Engineering vascularized tissue in vivo using postnatal progenitor cells

描述(申请人提供)：我的目标是开发一种基于细胞的技术，通过使用出生后可以通过非侵入性手段从患者身上获得的祖细胞，在体内设计血管组织。特别是，这项建议将重点放在联合使用骨髓间充质祖细胞(MPC)和人血源性内皮祖细胞(EPC)在体内形成脂肪组织。要测试的概念是，含有高度纯化和定义的MPC和EPC的植入物在体内植入后是否会导致专门的血管组织。我假设，要做到这一点，1)MPC和EPC都需要首先创建一个血管网络，允许通过植入物进行血液灌流；以及2)这种血管化将通过采用植入部位周围组织的表型，促进尚未组装到血管网络中的MPC经历适当的组织发育。在这项提案中，我将使用我们用人内皮祖细胞和间质干细胞建立的体内模型来确定关键参数，以将血管生成过程加速到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起源于人类，或者一旦功能血管网络被修复，它们是否从宿主被招募到植入物中作为内源性组织修复过程的一部分。 如果是这样的话。有了这些信息，我将实施策略，以增强体内分化过程，目的是拥有完全功能的组织。最后，我将测试这项基于细胞的技术，评估我们是否可以创造出长期存在的脂肪组织垫，随着时间的推移，脂肪组织垫的体积减少很少。解决这些目标的实验方法包括：1)使用基于荧光素酶的生物发光技术对植入物中的血液灌流进行活体成像；2)对移植细胞进行GFP标记，以跟踪它们对所产生的组织的特定贡献；3)共聚焦显微镜通过免疫荧光染色来评估植入物。我设想这个两个细胞、两个步骤的系统是一种使能技术，可以应用于许多不同的组织，在这些组织中，功能血管是必不可少的。 相关性：这是一项能够应用于组织/器官再生的技术，在这些组织/器官中，功能血管是必不可少的。这项建议将重点放在脂肪组织的形成上，但它将适用于再生医学的许多方面，包括组织工程和原位治疗性血管形成。

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.