Tracking dynamic interactions between haematopoietic stem cells and osteoblasts: molecular regulation of stem cell position and fate

大多数血细胞是短暂的，通过几个承诺和分化步骤，不断被血液干细胞产生的新后代的平衡生产所取代。众所周知，血细胞是静止的，相对于它们的后代来说，增殖的频率很低。当它们这样做时，它们会产生更多的干细胞(自我更新)或分化的后代。血液干细胞通过感知组织损伤并调整后代以适应生理和应激诱导的需求，微调静止、自我更新和分化之间的平衡。正如舒适的家有助于家庭幸福，工作环境影响员工的表现一样，血液干细胞在骨髓空间内特定的壁龛中的正确定位对于确保它们的正常运作至关重要。一些研究表明，造骨细胞，即成骨细胞，在调节血液干细胞的数量和功能方面具有重要作用；然而，少数旨在确定血液干细胞在骨髓中的位置的研究报告了广泛的位置，即使大多数观察到的细胞在成骨细胞附近，也只有少数明显地直接接触。由于在无脊椎动物模式生物或不同哺乳动物组织中观察到的许多干细胞生态位通过干细胞和生态位细胞之间的直接相互作用发挥作用，因此，如果成骨细胞真的存在这样的生态位，是否真的是血液干细胞生态位的主要组成部分，就引起了争议。我最近开发了一种体内成像方法，使我能够在移植的血液干细胞注入血液后立即可视化它们在骨髓空间内的情况，我可以在几天后‘观察’相同细胞的行为。我收集的证据表明，打开或关闭移植的血液干细胞内的某些分子信号可以通过影响它们的定位来调节它们的功能。目前的建议旨在应用和进一步发展血液干细胞的活体成像，以获得关于血液干细胞利基的存在、位置和功能的明确答案。我们将使用在所有成骨细胞中过度表达Wnt抑制物Dkk1的过度生理水平的转基因小鼠作为我们的工作模型，这些成骨细胞已被证明降低了血液干细胞功能，并特别是自我更新能力。我们知道移植的血液干细胞在DKK骨髓中定位异常，我们计划测试这种定位改变是否是它们故障的原因。我们将对移植的干细胞进行详细的时间推移跟踪，以询问离成骨细胞更近的细胞是否比离成骨细胞更远的细胞更静止(不太可能分裂)，我们将利用新的光转换技术来标记驻留在不同位置的干细胞及其后代，并随后比较它们的功能。此外，由于目前的体内成像技术不允许亚细胞分辨，我们将研究正常或DKK小鼠的血液干细胞和成骨细胞共培养是否可以作为替代系统，以更详细地研究至少在小鼠中观察到的一些事件，并使用它们来密切跟踪正常和不利条件下血液干细胞的行为和形态。我们将对来自自我更新允许或抑制培养或小鼠的基质和干细胞进行全基因组表达分析，并将重点放在调节细胞-细胞和细胞-环境相互作用的已知基因上。因此，我们将对成骨细胞和血液干细胞之间的关系有一个全面的了解，我们将调查干细胞位置是否直接影响功能，以及我们将了解这个过程涉及哪些分子。

Most blood cells are short lived and are constantly replaced by the balanced production of new progeny arising from blood stem cells through several commitment and differentiation steps. Blood cells are known to be quiescent and proliferate infrequently relative to their progeny. When they do so, they give rise to more stem cells (self-renewal) or to differentiating progeny. Blood stem cells finely tune the balance between quiescence, self-renewal and differentiation by sensing tissue damage and adjusting progeny generation to physiological and stress-induced demand. In the same way that a comfortable home contributes to family's happiness and the work environment influences employees' performance, correct positioning of blood stem cells in specific niches within the bone marrow space is crucial to ensure their proper functioning. Several studies indicate that bone-making cells, called osteoblasts, have an important role in regulating blood stem cell numbers and function; however, the few studies aimed at pinpointing blood stem cell location within the bone marrow have reported a wide range of positions and even when most observed cells are in the vicinity of osteoblasts only few are clearly in direct contact. As a number of stem cell niches observed in invertebrate model organisms or in different mammalian tissues function through direct interaction between the stem and niche cells, a controversy has arisen whether osteoblasts truly are the main component of the blood stem cell niche, provided such a niche exists at all. I have recently developed an in vivo imaging methodology that allows me to visualise transplanted blood stem cells within the bone marrow space immediately after their injection into the blood, and I can 'watch' the behaviour of the same cells until few days later. I have collected evidence that switching on or off certain molecular signals within transplanted blood stem cells can modulate their function by means of affecting their positioning. The current proposal seeks to apply and further develop in vivo imaging of blood stem cells in order to gain a definite answer on the existence, location and function of the blood stem cell niche. We will use as our working model transgenic mice expressing over-physiological levels of the Wnt inhibitor Dkk1 in all their osteoblasts, which have been shown to have decreased blood stem cell function and specifically self-renewal ability. We know that transplanted blood stem cells localize abnormally in Dkk bone marrow, and we plan to test whether this altered positioning is the cause of their malfunction. We will perform detailed time-lapse tracking of transplanted stem cells to ask whether cells closer to osteoblasts are more quiescent (less likely to divide) compared to those further away, and we will take advantage of novel photoconversion techniques to tag stem cells and their progeny residing in different locations and subsequently compare their function. Moreover, as current in vivo imaging technologies do not allow sub-cellular resolution, we will investigate whether co-culture of blood stem cells and osteoblasts from normal or Dkk mice can be used as a surrogate system to study in greater detail at least some of the events observed in the mice, and we will use them to closely track blood stem cells behaviour and morphology under normal and disadvantageous conditions. We will perform genome wide expression analysis of stroma and stem cells from self-renewal permissive or repressive cultures or mice, and we will focus on genes known to regulate cell-cell and cell-environment interactions. As a result, we will gain a comprehensive picture of the relationship between osteoblasts and blood stem cells, we will investigate whether stem cell position directly affects function, and we will learn what molecules are involved in the process.