Bonanno Giuseppina, Perillo Alessandro, Rutella Sergio, De Ritis Daniela Giovanna, Mariotti Andrea, Marone Maria, Meoni Franco, Scambia Giovanni, Leone Giuseppe, Mancuso Salvatore, Pierelli Luca
Department of Gynecology and Obstetrics, and UNICATT Cord Blood Bank, Catholic University Medical School, Rome, Italy.
Transfusion. 2004 Jul;44(7):1087-97. doi: 10.1111/j.1537-2995.2004.03252.x.
Human cord blood is a relevant source of CD133+ HPCs. Clinical-scale isolation of human umbilical cord blood (UCB) CD133+ HPCs using immunomagnetic microbeads and the CliniMACS clinical cell isolator is reported. CD133+ HPCs isolated after large-scale processing were functionally characterized.
Closed disposable sets were used to process nine different samples of RBC-reduced UCB nucleated cells. In-vitro hematopoietic assays and human xenografts in NOD/SCID mice were performed to assess the functional properties of isolated CD133+ cells. Different mixtures of human cytokines were tested for the ability to expand nascent CD133+ HPCs. Furthermore, freshly isolated CD133+ cells were conditioned in culture medium specifically tested to support in-vitro myogenesis or osteogenesis.
Isolation procedures yielded the recovery of an average of 2.53 x 10(6) CD133+ HPCs with a mean recovery of 96 percent (referred to as RBC-reduced samples) and a final sample purity of 82 percent. Purified CD133+ cells had high cloning efficiency, had relevant long-term activity, and were capable of repopulating irradiated NOD/SCID mice. In 10-day stroma-free cultures, a 2-fold and 8.3-fold expansion of colony-forming cells (CFCs) and extended long-term culture-initiating cells, respectively, was obtained. Freshly isolated CD133+ cells differentiated into large nucleated cells expressing either myosin D or osteopontin (as revealed by RT-PCR and immuno-cytochemistry), with a protein/mRNA expression comparable to or even higher than that observed in UCB CD133- nucleated cells in identical culture conditions.
Collectively, clinical-scale isolation of UCB CD133+ cells provides a relevant amount of primitive HPCs with high hematopoietic activity and in-vitro mesenchymal potential.
人脐带血是CD133+造血祖细胞的一个重要来源。据报道，使用免疫磁珠和CliniMACS临床细胞分离仪可进行临床规模的人脐带血（UCB）CD133+造血祖细胞分离。对大规模处理后分离得到的CD133+造血祖细胞进行了功能特性分析。
使用封闭的一次性装置处理9份不同的经红细胞去除的UCB有核细胞样本。进行体外造血分析和在NOD/SCID小鼠体内的人源异种移植，以评估分离得到的CD133+细胞的功能特性。测试了不同人细胞因子混合物促进新生CD133+造血祖细胞增殖的能力。此外，将新鲜分离的CD133+细胞置于经过专门测试以支持体外成肌或成骨的培养基中培养。
分离程序平均可回收2.53×10(6)个CD133+造血祖细胞，平均回收率为96%（称为经红细胞去除的样本），最终样本纯度为82%。纯化的CD133+细胞具有高克隆效率、相关的长期活性，并且能够重建经辐照的NOD/SCID小鼠的造血系统。在无基质培养10天的过程中，集落形成细胞（CFC）分别扩增了2倍和8.3倍，长期培养起始细胞数量也有所增加。新鲜分离的CD133+细胞分化为表达肌球蛋白D或骨桥蛋白的大核细胞（通过RT-PCR和免疫细胞化学检测），其蛋白质/ mRNA表达水平与相同培养条件下UCB CD133-有核细胞相当，甚至更高。
总体而言，临床规模分离UCB CD133+细胞可提供大量具有高造血活性和体外间充质潜能的原始造血祖细胞。
