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Tightly anchored tissue-mimetic matrices as instructive stem cell microenvironments

Nature Methods volume 10, pages 788–794 (2013)Cite this article

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Abstract

A major obstacle in defining the exact role of extracellular matrix (ECM) in stem cell niches is the lack of suitable in vitro methods that recapitulate complex ECM microenvironments. Here we describe a methodology that permits reliable anchorage of native cell–secreted ECM to culture carriers. We validated our approach by fabricating two types of human bone marrow–specific ECM substrates that were robust enough to support human mesenchymal stem cells (MSCs) and hematopoietic stem and progenitor cells in vitro. We characterized the molecular composition, structural features and nanomechanical properties of the MSC-derived ECM preparations and demonstrated their ability to support expansion and differentiation of bone marrow stem cells. Our methodology enables the deciphering and modulation of native-like multicomponent ECMs of tissue-resident stem cells and will therefore prepare the ground for a more rational design of engineered stem cell niches.

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Figure 1: Surface-anchored cell-derived ECM in vitro.
Figure 2: Properties of decellularized ECM.
Figure 3: Proteomic composition of bone marrow–mimetic ECM.
Figure 4: Human bone marrow MSCs cultured on MSC-derived ECM.
Figure 5: Human CD34+ cells cultured on MSC-derived ECM.

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References

  1. Schofield, R. The relationship between the spleen colony-forming cell and the haemopoietic stem cell. Blood Cells 4, 7–25 (1978).

    CAS  PubMed  Google Scholar 

  2. Wang, L.D. & Wagers, A.J. Dynamic niches in the origination and differentiation of haematopoietic stem cells. Nat. Rev. Mol. Cell Biol. 12, 643–655 (2011).

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  3. Discher, D.E., Mooney, D.J. & Zandstra, P.W. Growth factors, matrices, and forces combine and control stem cells. Science 324, 1673–1677 (2009).

    CAS  PubMed  PubMed Central  Google Scholar 

  4. Engler, A.J., Sen, S., Sweeney, H.L. & Discher, D.E. Matrix elasticity directs stem cell lineage specification. Cell 126, 677–689 (2006).

    Article  CAS  PubMed  Google Scholar 

  5. Holst, J. et al. Substrate elasticity provides mechanical signals for the expansion of hemopoietic stem and progenitor cells. Nat. Biotechnol. 28, 1123–1128 (2010).

    Article  CAS  PubMed  Google Scholar 

  6. Gupta, P. et al. Structurally specific heparan sulfates support primitive human hematopoiesis by formation of a multimolecular stem cell niche. Blood 92, 4641–4651 (1998).

    Article  CAS  PubMed  Google Scholar 

  7. Coulombel, L., Vuillet, M.H., Leroy, C. & Tchernia, G. Lineage- and stage-specific adhesion of human hematopoietic progenitor cells to extracellular matrices from marrow fibroblasts. Blood 71, 329–334 (1988).

    Article  CAS  PubMed  Google Scholar 

  8. Seib, F.P. et al. Engineered extracellular matrices modulate the expression profile and feeder properties of bone marrow-derived human multipotent mesenchymal stromal cells. Tissue Eng. Part A 15, 3161–3171 (2009).

    Article  CAS  PubMed  Google Scholar 

  9. Chen, X.-D., Dusevich, V., Feng, J.Q., Manolagas, S.C. & Jilka, R.L. Extracellular matrix made by bone marrow cells facilitates expansion of marrow-derived mesenchymal progenitor cells and prevents their differentiation into osteoblasts. J. Bone Miner. Res. 22, 1943–1956 (2007).

    Article  CAS  PubMed  Google Scholar 

  10. Klimanskaya, I. et al. Human embryonic stem cells derived without feeder cells. Lancet 365, 1636–1641 (2005).

    Article  CAS  PubMed  Google Scholar 

  11. Cukierman, E., Pankov, R., Stevens, D.R. & Yamada, K.M. Taking cell-matrix adhesions to the third dimension. Science 294, 1708–1712 (2001).

    Article  CAS  PubMed  Google Scholar 

  12. Vlodavsky, I. Preparation of extracellular matrices produced by cultured corneal endothelial and PF-HR9 endodermal cells. Curr. Protoc. Cell Biol. 1, 10.4 (2001).

    Google Scholar 

  13. Beacham, D.A., Amatangelo, M.D. & Cukierman, E. Preparation of extracellular matrices produced by cultured and primary fibroblasts. Curr. Protoc. Cell Biol. 33, 10.9 (2007).

