Embryoid Body-derived Hematopoietic Stem Cell Lines

从小鼠胚胎干细胞(ES)培养中建立造血细胞，为研究造血祖细胞由造血前中胚层发育而来的早期承诺步骤提供了有力的途径。对发育中的类胚体的分析表明，这些培养物中的造血分化概括了子宫内造血起始的许多方面，包括血管生成和从原始程序到最终程序的转换。我们以前用我们的MSCV逆转录病毒载体转导的CCE ES细胞过表达T细胞急性淋巴细胞白血病的同源异型盒基因Hox11，建立了阻止在原始和最终造血发育的新阶段的因子依赖的造血祖细胞系。这些结果证明了概念的正确性，为目前提出的扩展这一策略以获得具有各种分化潜能的条件性阻断的小鼠造血前体细胞系提供了理论基础。我们特别感兴趣的是有条件地阻止常见的内皮/造血祖细胞(即血管母细胞)以及具有长期体内再繁殖活性的造血干细胞的可能性。为此，我们的具体目标是开发两种条件永生化系统：第一种是在腺病毒介导的Cre重组酶瞬时表达的基础上定点切除Hox11；第二种是基于四环素可调节的Hox11逆转录病毒载体。虽然这两种方法都将允许明确地建立前体-后代关系，但我们假设后一种策略将允许前体细胞的克隆后代在造血层的连续阶段被阻止和扩增。可以预见，获得的多潜能前体细胞系将在造血分化进程和谱系限制的分子生物学研究中具有广泛的实用价值。为了实现这一目标，克隆相关前体细胞的同源群体将作为起始材料，利用差异表达基因的基因芯片技术鉴定作为决定造血细胞命运的候选调控基因。预计从这些研究中获得的信息还将深入了解那些与造血转录调控有关的同源框基因的潜在作用，并有助于更好地理解Hox11介导的白血病启动的潜在机制。这些目标的成功实现将为未来涉及灵长类胚胎干细胞的研究努力提供基础。

The establishment of hematopoiesis in culture from murine embryonic stem (ES) cells provides a powerful approach for studying early commitment steps as hematopoietic precursors develop from pre-hematopoietic mesoderm. Analysis of developing embryoid bodies has demonstrated that hematopoietic differentiation in these cultures recapitulates many aspects of the onset of hematopoiesis in utero, including vasculogenesis and the switch from the primitive to the definitive program. We previously used CCE ES cells transduced with our MSCV retroviral vector overexpressing the diverged homeobox gene HOX11 of T-cell acute lymphoblastic leukemia to establish factor-dependent hematopoietic precursor cell lines arrested at novel stages of primitive and definitive hematopoietic development. These results, demonstrating proof-of-concept, provide the rationale for the present proposal to extend this strategy to obtain conditionally-blocked murine hematopoietic precursor cell lines with a variety of differentiative potentials. We are particularly interested in the possibility of conditionally arresting common endothelial/hematopoietic precursors (i.e., hemangioblasts) as well as hematopoietic stem cells with long- term in vivo repopulating activity. To this end, our specific aims are to develop two systems for conditional immortalization: the first, based on site-specific excision of HOX11 by adenovirus-mediated transient expression of Cre recombinase; and the second, based on a tetracycline-regulatable HOX11 retroviral vector. While both approaches will permit precursor-progeny relationships to be unequivocally established, we hypothesize that the latter strategy will allow clonal descendants of precursor cells to be arrested and amplified at successive stages of the hematopoietic hierarchy. It is envisioned that the multipotent precursor lines obtained will have broad utility for molecular biological investigations of hematopoietic differentiative progression and lineage restriction. Toward this goal, homogenous populations of clonally-related precursor cells will be used as starting material for identification by cDNA microarray technology of differentially expressed genes as candidate regulators of hematopoietic cell fate decisions. It is anticipated that the information gained from these studies will also provide insight into the potential roles of those homeobox genes implicated in the transcriptional control of hematopoiesis as well as lead to a better understanding of the underlying mechanism of leukemia initiation mediated by HOX11. Successful realization of these goals will provide a basis for future research endeavors involving primate ES cells.