Advertisement

Cell and Tissue Research

, Volume 364, Issue 3, pp 573–584 | Cite as

Microcavity arrays as an in vitro model system of the bone marrow niche for hematopoietic stem cells

  • Patrick Wuchter
  • Rainer Saffrich
  • Stefan Giselbrecht
  • Cordula Nies
  • Hanna Lorig
  • Stephanie Kolb
  • Anthony D. Ho
  • Eric Gottwald
Regular Article

Abstract

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.

Keywords

Hematopoietic progenitor cells Mesenchymal stromal cells Stem cell niche Microcavity array Bioreactor 

Notes

Acknowledgments

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

Disclosures

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.

References

  1. Altmann B, Lochner A, Swain M, Kohal RJ, Giselbrecht S, Gottwald E, Steinberg T, Tomakidi P (2014) Differences in morphogenesis of 3D cultured primary human osteoblasts under static and microfluidic growth conditions. Biomaterials 35:3208–3219CrossRefPubMedGoogle Scholar
  2. Bauer N, Wilsch-Brauninger M, Karbanova J, Fonseca AV, Strauss D, Freund D, Thiele C, Huttner WB, Bornhauser M, Corbeil D (2011) Haematopoietic stem cell differentiation promotes the release of prominin-1/CD133-containing membrane vesicles--a role of the endocytic-exocytic pathway. EMBO Mol Med 3:398–409CrossRefPubMedPubMedCentralGoogle Scholar
  3. Chabannon C, Wood P, Torok-Storb B (1992) Expression of CD7 on normal human myeloid progenitors. J Immunol 149:2110–2113PubMedGoogle Scholar
  4. Cheng T, Rodrigues N, Shen M, Yang Y-G, Dombkowksi D, Sykes M, Scadden DT (2000) Hematopoietic Stem Cell Quiescence Maintained by p21cip1/waf1. Science 287:1804–1808CrossRefPubMedGoogle Scholar
  5. Christophis C, Taubert I, Meseck GR, Schubert M, Grunze M, Ho AD, Rosenhah A (2011) Shear Stress Regulates Adhesion and Rolling of CD44+ Leukemic and Hematopoietic Progenitor Cells on Hyaluronan. Biophys J 101:585–593CrossRefPubMedPubMedCentralGoogle Scholar
  6. Dang LT, Feric NT, Laschinger C, Chang WY, Zhang B, Wood GA, Stanford WL, Radisic M (2014) Inhibition of apoptosis in human induced pluripotent stem cells during expansion in a defined culture using angiopoietin-1 derived peptide QHREDGS. Biomaterials 35:7786–7799CrossRefPubMedPubMedCentralGoogle Scholar
  7. de Peppo GM, Vunjak-Novakovic G, Marolt D (2014) Cultivation of human bone-like tissue from pluripotent stem cell-derived osteogenic progenitors in perfusion bioreactors. Methods Mol Biol 1202:173–184CrossRefPubMedPubMedCentralGoogle Scholar
  8. Dexter TM, Laijtha LG (1975) Proliferation of hemopoietic stem cells and development of potentially leukemic cells in vitro. Bibl Haematol 1975 Oct (43):1–5Google Scholar
  9. Dexter TM, Allen TD, Lajtha LG (1976) Conditions controlling the proliferation of haematopoietic stem cells in vitro. J Cell Physiol 91:335–344CrossRefGoogle Scholar
  10. Dominici M, Le Blanc K, Mueller I, Slaper-Cortenbach I, Marini F, Krause D, Deans R, Keating A, Prockop D, Horwitz E (2006) Minimal criteria for defining multipotent mesenchymal stromal cells. Int Soc Cell Ther Pos Statement Cytotherapy 8:315–317Google Scholar
  11. Ehninger A, Trumpp A (2011) The bone marrow stem cell niche grows up: mesenchymal stem cells and macrophages move in. J Exp Med 208:421–428CrossRefPubMedPubMedCentralGoogle Scholar
  12. Giselbrecht S, Gietzelt T, Gottwald E, Guber A, Trautmann C, Truckenmüller R, Weibezahn K-F (2004) Microthermoforming as a novel technique for manufacturing scaffolds in tissue engineering. IEE Proc Nanobiotechnol 151:151–157CrossRefPubMedGoogle Scholar
  13. Giselbrecht S, Gietzelt T, Gottwald E, Trautmann C, Truckenmüller R, Weibezahn K-F, Welle A (2006a) 3D tissue culture substrates produced by microthermoforming of pre-processed polymer films. Biomed Microdevices 8:191–199CrossRefPubMedGoogle Scholar
