Notch pathway activation enhances cardiosphere in vitro expansion

Abstract Cardiospheres (CSps) are self‐assembling clusters of a heterogeneous population of poorly differentiated cells outgrowing from in vitro cultured cardiac explants. Scanty information is available on the molecular pathways regulating CSp growth and their differentiation potential towards cardiac and vascular lineages. Here we report that Notch1 stimulates a massive increase in both CSp number and size, inducing a peculiar gene expression programme leading to a cardiovascular molecular signature. These effects were further enhanced using Adeno‐Associated Virus (AAV)‐based gene transfer of activated Notch1‐intracellular domain (N1‐ICD) or soluble‐Jagged1 (sJ1) ligand to CSp‐forming cells. A peculiar effect was exploited by selected pro‐proliferating miRNAs: hsa‐miR‐590‐3p induced a cardiovascular gene expression programme, while hsa‐miR‐199a‐3p acted as the most potent stimulus for the activation of the Notch pathway, thus showing that, unlike in adult cardiomyocytes, these miRNAs involve Notch signalling activation in CSps. Our results identify Notch1 as a crucial regulator of CSp growth and differentiation along the vascular lineage, raising the attracting possibility that forced activation of this pathway might be exploited to promote in vitro CSp expansion as a tool for toxicology screening and cell‐free therapeutic strategies.

cardiac-specific transcription factors such as Gata4 or Nkx2.5 (for a review see 3 ). These cells have been used in several cell therapy approaches aiming at promoting regeneration of damaged hearts (for a review see 4,5 ). The functional benefits of transplanted CPCs have been mainly ascribed to indirect paracrine mechanisms mediated by secreted factors, as opposed to direct generation of new cardiomyocytes 6,7 ).
In the context of cardiac progenitor cell proliferation and expansion, a crucial role is played by the Notch signalling pathway, a wellconserved cell-to-cell communication system that critically regulates embryonic heart development. 8,9 Most specifically, Notch regulates cardiomyocyte proliferation during development and in the early postnatal life. In particular, we and others have shown that the Notch pathway promotes the proliferation of committed precursor cells and immature cardiomyocytes 10,11 and is essential for the maintenance of the heart structural and functional integrity after damage in mammals as well as in lower vertebrates, 12,13 in which it has been identified as a central mechanism in heart regeneration. 14,15 In CPCs, Notch activation prevents premature cardiomyogenic differentiation, while promoting proliferation in transient amplifying populations; 16,17 conversely, inhibition of the Notch pathway is required to promote cardiac mesoderm differentiation in embryonic stem cells. 18 Committed undifferentiated progenitor cells can be clonally expanded from myocardial biopsy specimens and cultured in vitro as tridimensional spheroids. 19,20 Cardiospheres (CSps) are cardiac-derived multicellular clusters, considered a reliable in vitro model of the cardiac microenvironment, hosting a reservoir of both immature and committed progenitors towards the three major cardiac cell lineages: cardiomyocytes, endothelial cells (ECs) and smooth muscle cells (SMCs). 21 CSps transplantation into infarcted hearts results in enhanced in vivo cell survival and cardioprotection. 22 Recent data have shown that activation of the Notch signalling pathway promotes cell differentiation along the vascular smooth muscle lineage in CSp-derived cells and is sensitive to oxygen tension in the tridimensional cellular array. 23 In this work, we aimed at dissecting the specific role of Notch1 signalling in CSps. We report that Notch1 stimulation by soluble Jagged-1 triggers a massive increase in both number and size of in vitro cultured CSps. This effect was paralleled by a significant increase in vascular developmental markers such as Flt-1, Kdr and CD31 and the adoption of a specific gene expression signature.
Conversely, the γ-secretase inhibitor, DAPT, which impairs Notch1 activation, effectively blunted in vitro growth of sJ1-stimulated CSps, as well as modulated their gene expression pattern. These effects were further enhanced using Adeno-associated virus (AAV)based gene transfer of activated Notch1-intracellular domain (N1-ICD) or the soluble-Jagged1 ligand (sJ1) to CSp-forming cells. These viral vectors possess the exquisite capacity to transduce cardiacderived cells at very high efficiency and drive sustained transgene expression. 24,25 Finally, a whole-genome, synthetic miRNA screening previously conducted in our laboratory has identified a pool of human miRNAs capable of inducing significant proliferation in rodents cardiomyocytes. 26 Notably, the molecular mechanisms by which these miRNAs function in adult cardiomyocytes do not involve reactivation of the Notch pathway. 24 We found that, among all tested miRNAs, hsa-miR-590-3p was the most powerful inducer of a cardiovascular gene expression programme in CSps, leading to a significant increase in both early (Gata4, Nkx2.5) and late cardiac markers (Myh6), as well as of vascular markers (CD31).
Collectively, our results identify Notch1 as a crucial regulator of CSps growth and differentiation along the vascular lineage, raising the possibility that forced activation of this signalling pathway might be exploited to promote in vitro CSps growth not only for therapeutic purposes but also as a tool for evaluating novel cell-free therapeutic strategies (for example, those based on the delivery of exosomes or miRNAs).
Isolation of the CSp-forming cells was performed from the same explant at least 4 times at 6-10 days intervals.

