Gronthos S, Zannettino A C, Graves S E, Ohta S, Hay S J, Simmons P J
Matthew Roberts Laboratory, Leukemia Research Unit, Hanson Center for Cancer Research, I.M.V.S., Adelaide, South Australia, Australia.
J Bone Miner Res. 1999 Jan;14(1):47-56. doi: 10.1359/jbmr.1999.14.1.47.
Human osteoblast-like cells can be readily cultured from explants of trabecular bone, reproducibly expressing the characteristics of cells belonging to the osteoblastic lineage. Dual-color fluorescence-activated cell sorting was employed to develop a model of bone cell development in primary cultures of normal human bone cells (NHBCs) based on the cell surface expression of the stromal precursor cell marker STRO-1 and the osteoblastic marker alkaline phosphatase (ALP). Cells expressing the STRO-1 antigen exclusively (STRO-1+/ALP-), were found to exhibit qualities preosteoblastic in nature both functionally by their reduced ability to form a mineralized bone matrix over time, as measured by calcium release assay, and in the lack of their expression of various bone-related markers including bone sialoprotein, osteopontin, and parathyroid hormone receptor based on reverse trancriptase polymerase chain reaction (PCR) analysis. The majority of the NHBCs which expressed the STRO-1-/ALP+ and STRO-1-/ALP- phenotypes appeared to represent fully differentiated osteoblasts, while the STRO-1+/ALP+ subset represented an intermediate preosteoblastic stage of development. All STRO-1/ALP NHBC subsets were also found to express the DNA-binding transcription factor CBFA-1, confirming that these cultures represent committed osteogenic cells. In addition, our primer sets yielded four distinct alternative splice variants of the expected PCR product for CBFA-1 in each of the STRO-1/ALP subsets, with the exception of the proposed preosteoblastic STRO-1+/ALP- subpopulation. Furthermore, upon re-culture of the four different STRO-1/ALP subsets only the STRO-1+/ALP- subpopulation was able to give rise to all of the four subsets yielding the same proportions of STRO-1/ALP expression as in the original primary cultures. The data presented in this study demonstrate a hierarchy of bone cell development in vitro and facilitate the study of bone cell differentiation and function.
人成骨样细胞可轻松从小梁骨外植体培养获得，可重复性地表达成骨细胞谱系细胞的特征。采用双色荧光激活细胞分选技术，基于基质前体细胞标志物STRO-1和成骨细胞标志物碱性磷酸酶（ALP）的细胞表面表达，建立正常人骨细胞（NHBC）原代培养中的骨细胞发育模型。仅表达STRO-1抗原的细胞（STRO-1+/ALP-），通过钙释放试验测定，发现其随着时间推移形成矿化骨基质的能力降低，在功能上表现出前成骨细胞的性质，并且基于逆转录聚合酶链反应（PCR）分析，其缺乏包括骨唾液蛋白、骨桥蛋白和甲状旁腺激素受体在内的各种骨相关标志物的表达。大多数表达STRO-1-/ALP+和STRO-1-/ALP-表型的NHBC似乎代表完全分化的成骨细胞，而STRO-1+/ALP+亚群代表发育的中间前成骨细胞阶段。还发现所有STRO-1/ALP NHBC亚群均表达DNA结合转录因子CBFA-1，证实这些培养物代表定向成骨细胞。此外，我们的引物组在每个STRO-1/ALP亚群中产生了CBFA-1预期PCR产物的四种不同可变剪接变体，但提议的前成骨细胞STRO-1+/ALP-亚群除外。此外，在对四个不同的STRO-1/ALP亚群进行再培养时，只有STRO-1+/ALP-亚群能够产生所有四个亚群，产生与原始原代培养中相同比例的STRO-1/ALP表达。本研究中呈现的数据证明了体外骨细胞发育的层次结构，并有助于骨细胞分化和功能的研究。
