Genomic approach to investigating ex vivo hematopoiesis

描述（由申请人提供）：自体或同种异体干细胞挽救的高剂量化疗导致全血细胞减少症延长，伴有感染和出血并发症，需要抗生素和输血治疗，有时延长住院时间。输注大量离体扩增的造血细胞-作为常规自体或同种异体移植物的补充-具有关闭中性粒细胞减少症和/或血小板减少症的窗口的潜力。此外，最近发现的造血干细胞的可塑性表明，这些容易获得的干细胞可用于产生用于非造血细胞和基因治疗的自体或同种异体细胞和组织。这种疗法的成功取决于产生大量具有所需的治疗依赖性细胞分化状态的细胞的能力。尽管基础生物学和应用生物学取得了很大的进展，但这仍然是一个难以捉摸的任务。培养条件，如细胞因子组合和呈递、氧张力（pC 2）和pH，改变干细胞和祖细胞的分化和增殖，具有显著的患者间变异性。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，我们知之甚少。这种知识对于离体和体内结果都具有很大的预测和诊断潜力。因此，将使用8，300个基因的DNA微阵列对体外扩增的人原代髓系细胞的转录程序（由CD 34+细胞启动）进行全面检查，并使用标准分子生物学工具进一步探索关键发现。研究包括检查的时间和差异转录程序的G和Mk细胞培养下的高或低的pC 2和/或pH值，并与不同的细胞因子组合。具体的问题，以审查包括在何种程度上细胞凋亡与Mk分化;如果Mk细胞凋亡采用类似的机制，一般的细胞凋亡;为什么，在Mk文化相反，有这样一个低水平的细胞凋亡在G文化。此外，基因聚类和调节网络技术应用于DNA微阵列数据可能会导致发现未知的MK和G分化基因。这些实验将为将来的研究提供基础，在这些研究中，临床标本可以在临床干细胞移植的背景下进行检查。

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.