Wang Heng-Xiang, Li Zhi-Yong, Guo Zhi-Kun, Guo Zi-Kuan
Heng-Xiang Wang, Department of Hematology, General Hospital of Air Force, Beijing 100042, China.
World J Stem Cells. 2015 Sep 26;7(8):1137-44. doi: 10.4252/wjsc.v7.i8.1137.
To establish an easily-handled method to isolate mesenchymal stem cells (MSCs) from coagulated human bone marrow samples.
Thrombin was added to aliquots of seven heparinized human bone marrow samples to mimic marrow coagulation. The clots were untreated, treated with urokinase or mechanically cut into pieces before culture for MSCs. The un-coagulated samples and the clots were also stored at 4 °C for 8 or 16 h before the treatment. The numbers of colony-forming unit-fibroblast (CFU-F) in the different samples were determined. The adherent cells from different groups were passaged and their surface profile was analyzed with flow cytometry. Their capacities of in vitro osteogenesis and adipogenesis were observed after the cells were exposed to specific inductive agents.
The average CFU-F number of urokinase-treated samples (16.85 ± 11.77/10(6)) was comparable to that of un-coagulated control samples (20.22 ± 10.65/10(6), P = 0.293), which was significantly higher than those of mechanically-cut clots (6.5 ± 5.32/10(6), P < 0.01) and untreated clots (1.95 ± 1.86/10(6), P < 0.01). The CFU-F numbers decreased after samples were stored, but those of control and urokinase-treated clots remained higher than the other two groups. Consistently, the numbers of the attached cells at passage 0 were higher in control and urokinase-treated clots than those of mechanically-cut clots and untreated clots. The attached cells were fibroblast-like in morphology and homogenously positive for CD44, CD73 and CD90, and negative for CD31 and CD45. Also, they could be induced to differentiate into osteoblasts and adipocytes in vitro.
Urokinase pretreatment is an optimal strategy to isolate MSCs from human bone marrow samples that are poorly aspirated and clotted.
建立一种易于操作的方法，从凝固的人骨髓样本中分离间充质干细胞（MSCs）。
向7份肝素化人骨髓样本的等分试样中加入凝血酶以模拟骨髓凝固。凝块在培养MSCs之前不进行处理、用尿激酶处理或机械切成小块。未凝固的样本和凝块在处理前也在4℃下储存8或16小时。测定不同样本中集落形成单位成纤维细胞（CFU-F）的数量。将不同组的贴壁细胞传代，并用流式细胞术分析其表面特征。在细胞暴露于特定诱导剂后，观察其体外成骨和成脂能力。
尿激酶处理样本的平均CFU-F数量（16.85±11.77/10(6)）与未凝固对照样本的平均CFU-F数量（20.22±10.65/10(6)，P = 0.293）相当，显著高于机械切割凝块（6.5±5.32/10(6)，P < 0.01）和未处理凝块（1.95±1.86/10(6)，P < 0.01）。样本储存后CFU-F数量减少，但对照和尿激酶处理凝块的CFU-F数量仍高于其他两组。同样，第0代贴壁细胞的数量在对照和尿激酶处理凝块中高于机械切割凝块和未处理凝块。贴壁细胞形态呈成纤维细胞样，CD44、CD73和CD90均呈均匀阳性，CD31和CD45呈阴性。此外，它们在体外可被诱导分化为成骨细胞和脂肪细胞。
尿激酶预处理是从抽吸不良且凝固的人骨髓样本中分离MSCs的最佳策略。
