Differentiation capacity and extracellular matrix production of cardiomyocyte progenitor cells
Citation for published version (APA):
Bax, N. A. M., Marion, van, M. H., Goumans, M. J. T. H., Bouten, C. V. C., & Schaft, van der, D. W. J. (2011). Differentiation capacity and extracellular matrix production of cardiomyocyte progenitor cells. Poster session presented at Mate Poster Award 2011 : 16th Annual Poster Contest.
Document status and date: Published: 01/01/2011
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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. The financial contribution of the Nederlandse Hartstichting is gratefully acknowledged
Introduction
Despite major improvements in therapy, heart diseases, such as myocardial infarction (MI), remain to be the leading cause of death in Western society [1]. MI leads to a reduction of
contractile force of the heart due to loss of cardiomyocytes. The discovery of cardiomyocyte progenitor cells (CMPCs) residing in the heart, which have been demonstrated to form new cardiac tissue and contribute to improvement of cardiac function, opens new possibilities for cardiac therapy [2,3]. To
repair the infarcted heart, not only lost cells need to be replaced, but also the scar tissue needs to be remodeled and be replaced with “healthy” extracellular matrix (ECM). The objective of this present study was to investigate whether CMPCs synthesize ECM. Therefore, the expression and production of ECM proteins was investigated in undifferentiated CMPCs and CMPCs at different time points in the differentiation process into the cardiomyogenic lineage.
Methods
Human L9TB cardiomyocyte progenitor cells
Human fetal CMPCs were isolated by magnetic cell sorting from fetal heart tissue using α-mouse-Sca-1-FITC antibody followed by binding to α-FITC-labeled iron beads. CMPCs are characterized by their expression of cardiac lineage markers, Nkx2.5 and Gata4 (Figure 2). Via RNA interference the human L9TB CMPCs cell line was created, which was used in this study.
Differentiation of CMPCs
To induce cardiac differentiation of L9TB CMPCs, cells were treated with 5 µM 5-azacytidine (5-aza) for 72hr in differentiation medium, containing vitamin C followed by transforming growth factor beta (TGFβ) treatment after one week (Figure 1).
Figure 1: Timeline of differentiation procedure
Results
CMPCs are able to differentiate into cardiomyocytes
Human CMPCs start to express the cardiomyocyte markers myocardin and cardiac troponin T (cTnT) after stimulation with 5-aza and TGFβ, which suggest differentiation into the cardiomyogenic lineage (Figure 3).
Figure 2: Cardiac lineage markers
Figure 3: Cardiomyocyte markers
Differentiation capacity and
extracellular matrix production of
cardiomyocyte progenitor cells
Before the onset of differentiation and after differentiation periods of 1, 2, 3 and 4 weeks the cells were isolated for qPCR and immunohistochemistry analysis to determine the state of differentiation and the production of ECM proteins.
4 1 0 2 3 5 6 7 8 differen1a1on medium 5-‐azacy1dine vitamin C TGFβ 1me [days] undifferen1ated CMPCs partly differen1ated CMPCs
[1]: Lloyd-Jones et al. Circulation 2009: 119: e21-181 [2]: Goumans et al. Stem Cell Res 2007: 1: 138-49 [3]: Smits et al. Nature Protols 2009: 4:232-43
N.A.M. Bax1#, M.H. van Marion1#, M.J.T.H Goumans2, C.V.C Bouten1, D.W.J van der Schaft1
1) Dep. of BioMedical Engineering, Eindhoven University of Technology, Eindhoven, The Netherlands
2) Dep. of Molecular Cell Biology, and Centre for Biomedical Genetics, Leiden University Medical Center, Leiden, The Netherlands
#Authors contributed equally.
Undifferentiated CMPCs as well as differentiating CMPCs produce ECM proteins
Undifferentiated CMPCs express Collagen I, III , Elastin and Fibronectin at low levels. During differentiation this expression was significantly upregulated (Figure 4).
Figure 4: Extracellular matrix proteins
Conclusion and Future directions
These results demonstrate that cardiomyogenic differentiation of human CMPCs was accompanied by the production of ECM matrix proteins. In addition to matrix production also matrix remodeling capacity needs to be investigated. For this purpose production of matrix metalloproteinases and their inhibitors by CMPCs are currently under investigation.
