Objectives: Therapeutic attempts based on transplantation of mesenchymal stem cells (MSCs) isolated from various sources showed inconsistent results. An ongoing investigation on resident cardiac stem cells reveals their regenerative role when specifically stimulated. In our recent study, we have demonstrated cardiomyogenic potential of the cardiac CD45 CD44+ DDR2+ MSC subpopulation isolated from rat hearts 24 hours after myocardial infarction (MI).
The aim of the present study was further molecular characterization of the MI-primed cardiac CD45 CD44+ DDR2+ subset relying on selective modulation of Wnt-signaling as well as evaluation of their functionality in a co-culture model within a cardiomyogenic environment.
Methods: Cardiac mononuclear cells were enzymatically isolated from rat hearts 24 hours post MI. Following a specific culture setup and using fluorescence-activated cell sorting, CD45 CD44+ DDR2+ MSCs were isolated. We adapted an additional cardiac differentiation protocol to these MI-primed MSCs which is commonly used for cardiac differentiation of pluripotent stem cells. Respective samples for qRT-PCR and for immunofluorescence (IF) staining were collected: before (day 0), after (day 3) the mesodermal induction and following cardiac activation (day 9). qRT-PCR toward cardiac transcription factors as well as IF detection of early cardiac proteins were performed. Moreover, for functional characterization, this cardiac differentiated and MI-primed MSC subset was analyzed in co-culture model with stem cell derived sinoatrial pacemaker cells (iSABs) and murine neonatal cardiomyocytes.
Result: When MI-primed cardiac CD45 CD44+ DDR2+ MSCs were induced toward a mesodermal fate (d0–d3), a highest peak in expression of the cardiovascular markers MEF2c and GATA4 was achieved on day 6. Surprisingly, mRNA levels of NKx2.5 slightly dropped by day 6, but increased again on day 9. While Mesp1 gene expression constantly increased over time, similarly to Eomes, mRNA encoding Brachyury was upregulated only on day 6. After nine days of differentiation, cardiac MI-primed MSCs showed successful integration into a cardiac cell environment, particularly associated by an expression of the sarcomere-marker, ACTN2.
Conclusion: Selective stimulation and inhibition of the canonical WNT-pathway supported a cardiac progenitor cell fate in the resident MI-primed CD45 CD44+ DDR2+ MSCs. It appears likely that an additional priming of this MSC subset toward cardiovascular fate may potentiate cardiovascular lineages. In summary, our findings may become of great importance for our understanding and optimization of cardiovascular regeneration and therapy options.
