Microcavity arrays as an in vitro model system of the bone marrow niche for hematopoietic stem cells
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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 3D-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 3D-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 RT2-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.
KeywordsHematopoietic progenitor cells Mesenchymal stromal cells Stem cell niche Microcavity array Bioreactor
The authors thank Angela Lenze and Anke Diehlmann for isolation and preparation of HPCs and MSCs, as well as David Thiele and Anke Dech for technical assistance. We also thank Siegfried Horn, Jörg Bohn and Hartmut Gutzeit for the construction and manufacturing of the bioreactors and periphery. This work was supported by the German Ministry of Education and Research (BMBF) within the supporting program “Cell Based Regenerative Medicine” (START-MSC2; funding code 01GN0940 to ADH and PW) and within the collaborative research project “Systems Biology of Erythropoietin” (SBEpo; funding code 0316182D to ADH and PW). This work was also supported by the HEiKA Research Alliance (funding to EG and PW) and the German Research Foundation DFG (SFB 873, funding to ADH and PW). The authors also thank the Karlsruhe Nano and Micro Facility (KNMF) for the support of the project.
We acknowledge the Karlsruhe Nano Micro Facility (KNMF, www.kit.edu/knmf ) of KIT for access to instruments at their laboratories and we would like to thank Dr. Matthias Worgull and his team for the manufacturing of the KITChips.
Compliance with ethical standards
The first author and all co-authors confirm that there are no relevant conflicts of interest to disclose, except for the following:
Patrick Wuchter: Honoraria and Membership on Advisory Boards of Sanofi-Aventis. Travel grants from Hexal AG.
Anthony D. Ho: Research funding from and Membership on Advisory Board of Genzyme/Sanofi-Aventis.
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