Serum-Induced Proliferation of Human Cardiac Stem Cells Is Modulated via TGFβRI/II and SMAD2/3

Abstract

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.

Keywords: RNA-Seq; TGFβ1; ageing; human blood serum; human cardiac stem cells; proliferation; senescence.

Figure 1

Human blood serum induces differential gene expression. Volcano plot shows upregulation of 213 differentially expressed genes after serum treatment and downregulation of 31 genes.

Figure 2

Human blood serum induces proliferation-related GO term enrichment. Upregulated GO terms regarding to (A) biological process, (B) cellular component and (C) molecular function.

Figure 2

Human blood serum induces proliferation-related GO term enrichment. Upregulated GO terms regarding to (A) biological process, (B) cellular component and (C) molecular function.

Figure 3

50 µM LY2109761 is effective for induction of proliferation in hCSCs. (A) After starvation, hCSCs were treated with human blood serum and TGFβR/II inhibitor. (B) Increasing concentrations of the selective TGFβRI/II inhibitor LY2109761 upon serum treatment indicate a proliferation enhancing effect in hCSCs with an effective concentration at 50 µM. (C) The IC50 value of the TGFβ RI/II inhibitor LY2109761 is 6.252 µM.

Figure 4

The dual role of TGFβ signalling in serum-mediated effects in hCSCs. (A–C) Upon serum treatment, the supplementation with LY2109761 leads to morphological changes of hCSCs. (D) The application of LY2109761 alone increases the proliferation higher than treatment with serum alone, while the proliferative effect of the serum is enhanced by LY210976. (E) TGFβ1 mediates proliferation in concentrations from 1 ng/mL to 10 ng/mL. (F–H) Simultaneous application of LY2109761 and TGFβ1 highly increases the cell proliferation of hCSCs regardless of the applied concentration of TGFβ1. (I) Serum reduces the SA-β-Gal activity in hCSCs, and this effect is further enhanced after addition of LY2109761. Application of TGFβ1 reduces senescence in hCSCs compared to untreated cells while it is significantly increased compared to serum-treated cells. (Mann–Whitney U two-tailed, ** p < 0.01; * p < 0.05).

Figure 5

Phosphorylation of SMAD2/3 after serum treatment and inhibition of TGFβRI/II in hCSCs. (A) Immunocytochemical staining of hCSCs shows enriched amounts of pSMAD2/3 in the nucleus after treatment with serum or TGFβ1 which further increases after inhibition of TGFβRI/II, magnification ×63. (B) Quantification of the fluorescence signal reveals a significant decrease of nuclear pSMAD2/3 after application of TGFβRI/II inhibitor. (Kruskal–Wallis, *** p < 0.001; **** p < 0.0001).

Figure 6

TGFβRI inhibition reduces serum-induced hCSC proliferation. (A) Application of TGFβRI inhibitor SB431542 reduces serum-mediated hCSC proliferation with an (B) IC₅₀ of 0.5339 µM. (Ordinary one-way ANOVA, **** p < 0.0001; *** p < 0.001).

Figure 7

Gene expression analysis of serum- and LY2109761-treated hCSCs. (A) Volcano plot of differentially expressed genes in serum-treated hCSCs after TGFβ RI/II inhibition shows upregulation of ten genes and downregulation of two genes. Proliferation-related and Ras-inactivating genes are significantly upregulated after serum and inhibitor treatment in hCSCs. (B) The genes FOS, FOSB, NR4A and EGR are similarly upregulated in hCSCs treated with serum or serum with LY2109761 compared to untreated hCSCs.