基质细胞,有别于造血细胞,为骨髓微环境中的必要组成,并且是长期维持生物体内造血干细胞所不可或缺的。先前的研究指出,基质细胞藉由产生造血调节因子与胞外基质,以及藉由调控细胞间的物理性接触和黏合分子与细缝连结为基础的细胞间讯息传导来控制造血细胞的生长及分化。然而,大多数关于基质细胞调控造血细胞生长及自我更新能力的研究都是利用平面骨髓培养系统所进行的,而且大部分的结果显示培养四至八周后,造血细胞即会开始减少。
Hirabayashi与来自日本大学医学院、大阪府立大学与挪威科技大学的共同研究者们,于2011年11月的《实验生物与医学》期刊中发表了立体骨髓培养系统的论文。正如同文章的共同作者Isao Tsuboi所述:“平面骨髓培养系统无法长期培养造血细胞,因此无法用来进行基质细胞的功能性研究。所以,我们发展了三维立体的骨髓培养系统,并且可以成功地长期培养造血细胞。”
此一新型态的立体培养系统由特殊的粒子所构成。论文的共同作者Yukio Hirabayashi解释:“此一由接合环氧树脂所组成聚合物粒子为立体骨髓培养系统中最重要的一部分。我们由二十多种具有不同接合聚合物链长度、表面密度、聚合物网状结构以及接合聚合物侧链的粒子中,选出了最适合用于细胞培养的一种,我们将其命名为G-02。”另一位共同作者Tomonori Harada亦指出:“除了老鼠纤维母细胞株(MS-5 cell)外,表皮细胞株(HeLa cell)、骨源母细胞株(MC3T3E1 cell)与软骨细胞株(ch-8 cell)皆可附着于G-02粒子并于其表面上快速生长。此一优势使的我们可以将G-02粒子用于三维立体骨髓培养系统,亦可应用于中央神经系统、心脏与肝脏的立体组织培养。
CD34是一个广为人知的人类造血先驱细胞表面标志分子,不过带有CD34分子的细胞亦可分化成基质细胞。因此,当带有CD34标志分子的细胞与基质细胞共同培养时,对于事先建立的基质细胞层的功能将十分难以厘清。基质细胞研究专家Shin Aizawa教授指出:“于此研究中我们利用老鼠基质细胞株(MS-5)而非人类的基质细胞,因此可以排除带有CD34标志分子的基质细胞所造成的影响。此一共培养系统使的我们可以清楚的区分MS-5基质细胞层与带有CD34基质细胞的功能。我们的研究团队正利用具有特异性的引子与探针来研究多种人类与老鼠细胞生长素在基质细胞中的基因表现。
《实验生物与医学》期刊主编Steven R. Goodman博士指出,由Hirabayashi与共同作者们所发展的立体骨髓培养系统为基质细胞功能性研究中的杰出且影响深远的工具。(生物谷 Bioon.com)
doi:10.1258/ebm.2011.011075
PMC:
PMID:
Novel three-dimensional long-term bone marrow culture system using polymer particles with grafted epoxy-polymer-chains supports the proliferation and differentiation of hematopoietic stem cells
Yukio Hirabayashi, Yoshihiro Hatta, Jin Takeuchi, Isao Tsuboi, Tomonori Harada, Kentaro Ono, Wilhelm Robert Glomm, Masahiro Yasuda and Shin Aizawa
Hematopoiesis occurs in the bone marrow, where primitive hematopoietic cells proliferate and differentiate in close association with a three-dimensional (3D) hematopoietic microenvironment composed of stromal cells. We examined the hematopoietic supportive ability of stromal cells in a 3D culture system using polymer particles with grafted epoxy polymer chains. Umbilical cord blood-derived CD34+ cells were co-cultivated with MS-5 stromal cells. They formed a 3D structure in the culture dish in the presence of particles, and the total numbers of cells and the numbers of hematopoietic progenitor cells, including colony-forming unit (CFU)-Mix, CFU-granulocyte-macrophage, CFU-megakaryocyte and burst-forming unit-erythroid, were measured every seven days. The hematopoietic supportive activity of the 3D culture containing polymer particles and stromal cells was superior to that of 2D culture, and allowed the expansion and maintenance of hematopoietic progenitor cells for more than 12 weeks. Various types of hematopoietic cells, including granulocytes, macrophages and megakaryocytes at different maturation stages, appeared in the 3D culture, suggesting that the CD34+ cells were able to differentiate into a range of blood cell types. Morphological examination showed that MS-5 stromal cells grew on the surface of the particles and bridged the gaps between them to form a 3D structure. Hematopoietic cells slipped into the 3D layer and proliferated within it, relying on the presence of the MS-5 cells. These results suggest that this 3D culture system using polymer particles reproduced the hematopoietic phenomenon in vitro, and might thus provide a new tool for investigating hematopoietic stem cell–stromal cell interactions.