The analysis of cellular mechanisms underlying haematopoietic stem cell (HSC) development using a novel in vitro system

哺乳动物的各种红细胞和白细胞都是由骨髓中的一小部分细胞产生的。在这个项目中，我们将研究血液干细胞是如何在哺乳动物胚胎中产生的。血液干细胞在被称为AGM区的区域发育，该区域包括一条大血管(背主动脉)以及胚胎性腺和肾脏。这里将提出两个主要问题：i)胚胎中发育成血液干细胞的细胞是什么？以及ii)诱导或促进这一过程的辅助细胞是什么？直到最近，由于缺乏适当的实验模型，还不可能解决这些问题。我们已经开发了一种实验系统，允许我们培养和操纵小鼠AGM区域的未成熟细胞，并产生血液干细胞。该系统将被用来测试不同类型的细胞发育成血液干细胞或支持干细胞形成的能力。为此，来自胚胎的不同类型的细胞将被特定的试剂(抗体)染色，并由一种名为荧光激活细胞分类器(FACS)的机器进行分离。然而，某些重要的细胞类型不能用抗体来分离。因此，我们将使用实验室产生的转基因小鼠。在两个这样的转基因小鼠模型中，已知与干细胞发育相关的AML1和HOXB4基因被绿色和黄色荧光报告分子标记。这些荧光标记将使我们能够通过流式细胞仪分离AML1和HOXB4细胞，并分析它们在培养系统中干细胞中的发育能力。该项目的一个补充和重要方面是研究使用我们的培养系统从胚胎干细胞(称为ES细胞)生成血液干细胞的可能性。胚胎干细胞可以用来培育完整的成年小鼠。因此，它们能够在小鼠体内产生所有类型的细胞，包括血液干细胞。在研究人员以前使用的传统培养条件下，ES细胞只能产生成熟的血细胞，而不能产生血液干细胞。使用我们的新系统，通过共培养ES细胞和AGM细胞，我们将能够确定ES细胞是否能够在AGM细胞的影响下产生血液干细胞。在这里，我们将再次通过流式细胞仪从培养的ES细胞中分离出细胞群，并在该系统中进行测试。该项目不仅将更好地了解血液干细胞的生物学，而且还将更好地了解其他干细胞。这也将对血液干细胞在培养中的产生和繁殖具有重要的实际意义。

The entire variety of red and white blood cells in mammals is generated by a small number of cells in the bone marrow. In this project we will investigate how blood stem cells are generated in the mammalian embryo. Blood stem cells develop in the area called the AGM region which encompasses a large vessel (the dorsal aorta) and embryonic gonads and kidneys. Two main questions will be asked here: i) what are the cells in the embryo which develop into blood stem cells? and ii) what are the helper cells which induce or facilitate this process? Until recently, owing to lack of an adequate experimental model it has not been possible to address these issues. We have developed an experimental system which allows us to culture and manipulate immature cells from the mouse AGM region and generate blood stem cells. This system will be used to test the ability of different cell types to develop into blood stem cells or to support stem cell formation. To this end different cell types from the embryo will be stained with specific reagents (antibodies) and isolated by a machine called fluorescence-activated cell sorter (FACS). However, certain important cell types cannot be isolated using antibodies. Therefore we will use genetically modified transgenic mice generated in the laboratory. In two such transgenic mouse models AML1 and HoxB4 genes known to be associated with stem cell development are labelled with green and yellow fluorescent reporter molecules. These fluorescent tags will allow us to isolate AML1 and HoxB4 cells by FACS and analyse their ability to develop in stem cells in the culture system. A complementary and important aspect of this project is to investigate the possibility of using our culture system for the generation of blood stem cells from embryonic stem cells (known as ES cells). ES cells can be used to produce entire adult mice. They are thus capable of generating all cell types in the mouse including blood stem cells. In traditional culture conditions previously used by researchers ES cells could only produce mature blood cells but not blood stem cells. Using our novel system by co-culturing ES cells and AGM cells we will be able to determine whether ES cells can generate blood stem cells under the influence of AGM cells. Here again we will isolate cell populations from cultured ES cells by FACS and test them in this system. This project will provide better understanding not only of biology of blood stem cells but also of other stem cells. It will also have important practical implications for generation and propagation of blood stem cells in culture.