    Google Scholar 

  14. Calvi, L.M. et al. Osteoblastic cells regulate the haematopoietic stem cell niche. Nature 425, 841–846 (2003).

    Article  CAS  PubMed  Google Scholar 

  15. Méndez-Ferrer, S. et al. Mesenchymal and haematopoietic stem cells form a unique bone marrow niche. Nature 466, 829–834 (2010).

    Article  PubMed  PubMed Central  CAS  Google Scholar 

  16. Pompe, T. et al. Maleic anhydride copolymers—a versatile platform for molecular biosurface engineering. Biomacromolecules 4, 1072–1079 (2003).

    Article  CAS  PubMed  Google Scholar 

  17. Gordon, M.Y. Extracellular matrix of the marrow microenvironment. Br. J. Haematol. 70, 1–4 (1988).

    Article  CAS  PubMed  Google Scholar 

  18. Ren, J. et al. Global transcriptome analysis of human bone marrow stromal cells (BMSC) reveals proliferative, mobile and interactive cells that produce abundant extracellular matrix proteins, some of which may affect BMSC potency. Cytotherapy 13, 661–674 (2011).

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  19. Lanfer, B. et al. The growth and differentiation of mesenchymal stem and progenitor cells cultured on aligned collagen matrices. Biomaterials 30, 5950–5958 (2009).

    Article  CAS  PubMed  Google Scholar 

  20. Shultz, L.D., Ishikawa, F. & Greiner, D.L. Humanized mice in translational biomedical research. Nat. Rev. Immunol. 7, 118–130 (2007).

    Article  CAS  PubMed  Google Scholar 

  21. Eppert, K. et al. Stem cell gene expression programs influence clinical outcome in human leukemia. Nat. Med. 17, 1086–1093 (2011).

    Article  CAS  PubMed  Google Scholar 

  22. Flaim, C.J., Chien, S. & Bhatia, S.N. An extracellular matrix microarray for probing cellular differentiation. Nat. Methods 2, 119–125 (2005).

    Article  CAS  PubMed  Google Scholar 

  23. Ehninger, A. & Trumpp, A. The bone marrow stem cell niche grows up: mesenchymal stem cells and macrophages move in. J. Exp. Med. 208, 421–428 (2011).

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  24. Bianco, P., Robey, P.G. & Simmons, P.J. Mesenchymal stem cells: revisiting history, concepts, and assays. Cell Stem Cell 2, 313–319 (2008).

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  25. Dexter, T.M., Spooncer, E., Simmons, P. & Allen, T.D. Long-term marrow culture: an overview of techniques and experience. Kroc Found. Ser. 18, 57–96 (1984).

    CAS  PubMed  Google Scholar 

  26. Baxter, M.A. et al. Study of telomere length reveals rapid aging of human marrow stromal cells following in vitro expansion. Stem Cells 22, 675–682 (2004).

    Article  CAS  PubMed  Google Scholar 

  27. Wagner, W. et al. Replicative senescence of mesenchymal stem cells: a continuous and organized process. PLoS ONE 3, e2213 (2008).

    Article  PubMed  PubMed Central  CAS  Google Scholar 

  28. Matsubara, T. et al. A new technique to expand human mesenchymal stem cells using basement membrane extracellular matrix. Biochem. Biophys. Res. Commun. 313, 503–508 (2004).

    Article  CAS  PubMed  Google Scholar 

  29. Mauney, J.R., Volloch, V. & Kaplan, D.L. Matrix-mediated retention of adipogenic differentiation potential by human adult bone marrow-derived mesenchymal stem cells during ex vivo expansion. Biomaterials 26, 6167–6175 (2005).

    Article  CAS  PubMed  Google Scholar 

  30. Chen, X.-D. Extracellular matrix provides an optimal niche for the maintenance and propagation of mesenchymal stem cells. Birth Defects Res. C Embryo Today 90, 45–54 (2010).

    Article  CAS  PubMed  Google Scholar 

  31. Sun, Y. et al. Rescuing replication and osteogenesis of aged mesenchymal stem cells by exposure to a young extracellular matrix. FASEB J. 25, 1474–1485 (2011).

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  32. Bruckner, P. Suprastructures of extracellular matrices: paradigms of functions controlled by aggregates rather than molecules. Cell Tissue Res. 339, 7–18 (2010).

    Article  CAS  PubMed  Google Scholar 

  33. Hines, M., Nielsen, L. & Cooper-White, J. The hematopoietic stem cell niche: what are we trying to replicate? J. Chem. Technol. Biotechnol. 83, 421–443 (2008).

