Khan Mohammad R, Chandrashekran Anil, Smith Roger K W, Dudhia Jayesh
Clinical Sciences and Services, Royal Veterinary College, North Mymms, Hatfield, Herts, AL9 7TA, United Kingdom.
Cytometry A. 2016 May;89(5):443-50. doi: 10.1002/cyto.a.22849. Epub 2016 Apr 14.
The clinical potential of multipotent mesenchymal stem cells (MSCs) has led to the essential development of analytical tools such as antibodies against membrane-bound proteins for the immunophenotypic characterization of human and rodent cells. Such tools are frequently lacking for emerging large animal models like the sheep that have greater relevance for the study of human musculoskeletal diseases. The present study identified a set of commercial nonspecies specific monoclonal antibodies for the immunophenotypic characterization of ovine MSCs. A protocol combining the less destructive proteolytic activity of accutase and EDTA was initially developed for the detachment of cells from plastic with minimum loss of cell surface antigens. A range of commercially available antibodies against human or rodent MSC antigens were then tested in single and multistain-based assays for their cross-reactivity to bone marrow derived ovine MSCs. Antibody clones cross-reactive to ovine CD73 (96.9% ± 5.9), CD90 (99.6% ± 0.3), CD105 (99.1 ± 1.5), CD271 (97.7 ± 2.0), and MHC1 (94.0% ± 7.2) antigens were identified using previously reported CD29, CD44, and CD166 as positive controls. Multistaining analysis indicated the colocalization of these antigens on MSCs. Furthermore, antibody clones identified to cross-react against white blood cell antigens exhibited either negative (CD117 (0.1% ± 0.1)) or low (MHCII (10.5% ± 16.0); CD31 (14.6% ± 4.2), and CD45 (39.4% ± 31.8)) cross-reactivity with ovine MSCs. The validation of these antibody clones to sheep MSC antigens is essential for studies utilizing this large animal model for stem cell-based therapies. © 2016 International Society for Advancement of Cytometry.
多能间充质干细胞（MSCs）的临床潜力推动了分析工具的重要发展，例如用于人和啮齿动物细胞免疫表型特征分析的抗膜结合蛋白抗体。对于新兴的大型动物模型（如绵羊）而言，此类工具常常缺失，而绵羊在人类肌肉骨骼疾病研究中具有更高的相关性。本研究鉴定出一组用于绵羊MSCs免疫表型特征分析的商业化非物种特异性单克隆抗体。最初开发了一种结合了Accutase和EDTA较低破坏性蛋白水解活性的方案，用于从塑料培养皿中分离细胞，同时将细胞表面抗原的损失降至最低。随后，在基于单染色和多染色的检测中，测试了一系列针对人或啮齿动物MSC抗原的市售抗体与骨髓来源的绵羊MSCs的交叉反应性。使用先前报道的CD29、CD44和CD166作为阳性对照，鉴定出与绵羊CD73（96.9%±5.9）、CD90（99.6%±0.3）、CD105（99.1±1.5）、CD271（97.7±2.0）和MHC1（94.0%±7.2）抗原具有交叉反应性的抗体克隆。多染色分析表明这些抗原在MSCs上共定位。此外，鉴定出与白细胞抗原发生交叉反应的抗体克隆与绵羊MSCs的交叉反应性为阴性（CD117（0.1%±0.1））或较低（MHCII（10.5%±16.0）；CD31（14.6%±4.2）和CD45（39.4%±31.8））。这些抗体克隆对绵羊MSC抗原的验证对于利用这种大型动物模型进行基于干细胞的治疗研究至关重要。©2016国际细胞计量学协会。
