Garba Abubakar, Acar Delphine D, Roukaerts Inge D M, Desmarets Lowiese M B, Devriendt Bert, Nauwynck Hans J
Department of Virology, Parasitology and Immunology, Faculty of Veterinary Medicine, Ghent University, Salisburylaan 133, B-9820 Merelbeke, Belgium.
Department of Virology, Parasitology and Immunology, Faculty of Veterinary Medicine, Ghent University, Salisburylaan 133, B-9820 Merelbeke, Belgium.
Vet Immunol Immunopathol. 2017 Sep;191:44-50. doi: 10.1016/j.vetimm.2017.08.002. Epub 2017 Aug 7.
Mesenchymal cells are multipotent stromal cells with self-renewal, differentiation and immunomodulatory capabilities. We aimed to develop a co-culture model for differentiating hematopoietic cells on top of immortalized mesenchymal cells for studying interactions between hematopoietic and mesenchymal cells, useful for adequately exploring the therapeutic potential of mesenchymal cells. In this study, we investigated the survival, proliferation and differentiation of porcine red bone marrow hematopoietic cells co-cultured with immortalized porcine bone marrow mesenchymal cells for a period of five weeks. Directly after collection, primary porcine bone marrow mesenchymal cells adhered firmly to the bottom of the culture plates and showed a fibroblast-like appearance, one week after isolation. Upon immortalization, porcine bone marrow mesenchymal cells were continuously proliferating. They were positive for simian virus 40 (SV40) large T antigen and the mesenchymal cell markers CD44 and CD55. Isolated red bone marrow cells were added to these immortalized mesenchymal cells. Five weeks post-seeding, 92±6% of the red bone marrow hematopoietic cells were still alive and their number increased 3-fold during five weekly subpassages on top of the immortalized mesenchymal cells. The red bone marrow hematopoietic cells were originally small and round; later, the cells increased in size. Some of them became elongated, while others remained round. Tiny dendrites appeared attaching hematopoietic cells to the underlying immortalized mesenchymal cells. Furthermore, weekly differential-quick staining of the cells indicated the presence of monoblasts, monocytes, macrophages and lymphocytes in the co-cultures. At three weeks of co-culture, flow cytometry analysis showed an increased surface expression of CD172a, CD14, CD163, CD169, CD4 and CD8 up to 37±0.8%, 40±8%, 41±4%, 23±3% and 19±5% of the hematopoietic cells, respectively. In conclusion, continuous mesenchymal cell cultures were successfully established and characterized and they supported the proliferation of red bone marrow hematopoietic cells, which finally differentiated into monocytic cells and CD4 and CD8 cells.
间充质细胞是具有自我更新、分化和免疫调节能力的多能基质细胞。我们旨在建立一种共培养模型，使永生化间充质细胞上的造血细胞得以分化，用于研究造血细胞与间充质细胞之间的相互作用，这有助于充分探索间充质细胞的治疗潜力。在本研究中，我们调查了与永生化猪骨髓间充质细胞共培养五周的猪红骨髓造血细胞的存活、增殖和分化情况。收集后，原代猪骨髓间充质细胞立即牢固地附着在培养板底部，分离一周后呈现成纤维细胞样外观。永生化后，猪骨髓间充质细胞持续增殖。它们对猿猴病毒40（SV40）大T抗原以及间充质细胞标志物CD44和CD55呈阳性。将分离的红骨髓细胞添加到这些永生化间充质细胞中。接种五周后，92±6%的红骨髓造血细胞仍然存活，并且在永生化间充质细胞上进行的五次每周传代培养期间，其数量增加了3倍。红骨髓造血细胞最初小而圆；后来，细胞体积增大。其中一些细胞变长，而其他细胞仍保持圆形。出现微小的树突将造血细胞附着于下面的永生化间充质细胞。此外，对细胞进行的每周一次的鉴别快速染色表明，共培养物中存在原单核细胞、单核细胞、巨噬细胞和淋巴细胞。共培养三周时，流式细胞术分析显示，造血细胞表面CD172a、CD14、CD163、CD169、CD4和CD8的表达分别增加至37±0.8%、40±8%、41±4%、23±3%和19±5%。总之，成功建立并鉴定了连续的间充质细胞培养物，它们支持红骨髓造血细胞的增殖，这些造血细胞最终分化为单核细胞以及CD4和CD8细胞。
