Overcoming Barriers to Bioengineering 3-D Human Lung

描述（由申请人提供）： 本申请的总体目标是定义有利于生成可移植的3-D人肺的条件。肺移植的主要障碍包括可用供体肺的缺乏、慢性排斥反应、需要终身免疫抑制和高死亡率。需要一种不会受到排斥的可移植肺组织的新来源。使用脱细胞肺基质（DLM）作为天然基础支架，我们的中心假设是，构建功能性的可移植肺需要几何结构的组合，该几何结构允许适当的细胞定位和仿生线索叠加在细胞上，因为它们对齐以驱动功能整合和成熟。本申请的PI开发了一种“脱细胞、通气肺生物反应器”系统，该系统支持胎肺细胞的生长，并可用于评价重建肺的潜力。除了使用DLM之外，该提议的新奇还需要使用来自相同BM来源的具有肺再细胞化潜力的自体细胞，即所有细胞彼此是自体的并且具有用于转化临床研究的潜力。纳塔尔后/成年人iPS细胞是已被重编程为具有胚胎干细胞特征的细胞。我们有证据表明，DLMs可以接种诱导多能干细胞（iPS细胞），产生肺泡II型样细胞。我们希望评估使用自体骨髓间充质干细胞（MSC）和iPS衍生的内皮细胞（EC）完成DLM的再细胞化。一个代表生物工程、肺生物学、生理学、干细胞、血管生物学和胸外科等领域的研究小组已经成立。我们的目标包括以发育阶段适当的方式使DLM再细胞化，从而产生具有紧密连接屏障、气体交换和流体清除功能特性的正确细胞模式。我们将采用逐步的方法来确定基质几何形状、生理拉伸和血管化对使用人自体祖细胞的再细胞化构建体的再上皮化和功能的贡献，并与天然肺进行比较。我们的理由是，通过比较不同的条件头对头和组合，几个基本的“规则”的生物工程自体功能肺将被定义。这个提案有三个具体目标。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分化为肺细胞的贡献。在目标2中，我们将评估使用BM衍生的MSC的间充质诱导是否最好地实现DLMs的再细胞化。目的3将确定DLM血管网络的先前内皮化是否增强随后的上皮祖细胞附着于DLM、分化和功能。我们将使用BM衍生的内皮细胞（EC）通过血管通路输注，使DLM再内皮化。由于血管是由MSC来源的周细胞稳定的，我们将使用人骨髓来源的MSC来研究它们是否可以增强EC的附着。(End摘要）

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)