Engineering vascularized tissue in vivo using postnatal progenitor cells

描述（由申请人提供）：我的目标是开发一种基于细胞的技术，通过使用可通过非侵入性手段从患者身上获得的出生后祖细胞，在体内制造血管化组织。特别是，本提案将集中于联合使用骨髓源性间充质祖细胞（MPCs）和人血源性内皮祖细胞（EPCs）在体内形成脂肪组织。要测试的概念是含有高度纯化和明确的MPCs和EPCs的植入物在体内植入后是否会产生特化的血管化组织。我假设要实现这一点，1)MPCs和EPCs首先需要创建一个血管网络，允许血液通过植入物灌注；2)这种血管化将通过在着床部位采用周围组织的表型，促进尚未组装成血管网络的MPCs进行适当的组织发育。在本提案中，我将使用我们用人类EPCs和MPCs建立的体内模型确定关键参数，以加速血管生成过程到24-48小时的时间框架。接下来，我将确定在植入物中发现的脂肪细胞是否来自人类，或者它们是否作为内源性组织修复过程的一部分从宿主中招募到植入物中，一旦功能血管网络存在

DESCRIPTION (provided by applicant): My goal is to develop a cell-based technology that will allow engineering vascularized tissues in vivo by using postnatal progenitor cells that can be obtained from patients by non-invasive means. In particular, this proposal will focus on the combined use of bone marrow-derived mesenchymal progenitor cells (MPCs) and human blood-derived endothelial progenitor cells (EPCs) to form adipose tissue in vivo. The concept to test is whether implants containing highly purified and defined MPCs and EPCs will result in specialized vascularized tissues upon implantation in vivo. I hypothesize that for this to happen, 1) both MPCs and EPCs need first to create a vascular network that will allow blood perfusion through the implants; and 2) this vascularization will promote MPCs that have not assembled into the vascular network to undergo appropriate tissue development by adopting the phenotype of the surrounding tissue at the site of implantation. In this proposal, I will determine key parameters to accelerate the vasculogenic process to a time frame of 24-48 hours using the in vivo model we have established with human EPCs and MPCs. Next, I will determine whether the adipocytes found in the implants are human in origin or whether they are recruited into the implants from the host as part of an endogenous tissue repair process once a functional vascular network is provided. With this information, I will implement strategies to enhance the process of in vivo differentiation with the aim of having fully functional tissues. Finally, I will test this cell-based technology by evaluating whether we can create long-lasting adipose tissue pads that present low volume reduction over time. The experimental approaches to address these aims include: 1) in vivo imaging of blood perfusion in the implants using luciferase-based bioluminescence 2) GFP labeling of implanted cells to track their specific contribution to the resulting tissues; 3) confocal microscopy to evaluate implants through immunofluorescence staining. I envision this two-cell, two-step system as an enabling technology that can be applied to many different tissues wherein functional blood vessels are essential. RELEVANCE: This is an enabling technology that can be applied to regeneration of tissues/organs wherein functional blood vessels are essential. This proposal will focus on formation of adipose tissue, but it will be applicable to many aspects of regenerative medicine, including tissue-engineering and in situ therapeutic vascularization.