Rapid clinical-scale propagation of mesenchymal stem cells using cultures initiated with immunoselected bone marrow CD105+ cells
Rapid clinical-scale propagation of mesenchymal stem cells using cultures initiated with immunoselected bone marrow CD105+ cells
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DOI：
10.1111/j.1582-4934.2010.01157.x
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发表时间：
2011-09-01
影响因子：
5.3
通讯作者：
Goussetis, Evgenios
中科院分区：
文献类型：
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作者：
Spiropoulos, Antonia;Theodosaki, Maria;Goussetis, Evgenios
目前用于分离和纯化间充质干细胞（MSC）的临床方案是基于从骨髓（BM）单核细胞开始的长期培养。使用市售的免疫选择试剂盒富集MSC，我们研究了富集的BM-CD 105（+）细胞培养物是否可以在短时间内提供足够数量的纯MSC，用于移植物抗宿主病和移植物衰竭/排斥的临床应用。我们从10个小体积（10-25 ml）BM样品中分离出平均5.4 x 10（5）+/- 0.9 x 10（5）CD 105（+）细胞，在MSC中实现了> 100倍的富集。接种2 × 10（3）免疫选择细胞/cm（2），我们能够在3周内从自体血清富集培养基的培养物中产生2.5 × 10（8）+/- 0.7 × 10（8）MSC。即使在原代培养细胞产物中也未检测到造血细胞或内皮细胞。扩增的细胞符合MSC的表型和功能当前标准;它们是CD 29（+）、CD 90（+）、CD 73（+）、CD 105（+）、CD 45（-）;它们抑制混合淋巴细胞培养物中的同种异体T细胞反应并保留体外分化潜力。此外，比较基因组杂交分析显示培养的MSC的染色体稳定性。我们的数据表明，使用富集的BM-CD 105（+）细胞可以在短时间内产生足够数量的适合临床应用的纯MSC。
Current clinical protocols used for isolation and purification of mesenchymal stem cells (MSC) are based on long-term cultures starting with bone marrow (BM) mononuclear cells. Using a commercially available immunoselection kit for enrichment of MSC, we investigated whether culture of enriched BM-CD105(+) cells could provide an adequate number of pure MSC in a short time for clinical use in the context of graft versus host disease and graft failure/rejection. We isolated a mean of 5.4 x 10(5) +/- 0.9 x 10(5) CD105(+) cells from 10 small volume (10-25 ml) BM samples achieving an enrichment > 100-fold in MSC. Seeding 2 x 10(3) immunoselected cells/cm(2) we were able to produce 2.5 x 10(8) +/- 0.7 x 10(8) MSC from cultures with autologous serum enriched medium within 3 weeks. Neither haematopoietic nor endothelial cells were detectable even in the primary culture cell product. Expanded cells fulfilled both phenotypic and functional current criteria for MSC; they were CD29(+), CD90(+), CD73(+), CD105(+), CD45(-); they suppressed allogeneic T-cell reaction in mixed lymphocyte cultures and retained in vitro differentiation potential. Moreover, comparative genomic hybridization analysis revealed chromosomal stability of the cultured MSC. Our data indicate that adequate numbers of pure MSC suitable for clinical applications can be generated within a short time using enriched BM-CD105(+) cells.