Overcoming Barriers to Bioengineering 3-D Human Lung

描述(由申请人提供)： 这项应用的总体目标是定义有利于产生可移植的3-D人类肺的条件。肺移植的主要障碍包括可用的供肺稀少、慢性排斥、需要终身免疫抑制以及高死亡率。需要一种不会发生排斥反应的新的可移植肺组织来源。使用脱细胞肺基质(DLM)作为天然的基础支架，我们的中心假设是，构建功能性的、可移植的肺需要适当的细胞定位和细胞排列时叠加的仿生信号的组合，以推动功能整合和成熟。该应用程序的PI开发了一种“去细胞、通风的肺生物反应器”系统，该系统支持胎儿肺细胞的生长，并服从于对重建肺的潜力的评估。除了使用DLM外，该方案的创新之处在于使用了具有肺再细胞化潜力的自体细胞，这些细胞都来自相同的BM来源，即所有细胞都是彼此自体的，并有可能用于转译临床研究。出生后/成人的iPS细胞是经过重新编程的细胞，具有胚胎干细胞的特征。我们有证据表明，DLMS可以接种诱导的多能干细胞(iPS细胞)，从而产生肺泡II型细胞。我们希望评估自体骨髓间充质基质细胞(MSCs)和iPS来源的内皮细胞(ECs)在完成DLm再细胞化中的应用。一支代表生物工程、肺部生物学、生理学、干细胞、血管生物学和胸外科领域的研究团队已经组建完毕。我们的目标包括以发育阶段适当的方式使DLm重新细胞化，从而产生具有紧密连接屏障、气体交换和液体清除功能特性的正确的细胞图案。我们将采取循序渐进的方法来确定基质几何形状、生理拉伸和血管形成对使用人类自体祖细胞的再上皮化和再细胞化结构的功能的贡献，并与自然肺进行比较。我们的理由是，通过比较不同的条件，面对面和组合，将定义几个基本的“规则”的生物工程自体功能肺。这项建议有三个具体目标。在目标1中，我们将确定基质几何形状和生理机械拉伸对人祖细胞向肺细胞分化的贡献。在目标2中，我们将评估使用骨髓来源的MSCs进行间充质诱导是否能最好地实现DLMS的再细胞化。目的3将确定DLm血管网络的预先内皮化是否增强随后的上皮祖细胞与DLm的附着、分化和功能。我们将使用骨髓来源的内皮细胞(ECs)通过血管通路输注来重新内皮化DLM。由于血管是由来源于MSC的周细胞稳定的，我们将使用人骨髓来源的MSCs来研究它们是否能够增强ECs的附着。(摘要结束)

DESCRIPTION (provided by applicant): The overall goal of this application is to define conditions that are conducive to generating transplantable, 3-D human lung. Major obstacles to lung transplantation include the paucity of donor lungs available, chronic rejection the requirement for lifelong immunosuppression and high mortality. A new source of transplantable lung tissue that will not be subject to rejection is needed. Using decellularized lung matrix (DLM) as the natural foundational scaffold, our central hypothesis is that building functional, transplantable lung requires a combination of geometry that allows for appropriate cell localization and biomimetic cues superimposed on cells as they align to drive functional integration and maturation. The PI of this application developed a "decellularized, ventilated lung bioreactor" system that supports the growth of fetal lung cells and is amenable to evaluation of the potential to rebuild the lung. In addition to the use of DLM, the novelty of this proposal entails the use of autologous cells with lung recellularization potential all from the same BM source, i.e. all cells are autologous to each other and have the potential to be used in translational clinical studies. Post natal/adult human iPS cells are cells that have been reprogrammed to have characteristics of embryonic stem cells. We have evidence that DLMs can be seeded with induced pluripotent stem cells (iPS cells) that give rise to alveolar type II-like cells. We wish to evaluate the use of autologous BM-derived mesenchymal stromal cells (MSCs) and iPS-derived endothelial cells (ECs) in completing the recellularization of DLM. A team of investigators representing a convergence of the fields of bioengineering, pulmonary biology, physiology, stem cells, vascular biology and thoracic surgery has been assembled. Our aims encompass the recellularization of DLM in a developmental stage-appropriate manner that results in correct cell patterning with tight junctional barriers, gas exchange and fluid clearance functional properties. We will take a stepwise approach to determine the contribution of matrix geometry, physiological stretch and vascularization on re-epithelialization and function of recellularized constructs using human autologous progenitors and compare to native lung. We reason that by comparing different conditions head-to-head and in combination, several fundamental "rules" for bioengineering an autologous functional lung will be defined. There are 3 Specific Aims in this proposal. In Aim 1 we will determine the contribution of matrix geometry and physiological mechanical stretch on the differentiation of human progenitor cells into pulmonary cells. In Aim 2, we will evaluate if recellularization of DLMs is best achieved with mesenchymal induction using BM-derived MSCs. Aim 3 will determine if prior endothelialization of the DLM vascular network potentiates subsequent epithelial progenitor cell attachment to DLM, differentiation and function. We will use BM-derived endothelial cells (ECs) infused through the vasculature access to re-endothelialize DLM. Since vessels are stabilized by pericytes that are of MSC origin, we will use human BM-derived MSCs to study if they can potentiate the attachment of ECs. (End of Abstract)