Embryoid Body-derived Hematopoietic Stem Cell Lines

从小鼠胚胎干（ES）细胞培养中建立造血提供了一个强大的方法来研究早期承诺的步骤，造血前体细胞从前造血中胚层发展。 对发育中的胚状体的分析表明，这些培养物中的造血分化概括了子宫内造血发生的许多方面，包括血管发生和从原始程序到最终程序的转换。 我们先前使用过表达T细胞急性淋巴细胞白血病的趋异同源盒基因HOX 11的MSCV逆转录病毒载体转导的CCE ES细胞，以建立在原始和永久造血发育的新阶段被捕的因子依赖性造血前体细胞系。 这些结果证明了概念验证，为本提议提供了基本原理，以扩展该策略以获得具有多种分化潜能的条件阻断的鼠造血前体细胞系。 我们特别感兴趣的是有条件地阻止常见的内皮/造血前体（即，成血管细胞）以及具有长期体内再增殖活性的造血干细胞。 为此，我们的具体目标是开发两个系统的条件永生化：第一，基于位点特异性切除HOX 11的腺病毒介导的Cre重组酶的瞬时表达;和第二，基于四环素可调控的HOX 11逆转录病毒载体。 虽然这两种方法都将允许明确建立克隆-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逮捕和扩增。 可以预见，获得的多能前体细胞系将具有广泛的用途，造血分化进程和谱系限制的分子生物学研究。 为了实现这一目标，克隆相关前体细胞的同质群体将被用作通过cDNA微阵列技术鉴定差异表达基因的起始材料，所述差异表达基因作为造血细胞命运决定的候选调节因子。 预计从这些研究中获得的信息也将提供深入了解那些参与造血转录控制的同源框基因的潜在作用，并导致更好地理解HOX 11介导的白血病起始的潜在机制。 这些目标的成功实现将为灵长类ES细胞的未来研究奠定基础。

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.