  14. Giselbrecht S, Gietzelt T, Gottwald E, Trautmann C, Truckenmüller R, Weibezahn K-F, Welle A (2006b) 3D tissue culture substrates produced by microthermoforming of pre-processed polymer films. Biomed Microdev 8:191–199CrossRefGoogle Scholar
  15. Giselbrecht S, Gottwald, E., Truckenmüller, R., Trautmann, C., Welle, A., Guber, A., Saile, V., Gietzelt, T., Weibezahn, K.-F. (2008) Microfabrication of chip-sized scaffolds for the three-dimensional cell cultivation J Vis Exp 15:e699Google Scholar
  16. Gottwald E, Giselbrecht S, Augspurger C, Lahni B, Dambrowsky N, Truckenmüller R, Piotter V, Gietzelt T, Wendt O, Pfleging W, Welle A, Rolletschek A, Wobus AM, Weibezahn K-F (2007a) A chip-based platform for the in vitro generation of tissues in three-dimensional organization. Lab Chip 7:777–785CrossRefPubMedGoogle Scholar
  17. Gottwald E, Giselbrecht S, Lahni B, Hiebl B, Weibezahn K-F (2007b) Cell Chip-basierte Bioreaktoren für die extrakorporale Organunterstützung. Galvanotechnik 4:974–978Google Scholar
  18. Gottwald E. LB, Thiele D., Giselbrecht S., Welle A., Weibezahn K.F. (2008) Chip-based three-dimensional cell culture in perfused micro-bioreactors. J Vis Exp 15:e564Google Scholar
  19. Hamblin TJ (2003) CD38: what is it good for? Blood 102:1939–1940CrossRefGoogle Scholar
  20. Handgretinger R, Kuci S (2013) CD133-Positive Hematopoietic Stem Cells: From Biology to Medicine. Adv Exp Med Biol 777:99–111CrossRefPubMedGoogle Scholar
  21. Hanke M, Hoffmann I, Christophis C, Schubert M, Hoang VT, Zepeda-Moreno A, Baran N, Eckstein V, Wuchter P, Rosenhahn A, Ho AD (2014) Differences between healthy hematopoietic progenitors and leukemia cells with respect to CD44 mediated rolling versus adherence behavior on hyaluronic acid coated surfaces. Biomaterials 35:1411–1419CrossRefPubMedGoogle Scholar
  22. Hunt P, Robertson D, Weiss D, Rennick D, Lee F, Witte ON (1987) A single bone marrow-derived stromal cell type supports the in vitro growth of early lymphoid and myeloid cells. Cell 48:997–1007CrossRefPubMedGoogle Scholar
  23. Jing D, Fonseca AV, Alakel N, Fierro FA, Muller K, Bornhauser M, Ehninger G, Corbeil D, Ordemann R (2009) Hematopoietic stem cells in co-culture with mesenchymal stromal cells--modeling the niche compartments in vitro. Haematologica 95:542–550CrossRefGoogle Scholar
  24. Jing DH, Fonseca AV, Alakel N, Fierro FA, Muller K, Bornhauser M, Ehninger G, Corbeil D, Ordemann R (2010) Hematopoietic stem cells in co-culture with mesenchymal stromal cells - modeling the niche compartments in vitro. Haematol-Hematol J 95:542–550CrossRefGoogle Scholar
  25. Kodama H, Hagiwara H, Sudo H, Amagai Y, Yokota T, Arai N, Kitamura Y (1986) MC3T3-G2/PA6 preadipocytes support in vitro proliferation of hemopoietic stem cells through a mechanism different from that of interleukin 3. J Cell Physiol 129:20–26CrossRefPubMedGoogle Scholar
  26. Lapidot T, Kollet O (2002) The essential roles of the chemokine SDF-1 and its receptor CXCR4 in human stem cell homing and repopulation of transplanted immune-deficient NOD/SCID and NOD/SCID/B2m(null) mice. Leukemia 16:1992–2003CrossRefPubMedGoogle Scholar
  27. Levesque JP, Helwani FM, Winkler IG (2010) The endosteal 'osteoblastic' niche and its role in hematopoietic stem cell homing and mobilization. Leukemia 24:1979–1992CrossRefPubMedGoogle Scholar
  28. Liu M, Liu N, Zang R, Li Y, Yang ST (2013) Engineering stem cell niches in bioreactors. World J Stem Cell 5:124–135CrossRefGoogle Scholar
  29. Ludwig A, Saffrich R, Eckstein V, Bruckner T, Wagner W, Ho AD, Wuchter P (2014) Functional potentials of human hematopoietic progenitor cells are maintained by mesenchymal stromal cells and not impaired by plerixafor. Cytotherapy 16:111–121CrossRefPubMedGoogle Scholar
