Liver Tissue Engineering Through Three-Dimensional Hepatocyte Culture

描述（由申请人提供）：我申请K08职业发展奖的目标是成为肝组织工程领域的独立研究者和领导者。我已经完成了医学博士学位，证明了我对成为一名临床医生和科学家的持续承诺。他在哈佛医学院获得联合学位，在加州大学旧金山分校（UCSF）获得普通外科住院医师学位，并进行了两年的博士后研究，研究三维（3D）培养作为组织工程的一种新方法。我完成了微创外科的临床奖学金，并被UCSF招募为外科助理教授，有75%的保护研究时间。作为一名外科科学家，我的长期职业目标是通过微创技术，为治疗性植入设计一个功能肝脏单元。这个奖项将帮助我实现这一目标，通过支持我在固体旋转生物反应器中3D肝细胞培养的博士后工作基础上的独立研究项目的发展。肝移植是终末期肝病（ESLD）患者目前唯一的治疗方法，终末期肝病是美国第12大疾病死亡原因。供体器官短缺仍然是主要的治疗限制。肝细胞移植等替代疗法已显示出治疗代谢性肝脏疾病的希望，但移植效率低，长期疗效差，是广泛临床应用的障碍。另一种策略是可植入的功能性活体单位的离体组织工程。然而，迄今为止，利用生物相容性支架构建3D组织结构的进展一直受到阻碍，因为这些材料的缓慢降解率阻碍了正常的细胞间和细胞-细胞外基质（ECM）相互作用。我的博士后研究表明，在旋转壁血管（RWV）生物反应器中培养的肝细胞，提供最小的剪切应力和最大的三维空间自由度，与在二维单层中培养的肝细胞相比，产生具有最佳代谢和合成基因表达和功能的自聚集球体。本研究以这些发现为基础，旨在确定基质细胞和基质细胞在维持肝细胞功能中的作用，并确定其潜在机制。该研究基于以下假设：1)3D细胞形状和基质刚度是相互关联的因素，它们通过Rho-ROCK和整合素信号级联介导的细胞骨架张力共同调节肝细胞功能；2)肝细胞与星状细胞的3D共培养将产生具有最佳支架基质刚度、ECM组成和旁分泌串扰的类器官，以维持肝细胞功能。具体而言，本提案将1)确定基质刚度对肝细胞功能的影响及其潜在的分子机制；2)表征肝细胞-星状细胞3D聚集体的基质刚度、ECM组成和旁分泌串扰，以及维持肝细胞特异性功能所需的潜在机制。这项研究的结果有望通过增加我们对肝细胞对基质刚度和3D基质细胞相互作用的反应的理解来推进组织工程领域。这项研究将为我启动r01资助的肝组织工程研究提供必要的初步数据。重要的是，其他培训活动将被用来增加我在各个学科的知识，这对我成为肝组织工程独立研究者的职业目标很重要。研究和培训将在UCSF进行，UCSF是一所排名第一的三级转诊医院，也是国际知名的生物医学研究机构，拥有丰富的科学调查和合作环境。我将通过加州大学旧金山分校生物医学科学研究生项目继续学习细胞外基质支架和生物工程方面的教学课程。我将参加加州大学旧金山分校胃肠内科肝病奖学金项目的研讨会，并与世界知名的肝脏病理学家一起学习肝脏病理学。我的主要导师是霍巴特·哈里斯医生，他是普通外科部门的主任，也是一位有成就的外科科学家，拥有一个校外资助的基础科学实验室。哈里斯博士是一位非常合适的科学导师，在初级肝细胞生物学方面有着广泛的研究背景，也是一位宝贵的整体职业导师，指导我度过成为外科医生-科学家的挑战。拟议的项目是跨学科的，我的共同导师瓦莱丽·韦弗博士将提供细胞外基质、细胞骨架和Rho-ROCK/整合素信号操纵方面的专业知识。我的另一位共同导师，Holger Willenbring博士，将在原代肝细胞和星状细胞培养和功能分析方面提供额外的专业知识。此外，我将与Jacquelyn Maher博士合作，她是我的顾问委员会成员兼UCSF肝脏中心主任。Maher博士和UCSF肝脏中心将是支持本研究中提出的原代肝细胞和星状细胞分离和分析的重要资源。有了这个导师团队和UCSF丰富的研究环境，我所提出的研究和职业发展的成功率非常高。通过K08职业发展奖的支持将是非常宝贵的，帮助我实现我的长期职业目标，成为一名独立的外科医生，研究重点是肝组织工程。

DESCRIPTION (provided by applicant): My goal in seeking a K08 Career Development Award is to become an independent investigator and leader in the field of liver tissue engineering. I have demonstrated a sustained commitment to become a clinician- scientist, having completed an M.D.-Ph.D. combined degree at Harvard Medical School, General Surgery Residency at the University of California, San Francisco (UCSF), and two years of post-doctoral research investigating three-dimensional (3D) culture as a novel approach to tissue engineering. I completed a clinical fellowship in Minimally Invasive Surgery and have been recruited to UCSF as Assistant Professor of Surgery with 75% protected research time. As a surgeon-scientist, my long-term career goal is to tissue engineer a functional liver unit for therapeutic implantation, preferably by minimally invasive techniques. This award will help me achieve that goal by supporting development of my independent research program based on my post- doctoral work on 3D hepatocyte culture in solid-body rotational bioreactors. Liver transplantation is currently the only treatment for patients with end-stage liver disease (ESLD), which is the 12th leading cause of death by disease in the U.S. The shortage of donor organs remains a major treatment limitation. Alternatives such as hepatocyte transplantation have shown promise in treating metabolic liver disorders, but low engraftment efficiency and poor long-term efficacy are barriers to broader clinical application. Another strategy is ex vivo tissue engineering of a functional livr unit that can be implanted. However, progress thus far towards building 3D tissue structure with biocompatible scaffolds has been hindered because the slow degradation rates of these materials prevent normal cell-cell and cell-to- extracellular matrix (ECM) interactions. My post-doctoral research demonstrated that hepatocytes cultured in Rotating Wall Vessel (RWV) bioreactors, which provide minimal shear stress with maximal 3D spatial freedom, produced self-aggregated spheroids with optimal metabolic and synthetic gene expression and function as compared to those cultured in 2D