Embryoid Body-derived Hematopoietic Stem Cell Lines

小鼠胚胎干细胞（ES）培养中造血功能的建立为研究造血前体从造血前中胚层发育的早期承诺步骤提供了有力的方法。对发育中的胚状体的分析表明，这些培养中的造血分化概括了子宫内造血开始的许多方面，包括血管发生和从原始程序到最终程序的转换。我们之前使用过表达t细胞急性淋巴细胞白血病分化同源盒基因HOX11的MSCV逆转录病毒载体转导的CCE ES细胞，建立了在原始和最终造血发育的新阶段被阻止的因子依赖性造血前体细胞系。这些结果证明了概念验证，为目前提出的扩展该策略以获得具有多种分化潜能的条件阻断小鼠造血前体细胞系提供了基本原理。我们对有条件抑制常见内皮/造血前体细胞（即成血管细胞）以及具有长期体内再生活性的造血干细胞的可能性特别感兴趣。为此，我们的具体目标是开发两种条件永生化系统：第一种，基于腺病毒介导的Cre重组酶的瞬时表达对HOX11进行定点切除；第二种是基于四环素可调节的HOX11逆转录病毒载体。虽然这两种方法都可以明确地建立前体-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次的连续阶段被捕获和扩增。预计所获得的多能前体系将在造血分化进展和谱系限制的分子生物学研究中具有广泛的用途。为了实现这一目标，同源的克隆相关前体细胞群体将作为起始材料，通过cDNA微阵列技术鉴定作为造血细胞命运决定候选调节因子的差异表达基因。预计从这些研究中获得的信息也将有助于深入了解那些参与造血功能转录控制的同源盒基因的潜在作用，并有助于更好地理解由HOX11介导的白血病起始的潜在机制。这些目标的成功实现将为未来灵长类动物胚胎干细胞的研究奠定基础。

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.