Wuchter Patrick, Saffrich Rainer, Giselbrecht Stefan, Nies Cordula, Lorig Hanna, Kolb Stephanie, Ho Anthony D, Gottwald Eric
Department of Medicine V, Heidelberg University, 69120, Heidelberg, Germany.
HEiKA - Heidelberg Karlsruhe Research Partnership, Heidelberg University and Karlsruhe Institute of Technology, Heidelberg and Karlsruhe, Germany.
Cell Tissue Res. 2016 Jun;364(3):573-584. doi: 10.1007/s00441-015-2348-8. Epub 2016 Jan 30.
In previous studies human mesenchymal stromal cells (MSCs) maintained the "stemness" of human hematopoietic progenitor cells (HPCs) through direct cell-cell contact in two-dimensional co-culture systems. We establish a three-dimensional (3D) co-culture system based on a custom-made chip, the 3(D)-KITChip, as an in vitro model system of the human hematopoietic stem cell niche. This array of up to 625 microcavities, with 300 μm size in each orientation, was inserted into a microfluidic bioreactor. The microcavities of the 3(D)-KITChip were inoculated with human bone marrow MSCs together with umbilical cord blood HPCs. MSCs used the microcavities as a scaffold to build a complex 3D mesh. HPCs were distributed three-dimensionally inside this MSC network and formed ß-catenin- and N-cadherin-based intercellular junctions to the surrounding MSCs. Using RT(2)-PCR and western blots, we demonstrate that a proportion of HPCs maintained the expression of CD34 throughout a culture period of 14 days. In colony-forming unit assays, the hematopoietic stem cell plasticity remained similar after 14 days of bioreactor co-culture, whereas monolayer co-cultures showed increasing signs of HPC differentiation and loss of stemness. These data support the notion that the 3D microenvironment created within the microcavity array preserves vital stem cell functions of HPCs more efficiently than conventional co-culture systems.
在先前的研究中，人间充质基质细胞（MSCs）在二维共培养系统中通过直接的细胞间接触维持了人类造血祖细胞（HPCs）的“干性”。我们基于定制芯片3(D)-KITChip建立了一种三维（3D）共培养系统，作为人类造血干细胞龛的体外模型系统。这个由多达625个微腔组成的阵列，每个方向的尺寸为300μm，被插入到一个微流控生物反应器中。在3(D)-KITChip的微腔中接种了人骨髓间充质干细胞和脐带血造血祖细胞。间充质干细胞利用微腔作为支架构建了一个复杂的三维网状结构。造血祖细胞三维分布在这个间充质干细胞网络内部，并与周围的间充质干细胞形成基于β-连环蛋白和N-钙黏蛋白的细胞间连接。使用RT(2)-PCR和蛋白质印迹法，我们证明一部分造血祖细胞在14天的培养期内维持了CD34的表达。在集落形成单位测定中，生物反应器共培养14天后造血干细胞的可塑性保持相似，而单层共培养显示造血祖细胞分化迹象增加且干性丧失。这些数据支持了这样一种观点，即微腔阵列内创建的三维微环境比传统共培养系统更有效地保留了造血祖细胞的重要干细胞功能。
