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The human amniotic fluid stem cell secretome triggers intracellular Ca²⁺ oscillations, NF-κB nuclear translocation and tube formation in human endothelial colony-forming cells.

ABSTRACT: Second trimester foetal human amniotic fluid-derived stem cells (hAFS) have been shown to possess remarkable cardioprotective paracrine potential in different preclinical models of myocardial injury and drug-induced cardiotoxicity. The hAFS secretome, namely the total soluble factors released by cells in their conditioned medium (hAFS-CM), can also strongly sustain in vivo angiogenesis in a murine model of acute myocardial infarction (MI) and stimulates human endothelial colony-forming cells (ECFCs), the only truly recognized endothelial progenitor, to form capillary-like structures in vitro. Preliminary work demonstrated that the hypoxic hAFS secretome (hAFS-CM^Hypo ) triggers intracellular Ca²⁺ oscillations in human ECFCs, but the underlying mechanisms and the downstream Ca²⁺ -dependent effectors remain elusive. Herein, we found that the secretome obtained by hAFS undergoing hypoxic preconditioning induced intracellular Ca²⁺ oscillations by promoting extracellular Ca²⁺ entry through Transient Receptor Potential Vanilloid 4 (TRPV4). TRPV4-mediated Ca²⁺ entry, in turn, promoted the concerted interplay between inositol-1,4,5-trisphosphate- and nicotinic acid adenine dinucleotide phosphate-induced endogenous Ca²⁺ release and store-operated Ca²⁺ entry (SOCE). hAFS-CM^Hypo -induced intracellular Ca²⁺ oscillations resulted in the nuclear translocation of the Ca²⁺ -sensitive transcription factor p65 NF-κB. Finally, inhibition of either intracellular Ca²⁺ oscillations or NF-κB activity prevented hAFS-CM^Hypo -induced ECFC tube formation. These data shed novel light on the molecular mechanisms whereby hAFS-CM^Hypo induces angiogenesis, thus providing useful insights for future therapeutic strategies against ischaemic-related myocardial injury.

SUBMITTER: Balducci V

PROVIDER: S-EPMC8358861 | biostudies-literature |

REPOSITORIES: biostudies-literature

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Project description:We previously reported that c-KIT+ human amniotic-fluid derived stem cells obtained from leftover samples of routine II trimester prenatal diagnosis (fetal hAFS) are endowed with regenerative paracrine potential driving pro-survival, anti-fibrotic and proliferative effects. hAFS may also be isolated from III trimester clinical waste samples during scheduled C-sections (perinatal hAFS), thus offering a more easily accessible alternative when compared to fetal hAFS. Nonetheless, little is known about the paracrine profile of perinatal hAFS. Here we provide a detailed characterization of the hAFS total secretome (i.e., the entirety of soluble paracrine factors released by cells in the conditioned medium, hAFS-CM) and the extracellular vesicles (hAFS-EVs) within it, from II trimester fetal- versus III trimester perinatal cells. Fetal- and perinatal hAFS were characterized and subject to hypoxic preconditioning to enhance their paracrine potential. hAFS-CM and hAFS-EV formulations were analyzed for protein and chemokine/cytokine content, and the EV cargo was further investigated by RNA sequencing. The phenotype of fetal- and perinatal hAFS, along with their corresponding secretome formulations, overlapped; yet, fetal hAFS showed immature oxidative phosphorylation activity when compared to perinatal ones. The profiling of their paracrine cargo revealed some differences according to gestational stage and hypoxic preconditioning. Both cell sources provided formulations enriched with neurotrophic, immunomodulatory, anti-fibrotic and endothelial stimulating factors, and the immature fetal hAFS secretome was defined by a more pronounced pro-vasculogenic, regenerative, pro-resolving and anti-aging profile. Small RNA profiling showed microRNA enrichment in both fetal- and perinatal hAFS-EV cargo, with a stably- expressed pro-resolving core as a reference molecular signature. Here we confirm that hAFS represents an appealing source of regenerative paracrine factors; the selection of either fetal or perinatal hAFS secretome formulations for future paracrine therapy should be evaluated considering the specific clinical scenario.

Project description:Uncultured mesenchymal stromal cells (MSCs) are increasingly used in therapies; however, the effects of donor age on their biological characteristics and gene expression remain unclear. The aim of this study was to investigate age-related changes in bone marrow (BM) MSCs following minimal or no culture manipulation. Iliac crest BM was aspirated from 67 healthy donors (19-89 years old) and directly used for the colony-forming unit-fibroblast (CFU-F) assay or CD45lowCD271+ cell enumeration. The colonies were analysed for colony area and integrated density (ID) when grown in standard MSC media or media supplemented with human serum from young (YS) or old (OS) donors. There was a notable age-related decline in the number of MSCs per millilitre of BM aspirate revealed by the CFU-F assay (r = -0.527, p < 0.0001) or flow cytometry (r = -0.307, p = 0.0116). Compared to young donors (19-40 years old), colony IDs were significantly lower in older donors (61-89 years old), particularly for smaller-sized colonies (42% lower, p < 0.01). When cultured in media supplemented with OS, young and old donor MSCs formed colonies with lower IDs, by 21%, p < 0.0001, and 27%, p < 0.05, respectively, indicating the formation of smaller sparser colonies. No significant differences in the expression of selected adipogenic, osteogenic, stromal, and bone remodelling genes as well as CD295, CD146, CD106, and connexin 43 surface molecules were found in sorted CD45lowCD271+ MSCs from young and old donors (n = 8 donors each). Altogether, these results show similar trends for age-related decline in BM MSC numbers measured by the CFU-F assay and flow cytometry and reveal age-related effects of human serum on MSC colony formation. No significant differences in selected gene expression in uncultured CD45lowCD271+ MSCs suggest that old donor MSCs may not be inferior in regard to their multipotential functions. Due to large donor-to-donor variation in all donor groups, our data indicate that an individual's chronological age is not a reliable predictor of their MSC number or potency.

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The human amniotic fluid stem cell secretome triggers intracellular Ca²⁺ oscillations, NF-κB nuclear translocation and tube formation in human endothelial colony-forming cells.

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The human amniotic fluid stem cell secretome triggers intracellular Ca2+ oscillations, NF-κB nuclear translocation and tube formation in human endothelial colony-forming cells.

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OmicsDI is part of the ELIXIR infrastructure

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The human amniotic fluid stem cell secretome triggers intracellular Ca²⁺ oscillations, NF-κB nuclear translocation and tube formation in human endothelial colony-forming cells.