Author(s) Institution Fibroblasts facilitate the engraftment of embryonic stem cell-derived cardiomyocytes on three-dimensional collagen matrices Matrices and aggregation in hanging drops Pfannkuche K, Neuss S, Pillekamp F, Frenzel L, Attia W, Hannes T, Salber J, Hoss M, Zenke M, Fleischmann B, Hescheler J, Saric T. University of Cologne, Neurophysiology, Robert Koch Str. 39, Cologne, Germany, 50931, 00492214786960; kurt.pfannkuche@uni-koeln.de Stem Cells Dev. 2010 Feb 22 Abstract Aim of the Work There is growing interest in use of cardiomyocytes purified from embryonic stem (ES) cells for tissue engineering and cardiomyoplasty. However, most of transplanted cells are lost shortly after transplantation due to the lack of integration into the host tissue and subsequent apoptosis. Here we examine whether murine embryonic fibroblasts (MEFs) can support the integration of purified murine ES cell-derived cardiomyocytes in a three-dimensional tissue culture model based on a freezed-dryed collagen matrix with tubular structure. Collagen matrix was seeded either with cardiomyocytes alone or in combination with MEFs. The collagen sponges that were transplanted with cardiomyocytes alone did neither show morphological nor functional integration of viable cells. Cardiomyocytes also did not appear to be capable of attaching quantitatively to any of 16 different two-dimensional biomaterials. However, cardiomyocytes co-cultured with MEFs formed fibre-like structures of rod-shaped cells with organized sarcomeric structure that contracted spontaneously. Electrical coupling between cardiomyocytes was suggested by strong expression of connexin 43. In addition, MEFs as well as cardiac fibroblasts supported re-aggregation of dissociated cardiomyocytes in hanging drops in the absence of collagen matrix. We conclude that fibroblasts promote cardiomyocyte engraftment and formation of functional three-dimensional tissue in vitro. Elucidation of the mechanism of this phenomenon may help improving the integration of cardiomyocytes in vivo. To assess the role of fibroblasts in engraftment of ESC-CM we have developed an in vitro model based on a synthetic three-dimensional extracellular matrix composed of collagen type I. In addition, we used the hanging drop method to assess the ability of MEFs to support reaggregation of dissociated ESC-CM into beating cell clusters. We demonstrate that ESC-CM alone were incapable of populating the collagen matrix but when seeded together with MEFs they integrated well and formed three-dimensional spontaneously beating structures. This effect was independent of the collagen matrix as demonstrated by the ability of MEFs and cardiac fibroblasts to support the aggregation of ESC-CM in hanging drops. These findings suggest that fibroblasts provide mechanical support and/or surface-bound or soluble factors that promote the organisation of cardiomyocytes into three-dimensional structures. Methods and Results ES cells: αPIG44 ES cells express the puromycin resistance gene and enhanced green fluorescent protein (eGFP) gene under the control of a cardiospecific α-myosin heavy chain promoter . On day 9, puromycin was added to a final concentration of 10 Qg/ml. From day 14 on, purified cardiac bodies were incubated without puromycin and used for experiments within one week Fig 1 Assessment of purity and viability of lineage selected ESC-CM II-Engraftment of ES-CMCs into Collagen matrix The engraftment into collagen matrix of ESC-CM seeded alone or in combination with MEFs was monitored daily by fluorescence microscopy One week after seeding the collagen sponges were fixed with PFA and stained with propidium iodide to visualize all seeded cells. Figure 2: Effect of fibroblasts on cardiomyocytes engraftment into collagen matrix A) Cardiomyocytes and fibroblasts in coculture seven days after seeding into Matricel. Intact sponge was labelled with propidium iodide (PI, pink) after fixation to mark all cells present. Cardiomyocytes can be additionally identified as eGFP-positive cells (green). Scale bar: 20 Qm. B) Cross-section (300 Qm thick) through a PFA-fixed PI-stained collagen sponge seeded with MEFs and GFP- expressing ESC-CMs seven days after seeding al lower magnification. Bar: 200 C and D) GFP-positive cardiomyocytes depicted in intact collagen sponge in the presence of MEFs as elongated cells at day 7 (C) and 14 (D) after seeding. Scale bar: 200 Qm. E) eGFPexpressing ESC-CMs directly plated on MEFs in a two-dimensional tissue culture plate can be easily distinguished from MEFs by their strong