Platelet released growth factors boost expansion of bone marrow derived CD34+ and CD133+ endothelial progenitor cells for autologous grafting
Platelet released growth factors boost expansion of bone marrow derived CD34+ and CD133+ endothelial progenitor cells for autologous grafting
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DOI：
10.3109/09537104.2011.559559
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发表时间：
2011-01-01
期刊：
影响因子：
3.3
通讯作者：
Verrier, Sophie
中科院分区：
文献类型：
--
作者：
Lippross, Sebastian;Loibl, Markus;Verrier, Sophie
基于干细胞的自体移植最近在各种外科领域获得了广泛的组织缺损治疗的主要兴趣。可以从骨髓单个核细胞（BMC）池中分离的CD 34（+）和CD 133（+）细胞能够在体内分化为成熟的内皮细胞。这些内皮祖细胞（EPC）被认为是当组织损伤发生时从骨髓释放的血管生成再生细胞的主要部分。近年来，组织工程师越来越多地关注血管新生过程，因为它对于成功的细胞移植以替换受损组织至关重要。到目前为止，阻止临床应用的最大问题之一是为此目的所需的大规模扩展。我们建立了一种通过使用血小板释放生长因子（PRGF）作为培养基补充物来有效增强CD 34（+）和CD 133（+）细胞扩增的方法。从血小板浓缩物制备PRGF，并用作iscove改良的dulbecco培养基（IMDM）的培养基补充剂。采用免疫磁性方法从人骨髓单核细胞中分离出内皮祖细胞，并在IMDM + 10%胎牛血清（FCS）、IMDM +5%、FCS +5%PRGF和IMDM +10%PRGF中培养。我们清楚地证明了在培养的第7、14、21和28天，当PRGF和FCS两者都添加到培养基中时，与单独的10%FCS或10%PRGF相比，细胞增殖率在统计学上显著更高和更快。在没有FCS的情况下，向IMDM中加入10%PRGF导致从第14天开始生长停滞。在组织化学、免疫细胞化学和基因表达分析中，我们发现血管生成和CD 34（+）和CD 133（+）细胞的前体标记在长期培养中得以维持。综上所述，我们建立了一个方案，以加强CD34（+）和CD133（+）细胞的扩增。从而为自体干细胞移植的临床应用提供了技术基础。
Stem cell based autologous grafting has recently gained mayor interest in various surgical fields for the treatment of extensive tissue defects. CD34(+) and CD133(+) cells that can be isolated from the pool of bone marrow mononuclear cells (BMC) are capable of differentiating into mature endothelial cells in vivo. These endothelial progenitor cells (EPC) are believed to represent a major portion of the angiogenic regenerative cells that are released from bone marrow when tissue injury has occurred. In recent years tissue engineers increasingly looked at the process of vessel neoformation because of its major importance for successful cell grafting to replace damaged tissue. Up to now one of the greatest problems preventing a clinical application is the large scale of expansion that is required for such purpose. We established a method to effectively enhance the expansion of CD34(+) and CD133(+) cells by the use of platelet-released growth factors (PRGF) as a media supplement. PRGF were prepared from thrombocyte concentrates and used as a media supplement to iscove's modified dulbecco's media (IMDM). EPC were immunomagnetically separated from human bone morrow monocyte cells and cultured in IMDM + 10% fetal calf serum (FCS), IMDM + 5%, FCS + 5% PRGF and IMDM + 10% PRGF. We clearly demonstrate a statistically significant higher and faster cell proliferation rate at 7, 14, 21, and 28 days of culture when both PRGF and FCS were added to the medium as opposed to 10% FCS or 10% PRGF alone. The addition of 10% PRGF to IMDM in the absence of FCS leads to a growth arrest from day 14 on. In histochemical, immunocytochemical, and gene-expression analysis we showed that angiogenic and precursor markers of CD34(+) and CD133(+) cells are maintained during long-term culture. In summary, we established a protocol to boost the expansion of CD34(+) and CD133(+) cells. Thereby we provide a technical step towards the clinical application of autologous stem cell transplantation.