    Article  CAS  Google Scholar 

  34. Lapidot, T. & Kollet, O. 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/B2mnull mice. Leukemia 16, 1992–2003 (2002).

    Article  CAS  PubMed  Google Scholar 

  35. Fleming, H.E. et al. Wnt signaling in the niche enforces hematopoietic stem cell quiescence and is necessary to preserve self-renewal in vivo. Cell Stem Cell 2, 274–283 (2008).

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  36. Janowska-Wieczorek, A., Majka, M., Ratajczak, J. & Ratajczak, M.Z. Autocrine/paracrine mechanisms in human hematopoiesis. Stem Cells 19, 99–107 (2001).

    Article  CAS  PubMed  Google Scholar 

  37. Tsuji, K., Ueda, T. & Ebihara, Y. Cytokine-mediated expansion of human NOD-SCID-repopulating cells. Methods Mol. Biol. 215, 387–395 (2003).

    CAS  PubMed  Google Scholar 

  38. Alakel, N. et al. Direct contact with mesenchymal stromal cells affects migratory behavior and gene expression profile of CD133+ hematopoietic stem cells during ex vivo expansion. Exp. Hematol. 37, 504–513 (2009).

    Article  CAS  PubMed  Google Scholar 

  39. Kurth, I., Franke, K., Pompe, T., Bornhäuser, M. & Werner, C. Hematopoietic stem and progenitor cells in adhesive microcavities. Integr. Biol. (Camb.) 1, 427–434 (2009).

    Article  CAS  Google Scholar 

  40. Franke, K., Pompe, T., Bornhäuser, M. & Werner, C. Engineered matrix coatings to modulate the adhesion of CD133+ human hematopoietic progenitor cells. Biomaterials 28, 836–843 (2007).

    Article  CAS  PubMed  Google Scholar 

  41. Melkoumian, Z. et al. Synthetic peptide-acrylate surfaces for long-term self-renewal and cardiomyocyte differentiation of human embryonic stem cells. Nat. Biotechnol. 28, 606–610 (2010).

    Article  CAS  PubMed  Google Scholar 

  42. Rodin, S. et al. Long-term self-renewal of human pluripotent stem cells on human recombinant laminin-511. Nat. Biotechnol. 28, 611–615 (2010).

    Article  CAS  PubMed  Google Scholar 

  43. Lanfer, B. et al. Aligned fibrillar collagen matrices obtained by shear flow deposition. Biomaterials 29, 3888–3895 (2008).

    Article  CAS  PubMed  Google Scholar 

  44. Oswald, J. et al. Mesenchymal stem cells can be differentiated into endothelial cells in vitro. Stem Cells 22, 377–384 (2004).

    Article  PubMed  Google Scholar 

  45. Gilbert, T.W., Sellaro, T.L. & Badylak, S.F. Decellularization of tissues and organs. Biomaterials 27, 3675–3683 (2006).

    CAS  PubMed  Google Scholar 

  46. Pittenger, M.F., Mbalaviele, G., Black, M., Mosca, J.D. & Marshak, D.R. Mesenchymal stem cells. in Human Cell Culture Vol. 5 (eds. Koller, M.R., Palsson, B.O. & Masters, J.R.W.) Ch. 9, 189–207 (Kluwer, 2001).

  47. Sen, A. et al. Adipogenic potential of human adipose derived stromal cells from multiple donors is heterogeneous. J. Cell. Biochem. 81, 312–319 (2001).

    Article  CAS  PubMed  Google Scholar 

  48. Waskow, C. et al. The receptor tyrosine kinase Flt3 is required for dendritic cell development in peripheral lymphoid tissues. Nat. Immunol. 9, 676–683 (2008).

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  49. Wight, T.N., Kinsella, M.G., Keating, A. & Singer, J.W. Proteoglycans in human long-term bone marrow cultures: biochemical and ultrastructural analyses. Blood 67, 1333–1343 (1986).

    Article  CAS  PubMed  Google Scholar 

  50. Hutter, J.L. & Bechhoefer, J. Calibration of atomic-force microscope tips. Rev. Sci. Instrum. 64, 1868–1873 (1993).

    Article  CAS  Google Scholar 

  51. Krieg, M. et al. Tensile forces govern germ-layer organization in zebrafish. Nat. Cell Biol. 10, 429–436 (2008).

    Article  CAS  PubMed  Google Scholar 

  52. Shevchenko, A., Tomas, H., Havlis, J., Olsen, J.V. & Mann, M. In-gel digestion for mass spectrometric characterization of proteins and proteomes. Nat. Protoc. 1, 2856–2860 (2006).

