Constrainment is crucial for the survival, alignment and differentiation of cardiomyocyte progenitor cells
Citation for published version (APA):
Marion, van, M. H., Schaft, van der, D. W. J., Goumans, M. J. T. H., Baaijens, F. P. T., & Bouten, C. V. C. (2011). Constrainment is crucial for the survival, alignment and differentiation of cardiomyocyte progenitor cells. Poster session presented at Mate Poster Award 2011 : 16th Annual Poster Contest.
Document status and date: Published: 01/01/2011 Document Version:
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Constrainment is crucial for the survival,
alignment and differentation of cardiomyocyte
progenitor cells in 3D tissue culture
M.H. van Marion1, D.W.J. van der Schaft1, M.J. Goumans2, F.P.T. Baaijens1
and C.V.C. Bouten1
1Soft Tissue Biomechanics and Engineering, TU/e; 2Molecular Cell Biology, LUMC
Introduction
Stem cell therapy has emerged as promising treatment for myocardial infarction. A population of stem cells that resides in the human heart (cardiomyocyte progenitor cells, CMPCs) can be isolated and differentiated into beating cardiomyocytes using biochemical factors in
vitro [1]. Upon injection in the heart, these cells will
experience a three dimensional (3D) rather than a 2D environment and are exposed to mechanical forces. We therefore investigate how these CMPCs behave in 3D constrained environment.
Methods
Human fetal CMPCs were encapsulated in A) longitudinally constrained and B) free floating hydrogels composed of collagen-Matrigel (n=3 each) (fig. 1). Resulting constructs were cultured for 9 days in control or differentiation medium, and analyzed for compaction cell survival, proliferation, alignment and cardiac differentiation using immunofluorescent stainings.
Results
Cell survival and alignment
Cells survival was better in constrained constructs. Cells aligned in the direction of the constraint (fig. 2).
Construct compaction
Constrained constructs showed strong compaction and internal stress fiber formation (fig. 3), probably resulting in cell alignment. Compaction was greatly enhanced after culture in differentiation medium.
Cell proliferation
Proliferation in constrained constructs was decreased, but 14% of the cells was still able to divide (fig. 4). In contrast, in free floating gels proliferation was abolished.
Cardiac differentiation
Cells cultured in constrained constructss expressed the cardiac markers Nkx2.5 and α-actinin, independent of culture medium (fig. 5). GATA4 was only expressed in constructs cultured in differentiated medium.
Conclusion
Constraining appeared to be a crucial factor for the survival, proliferation and cardiac differentiation of CMPCs cultured in 3D hydrogel-based constructs. Moreover, constraining resulted in cell alignment, which is important for proper tissue integration. Optimization of the culture system with e.g. synthetic materials may be of great relevance for cardiac regeneration.
Figure 1: Macroscopic pictures of (A) longitudinally constrained
and (B) free floating constructs.
constrained free floating number of counts degrees number of counts degrees Aa Ab Ba Bb
Figure 2: A: confocal images of life (green) and dead (red) cells. B:
Histogram showing the direction of the long axes of living cells. (a) constrained hydrogels; (b) and free floating constructs.
constrained
100 μm
Figure 3: A: Immunofluorescent image of cells stained for stress fibers
(red) in constrained constructs (nuclei in blue, arrow denotes constrained direction). B: Bar plot showing the percentage of compaction in constrained constructs after 1 and 9 days of culture.
A B
Figure 4: Bar plot showing
the percentage of BrdU positive cells, as measure for cell proliferation, in
constrained and free
floating constructs after 1 and 9 days of culture.
100 μm α-actinin Nkx2.5 DAPI GATA4 vimentin DAPI 100 μm Figure 5: Immunofluorescent images of Nkx 2.5,
GATA4, α-actinin, and
vimentin expression in
9 days cultured
constrained hydrogels. cell proliferation in constrained and
free floating hydrogels
% B rd U p o si ti v e cel ls
day 1 day 9, control day 9, differentiated
compaction in constrained constructs
% o f co mp acti o n day 1 day 9
References: [1] MJ Goumans et al. Stem Cell Research 2007,1: 138-149.
This research forms part of the Project P1.04 SMARTCARE of the research program of the BioMedical
Materials institute, co-funded by the Dutch Ministry of Economic Affairs, Agriculture and Innovation.
