Tissue Engineering Group, National Orthopaedics Centre of Excellence in Research & Learning, Department of Orthopaedic Surgery, Faculty of Medicine, University of Malaya, Kuala Lumpur, Malaysia.
J Anat. 2013 Apr;222(4):437-50. doi: 10.1111/joa.12032.
Mesenchymal stem cells (MSCs) are recognized by their plastic adherent ability, fibroblastic-like appearance, expression of specific surface protein markers, and are defined by their ability to undergo multi-lineage differentiation. Although rabbit bone marrow-derived MSCs (rbMSCs) have been used extensively in previous studies especially in translational research, these cells have neither been defined morphologically and ultrastructurally, nor been compared with their counterparts in humans in their multi-lineage differentiation ability. A study was therefore conducted to define the morphology, surface marker proteins, ultrastructure and multi-lineage differentiation ability of rbMSCs. Herein, the primary rbMSC cultures of three adult New Zealand white rabbits (at least 4 months old) were used for three independent experiments. rbMSCs were isolated using the gradient-centrifugation method, an established technique for human MSCs (hMSCs) isolation. Cells were characterized by phase contrast microscopy observation, transmission electron microscopy analysis, reverse transcriptase-polymerase chain reaction (PCR) analysis, immunocytochemistry staining, flow cytometry, alamarBlue(®) assay, histological staining and quantitative (q)PCR analysis. The isolated plastic adherent cells were in fibroblastic spindle-shape and possessed eccentric, irregular-shaped nuclei as well as rich inner cytoplasmic zones similar to that of hMSCs. The rbMSCs expressed CD29, CD44, CD73, CD81, CD90 and CD166, but were negative (or dim positive) for CD34, CD45, CD117 and HLD-DR. Despite having similar morphology and phenotypic expression, rbMSCs possessed significantly larger cell size but had a lower proliferation rate as compared with hMSCs. Using established protocols to differentiate hMSCs, rbMSCs underwent osteogenic, adipogenic and chondrogenic differentiation. Interestingly, differentiated rbMSCs demonstrated higher levels of osteogenic (Runx2) and chondrogenic (Sox9) gene expressions than that of hMSCs (P < 0.05). There was, however, no difference in the adipogenic (Pparγ) expressions between these cell types (P > 0.05). rbMSCs possess similar morphological characteristics to hMSCs, but have a higher potential for osteogenic and chondrogenic differentiation, despite having a lower cell proliferation rate than hMSCs. The characteristics reported here may be used as a comprehensive set of criteria to define or characterize rbMSCs.
间充质干细胞（MSCs）的特征为贴壁生长能力、成纤维细胞样外观、表达特定的表面蛋白标志物，并可多向分化。虽然兔骨髓来源的间充质干细胞（rbMSCs）已在之前的研究中被广泛应用，尤其是在转化研究中，但这些细胞的形态和超微结构尚未被定义，其多向分化能力也尚未与人类细胞进行比较。因此，本研究旨在对 rbMSCs 的形态、表面标志物蛋白、超微结构和多向分化能力进行定义。本研究使用了 3 只成年新西兰白兔（至少 4 月龄）的原代 rbMSC 培养物进行了 3 项独立的实验。采用梯度离心法分离 rbMSCs，该方法已被用于分离人 MSCs（hMSCs）。通过相差显微镜观察、透射电子显微镜分析、逆转录聚合酶链反应（PCR）分析、免疫细胞化学染色、流式细胞术、alamarBlue（®）检测、组织学染色和定量（q）PCR 分析对细胞进行了鉴定。分离的贴壁细胞呈成纤维细胞样梭形，具有偏心、不规则形状的核以及富含细胞质的内区，类似于 hMSCs。rbMSCs 表达 CD29、CD44、CD73、CD81、CD90 和 CD166，但 CD34、CD45、CD117 和 HLA-DR 呈阴性（或弱阳性）。尽管 rbMSCs 具有相似的形态和表型表达，但与 hMSCs 相比，rbMSCs 细胞体积较大，增殖率较低。使用已建立的分化方案使 hMSCs 向成骨细胞、脂肪细胞和软骨细胞分化，rbMSCs 也发生了向成骨细胞、脂肪细胞和软骨细胞的分化。有趣的是，与 hMSCs 相比，分化后的 rbMSCs 的成骨基因（Runx2）和软骨基因（Sox9）表达水平更高（P＜0.05）。然而，这些细胞类型的脂肪基因（Pparγ）表达水平无差异（P＞0.05）。rbMSCs 具有与 hMSCs 相似的形态特征，但成骨和软骨分化潜能更高，尽管增殖率低于 hMSCs。本研究报道的这些特征可作为一套全面的标准，用于定义或鉴定 rbMSCs。
