Zhou Dun-hua, Huang Shao-lfang, Wu Yan-Feng, Wei Jing, Chen Ge-yu, Li Yang, Bao Rong
Department of Pediatrics, Second Hospital, Sun Yat-Sen University, GuangZhou 510120, China.
Zhonghua Er Ke Za Zhi. 2003 Aug;41(8):607-10.
Mesenchymal stem cells (MSCs) were adult stem cells which contribute to the regeneration of mesenchymal tissues such as bone cartilage, muscle, ligament, tendon, adipose and stroma. Due to the multipotential ability and self-renewal capacity, the mesenchymal stem cells can be applied in many fields, such as the seed cells in tissues engineering, cell therapy and gene therapy. To enhance the clinical use of MSCs, the investigators studied the isolation and expansion of MSCs from adult bone marrow, fetal bone marrow and human umbilical cord blood, and investigated their biological identities.
Two kinds of incubation systems containing L-DMEM or MSC special culture medium were used to purify and expand MSCs. The growth, purification and proliferative abilities of 3 kinds of MSCs were observed and their immunophenotypes were determined by flow-cytometry.
(1) The shapes of 3 kinds of cells were same. There was no difference in number and size. The colonies formed early in adult bone marrow MSCs. (2) There was no difference in the expansion speed of the 3 kinds of MSCs, but after the colonies confluenced there had no touching constrain in MSCs from umbilical cord blood and fetal bone marrow. When the colonies confluenced, the cells also had proliferation ability. But in adult bone marrow, the touching constrain was significant. (3) MSCs had strong self-renewal capacity. After primary culture approximately 5 - 6 x 10(5) MSCs were obtained from 8 x 10(6) MNC of bone marrow and 25 x 10(6) MNC of umbilical cord blood. After passage 3, passage 5 and passage 10, the investigators could get 10(7), 10(8) and 10(10) MSCs, respectively. (4) Along with the increase in the passage and prolonging of culture time, the ability of expansion decreased, but they maintained good puripotentiality. After passage 2, passage 3 and passage 5, the purity of MSCs was 90%, 95% and 99%, respectively. (5) Three kinds of MSCs were all positive for CD(29), CD(44), CD(59), CD(90), CD(105), CD(166) and all negative for the markers of hematopoietic cells such as CD(11a), CD(14), CD(33), CD(34), CD(28), CD(45). All the important GVHD correlation markers were negative, such as HLA-DR, B7-1 (CD(80)), B7-2 (CD(86)), CD(40) and CD(40L). There were no differences in the phenotype among the 3 kinds of MSCs cells. (6) The 2 kinds of culture mediums used did not markedly affect isolation and expansion of MSCs, and the biological properties of MSCs.
(1) Human MSCs could be isolated from many kinds of human tissues, and they had no difference in their origin; (2) Human MSCs maintained good puripotentiality and self-renewal capacity. Therefore, they could meet with the need of clinical tissue engineering. (3) The negative GVHD correlated markers might result from the fact that MSCs had no HLA barrier but had broad clinical use.
间充质干细胞（MSCs）是成体干细胞，有助于骨、软骨、肌肉、韧带、肌腱、脂肪和基质等间充质组织的再生。由于其多能性和自我更新能力，间充质干细胞可应用于许多领域，如组织工程中的种子细胞、细胞治疗和基因治疗。为提高间充质干细胞的临床应用价值，研究人员对从成人骨髓、胎儿骨髓和人脐带血中分离和扩增间充质干细胞进行了研究，并对其生物学特性进行了研究。
采用含L-DMEM或间充质干细胞专用培养基的两种培养体系纯化和扩增间充质干细胞。观察3种间充质干细胞的生长、纯化和增殖能力，并用流式细胞术检测其免疫表型。
（1）3种细胞形态相同，数量和大小无差异。成人骨髓间充质干细胞早期形成集落。（2）3种间充质干细胞的扩增速度无差异，但集落汇合后，脐带血和胎儿骨髓来源的间充质干细胞无接触抑制。集落汇合时，细胞仍有增殖能力。但在成人骨髓中，接触抑制明显。（3）间充质干细胞具有较强的自我更新能力。原代培养后，从8×10⁶骨髓单个核细胞和25×10⁶脐带血单个核细胞中分别获得约5 - 6×10⁵个间充质干细胞。传代3、5、10代后，分别可获得10⁷、10⁸和10¹⁰个间充质干细胞。（4）随着传代次数增加和培养时间延长，扩增能力下降，但仍保持良好的多能性。传代2、3、5代后，间充质干细胞的纯度分别为90%、95%和99%。（5）3种间充质干细胞CD(29)、CD(44)、CD(59)、CD(90)、CD(105)、CD(166)均为阳性，造血细胞标志物如CD(11a)、CD(14)、CD(33)、CD(34)、CD(28)、CD(45)均为阴性。所有重要的移植物抗宿主病相关标志物均为阴性，如HLA-DR、B7-1(CD(80))、B7-2(CD(86))、CD(40)和CD(40L)。3种间充质干细胞的表型无差异。（6）所用的两种培养基对间充质干细胞的分离、扩增及生物学特性无明显影响。
（1）人源间充质干细胞可从多种人体组织中分离得到，其来源无差异；（2）人源间充质干细胞保持良好的多能性和自我更新能力。因此，可满足临床组织工程的需求；（3）移植物抗宿主病相关标志物阴性可能是因为间充质干细胞无HLA屏障但临床应用广泛。