green fluorescence. Bar: 200 Qm. G)Typical cross-striations in cardiomyocyte fibres formed in cocultures with MEFs one week after seeding into collagen matrix (α-actinin staining). Bar: 20 Qm. H) Cardiomyocytes cocultured with MEFs in collagen sponges communicate through gap junctions stained with connexin 43 antibodies (red). Cross-striations in cardiomyocytes were visualized by α-actinin staining (yellow) and nuclei were stained with Hoechst 33432 (blue). Bar: 20 Qm. I) Three dimensional reconstructed view of the figure in panel H shows ordered distribution of connexin 43-positive gap junctions on the surface of cardiomyocytes. Bar: 20 Qm. Fig 3: ES-CMs Engraftment in the absence of MEFs J) Low magnification view of GFPpositive ESC-CMs cultured in the absence of MEFs on collagen sponges for 1 week. Bar: 200 Qm. K, L and M) Transmission light (K) and epifluorescence image (L) of collagen matrix seeded with pure ESC-CMs for 14 days. GFP-positive ESC-CMs are still trapped in the collagen matrix after 14 days of culture but remain round- shaped. Panel M is an overlay of images K and L. Bars: 50 Qm. N) GFP-positive cardiomyocytes cultured in the absence of MEFs on collagen sponges for 3 weeks failed to elongate and still do not show any structural maturation. Bar: 100 Qm. O) High resolution view of a fixed sample of a collagen sponge that was seeded with pure cardiomyocytes after seven days in culture. Sarcomeric α-actinin staining (red) reveals only a spotted pattern of α-actinin protein expression. Nuclei were stained with Hoechst 33432 (blue). Lack of clear overlap between α-actinin and nuclear staining is due to nuclei being partially out of focus in this three-dimensional structure. Magnification bar: 20 Qm. III-Formation of secondary cardiac bodies in hanging drops Hanging drops (20 μl) were formed with single cell suspensions of MEFs alone (1000 cells/drop), ESC-CMs alone (1000 cells/drop), or admixtures of 200 MEFs and 1000 ESC- CMs per drop. On day 2 of culture, cell aggregates formed in hanging drops were transferred onto the bottom of a corresponding 96-well plate by centrifugation and the same cell cluster was imaged on days indicated on the left side of each panel row. MEFs were labelled with Cell Tracker Red CMTPX and can be easily identified by their red fluorescence. Viable ESC-CMs express eGFP and are depicted as green fluorescent cells. Dead cardiomyocytes lost their eGFP expression and are seen as dark cells scattered around the clusters, mostly in “CM only” aggregates. Conclusions Our data indicate that it is crucial to provide the cardiomyocytes with an environment that supports their integration and structural maturation. This may include the addition of supportive cells or other types of substrates and substances that promote cell adhesion and prevent cell death upon transplantation. In vitro models, such as those employed in this study, may allow for controlled investigation of interaction between different cell types and provide valuable information that could be tested in a more complex in vivo system. These in vitro assays may also facilitate the rational design of safer and more efficient cardiac cell therapies. Acknowledgement Collagen matrices were a generous gift from Dr. Ingo Heschel, Matricel GmbH, Herzogenrath, Germany. We thank Katja Martina Eckl and Damir Jacob Illich for isolation of cardiac fibroblasts. We thank Nadin Lange, Rebecca Dieterich and Stephanie Brosig for excellent technical support. This study was supported by grants from the Federal Ministry for Education and Research to T.Š. and H.J. (Grant Nr. 01 GN 0947), and the Else-Kroner Fresenius Stiftung (Grant Nr. 85/2009) and the Koln Fortune Program to T. Š. (Grant Nr. 85/2009). LPF was supported by a stipend from the Koln Fortune Programm (KF133/2007). Addition of puromycin to differentiating embryoid bodies at day 8 of differentiation gives rise to pure cardiac bodies (A) that are composed almost exclusively of eGFP-expressing cardiomyocytes at day 12 of differentiation (B). Single cardiac myocytes generated by enzymatic digestion of cardiac bodies were plated on fibronectin-coated culture plates and incubated for 4 days (C and D) or for 30 days (E) prior to staining with antibodies against α-actinin (green fluorescence). Cardiomyocytes were also plated on inactivated murine embryonic fibroblasts and stained for α-actinin (red) 20 days later (F). Nuclei in images C-F were stained with Hoechst 33432 (blue fluorescence). Scale bars: panels A, B and F - 200 μm, panel C – 500 μm, and panels D and E – 100 μm.