| miRNA transfection
MicroRNAs were obtained from Dharmacon, Thermo Scientific and transfected into CSp-forming cells contextually to plating on poly-K coated dishes using a standard reverse transfection protocol. 26 Briefly, the transfection reagent (Lipofectamine RNAiMAX, Life Technologies, Carlsbad, Ca, USA) was diluted in OPTIMEM (Life Technologies) and added to the miR-NAs (at a final concentration of 25 nmol/L each nucleic acid), arrayed on 96-well plates. Thirty minutes later, 1 x 10 4 cells were seeded per well.
Twenty-four hours after transfection, culture medium was replaced by fresh medium. Cells were fixed 3, 5 and 7 days after plating and processed for further analyses. Experiments were performed in quadruplicate.

| Image acquisition and analysis
Images were acquired at room temperature with a DMLB upright fluorescence microscope (Leica, Wetzlar, Germany) equipped with a charge- Within each experiment, instrument settings were kept constant.

| AAV vector production and transduction
The AAV vectors used in this study were generated by the AAV Vector Unit (AVU) at ICGEB Trieste (http://www.icgeb.org/avu-corefacility.html) according to established procedures. All vectors were T A B L E 1 TaqMan real-time PCR assays used to analyse gene expression levels

| Statistical analysis
All data are presented as mean ± standard error of the mean (SEM).
Statistical analysis was carried out with Prism Software (GraphPad) using one-way ANOVA followed by Bonferroni's post hoc test for comparisons of 3 or more groups. Collectively, these evidences were consistent with the notion that CSps actually represent a valuable in vitro model of early cardiac development.

| Notch signalling components in CSps
To investigate a possible role of the Notch pathway during CSp formation, we analysed the expression levels of the Notch receptors receptors, as well as that of Jagged1 and Dll-1, was also observed in substrate-adherent CSp-forming cells ( Figure 2E).
We also investigated the subcellular localization of Hes1 and N1-ICD, two key players of the activated Notch pathway. Hes1 showed a diffuse staining in the inner part of CSps, while activated N1-ICD (detected by an antibody raised against the first amino acids of its cleaved intracellular domain) exhibited significant co-localization with Notch1 ( Figure 2D panels a and b). These results suggested an active role of Notch signalling in CSp formation and maturation.

CSp growth
In several models of stem cell in vitro culture, Notch expression colocalizes with pools of proliferating cells. 18,32,33 To directly assess this issue, we cultured CSps in the presence of the conditioned medium from NIH-3T3 cells secreting a soluble form of Jagged1 (sJ1; 34 ).
In a mirror experiment, we treated cells with the γ-secretase inhibitor, DAPT, to investigate whether blocking Notch activation impairs CSp formation and growth.
The effect of sJ1 has been extensively characterized in our laboratory. 10 Collectively, these results strongly support the conclusion that  We also assessed mRNA expression levels for endogenous Notch targets. Gene expression of Hes1 and, to a lesser extent, Hey2 (two major transducers of Notch signalling during heart development) paralleled the increase in CSp growth ( Figure S2). We next analysed the expression of genes related to stemness, blood vessel or heart development. Figure 3E shows that sJ1 stimulation markedly increased expression levels of the genes involved in angiogenesis, such as CD31 and VEGF receptors 1 and 2. In addition, sJ1 stimulation was associated with increased expression of Nkx2.5, the master gene for cardiac development, but decreased Gata4 expression, in agreement with published data. 16 To elucidate the gene expression programme activated upon viral transduction, we analysed gene expression of transduced CSps at 3, 5 and 7 days of culture. Both AAV vectors triggered a gene expression programme ( Figure 4D) superimposable to the one observed upon sJ1 stimulation (cf. Figure 3E), further supporting the role of Notch1 in the expansion of a pool of cells putatively committed to a cardiovascular fate.

