VALIDATION OF A HUMAN CD34+ STEM CELL TOXICITY BIOASSAY

描述（由申请人提供）：此SBIR申请是为了响应NIEHS安全评估计划预测测试系统的目标而提交的，该计划通过开发、标准化和验证敏感和特定的新的和新颖的测试或测试组来实现。原代人CD34+骨髓干细胞将被用作一种新型生物系统，以识别改变的免疫系统分化、细胞毒性和遗传毒性的预测性生物标志物。人CD34+骨髓干细胞是具有自我更新、增殖和向不同血细胞谱系分化的潜能的多能细胞。人CD34+细胞可在体外定向分化并沿着免疫细胞的特定细胞谱系扩增。CD34+细胞的这种谱系特异性分化是一个复杂的生物学过程，包括驱动细胞增殖和获得专门细胞功能的遗传途径的组合和协调表达。基于人原代细胞的筛选测定可用于评估基于肿瘤细胞系的高通量筛选的预测能力或体外体内相关性。由于来源于肿瘤的细胞系通常缺乏调节细胞对应激源的反应的许多关键基因，因此在基于肿瘤细胞系的测定中测试的环境因子的结果可能与人类健康和疾病的相关性有限，并且需要基于人类原代细胞的筛选测定。第一阶段SBIR有三个具体目标：微型化人TK6细胞试验作为原型试验，以建立基于FCM的细胞毒性、细胞凋亡、细胞周期阻滞、氧化性DNA损伤和DNA双链断裂的测量，调整在人TK6细胞中建立的具有人CD 34+干细胞的生物学终点，建立人CD 34+干细胞沿沿着特定谱系分化的细胞和分子生物标志物，评估毒物对CD 34+健康状况和分化的影响。由于骨髓干细胞在造血和免疫系统的功能中起关键作用，并且是宿主人类癌症和疾病所关注的假定靶细胞群体，因此从这些系统获得的结果与人类疾病具有生物学相关性，并且可以外推至人类。本文提出的人CD34+干细胞多重检测非常适合作为评估环境因子和临床前候选药物毒性的平台，以及高通量检测计划的后续检测系统。公共卫生相关性：干细胞在人体所有细胞中具有独特的自我更新能力，也就是说，它们可以保持在原始的非特化状态。在适当的条件下，它们可以产生身体的专门细胞（分化），如心脏，肝脏或胰腺。CD34+干细胞是骨髓的干细胞，可分化为血液中的所有细胞（白色和红细胞）。因此，这些细胞提供了一个独特的模型系统来理解和评估环境因子和新药候选物的影响，以预测或预测人类的毒性。

DESCRIPTION (provided by applicant): This SBIR application is submitted in response to the objectives of the NIEHS Predictive Test Systems for Safety Evaluation Program by developing, standardizing, and validating sensitive and specific new and novel tests or batteries of tests . Primary human CD34+ bone marrow stem cells will be used as a novel biological system to identify predictive biomarkers of altered immune system differentiation, cellular toxicity, and genotoxicity. Human CD34+ bone marrow stem cells are pluripotent cells that possess the potential of self-renewal, proliferation, and differentiation toward different lineages of blood cells. Human CD34+ cells can be directed to differentiate in vitro and expand along specific cell lineages of immune cells. This lineage-specific differentiation of CD34+ cells is a complex biological process that includes the combinatorial and coordinated expression of genetic pathways that drive cellular proliferation and the acquisition of specialized cell functions. A screening assay based on human primary cells would be useful to assess the predictive power, or in vitro in vivo correlation, of high-throughput screens based on tumor cell lines. As cell lines derived from tumors often lack many critical genes that regulate cellular responses to stressors, the results of environmental agents tested in tumor cell line-based assays may have limited relevance to human health and disease, and there is a need for a human primary cell- based screening assay. This Phase I SBIR has three specific aims: Miniaturize human TK6 cell assay as a prototype assay to establish FCM-based measures of cytotoxicity, apoptosis, cell cycle arrest, oxidative DNA damage and DNA double strand breaks, adapt biological endpoints established in human TK6 cells with human CD34+ stem cells, establish cellular and molecular biomarkers of human CD34+ stem cell differentiation along specific lineages, assess impact of toxicants on CD 34+ health status and differentiation. Since bone marrow stem cells play a pivotal role in the function of the hematopoietic and immune systems and are the putative target cell population of concern for a host human cancers and diseases, the results obtained from these systems are biologically relevant to human disease and can be extrapolated to humans. The human CD34+ stem cell multiplex assay proposed here is ideally suited as platform to assess toxicity for environmental agents and pre-clinical drug candidates, as well a follow-up test system for high-throughput testing initiatives. PUBLIC HEALTH RELEVANCE: Stem cells have the unique ability among all of the cells of the human body of self- renewal, that is, they can remain in a primitive unspecialized state. Under the right conditions, they can give rise to specialized cells of the body (differentiation) like the heart, liver, or pancreas. CD34+ stem cells are the stem cell of bone marrow that differentiates into all of the cells in the blood (white and red blood cells). Therefore, these cells present a unique model system to understand and assess the effects of environmental agents and new drug candidates to predict or anticipate toxicity in humans.