Cardiovascular diseases (CVDs) are often linked to ageing and are the major cause of death worldwide. The declined proliferation of adult stem cells in the heart often impedes its regenerative potential. Thus, an investigation of the proliferative potential of adult human cardiac stem cells (hCSCs) might be of great interest for improving cell-based treatments of cardiovascular diseases. The application of human blood serum was already shown to enhance hCSC proliferation and reduce senescence. Here, the underlying signalling pathways of serum-mediated hCSC proliferation were studied. We are the first to demonstrate the involvement of the transcription factor NF-κB in the serum-mediated proliferative response of hCSCs by utilizing the NF-κB inhibitor pyrrolidine dithiocarbamate (PDTC). RNA-Sequencing (RNA-Seq) revealed ATF6B, COX5B, and TNFRSF14 as potential targets of NF-κB that are involved in serum-induced hCSC proliferation.

The ageing phenotype is strongly driven by the exhaustion of adult stem cells (ASCs) and the accumulation of senescent cells. Cardiovascular diseases (CVDs) and heart failure (HF) are strongly linked to the ageing phenotype and are the leading cause of death. As the human heart is considered as an organ with low regenerative capacity, treatments targeting the rejuvenation of human cardiac stem cells (hCSCs) are of great interest. In this study, the beneficial effects of human blood serum on proliferation and senescence of hCSCs have been investigated at the molecular level. We show the induction of a proliferation-related gene expression response by human blood serum at the mRNA level. The concurrent differential expression of the TGFβ target and inhibitor genes indicates the participation of TGFβ signalling in this context. Surprisingly, the application of TGFβ1 as well as the inhibition of TGFβ type I and type II receptor (TGFβRI/II) signalling strongly increased the proliferation of hCSCs. Likewise, both human blood serum and TGFβ1 reduced the senescence in hCSCs. The protective effect of serum on senescence in hCSCs was enhanced by simultaneous TGFβRI/II inhibition. These results strongly indicate a dual role of TGFβ signalling in terms of the serum-mediated effects on hCSCs. Further analysis via RNA sequencing (RNA-Seq) revealed the participation of Ras-inactivating genes wherefore a prevention of hyperproliferation upon serum-treatment in hCSCs via TGFβ signalling and Ras-induced senescence is suggested. These insights may improve treatments of heart failure in the future.

Migratory capabilities of adult human stem cells are vital for assuring endogenous tissue regeneration and stem cell-based clinical applications. Although human blood serum has been shown to be beneficial for cell migration and proliferation, little is known about its impact on the migratory behavior of cardiac stem cells and underlying signaling pathways. Within this study, we investigated the effects of human blood serum on primary human cardiac stem cells (hCSCs) from the adult heart auricle. On a technical level, we took advantage of a microfluidic cultivation platform, which allowed us to characterize cell morphologies and track migration of single hCSCs via live cell imaging over a period of up to 48 h. Our findings showed a significantly increased migration distance and speed of hCSCs after treatment with human serum compared to control. Exposure of blood serum-stimulated hCSCs to the p38 mitogen-activated protein kinase (p38-MAPK) inhibitor SB239063 resulted in significantly decreased migration. Moreover, we revealed increased phosphorylation of heat shock protein 27 (Hsp27) upon serum treatment, which was diminished by p38-MAPK-inhibition. In summary, we demonstrate human blood serum as a strong inducer of adult human cardiac stem cell migration dependent on p38-MAPK/Hsp27-signalling. Our findings further emphasize the great potential of microfluidic cultivation devices for assessing spatio-temporal migration dynamics of adult human stem cells on a single-cell level.

