Heart failure is a leading cause of death in developed countries worldwide. It is typically caused by a heart attack that irreversibly damages the muscle of the heart. Damaged heart muscle is replaced by a non-functional scar tissue that limits the ability of the heart to pump enough blood to meet the body’s demand.
The only cure for heart failure is to have a heart transplant; however, there is a lack of available donor hearts to meet the demand. Not surprisingly then, over the last decades, researchers within the field of regenerative medicine and tissue engineering have turned to stem cells for their ability to replace damaged cardiac muscle.
A number of endogenous cardiac stem cell populations have been identified recently and are being characterized for their ability to be converted into cardiac muscle cells (cardiomyocytes) both in vitro and in vivo. In our manuscript, we study a population of cardiac stem cells with mesenchymal stem cell characteristics that are functionally defined by their ability to form colonies in vitro.
This population, referred to as cardiac colony forming unit-fibroblasts (cCFU-F) are thought to play a role in remodeling cardiac tissue during homeostasis and in response to injury. cCFU-F are rare cells (<1% of the cardiac interstitial population), which makes it difficult to study them in vivo.
In vitro, however, cCFU-F and their descendants rapidly proliferate, which makes it easier to study their function. While cCFU can self-renew in vitro, the common use of serum has made it difficult to identify cytokines that maintain their lineage identity and self-renewal ability. Therefore, we addressed this limitation by developing serum-free medium (SFM) for isolating, expanding, and characterizing cCFU-F in vitro.
In this study, we used PDGFRα-GFP expression as a marker of self-renewing cells to investigate how SFM and SCM influenced cCFU-F self-renewal. We isolated cells from adult PdgfraGFP/+ mice hearts and cultured them in SCM, or SFM containing an array of different cytokines. By employing time-lapse imaging and single-cell tracking technologies, we recorded fate outcomes, morphology, and PDGFRα expression for hundreds of single cells over time.
We found that SFM supplemented with basic fibroblast growth factor (bFGF), transforming growth factor-β (TGF-β) and platelet-derived growth factor (PDGF) enhanced cCFU-F colony formation and long-term self-renewal, with maintenance cCFU-F potency.
By flow cytometry, we observed that cCFU-F cultured in SFM supplemented with these factors (referred to as SFM-FTP) maintained a higher proportion of PDGFRα+ cells, a marker of self-renewing cCFU-F. By measuring cell cycle times and Pdgfra-GFP expression for mother and daughter cells, we found that the increased proportion of PDGFRα+ cells in SFM-FTP could be attributed to an increased probability of Pdgfra-GFP+ divisions. Using lineage-specific marker expression of single cells, we found that the increased probability of division of Pdgfra-GFP+ cells in SFM-FTP was accompanied by a reduction in the probability of myofibroblast differentiation.
Self-renewal and differentiation are dynamic processes that take place at the single-cell level. Therefore, they are best understood by an analysis of single-cell pedigrees. However, it is usually the case that self-renewal and differentiation can only be assessed retrospectively using functional assays. Therefore, to analyze renewal/differentiation dynamics within cell lineages requires predictive readouts of these traits.
In this study, we used this approach to show that SFM-FTP promoted a higher rate of PDGFRα-GFP+ self-renewal and a lower rate of myofibroblast differentiation. These findings may help us to understand the role cCFU-F play in cardiac repair given the pro-reparative and profibrotic functions of fibroblasts and myofibroblasts in remodeling cardiac tissue after injury.
These findings are described in the article entitled, Analysis of cardiac stem cell self-renewal dynamics in serum-free medium by single cell lineage tracking, recently published in the journal Stem Cell Research. This work was conducted by J.A. Cornwell (University of New South Wales, Australian Research Council Special Research Initiative in Stem Cell Science – Stem Cells, Victor Chang Cardiac Research Institute, University of Sydney), R.E. Nordon (University of New South Wales, Australian Research Council Special Research Initiative in Stem Cell Science – Stem Cells), and R.P. Harvey (Australian Research Council Special Research Initiative in Stem Cell Science – Stem Cells, Victor Chang Cardiac Research Institute, University of New South Wales).
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