍 HLC 治疗的缺点包括细胞植入不良和成熟不足。因此，为了填补这一空白，我们提出了一种多 模块化方法，包括 (1) 由 HLC 和支持性内皮细胞 (EC) 组成的双细胞疗法， 两者均源自 hiPSC (2) 使用新颖的基因电路技术对 HLC 进行改造，以促进其 植入和成熟；(3) 工程化 EC 以支持 HLC 介导的肝脏修复。这种方法是 基于我们在 PSC 定向分化为 HLC 方面的长期专业知识，并率先使用 BMP4 诱导肝脏特异性，这种方法现已在该领域广泛使用。我们最初观察到 EC 始终与小鼠 ESC 衍生的 HLC 簇相关，并且表明 EC 充当生态位 抑制 Wnt 和 Notch 信号传导以促进 HLC 规范。最近，我们发现这个EC 支持功能依赖于 VEGFR2 的激活。与我们的数据一致，已经表明 VEGFA-VEGFR2 轴激活受损小鼠肝脏中的正弦 EC，诱导以下因子的表达 如触发肝细胞增殖的 WNT2 和 HGF。类似地，WNT2和WNT9b由正弦曲线分泌 ECs 显示可在部分肝切除术后诱导肝细胞增殖。因此，使用 EC 进行双细胞疗法 代表了克服 HLC 疗法障碍的潜在策略。此外，我们将加强EC 通过调节 VEGFR2 和 HGF、WNT2 和 WNT9b 等下游因子来支持功能。 与此同时，我们将改造 HLC 来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） 重新编程的成纤维细胞衍生的 HLC。我们将测试这些途径和 TF 是否加速 HLC 植入 和双细胞疗法后的成熟。该提案可能具有重大临床意义，如 VEGFR2、 使用临床安全的脂质纳米颗粒复合非-cMET、EGFR和IL6R将在体内被激活 整合核苷修饰 mRNA (mRNA-LNP)。我们的初步数据表明 mRNA-LNP 编码 HGF、EGF 和 IL6 显着提高 HLC 移植到肝损伤小鼠模型后的存活率。我们的 因此，中心假设是：采用 HLC 和 EC 进行双细胞疗法，旨在促进 EC 生态位-HLC 串扰与前所未有的 mRNA-LNP 工具和遗传电路 hiPSC 系相结合，这 该应用将成为肝细胞治疗领域的先驱，并将带来基于 HLC 的肝病治疗的成功。