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.

项目摘要 肝移植是目前治疗急性和慢性肝功能衰竭的唯一方法。由于缺乏 肝细胞移植被认为是肝脏疾病的潜在治疗方法， 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供应 细胞替代疗法然而，产生hiPSC衍生的肝细胞样细胞（HLC）， 修复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疗法的缺点包括细胞移植不良和成熟不足。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（EC）组成的双细胞疗法， 两者都来源于hiPSC（2）用新的基因电路技术工程化HLC，以促进其 移植和成熟和（3）工程化EC以支持HLC介导的肝修复。这种方法 基于我们在PSC向HLC定向分化方面的长期专业知识， 的BMP 4诱导肝特化，这是一种目前在该领域广泛使用的方法。我们最初观察到， EC总是与小鼠ESC衍生的HLC簇相关联，并显示EC作为生态位发挥作用 抑制Wnt和Notch信号通路促进HLC的特化。最近，我们发现， 支持功能取决于VEGFR 2的激活。与我们的数据一致，已经表明， VEGFA-VEGFR 2轴激活损伤小鼠肝脏中的窦状内皮细胞，诱导诸如 如WNT 2和HGF，它们触发肝细胞增殖。类似地，由窦状核分泌的WNT 2和WNT 9 b 内皮细胞显示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，我们会加强电子商务 通过调节VEGFR 2和下游因子如HGF、WNT 2和WNT 9 b的支持功能。 与此同时，我们将设计HLC以激活促分裂原受体cMET、EGFR和IL 6 R并表达转录， 这些因子（ATF 5，PROX 1，CEBPA）对肝脏发育，再生和成熟至关重要。 重编程成纤维细胞衍生的HLC。我们将测试这些途径和TF加速HLC植入 以及双细胞疗法后的成熟。该提议可能具有重要的临床意义，如VEGFR 2， cMET、EGFR和IL 6 R将使用临床上安全的脂质纳米颗粒复合的非脂质纳米颗粒在体内活化。 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IL 6显著改善移植到肝损伤小鼠模型中的HLC存活。我们 因此，中心假设是：具有HLC和EC的双细胞疗法被工程化以促进EC小生境-HLC 串扰与前所未有的mRNA-LNP工具和遗传电路hiPSC系相结合， 应用将成为肝细胞治疗领域的先驱，将导致成功的基于HPLC的肝病治疗。