"In vivo reconstitution of the hematopoietic niche"

描述(由申请人提供) 摘要：尽管造血干细胞(HSCs)具有通过骨髓移植重建造血系统的能力，具有巨大的临床应用价值，但由于HSCs的稀缺性，其应用受到限制。因此，体外扩增造血干细胞的能力将具有重大的临床影响，但到目前为止，大多数培养系统倾向于扩增承诺的祖细胞，而不是增加具有自我更新能力的干细胞数量。在体内，HSCs严重依赖于骨髓微环境，而成骨细胞现在被认为是其中的重要组成部分。成骨细胞系是一个异质性的群体，越来越多的证据表明，处于成骨分化特定阶段的细胞可能参与了不同的造血生态位。此外，尽管一些研究指出成熟的成骨细胞是HSC生态位的关键组成部分，但其他报告强调了间充质前体细胞的支持潜力。准确识别支持造血干细胞的细胞成分将增加在共培养系统中成功体外扩增造血干细胞的可能性。然而，存在两个主要障碍：1)由于缺乏表征良好的细胞表面标志物，无法分离大量在成骨细胞分化的特定阶段浓缩的细胞；2)缺乏对细胞群体重建骨和造血生态位能力的体内严格检测。我们现在提出了一种新的方法来规避这些问题，通过使用来自小鼠的诱导多能干细胞(IPS)携带成骨细胞分化的荧光报告来获取大量的成骨前体细胞和成熟的成骨细胞。此外，我们还开发了胚胎发育过程中体内骨骼互补的模型，利用该模型，我们可以分析成骨细胞谱系中特定阶段的成骨细胞在活生物体中支持造血干细胞的能力。我们预计，这些技术的成功应用将显著提高我们目前体外扩增造血干细胞的能力，最终目标是增加可以从干细胞移植中受益的患者数量。 公共卫生相关性：扩大造血干细胞(HSCs)数量的能力将极大地增加可从救命干细胞移植中受益的患者数量。在骨髓中，造血干细胞由成骨细胞支持。这项建议旨在将诱导的多能干细胞分化为不同阶段的成骨细胞，以确定哪个阶段对HSCs提供最大的支持。

DESCRIPTION (Provided by the applicant) Abstract: Although hematopoietic stem cells (HSCs) have tremendous clinical utility by virtue of their ability to reconstitute the hematopoietic system by bone marrow transplantation, their benefit is limited by the scarcity of HSCs. The ability to expand HSCs ex vivo would therefore be of significant clinical impact, but to date most culture systems favor expansion of committed progenitors rather than increasing numbers of stem cells with self-renewal capacity. In vivo, HSCs are critically dependent upon the bone marrow microenvironment, of which osteoblasts are now recognized as a crucial component. The osteoblast lineage is a heterogeneous population, and growing evidence suggests that cells at specific stages of osteogenic differentiation may participate in distinct hematopoietic niches. Furthermore, although several studies point to mature osteoblasts as a key component of the HSC niche, other reports highlight the supporting potential of mesenchymal progenitor cells. The identification of the precise cellular components within the niche that support HSCs would enhance the likelihood of successfully expanding HSCs ex vivo in a co-culture system. However, two major barriers exist: 1) the inability to isolate large numbers of cells enriched at defined stages of osteoblast differentiation, for lack of well-characterized cell surface markers, and 2) the absence of a rigorous in vivo assay for the capacity of a cellular population to reconstitute bone and the hematopoietic niche. We now propose a novel approach to circumvent these issues, by using induced pluripotent stem (iPS) cells derived from mice carrying fluorescent reporters of osteoblast differentiation to harvest abundant numbers of osteoblast progenitors and mature osteoblasts. Furthermore we have developed a model of in vivo skeletal complementation during embryonic development, with which we can assay specific stages of the osteoblast lineage for their ability to support HSCs in a living organism. We anticipate that the successful application of these techniques will offer significant improvement in our current abilities to expand HSCs ex vivo, with the ultimate goal of increasing the number of patients that can benefit from stem cell transplantation. Public Health Relevance: The ability to expand the number of hematopoietic stem cells (HSCs) would greatly increase the number of patients who can benefit from life-saving stem cell transplantation. Within the bone marrow HSCs are supported by bone-forming osteoblasts. This proposal seeks to differentiate induced pluripotent stem cells into different stages of osteoblasts to determine which provide the greatest support to HSCs.