Recellularization of Liver Bioscaffolds

描述(申请人提供)：美国约有3000万人因不同原因患有肝病，美国每年约有2.7万人死于肝病。目前，治疗肝功能衰竭的唯一有效方法是原位移植。然而，器官严重短缺，每年约有3000个肝脏短缺。类似的数字影响到大多数器官和组织，目前总的器官等待名单上有10万个请求，而且这个数字每年都在增加5%。鉴于只有处于原始状态的器官才能移植，原位移植将始终是一个有限的池。一个更好的长期解决方案是使用干细胞开发组织工程化的替代物。然而，尽管已经证明了许多体外实验的成功，但由于体内长期的细胞活性和功能低下，临床上的成功一直非常有限。主要的差距是缺乏理想的可移植支架，该支架具有细胞附着、分化、功能和血管形成所需的所有必要微结构和细胞外信号，到目前为止，这种支架很难在体外制造。我们的长期目标是设计可移植的肝移植，以治愈或治疗肝功能障碍和衰竭。这项研究的目的是开发功能性和可移植的大鼠肝移植。这里要检验的中心假设是，来自废弃肝脏的天然肝支架可以广泛地再生，能够为干细胞来源的肝细胞提供足够的成熟环境，并且这些移植物在体外和体内都发挥着基本的肝脏功能。这项研究的基本原理是，虽然大多数研究都集中在使用合成生物材料从头开始制造理想的支架，但本地的ECM可能包含必要的结构和环境线索，因此提供了一种有前途的、很少被探索的器官移植替代方法，可以垂直推进组织工程领域的发展。组建的研究团队在灌流系统、组织工程、干细胞工程和肝移植方面拥有专业知识，这些都是执行拟议研究所必需的。从干细胞来源的肝细胞和肝脏的自然基质中构建功能性肝移植是一项创新性的努力，因为它有可能成为肝脏组织工程的新平台。本文描述的工作有望导致一种新的移植物工程方法来提供辅助肝支持。虽然这项工作利用肝脏作为模型器官，但这项工作的结果也将产生积极的影响，为未来复杂的器官工程技术奠定基础，这些技术结合了几种不同的细胞类型，可以应用于其他器官(胰腺、肾脏等)，并最终可能导致整个器官的体外发育。在此开发的胚胎干细胞成熟方案有望对干细胞工程领域做出重大贡献。脱细胞基质中的肝细胞培养也可能被证明是药学研究的新平台。 与公共卫生相关：美国约有3000万人因不同原因患有肝病，美国每年约有2.7万人死于肝病。目前，治疗肝功能衰竭的唯一有效方法是原位移植。然而，器官严重短缺，每年约有3000个肝脏短缺。类似的数字影响到大多数器官和组织，目前总的器官等待名单上有10万个请求，而且这个数字每年都在增加5%。鉴于只有处于原始状态的器官才能移植，原位移植将始终是一个有限的池。这项研究的结果有望通过开发一种新的方法来利用干细胞来源的肝细胞和废弃肝脏的天然基质来设计辅助肝移植，从而直接改善公众健康，从而治疗肝功能衰竭患者。

DESCRIPTION (provided by applicant): About thirty million people in the US undergo a liver disorder for different causes and about 27,000 deaths are registered annually in the US due to liver disease. At this time, the only definitive treatment of hepatic failure is orthotopic transplantation. However, there is a critical shortage of organs, with a deficit of ~3,000 livers per year. Similar numbers affect most organs & tissues, with the total organ waiting list currently at 100,000 requests and the number increasing by 5% every year. Given that only organs in pristine condition are transplantable, orthotopic transplantation will always remain a limited pool. A more elegant, long-term solution is using stem cells to develop tissue-engineered replacements. However, while many in vitro successes have been demonstrated, clinical success has been very limited due to low cell viability and functionality in the long term in vivo. The major gap is the lack of an ideal transplantable scaffold that has all the necessary microstructure and extracellular cues for cell attachment, differentiation, function and vascularization, which has so far proven difficult to manufacture in vitro. Our long-term goal is to engineer transplantable liver grafts for curing or treating liver dysfunction and failure. The objective of the proposed study is to develop functional and transplantable rat liver grafts. The central hypothesis to be tested here is that the natural liver scaffold derived from discarded livers can be extensively repopulated, can provide an adequate maturation environment for stem cell derived hepatocytes, and that these grafts perform the essential hepatic functions in vitro and in vivo. The rationale of the study is that while most research focuses on producing the ideal scaffold from the ground up using synthetic biomaterials, the native ECM is likely to contain the necessary architecture and environmental cues, hence presents a promising, little explored alternative approach for producing organ grafts which can vertically advance the field of tissue engineering. The research team assembled has expertise on perfusion systems, tissue engineering, stem cell engineering, and liver transplantation (see Biographical Sketches), which are necessary to perform the proposed studies. Engineering of functional liver grafts from stem-cell derived hepatocytes and liver's natural matrix is an innovative endeavor, as it has the potential to become a novel platform for hepatic tissue engineering. The work described here is expected to lead to a novel graft engineering approach to provide auxiliary hepatic support. While this work utilizes liver as the model organ, the results of this work will also have a positive impact by establishing the basis of future sophisticated organ engineering techniques that incorporate several different cell types and can be applied to other organs (pancreas, kidney, etc.), and may ultimately lead to development of entire organs in vitro. The ESC maturation protocol developed here is expected to be a significant contribution to the field of stem cell engineering. Hepatocyte culture in the decellularized matrix may also prove to be a new platform for pharmaceutical studies. PUBLIC HEALTH RELEVANCE: About thirty million people in the US undergo a liver disorder for different causes and about 27,000 deaths are registered annually in the US due to liver disease. At this time, the only definitive treatment of hepatic failure is orthotopic transplantation. However, there is a critical shortage of organs, with a deficit of ~3,000 livers per year. Similar numbers affect most organs & tissues, with the total organ waiting list currently at 100,000 requests and the number increasing by 5% every year. Given that only organs in pristine condition are transplantable, orthotopic transplantation will always remain a limited pool. The results of this study are expected to directly improve public health by the developing a novel approach for engineering auxiliary liver grafts using stem cell derived hepatocytes and native matrices from discarded livers, in order to treat patients with liver failure.