Department of Hematology, Hematology Research Laboratory, West China Hospital, Sichuan University, 610041, Chengdu, Sichuan, People's Republic of China.
Laboratory of Stem Cell and Tissue Engineering, West China Hospital, Sichuan University, 610041, Chengdu, Sichuan, People's Republic of China.
Int J Hematol. 2009 Oct;90(3):281-291. doi: 10.1007/s12185-009-0392-4. Epub 2009 Aug 11.
We constructed a "biomimetic osteoblast niche" with bio-derived bone as a scaffold, on which we seeded marrow mesenchymal stem cells (MSCs) from CML patients, and induced the MSCs to differentiate into osteoblasts. Bone marrow mononuclear cells from CML patients were cultured in the biomimetic niche (3D culture system) or a 2D culture system with the induced MSCs/osteoblasts as a feeder cell layer for 2 and 5 weeks without adding exogenous cytokines. Cultured cells were analyzed regarding their phenotypes and functions using flow cytometry, colony-forming unit (CFU) assay and long-term culture-initiating cells (LTC-IC) assay. All cultured and colony cells in the LTC-IC assay were collected and analyzed by fluorescent in situ hybridization to identify Ph (bcr/abl)-positive cells. Our results showed that all parameters were higher in the 3D than in the 2D system, either at 2 or 5 weeks, i.e., regarding the number of CD34(+) cells (8,277.00 vs. 4,490.75 or 2,276.75 vs. 786.00 per well on average, respectively), number of CD34(+)/CD38(-) cells (1,207.50 vs. 474.25 or 497.25 vs. 114.25 per well on average, respectively), numbers of CFUs (103.33 vs. 79 or 47.0 vs. 21.67/10(5) MNCs; 189.33 vs. 131.00 or 10.33 vs. 3.67 per well on average, respectively), frequency of LTC-ICs (2.23 x 10(-5) vs. 1.40 x 10(-5) or 1.86 x 10(-5) vs. 0.64 x 10(-5), respectively) and number of remaining LTC-ICs (2.80 vs. 2.03 or 0.46 vs. 0.07 per well on average, respectively). The Ph (bcr/abl)-positive cell fraction was reduced in both systems during culture, but in the 3D system, it was not as rapid as in the 2D system and showed a leukemic predominance. In conclusion, our "biomimetic osteoblast niche" might provide a more adaptive microenvironment for leukemic stem/progenitor cell growth. The biological characteristics of leukemic stem/progenitor cells were partially maintained. It was suggested that the 3D biomimetic niche might be a new tool for studying the behaviors of leukemic hematopoietic stem cells/hematopoietic progenitor cells in vitro.
我们构建了一个“仿生成骨细胞龛”，以生物衍生骨作为支架，在其上接种来自 CML 患者的骨髓间充质干细胞（MSCs），并诱导 MSCs 分化为成骨细胞。CML 患者的骨髓单核细胞在仿生龛（3D 培养系统）或 2D 培养系统中培养，其中诱导的 MSCs/成骨细胞作为饲养细胞层，2 周和 5 周内不加外源性细胞因子。使用流式细胞术、集落形成单位（CFU）测定和长期培养起始细胞（LTC-IC）测定分析培养细胞的表型和功能。通过荧光原位杂交鉴定 LTC-IC 测定中所有培养和集落细胞中的 Ph（bcr/abl）-阳性细胞。我们的结果表明，无论是在 2 周还是 5 周时，3D 系统中的所有参数均高于 2D 系统，例如 CD34（+）细胞的数量（平均每个孔分别为 8,277.00、4,490.75 或 2,276.75、786.00），CD34（+）/CD38（-）细胞的数量（平均每个孔分别为 1,207.50、474.25 或 497.25、114.25），CFU 的数量（103.33、79 或 47.0、21.67/10(5)MNC；189.33、131.00 或 10.33、3.67 平均每个孔），LTC-IC 频率（2.23 x 10(-5)、1.40 x 10(-5) 或 1.86 x 10(-5)、0.64 x 10(-5)）和剩余 LTC-IC 的数量（平均每个孔分别为 2.80、2.03 或 0.46、0.07）。在培养过程中，两种系统中的 Ph（bcr/abl）-阳性细胞比例均降低，但在 3D 系统中降低速度不如 2D 系统快，并且表现出白血病优势。总之，我们的“仿生成骨细胞龛”可能为白血病干细胞/祖细胞的生长提供了更适应的微环境。白血病干细胞/祖细胞的生物学特性得到了部分维持。提示 3D 仿生龛可能是研究白血病造血干/祖细胞体外行为的新工具。
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