Delivery of stem cells to the failing heart is a promising therapeutic strategy. However, the improvement in cardiac function in animal studies has not fully translated to humans. To help bridge the gap between species, we investigated the effects of adult human cardiac stem cells (hCSCs) on contractile function of human engineered cardiac tissues (hECTs) as a species-specific model of the human myocardium.
Human induced pluripotent stem cell-derived cardiomyoctes (hCMs) were mixed with Collagen/Matrigel to fabricate control hECTs, with an experimental group of hCSC-supplemented hECT fabricated using a 9:1 ratio of hCM to hCSC. Functional testing was performed starting on culture day 6, under spontaneous conditions and also during electrical pacing from 0.25 to 1.0 Hz, measurements repeated at days 8 and 10. hECTs were then frozen and processed for gene analysis using a Nanostring assay with a cardiac targeted custom panel.
The hCSC-supplemented hECTs displayed a twofold higher developed force vs. hCM-only controls by day 6, with approximately threefold higher developed stress and maximum rates of contraction and relaxation during pacing at 0.75 Hz. The spontaneous beat rate characteristics were similar between groups, and hCSC supplementation did not adversely impact beat rate variability. The increased contractility persisted through days 8 and 10, albeit with some decrease in the magnitude of the difference of the force by day 10, but with developed stress still significantly higher in hCSC-supplemented hECT; these findings were confirmed with multiple hCSC and hCM cell lines. The force-frequency relationship, while negative for both, control (- 0.687 Hz- 1; p = 0.013 vs. zero) and hCSC-supplemented (- 0.233 Hz- 1;p = 0.067 vs. zero) hECTs, showed a significant rectification in the regression slope in hCSC-supplemented hECT (p = 0.011 vs. control). Targeted gene exploration (59 genes) identified a total of 14 differentially expressed genes, with increases in the ratios of MYH7/MHY6, MYL2/MYL7, and TNNI3/TNNI1 in hCSC-supplemented hECT versus controls.
For the first time, hCSC supplementation was shown to significantly improve human cardiac tissue contractility in vitro, without evidence of proarrhythmic effects, and was associated with increased expression of markers of cardiac maturation. These findings provide new insights about adult cardiac stem cells as contributors to functional improvement of human myocardium.

Aging of cardiac stem/progenitor cells (CSCs) impairs heart regeneration and leads to unsatisfactory outcomes of cell-based therapies. As the precise mechanisms underlying CSC aging remain unclear, the use of therapeutic strategies for elderly patients with heart failure is severely delayed. In this study, we used human cardiosphere-derived cells (CDCs), a subtype of CSC found in the postnatal heart, to identify secreted factor(s) associated with CSC aging. Human CDCs were isolated from heart failure patients of various ages (2-83 years old). Gene expression of key soluble factors was compared between CDCs derived from young and elderly patients. Among these factors, SFRP1, a gene encoding a Wnt antagonist, was significantly up-regulated in CDCs from elderly patients (≥65 years old). sFRP1 levels was increased significantly also in CDCs, whose senescent phenotype was induced by anti-cancer drug treatment. These results suggest the participation of sFRP1 in CSC aging. We show that the administration of recombinant sFRP1 induced cellular senescence in CDCs derived from young patients, as indicated by increased levels of markers such as p16, and a senescence-associated secretory phenotype. In addition, co-administration of recombinant sFRP1 could abrogate the accelerated CDC proliferation induced by Wnt3A. Taken together, our results suggest that canonical Wnt signaling and its antagonist, sFRP1, regulate proliferation of human CSCs. Furthermore, excess sFRP1 in elderly patients causes CSC aging.

Over the past decade, adult stem cells have attracted great attention because of their ability to potentially regenerate desired tissues or entire organs. With the emergence of nanomaterial-based gene therapy, adult stem cells have been considered as a proper tool for the biomedical field. In this study, we utilized organically modified silica (ORMOSIL) nanoparticles to deliver small interfering RNA (siRNA) against pigment epithelium-derived factor (PEDF) and induce the differentiation of human cardiac stem cells (CSCs). We found that the down-regulation of PEDF can inhibit the proliferation of human CSCs and induce cell differentiation. To further study the mechanism, we have tested the Notch signalling pathway genes, Hes1 and Hes5, and found that their expressions were inhibited by the PEDF down-regulation. Furthermore, with the restoration of PEDF, both the proliferation of human CSCs and expressions of Hes1 and Hes5 were recovered. Our results suggest for the first time the use of ORMOSIL as nanocarriers for the delivery of PEDF siRNA in human CSCs, and demonstrated the cooperation between PEDF and the Notch signalling pathway in maintaining the self-renewal and pluripotency of stem cells. PEDF as the essential controller in differentiation may be a promising target for the regulation of cardiac homeostasis and damage repair, which opens new treatment strategies using nanomaterials for heart disease therapy.

Human cardiac stem cells (hCSC) express a portfolio of plasma membrane receptors that are involved in the regulatory auto/paracrine feedback loop mechanism of activation of these cells, and consequently contribute to myocardial regeneration. In order to attain a comprehensive description of hCSC receptome and overcoming the inability demonstrated by other technologies applied in receptor identification, mainly due to the transmembrane nature, high hydrophobic character and relative low concentration of these proteins, we have exploited and improved a proteomics workflow. This approach was based on the enrichment of hCSC plasma membrane fraction and addition of prefractionation steps prior to MS analysis. More than 100 plasma membrane receptors were identified. The data reported herein constitute a valuable source of information to further understand cardiac stem cells activation mechanisms and the subsequent cardiac repair process. All MS data have been deposited in the ProteomeXchange with identifier PXD001117 (http://proteomecentral.proteomexchange.org/dataset/PXD001117).