  30. Marzesco AM, Janich P, Wilsch-Brauninger M, Dubreuil V, Langenfeld K, Corbeil D, Huttner WB (2005) Release of extracellular membrane particles carrying the stem cell marker prominin-1 (CD133) from neural progenitors and other epithelial cells. J Cell Sci 118:2849–2858CrossRefPubMedGoogle Scholar
  31. Mendez-Ferrer S, Michurina TV, Ferraro F, Mazloom AR, Macarthur BD, Lira SA, Scadden DT, Ma'ayan A, Enikolopov GN, Frenette PS (2010) Mesenchymal and haematopoietic stem cells form a unique bone marrow niche. Nature 466:829–834CrossRefPubMedPubMedCentralGoogle Scholar
  32. Moepps B, Frodl R, Rodewald HR, Baggiolini M, Gierschik P (1997) Two murine homologues of the human chemokine receptor CXCR4 mediating stromal cell-derived factor 1alpha activation of Gi2 are differentially expressed in vivo. Eur J Immunol 27:2102–2112CrossRefPubMedGoogle Scholar
  33. Mohty M, Hubel K, Kroger N, Aljurf M, Apperley J, Basak GW, Bazarbachi A, Douglas K, Gabriel I, Garderet L, Geraldes C, Jaksic O, Kattan MW, Koristek Z, Lanza F, Lemoli RM, Mendeleeva L, Mikala G, Mikhailova N, Nagler A, Schouten HC, Selleslag D, Suciu S, Sureda A, Worel N, Wuchter P, Chabannon C, Duarte RF (2014) Autologous haematopoietic stem cell mobilisation in multiple myeloma and lymphoma patients: a position statement from the European Group for Blood and Marrow Transplantation. Bone Marrow Transplant 49:865–872CrossRefPubMedGoogle Scholar
  34. O'Neill JD, Freytes DO, Anandappa AJ, Oliver JA, Vunjak-Novakovic GV (2013) The regulation of growth and metabolism of kidney stem cells with regional specificity using extracellular matrix derived from kidney. Biomaterials 34:9830–9841CrossRefPubMedGoogle Scholar
  35. Rieke M, Gottwald E, Weibezahn K-F, Layer PG (2008) Tissue reconstruction in 3D-spheroids from rodent retina in a motion-free, bioreactor-based microstructure. Lab Chip 8:1570–1579CrossRefGoogle Scholar
  36. Schajnovitz A, Itkin T, D'Uva G, Kalinkovich A, Golan K, Ludin A, Cohen D, Shulman Z, Avigdor A, Nagler A, Kollet O, Seger R, Lapidot T (2011) CXCL12 secretion by bone marrow stromal cells is dependent on cell contact and mediated by connexin-43 and connexin-45 gap junctions. Nat Immunol 12:391–398CrossRefPubMedGoogle Scholar
  37. Sharma MB, Limaye LS, Kale VP (2012) Mimicking the functional hematopoietic stem cell niche in vitro: recapitulation of marrow physiology by hydrogel-based three-dimensional cultures of mesenchymal stromal cells. Haematologica 97:651–660CrossRefPubMedPubMedCentralGoogle Scholar
  38. Siena S, Schiavo R, Pedrazzoli P, Carlo-Stella C (2000) Therapeutic relevance of CD34 cell dose in blood cell transplantation for cancer therapy. J Clin Oncol 18:1360–1377PubMedGoogle Scholar
  39. Spiller KL, Nassiri S, Witherel CE, Anfang RR, Ng J, Nakazawa KR, Yu T, Vunjak-Novakovic G (2015) Sequential delivery of immunomodulatory cytokines to facilitate the M1-to-M2 transition of macrophages and enhance vascularization of bone scaffolds. Biomaterials 37:194–207CrossRefPubMedPubMedCentralGoogle Scholar
  40. Truckenmüller R, Giselbrecht S, van Bitterswijk C, Dambrowsky N, Gottwald E, Mappes T, Rolletschek A, Saile V, Trautmann C, Weibezahn K-F (2008) Flexible fluidic microchips based on thermoformed and locally modified thin polymer films. Lab Chip 8:1570–1579CrossRefPubMedGoogle Scholar
  41. Truckenmuller R, Giselbrecht S, Rivron N, Gottwald E, Saile V, van den Berg A, Wessling M, van Blitterswijk C (2011) Thermoforming of film-based biomedical microdevices. Adv Mater 23:1311–1329CrossRefPubMedGoogle Scholar
  42. Tsai S, Emerson SG, Sieff CA, Nathan DG (1986) Isolation of a human stromal cell strain secreting hemopoietic growth factors. J Cell Physiol 127:137–145CrossRefPubMedGoogle Scholar
