Endothelial Progenitor Cells for Assessing Toxicological Response Variability in Humans

 说明(申请人提供)：毒理学风险评估缺乏对人类对环境毒物反应的大小和变异性的直接估计。具体地说，关于低剂量毒物对包括细胞分化在内的人类发育过程的影响的数据非常少。这是因为目前毒理学评估的基础，即动物和永生化细胞系，不能充分模拟低水平毒物暴露对人类种群的影响。替代检测平台应能够以高通量的方式将人类的可变性纳入大量毒物的筛选。为了建立这样一个平台，我们建议使用人类供者特有的细胞，这种细胞可以(A)以非侵入性方式分离，(B)易于在培养中扩增，以及(C)冷冻保存而不丧失活力。我们的初步数据表明，人类内皮祖细胞或内皮细胞集落形成细胞(ECFCs)，即在循环血液中发现的CD31/CD34多能细胞群，将满足这些要求。ECFC在体外高度增殖，可分化为成熟的内皮细胞。这项研究的总体目标是评估 化学毒物对来自不同个体的ECFC活性、增殖和分化的影响。在我们最近成功完成第一阶段计划之前，我们使用专有的细胞生长介质补充剂建立了ECFC的细胞培养参数，并使用低剂量电离辐射(LDIR)完成了初步的毒理学风险评估研究。这些研究证实了ECFC在毒理学风险评估筛选中的效用，以及它们广泛代表人类人口预期的不同反应的能力。第一阶段研究通过使用双酚A(BPA)、全氟辛酸(PFOA)和镉(Cd)进行ECFC毒理学测试，成功地扩大了这些发现。发现ECFC对双酚A和环糊精敏感，对全氟辛酸不敏感，表现出供体特定的增殖率差异。BPA和Cd还以供体特有的方式诱导活性氧(ROS)产生。ECFC在暴露于毒物后会经历氧化应激和细胞周期停滞。与毒物孵育更长的时间后，ROS产生的增加与细胞死亡相关。我们发现这些毒物 在一项新开发的分化试验中，我们还在蛋白水平上分析了内皮特异性细胞标志物的影响表达。第二阶段将专注于继续开发这一筛选平台，以开发一种经过验证的基于高内容成像的定量屏幕。还将把该平台作为一种服务进行商业化，并生产用于ECFC培养和随后的毒物分析的试剂盒。最后，筛选将扩大到包括毒物诱导的ECFC分化为成熟内皮细胞的影响。

 DESCRIPTION (provided by applicant): Toxicological risk assessment lacks direct estimation of the magnitude and variability of human responses to environmental toxicants. Specifically, there is very little data on the effects of low dose toxicants on human developmental processes, including cellular differentiation. That is because the current basis of toxicological assessment, animals and immortalized cell lines, cannot adequately model the impact of low level toxicant exposure on human populations. An alternative detection platform should enable incorporation of human variability in screening of a large number of toxicants in a high-throughput manner. To establish such a platform, we propose using human donor-specific cells that can be (a) isolated in a non-invasive manner, (b) easily expanded in culture, and (c) cryopreserved without loss of viability. Our preliminary data suggest that human endothelial progenitor cells, or endothelial colony forming cells (ECFCs), a population of CD31+/CD34+ pluripotent cells found in circulating blood, would fulfill these requirements. ECFCs are highly proliferative in vitro and ca differentiate into mature endothelial cells (ECs). The overall goal of this research is to evaluate the effect of chemical toxicants on viability, proliferation, and differentiation of ECFCs derived from different individuals. Prior to our recent and successful completion of a Phase I program, we established cell culture parameters for ECFCs using a proprietary cell growth media supplement and completed initial toxicological risk assessment studies using low-dose ionizing radiation (LDIR). These studies confirmed the utility of ECFCs in toxicological risk assessment screening as well as their ability to broadly represent varying responses expected in human populations. Phase I studies successfully expanded these findings by performing ECFC toxicology assays using Bisphenol A (BPA), perfluorooctanoic acid (PFOA) and cadmium (Cd). ECFCs were found to be sensitive to BPA and Cd and less sensitive to PFOA, exhibiting donor-specific variability in proliferation rates. BPA and Cd also induced reactive oxygen species (ROS) production in a donor-specific manner. ECFCs were shown to experience oxidative stress and cell cycle arrest after exposure to toxicants. Elevated ROS production was shown to correlate with cell death after longer incubation times with toxicants. We found that the toxicants we analyzed also affected expression of endothelial-specific cell markers at a protein level in a newly developed differentiation assay. Phase II will focus on the continued development of this screening platform toward a validated quantitative high content imaging-based screen. Commercialization of the platform as a service will also be pursued as well as the production of kits for ECFC culture and subsequent toxicological analysis. Finally, the screen will be expanded to include toxicant-induced effects on ECFC differentiation into mature endothelial cells.