"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）凭借其通过骨髓移植重建造血系统的能力而具有巨大的临床用途，但其益处受到HSCs稀缺的限制。因此，离体扩增HSC的能力将具有显著的临床影响，但迄今为止，大多数培养系统有利于扩增定向祖细胞，而不是增加具有自我更新能力的干细胞的数量。在体内，HSC严重依赖于骨髓微环境，成骨细胞现在被认为是骨髓微环境的重要组成部分。成骨细胞系是一个异质群体，越来越多的证据表明，在成骨分化的特定阶段的细胞可能参与不同的造血生态位。此外，尽管一些研究指出成熟成骨细胞是HSC生态位的关键组成部分，但其他报告强调了间充质祖细胞的支持潜力。识别支持HSC的小生境内的精确细胞组分将提高在共培养系统中成功地离体扩增HSC的可能性。然而，存在两个主要障碍：1）由于缺乏良好表征的细胞表面标志物，无法分离在成骨细胞分化的限定阶段富集的大量细胞，和2）缺乏对细胞群重建骨和造血生态位的能力的严格体内测定。我们现在提出了一种新的方法来规避这些问题，通过使用诱导多能干细胞（iPS）细胞来源于小鼠携带成骨细胞分化的荧光报告收获大量的成骨细胞祖细胞和成熟的成骨细胞。此外，我们已经开发了一个模型，在胚胎发育过程中，在体内骨骼互补，我们可以测定特定阶段的成骨细胞谱系的能力，以支持HSC在一个活的有机体。我们预计，这些技术的成功应用将大大提高我们目前体外扩增HSC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（HSC）数量的能力将大大增加可以从挽救生命的干细胞移植中受益的患者数量。在骨髓中，HSC由成骨细胞支持。该提案旨在将诱导多能干细胞分化为成骨细胞的不同阶段，以确定哪一个为HSC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.