Oswald Joachim, Steudel Christine, Salchert Katrin, Joergensen Brigitte, Thiede Christian, Ehninger Gerhard, Werner Carsten, Bornhäuser Martin
Med. Klinik und Poliklinik I, University Hospital Carl Gustav Carus, Liebniz Institute of Polymer Research Dresden, Germany.
Stem Cells. 2006 Mar;24(3):494-500. doi: 10.1634/stemcells.2005-0276. Epub 2005 Sep 15.
CD34+ hematopoietic stem/progenitor cells (HSCs) reside in the bone marrow in close proximity to the endosteal bone surface, surrounded by osteoblasts, stromal cells, and various extracellular matrix molecules. We used a bioartificial matrix of fibrillar collagen I, the major matrix component of bone, as a scaffold for ex vivo expansion of HSCs. CD34+ HSCs were isolated from umbilical cord blood and cultivated within reconstituted collagen I fibrils in the presence of fms-like tyrosine kinase-3 ligand, stem cell factor, and interleukin (IL)-3. After 7 days of culture, the cell number, number of colony-forming units (CFU-C), and gene-expression profile of the cultured cells were assessed. Although the total expansion factor of CD34+ cells was slightly lower when cells were cultivated in the collagen I gel, the frequency of CFU-C was greater than in control suspension cultures. Gene-expression analysis with microarray chip technology revealed the upregulation of more than 50 genes in the presence of collagen I. Among these, genes for several growth factors, cytokines, and chemokines (e.g., IL-8 and macrophage inhibitory protein 1alpha) could be confirmed using quantitative polymerase chain reaction. Furthermore, greater expression levels of the negative cell-cycle regulator BTG2/TIS21 and an inhibitor of the mitogen-activated protein kinase pathway, DUSP2, underline the regulatory role of the extracellular matrix. Together, these data show that the expansion of CD34+ cord blood cells in a culture system containing a three-dimensional collagen I matrix induces a qualitative change in the gene-expression profile of cultivated HSCs.
CD34+造血干/祖细胞（HSCs）位于骨髓中靠近骨内膜骨表面的位置，被成骨细胞、基质细胞和各种细胞外基质分子所包围。我们使用了骨的主要基质成分——I型纤维状胶原蛋白的生物人工基质，作为HSCs体外扩增的支架。从脐带血中分离出CD34+HSCs，并在重组I型胶原纤维中，于fms样酪氨酸激酶-3配体、干细胞因子和白细胞介素（IL）-3存在的情况下进行培养。培养7天后，评估培养细胞的细胞数量、集落形成单位（CFU-C）数量和基因表达谱。尽管当细胞在I型胶原凝胶中培养时，CD34+细胞的总扩增倍数略低，但CFU-C的频率高于对照悬浮培养。利用微阵列芯片技术进行的基因表达分析显示，在I型胶原存在的情况下，有50多个基因上调。其中，几种生长因子、细胞因子和趋化因子（如IL-8和巨噬细胞抑制蛋白1α）的基因可以通过定量聚合酶链反应得到证实。此外，负性细胞周期调节因子BTG2/TIS21和丝裂原活化蛋白激酶途径抑制剂DUSP2的更高表达水平，突出了细胞外基质的调节作用。总之，这些数据表明，在含有三维I型胶原基质的培养系统中扩增CD34+脐带血细胞会诱导培养的HSCs基因表达谱发生质的变化。