  43. Wagner W, Saffrich R, Wirkner U, Eckstein V, Blake J, Ansorge A, Schwager C, Wein F, Miesala K, Ansorge W, Ho AD (2005a) Hematopoietic progenitor cells and cellular microenvironment: behavioral and molecular changes upon interaction. Stem Cells 23:1180–1191CrossRefPubMedGoogle Scholar
  44. Wagner W, Wein F, Seckinger A, Frankhauser M, Wirkner U, Krause U, Blake J, Schwager C, Eckstein V, Ansorge W, Ho AD (2005b) Comparative characteristics of mesenchymal stem cells from human bone marrow, adipose tissue, and umbilical cord blood. Exp Hematol 33:1402–1416CrossRefPubMedGoogle Scholar
  45. Wagner W, Roderburg C, Wein F, Diehlmann A, Frankhauser M, Schubert R, Eckstein V, Ho AD (2007a) Molecular and secretory profiles of human mesenchymal stromal cells and their abilities to maintain primitive hematopoietic progenitors. Stem Cells 25:2638–2647CrossRefPubMedGoogle Scholar
  46. Wagner W, Wein F, Roderburg C, Saffrich R, Faber A, Krause U, Schubert M, Benes V, Eckstein V, Maul H, Ho AD (2007b) Adhesion of hematopoietic progenitor cells to human mesenchymal stem cells as a model for cell-cell interaction. Exp Hematol 35:314–325CrossRefPubMedGoogle Scholar
  47. Wagner W, Wein F, Roderburg C, Saffrich R, Faber A, Krause U, Schubert M, Benes V, Eckstein V, Maul H, Ho AD (2007c) Adhesion of hematopoietic progenitor cells to human mesenchymal stem cells as a model for cell-cell interaction. Exp Hematol 35:314–325CrossRefPubMedGoogle Scholar
  48. Walenda T, Bork S, Horn P, Wein F, Saffrich R, Diehlmann A, Eckstein V, Ho AD, Wagner W (2010) Co-culture with mesenchymal stromal cells increases proliferation and maintenance of haematopoietic progenitor cells. J Cell Mol Med 14:337–350CrossRefPubMedPubMedCentralGoogle Scholar
  49. Wein F, Pietsch L, Saffrich R, Wuchter P, Walenda T, Bork S, Horn P, Diehlmann A, Eckstein V, Ho AD, Wagner W (2010) N-cadherin is expressed on human hematopoietic progenitor cells and mediates interaction with human mesenchymal stromal cells. Stem Cell Res 4:129–139CrossRefPubMedGoogle Scholar
  50. Whitlock CA, Tidmarsh GF, Muller-Sieburg C, Weissman IL (1987) Bone marrow stromal cell lines with lymphopoietic activity express high levels of a pre-B neoplasia-associated molecule. Cell 48:1009–1021CrossRefPubMedGoogle Scholar
  51. Wuchter P, Boda-Heggemann J, Straub BK, Grund C, Kuhn C, Krause U, Seckinger A, Peitsch WK, Spring H, Ho AD, Franke WW (2007) Processus and recessus adhaerentes: giant adherens cell junction systems connect and attract human mesenchymal stem cells. Cell Tissue Res 328:499–514CrossRefPubMedGoogle Scholar
  52. Wuchter P, Leinweber C, Saffrich R, Hanke M, Eckstein V, Ho AD, Grunze M, Rosenhahn A (2014) Plerixafor induces the rapid and transient release of stromal cell-derived factor-1 alpha from human mesenchymal stromal cells and influences the migration behavior of human hematopoietic progenitor cells. Cell Tissue Res 355:315–326CrossRefPubMedGoogle Scholar

Copyright information

© Springer-Verlag Berlin Heidelberg 2015

Authors and Affiliations

  • Patrick Wuchter
    • 1
    • 3
  • Rainer Saffrich
    • 1
    • 3
  • Stefan Giselbrecht
    • 3
    • 4
  • Cordula Nies
    • 2
    • 3
  • Hanna Lorig
    • 2
    • 3
  • Stephanie Kolb
    • 2
    • 3
  • Anthony D. Ho
    • 1
    • 3
  • Eric Gottwald
    • 2
    • 3
  1. 1.Department of Medicine VHeidelberg UniversityHeidelbergGermany
  2. 2.Institute for Biological Interfaces-5Karlsruhe Institute of Technology (KIT)KarlsruheGermany
  3. 3.HEiKA - Heidelberg Karlsruhe Research PartnershipHeidelberg University and Karlsruhe Institute of TechnologyHeidelberg and KarlsruheGermany
  4. 4.Department of Complex Tissue Regeneration, MERLN Institute for Technology-Inspired Regenerative MedicineMaastricht UniversityMaastrichtThe Netherlands

Personalised recommendations