| miR-199a-3p induces Notch expression, while miR-590-3p enhances CSp growth and cardiovascular commitment
A previous whole-genome, synthetic miRNA screening conducted in our laboratory identified several human miRNAs capable of inducing significant proliferation of rodent cardiomyocytes. Of interest, at least two of these miRNAs, miR-199a-3p and miR-590-3p induced cardiac regeneration after myocardial infarction using AAV-mediated gene transfer 26 or delivery of naked miRNA mimics. 44 Notably, the molecular mechanism by which these miRNAs function in adult cardiomyocytes does not involve reactivation of the Notch pathway. 24 We therefore investigated whether the most

| DISCUSSION
The mammalian heart is unable to compensate for a massive loss of cardiomyocytes after injury. This is due to a dramatic decrease in the proliferative capability of mature cardiomyocytes, compared to neonatal conditions and to the poor regenerative potential of CPCs. Several approaches are under investigation to induce cardiomyocyte proliferation or progenitor activation and expansion.
Here we have exploited the CSp model to investigate the role of the Notch pathway in the cardiovascular differentiation programme. Previous gene expression studies have shown a phenotypic heterogeneity of the cellular components of CSps. 45,46 Consistent with these findings, we detected Sca-1 positive cells, along with cells that were more committed towards vasculoendothelial or cardiac fate specification. Previous evidence from our group and other laboratories have demonstrated a peculiar role of the Notch pathway in the expansion of the undifferentiated precursor cell pool in the heart and in the regulation of neonatal cardiomyocyte proliferation both in vitro and in vivo. 10,12,16,29,30 Given the poorly differentiated molecular signature of the cell components of CSps, we wondered whether Notch signalling was involved in the regulation of CSp growth. Interestingly, we found a peculiar spatial distribution of different key molecules in the Notch pathway: immunostaining for Notch 1 and 2 receptors revealed a patchy distribution, whereas Hes1 expression was localized in the deep core of the spheres. Conversely, the ligands Jagged1 and Delta-like1 were expressed by cells located in the outer layer of the spheres. We then either stimulated the Notch pathway with the soluble ligand Jagged1 (sJ1) or blocked its activation by inhibiting γ-secretase processing with DAPT. The number and size of CSps was significantly increased after sJ1 treatment, with an overall upregulation of genes involved in cardiovascular development.
DAPT, on the contrary, completely reversed this effect. We then decided to validate these results using a gene transfer approach involving AAV-mediated transduction of CSp-forming cells with either sJ1 or the activated intracellular domain of Notch1 (N1-ICD). In both cases, we detected a net increase in both numbers and size of transduced CSps, which was associated with activation of a peculiar genetic programme promoting cardiovascular differentiation.
Multiple evidence over the last few years has shown a pivotal role of the microRNA network in the control of most biological functions of heart cells. 47,48 In particular, a previous whole-genome, synthetic miRNA screening conducted in our laboratory revealed several human miRNAs capable of inducing significant proliferation of neonatal rat and mouse CMs. 26 Among these miRNAs, miR-199a-3p, miR-590-3p and miR-1825, have been shown to be involved in the regulation of embryonic cell proliferation 49 and were capable to induce cardiac regeneration after myocardial infarction (MI), either when expressed using AAV-mediated gene transfer 26 or delivered as naked miRNA mimics. 44 Remarkably, the molecular mechanisms by which the most effective pro-proliferative miRNAs function in adult cardiomyocytes do not involve reactivation of the Notch pathway. 24 We therefore wondered whether they could play a functional role in CSps, acting through multiple, specific pathways. All the miRNAs of interest shared a common effect on CSps, triggering a significant increase in their number and size. Gene expression analysis revealed increased levels of genes of the Notch pathway at the earliest time points of in vitro culture, as a result of each of the four miRNAs tested, suggesting this pathway as a possible target of either direct or indirect regulation by these miRNAs. While the overall mechanism of action of each miRNA is likely different, common pathways are likely to exist. Among Notch signalling negative regulators (GO:0045746), we found several different genes predicted to be differentially downregulated by each of the miRNAs of interest. In particular, we validated as possible targets Neurl1a, a ligand-specific ubiquitin ligase 50,51 and Dner, coding for a atypical Notch ligand. 28,52,53 In both cases, a net downregulation of the transcripts for these genes was observed after expression of all the tested miR-NAs.
Collectively, our data show that the Notch pathway drives the proliferation of CSp-forming cells. Exogenous activation of this pathway magnifies these effects and causes a shift of the gene expression profile towards genes involved in cardiovascular differentiation.

ACKNOWLEDG EMENTS
The authors are grateful to Marina Dapas and Michela Zotti for technical support in AAV production and to Suzanne Kerbavcic for editorial assistance.