Recellularization of Liver Bioscaffolds

描述（由申请人提供）：美国约有3000万人因不同原因患有肝脏疾病，美国每年约有27，000人因肝脏疾病而死亡。目前，肝衰竭的唯一确定性治疗方法是原位移植。然而，器官严重短缺，每年约缺少3，000个肝脏。类似的数字影响到大多数器官和组织，目前器官等待名单上有10万个请求，并且每年增加5%。鉴于只有原始状态的器官才可移植，原位移植将始终保持有限的库。一个更优雅、更长期的解决方案是使用干细胞来开发组织工程替代品。然而，虽然已经证明了许多体外成功，但由于长期体内细胞活力和功能性低，临床成功非常有限。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、功能和血管化所需的所有必要的微观结构和细胞外线索，迄今为止已证明难以在体外制造。 我们的长期目标是设计可移植的肝脏移植物，用于治疗或治疗肝功能障碍和衰竭。本研究的目的是开发功能性和可移植的大鼠肝移植物。这里要测试的中心假设是，来自废弃肝脏的天然肝脏支架可以被广泛地重新填充，可以为干细胞衍生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执行基本的肝脏功能。该研究的基本原理是，虽然大多数研究都集中在使用合成生物材料从头开始生产理想的支架，但天然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，很少探索的替代方法，用于生产器官移植物，可以垂直推进组织工程领域。组建的研究团队拥有灌注系统、组织工程、干细胞工程和肝移植方面的专业知识（见传记草图），这些都是进行拟议研究所必需的。 从干细胞衍生的肝细胞和肝脏的天然基质工程化功能性肝移植物是一项创新的奋进，因为它有可能成为肝组织工程的新平台。这里描述的工作有望导致一种新的移植物工程方法，以提供辅助肝脏支持。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通过建立未来复杂器官工程技术的基础而产生积极影响，这些技术包括几种不同的细胞类型，并可应用于其他器官（胰腺，肾脏等），并可能最终导致整个器官在体外发育。在这里开发的ESC成熟协议预计将是干细胞工程领域的重大贡献。脱细胞基质中的肝细胞培养也可能被证明是药物研究的新平台。 公共卫生相关性：美国约有三千万人因不同原因而患有肝病，美国每年约有27，000人因肝病而死亡。目前，肝衰竭的唯一确定性治疗方法是原位移植。然而，器官严重短缺，每年约缺少3，000个肝脏。类似的数字影响到大多数器官和组织，目前器官等待名单上有10万个请求，并且每年增加5%。鉴于只有原始状态的器官才可移植，原位移植将始终保持有限的库。这项研究的结果有望通过开发一种新的方法来直接改善公共健康，该方法使用干细胞衍生的肝细胞和来自废弃肝脏的天然基质来工程化辅助肝脏移植物，以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.