Genomic approach to investigating ex vivo hematopoiesis

描述(申请人提供)：大剂量化疗联合自体或异体干细胞挽救会导致长期的全血细胞减少，并伴有感染和出血并发症，需要抗生素和输血治疗，有时还会延长住院时间。大量体外扩增的造血细胞的输注--作为传统自体或异体移植的补充--有可能关闭中性粒细胞减少和/或血小板减少的窗口。此外，最近发现的造血干细胞的可塑性表明，这些现成的干细胞可能被用来产生自体或同种异体细胞和组织，用于非造血细胞和基因治疗。这种疗法的成功取决于产生大量细胞的能力，这些细胞具有所需的、依赖于治疗的细胞分化状态。尽管基础生物学和应用生物学取得了巨大进展，但这仍然是一项难以捉摸的任务。培养条件，如细胞因子组合和呈递、氧分压(PC2)和pH值，会改变干细胞和祖细胞的分化和增殖，患者之间的差异很大。关于这些效应的潜在分子生物学，特别是关于分化过程中的大规模转录程序，人们知之甚少。这种知识对体外和体内结果都有很大的预测和诊断潜力。因此，将使用8,300基因DNA微阵列对体外扩增的人类初级髓系细胞的转录程序进行全面检查-由CD34细胞启动-将使用标准分子生物学工具进一步探索关键发现。研究包括检测在高或低PC2和/或pH以及不同细胞因子组合下培养的G细胞和Mk细胞的时间和差异转录程序。要研究的具体问题包括：细胞凋亡与Mk分化有关的程度；Mk细胞凋亡是否采用了类似于一般细胞凋亡的机制；以及为什么与Mk细胞培养相反，G细胞培养中的细胞凋亡率如此低。此外，应用于DNA微阵列数据的Gone聚类和调控网络技术可能导致未知的Mk和G分化基因的发现。这些实验将为未来的研究提供基础，在这些研究中，可以在临床干细胞移植的背景下检查临床标本。

DESCRIPTION (provided by applicant): High-dose chemotherapy with autologous or allogeneic stem-cell rescue results in prolonged pancytopenia that is accompanied by infectious and bleeding complications requiring antibiotic and transfusion therapy and, at times, prolonged hospitalization. Infusion of large numbers of ex vivo expanded hematopoietic cells -as a supplement to the conventional auto- or allograft - has the potential to close the window of neutropenia and/or thrombocytopenia. Furthermore, the recently discovered plasticity of hematopoietic stem cells suggests that these readily available stem cells may be used for generating autologous or allogeneic cells and tissues for non-hematopoietic cell and gene therapies. Success of such therapies depends on the ability to generate large numbers of cells with the desired, therapy-dependent state of cell differentiation. This remains an elusive task despite the great progress in basic and applied biology. Culture conditions, such as cytokine combinations and presentation, oxygen tension (pC2) and pH, alter stem- and progenitor-cell differentiation and proliferation with substantial patient-to-patient variability. Little is known about the underlying molecular biology of these effects, and specifically, about the large-scale transcriptional program during differentiation. Such knowledge has large predictive and diagnostic potential for both ex vivo and in vivo outcomes. Thus, a comprehensive examination of the transcriptional program of ex vivo expanded human primary myeloid cells - initiated with CD34+ cells - will be examined using 8,300-gene DNA microarrays, and key findings further explored using standard molecular-biology tools. Studies include examination of the temporal and differential transcriptional program of G and Mk cells cultured either under high or low pC2 and/or pH, and with different cytokine combinations. Specific issues to be examined include the extent to which apoptosis is linked to Mk differentiation; if Mk apoptosis employs a machinery similar to that of general apoptosis; and why, in contrast to Mk cultures, there is such a low level of apoptosis in G cultures. Furthermore, gone-clustering and regulatory-network techniques applied to DNA-microarray data may lead to the discovery of unknown Mk- and G-differentiation genes. These experiments will provide the basis for future studies in which clinical specimens could be examined in the context of clinical stem cell transplantation.