    Article  CAS  PubMed  Google Scholar 

  53. Cox, J. & Mann, M. MaxQuant enables high peptide identification rates, individualized p.p.b.-range mass accuracies and proteome-wide protein quantification. Nat. Biotechnol. 26, 1367–1372 (2008).

    Article  CAS  PubMed  Google Scholar 

  54. Hubner, N.C. & Mann, M. Extracting gene function from protein-protein interactions using Quantitative BAC InteraCtomics (QUBIC). Methods 53, 453–459 (2011).

    Article  CAS  PubMed  Google Scholar 

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Acknowledgements

The authors thank A. Springer for help with electron microscopy analysis and K. Schneider and K. Heidel for technical assistance. This work was supported by the Deutsche Forschungsgemeinschaft within the Collaborative Research Center “From Cells to Tissues” SFB 655 (A12/B2, M.v.B. and C. Werner; B9, C. Waskow) and by the Research Consortium SkelMet (M.v.B.). F.P.S. was supported by a Mildred Scheel Postdoctoral fellowship from the German Cancer Aid, and M.v.B. was supported by the German Cancer Consortium and by the German Cancer Research Center.

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Author notes

  1. F Philipp Seib

    Present address: Present address: Strathclyde Institute of Pharmacy and Biomedical Sciences, University of Strathclyde, Glasgow, UK.,

Authors and Affiliations

  1. Leibniz Institute of Polymer Research Dresden, Max Bergmann Center of Biomaterials, Dresden, Germany

    Marina C Prewitz, F Philipp Seib, Jens Friedrichs, Aline Stißel & Carsten Werner

  2. Medical Clinic I, University Hospital Carl Gustav Carus, Dresden, Germany

    Malte von Bonin, Katrin Müller & Martin Bornhäuser

  3. Biotechnology Center, Dresden University of Technology (TU Dresden), Dresden, Germany

    Christian Niehage, Konstantinos Anastassiadis & Bernard Hoflack

  4. Center for Regenerative Therapies, TU Dresden, Dresden, Germany

    Claudia Waskow, Martin Bornhäuser & Carsten Werner

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Contributions

M.C.P. designed and performed experiments, analyzed data and wrote the manuscript; F.P.S. designed experiments and cosupervised parts of the study; M.v.B. designed, performed and analyzed transplantation experiments together with M.C.P.; C. Waskow established the transplantation experiments; J.F. performed and analyzed AFM measurements; A.S. performed experiments; K.M. isolated primary human MSCs; C.N. and B.H. performed mass spectroscopy and analyzed the proteomics data; K.A. gave conceptual advice; M.B. and C. Werner initiated and supervised the study and edited the manuscript.

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Correspondence to Carsten Werner.

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Supplementary information

Supplementary Text and Figures

Supplementary Figures 1–7 and Supplementary Table 1 (PDF 5477 kb)

Supplementary Data

Excel list of all detected proteins via Orbitrap mass spectrometry. Each mass spectrometrically analyzed sample comprises the pool of five different MSC-donor ECM extracts (for aaECM and osteoECM respectively). Data represent three independent experiments. (XLSX 406 kb)

Time-lapse visualization of MSC behavior in contact with aaECM

Time-lapse recordings of MSC in contact with aaECM for a total duration of 89 hours, starting from day 1 in culture. MSC align and migrate along the underlying ECM fibrillar structures. Several events of cell division can be observed during the recorded time frame. Scale bar at 100 μm. (MP4 5324 kb)

Time-lapse visualization of MSC behavior in contact with osteoECM

Time-lapse recordings of MSC in contact with osteoECM for a total duration of 89 hours, starting from day 1 in culture. MSC align and migrate along the underlying ECM fibrillar structures and actively pull and rearrange the matrix fibers. Several events of cell division can be observed during the recorded time frame. Scale bars at 100 μm. (MP4 5315 kb)

Time-lapse visualization of MSC behavior in contact with PTP

Time-lapse recordings of MSC in contact with PTP for a total duration of 89 hours, starting from day 1 in culture. MSC demonstrate very polygonal shaped morphology and make wide spread contacts with the underlying surface, thereby generating cytoskeletal stress fibers and long cell protrusions to contact other neighboring cells. Scale bars at 100 μm. (MP4 5640 kb)

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Prewitz, M., Seib, F., von Bonin, M. et al. Tightly anchored tissue-mimetic matrices as instructive stem cell microenvironments. Nat Methods 10, 788–794 (2013). https://doi.org/10.1038/nmeth.2523

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