Delalat Bahman, Pourfathollah Ali Akbar, Soleimani Masoud, Mozdarani Hossein, Ghaemi Soraya Rasi, Movassaghpour Ali Akbar, Kaviani Saeed
Department of Hematology, School of Medical Sciences Faculty, Tarbiat Modares University, PO Box 14115-111, Tehran, Iran.
Hematology. 2009 Jun;14(3):125-32. doi: 10.1179/102453309X402250.
Umbilical cord blood (UCB) contains a high number of primitive progenitor cells, allowing UCB to be used as a source of hematopoietic progenitors for clinical transplantation. However the rate of UCB CD34(+) stem cells graft is low. Mesenchymal stem cells (MSCs) have been implicated in playing an important role in hematopoietic stem cell engraftment. In this study we examined the effect of human MSC on engraftment of human UCB-derived CD34(+) cells in irradiated Balb/c mice. Human UCB CD34(+) cells were obtained from full-term normal deliveries by using an immunomagnetic separation technique and MSC were isolated by standard methodology from human bone marrow. Isolated CD34(+) cells were cultured in Stemline Hematopoietic stem cell expansion medium supplemented with 100 ng/ml stem cell factor (SCF), and 100 ng/ml thrombopoietin (TPO) in 24-well plates and incubated at 37 degrees C in a fully humidified atmosphere with 5% CO(2), and maintained over 3 weeks and half the medium was exchanged twice a week. Irradiated (7 Gy) Balb/c mice were transplanted intravenously with 0.1 x 10(6) to 10 x 10(6) human UCB CD34(+) cells in the presence or absence of 0.5 x 10(6) and 1 x 10(6) human bone marrow-derived MSC. After 11 days, in each group, the spleen was dissected and colony assay performed. Hematoxilin and eosin staining of the spleen colony was performed, and UCB CD34(+) cells labeled with super paramagnetic iron oxide (SPIO). After establishing the presence of colonies in spleen, Prussian blue staining was performed. Flow cytometry assay showed that up to 90% purity of CD34(+) cells and 96% for MSC. After 3 weeks the cell numbers showed a 1000-fold increase in CD34(+). Cotransplantation of low doses of UCB CD34(+) cells (0.2 x 10(6) and 0.3 x 10(6)) and MSC (0.5 x 10(6) and 1 x 10(6)) resulted in a significant increase in colony forming unit spleen, in comparison with engraftment of UCB CD34(+) stem cells without MSC after 11 days (p<0.01). In conclusion the results showed that two cytokines (SCF, TPO) were sufficient for expansion of UCB CD34(+) cells and cotransplantation of MSC with UCB CD34(+) cells, promoting engraftment of UCB CD34(+) cells.
脐带血（UCB）含有大量原始祖细胞，这使得脐带血能够用作临床移植的造血祖细胞来源。然而，脐带血CD34(+)干细胞的移植率较低。间充质干细胞（MSCs）在造血干细胞植入过程中发挥着重要作用。在本研究中，我们检测了人MSCs对经照射的Balb/c小鼠体内人脐带血来源的CD34(+)细胞植入的影响。通过免疫磁珠分离技术从足月正常分娩中获取人脐带血CD34(+)细胞，并采用标准方法从人骨髓中分离MSCs。将分离得到的CD34(+)细胞接种于24孔板中，在含有100 ng/ml干细胞因子（SCF）和100 ng/ml血小板生成素（TPO）的Stemline造血干细胞扩增培养基中培养，于37℃、5%二氧化碳的完全湿润环境中孵育，培养超过3周，每周更换一半培养基两次。将经照射（7 Gy）的Balb/c小鼠静脉注射0.1×10(6)至10×10(6)个人脐带血CD34(+)细胞，同时加入或不加入0.5×10(6)和1×10(6)个人骨髓来源的MSCs。11天后，每组解剖脾脏并进行集落分析。对脾脏集落进行苏木精和伊红染色，并用超顺磁性氧化铁（SPIO）标记脐带血CD34(+)细胞。在确定脾脏中存在集落后，进行普鲁士蓝染色。流式细胞术分析显示CD34(+)细胞纯度高达90%，MSCs纯度为96%。3周后，CD34(+)细胞数量增加了1000倍。与11天后未联合MSCs的脐带血CD34(+)干细胞植入相比，低剂量脐带血CD34(+)细胞（0.2×10(6)和0.3×10(6)）与MSCs（0.5×10(6)和1×10(6)）共移植导致脾集落形成单位显著增加（p<0.01）。总之，结果表明两种细胞因子（SCF、TPO）足以扩增脐带血CD34(+)细胞，且MSCs与脐带血CD34(+)细胞共移植可促进脐带血CD34(+)细胞的植入。
