Differentiation of Adult Stem Cells to Lung Ex Vivo

我们将使用最近开发的共培养系统和竞争性植入试验来确定最佳候选人。具体目标是：（1）在最近开发的与热休克或高氧损伤的小气道上皮细胞（SAEC）共培养系统中使用人和大鼠MSC的标准和未分级制备物，以产生（a）MSC的分化;（B）MSC与SAEC的融合;和（c）与SAEC融合的MSC的命运的定量测定。（2.）使用特定目标1中开发的共培养试验来确定最近在我们实验室中表征的三个MSC亚群中的哪一个在以下方面最有效： 区分SAEC：（a）小的和快速自我更新的细胞（RS细胞）;（B）大的和缓慢复制的成熟MSC（mMSC）;和（c）我们最近分离的并且暂时称为前RS细胞的细胞，因为它们比RS细胞更类似于胚胎细胞。为了定义解释在测定中观察到的细胞之间的任何差异的分子机制，我们将在加入共培养物之前和之后对细胞进行微阵列测定。（3.）使用特定目标1中的共培养试验，在靶细胞为大支气管上皮细胞的类似共培养系统中检测相同的MSC群体 细胞、肺泡上皮细胞或肺血管内皮细胞。（4.）通过在免疫缺陷小鼠中使用竞争性移植试验，确定来自特异性目的2的MSC亚群中的哪一个可以最有效地在肺中移植。

We will use a recently developed co-culture system and an assay for competitive engraftment to identify the best candidatesfor use. The Specific Aims are: (1.) Use standard and unfractionated preparations of human and rat MSCs in the recently developed co-culture system with small airway epithelial cells (SAECs) damaged by heat shock or hyperoxia to generate quantitative assays for (a) differentiation of MSCs; (b) fusion of MSCs with SAECs; and (c) the fate of MSCs that fuse with SAECs. (2.) Use the co-culture assay developed in Specific Aim 1 to determine which of three sub-populations of MSCs that have recently been characterized in our laboratory are the most effective in differentiating into SAECs: (a) small and rapidly self-renewing cells (RS cells); (b) large and slowly replicating mature MSCs (mMSCs); and (c) cells that we have recently isolated and tentatively referred to as pre-RS cells because they are more similar to embryonic cells than RS cells. To define molecular mechanisms that explain any observed differences among cells in the assay, we will carry out microarray assays on the cells before and after addition to the co-cultures. (3.) Use the co-culture assay from Specific Aim 1 to test the same populations of MSCs in analogous co-culture systems in which the target cells are large bronchial epithelial cells, alveolar epithelial cells, or pulmonary vascular endothelial cells. (4.) Determine which of the subpopulations of MSCs from Specific Aim 2 can most efficiently engraft in lung by using a competitive engraftment assay in immunodeficient mice.