Location of indigenous human HSCs reveals functional properties of HSCs that are dictated by anatomic and cellular architecture of bone marrow II

Demonstration of hematopoietic stem cells (HSCs) was first shown in the mouse and was dependent on recipient bone marrow (BM) to support in vivo multilineage hematopoietic reconstitution, thereby illustrating non-cell-autonomous requirements for HSC functions. Murine studies have defined microanatomic compartments in the BM comprised of osteoblasts, mesenchymal cells, subsets of vasculature, and innervating neural cells functioning as an HSC-supportive niche. Despite the potential clinical applications, analyses of putative HSCs in the BM of humans has not been examined. Here, using human bone biopsies, we provide evidence of HSC propensity to endosteal regions of Trabecular Bone Area (TBA). Independent of phenotypic definitions based on prospective isolation, functional studies indicate that human HSCs residing in the TBA of human and transplanted recipients had superior regenerative and self-renewal capacity and are molecularly distinct to those repopulating the Long Bone Area (LBA). Consistent with the non-cell-autonomous nature of HSC function, osteoblasts in the TBA possess unique characteristics and expressed a key network of factors including those involving Notch activity which could regulate TBA vs. LBA location of human HSCs in vivo. Our study illustrates that human-mouse xenografts provide a surrogate to indigenous human HSC in the BM, and demonstrates that BM architecture plays a critical role in defining functional properties of human HSCs. Total bone marrow cells were collected from NOD/SCID recipient adult mice. Osteoblast cells (OB) were sorted based on five paramater markers combination Lin- CD45- CD31- Sca1- CD51+ using a FACSAria II (BD). Total RNA from purified populations was extracted and amplified as described previously (Shojaei et al., 2005). Amplified-labeled RNA was hybridized to HG-U133Plus v2.0 chip.

造血干细胞（hematopoietic stem cells, HSCs）的功能首次在小鼠模型中得到证实，其依赖受体骨髓（bone marrow, BM）支持体内多谱系造血重建，由此揭示了HSC功能存在非细胞自主性需求。小鼠相关研究已明确骨髓内的显微解剖微区室，其由成骨细胞、间充质细胞、血管亚群及参与神经支配的神经细胞构成，作为支持HSC的造血微环境发挥功能。尽管该领域具有潜在临床应用价值，但针对人类骨髓内推定HSC的相关分析尚未得到系统开展。本研究通过人类骨活检样本，证实了HSC倾向定位于骨小梁区域（Trabecular Bone Area, TBA）的骨内膜区域。无需依赖基于前瞻性分离的表型定义，功能研究表明，定位于人类及移植受体骨小梁区域的人类HSC，其再生与自我更新能力更优，且分子特征与长骨区域（Long Bone Area, LBA）内的重植HSC存在显著差异。与HSC功能的非细胞自主性特性一致，骨小梁区域的成骨细胞具有独特特征，并表达包括Notch信号通路相关因子在内的关键调控网络，这些因子可在体内调控人类HSC在骨小梁区域与长骨区域的定位。本研究证实，人-小鼠异种移植模型可作为人类骨髓内原生HSC的替代研究体系，并证明骨髓结构在定义人类HSC功能特性中发挥关键作用。本研究收集了NOD/SCID成年受体小鼠的全骨髓细胞。基于Lin⁻ CD45⁻ CD31⁻ Sca1⁻ CD51⁺这五个参数的组合标记，通过FACSAria II（BD公司）分选得到成骨细胞（osteoblast cells, OB）。如既往研究所述（Shojaei等，2005），我们对纯化群体的总RNA进行了提取与扩增。将扩增标记后的RNA与HG-U133Plus v2.0芯片进行杂交。