monolayers. This proposal builds upon those findings and aims to define the role of matrix and stromal cells in maintaining hepatocyte functions and to determine the underlying mechanisms. The research is driven by the hypotheses that 1) 3D cell shape and matrix rigidity are interconnected factors that together modulate hepatocyte function through cytoskeletal tension mediated by the Rho-ROCK and integrin signal cascades, and 2) 3D co-culture of hepatocytes with stellate cells will generate organoids demonstrating optimal scaffold matrix rigidity, ECM composition, and paracrine crosstalk for maintaining hepatocyte functions. Specifically, this proposal will 1) Determine the effect of matrix rigidity on hepatocyt function and the underlying molecular mechanisms and 2) Characterize the matrix rigidity, ECM composition, and paracrine crosstalk of hepatocyte-stellate cell 3D aggregates and the underlying mechanisms required to maintain hepatocyte specific functions. The results of this study are expected to advance the field of tissue engineering by increasing our understanding of hepatocyte cellular response to matrix stiffness and 3D stromal cell interactions. This research will provide the necessary preliminary data for me to initiate an R01-funded study of liver tissue engineering. Importantly, other training activities will be utilized to increase my knowledge in various disciplines important to my career goal of becoming an independent investigator in liver tissue engineering. The research and training will be carried out at UCSF, a top-ranked tertiary referral hospital and internationally-known institution for biomedical research with an incredibly rich environment for scientific investigation and collaboration. I will pursue didactic coursework in extracellular matrix scaffolds and bioengineering through the UCSF Biomedical Sciences Graduate Program. I will attend seminars offered through UCSF's Hepatology Fellowship Program in the Division of Gastroenterology and study liver pathology with a world-renowned liver pathologist. My primary mentor is Dr. Hobart Harris, who is Chief of the Division of General Surgery and an accomplished surgeon-scientist with an extramurally-funded basic science laboratory. Dr. Harris is a well- suited scientific mentor with extensive research background in primary hepatocyte biology and an invaluable overall career mentor in guiding me through the challenges of becoming a surgeon-scientist. The proposed project is interdisciplinary and my co-mentor Dr. Valerie Weaver will provide expertise in extracellular matrix, cytoskeletal, and Rho-ROCK/integrin signaling manipulation. My other co-mentor, Dr. Holger Willenbring, will provide additional expertise in primary hepatocyte and stellate cell culture and functional assays. In addition, I will be working with Dr. Jacquelyn Maher, who is a member of my advisory committee and director of the UCSF Liver Center. Dr. Maher and the UCSF Liver Center will be important resources to support the isolation and analysis of primary hepatocytes and stellate cells proposed in this research. With this mentoring team and UCSF's rich research environment, success of my proposed research and career development are very high. Support through the K08 Career Development Award will be invaluable in helping me achieve my long-term career goal of becoming an independent surgeon-scientist with a research focus in liver tissue engineering. PUBLIC HEALTH RELEVANCE: Liver transplantation is currently the only treatment for patients with end-stage liver disease, but the shortage of donor organs remains a major treatment limitation. In order to develop functional ex vivo engineered liver tissues for organ replacement therapy, this research proposes to investigate the effect of matrix rigidity and three-dimensional stromal cell interactions on hepatocyte function.