Proteomic profiling of the secretome formulations of fetal and perinatal human amniotic fluid stem cells
Ontology highlight
ABSTRACT:
INSTRUMENT(S): LTQ Orbitrap XL ETD
ORGANISM(S): Homo Sapiens (ncbitaxon:9606)
SUBMITTER: 
Antonella De Palma
PROVIDER: MSV000087013 | MassIVE | Sun Mar 07 12:38:00 GMT 2021
REPOSITORIES: MassIVE
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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, pro-angiogenic, anti-fibrotic and proliferative effects on target cells. hAFS can be also isolated from III trimester clinical waste samples during C-section scheduled procedures (perinatal hAFS), thus offering an easily accessible alternative source. Nonetheless, little is known about their paracrine profile. Here we provide a detailed characterization of the hAFS total secretome (i.e. whole of the soluble paracrine factors released by the cells in their conditioned medium, hAFS-CM) and the extracellular vesicles (hAFS-EVs) within it as 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. Their secretome formulations were analyzed in terms of protein and chemokine/cytokine content, with the EV cargo further investigated by RNA sequencing. While the appearance of fetal and perinatal hAFS, along with their corresponding secretome formulations was overlapping, the profiling of their paracrine cargo revealed some differences according to gestational stage and hypoxic preconditioning. While both cell sources provided paracrine formulations enriched with neurotrophic, immunomodulatory and endothelial stimulating factors, the more immature fetal hAFS secretome was defined by a more pronounced pro-angiogenic, pro-resolving and anti-aging profile over the perinatal one, while the corresponfing EV…. Moreover, small RNA profiling showed a relevant enrichment of microRNA in both the fetal and perinatal hAFS-EV cargo, with a steadily expressed pro-resolving miRNA core as 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.
2021-04-26 | GSE168152 | GEO
Project description:The anthracycline, doxorubicin (Dox), is widely used in oncology, but it may it may cause a cardiomyopathy which has dismal prognosis and cannot be effectively prevented. The secretome of multipotent human amniotic fluid-derived stem cells (hAFS) has previously been demonstrated to reduce ischemic cardiac damage. Here, it is shown that the hAFS conditioned medium (hAFS-CM) antagonizes senescence and apoptosis of cardiomyocytes and cardiac progenitor cells, two major features of Dox cardiotoxicity. Mechanistic studies with primary mouse neonatal cardiomyocytes reveal that hAFS-CM inhibition of Dox-elicited senescence and apoptosis is paralleled by decreased DNA damage and is associated with nuclear translocation of NF-kB and upregulation of a set of genes controlled by NF-kB, namely Il6 and Cxcl1, which promote cardiomyocyte survival, and Cyp1b1 and Abcb1, which encode for proteins involved in Dox metabolism and efflux, respectively. The PI3K/Akt signaling cascade, upstream of NF-kB, is potently activated by the hAFS-CM and pre-treatment with a PI3K inhibitor abrogates NF-kB accumulation into the nucleus, modulation of its target genes, and prevention of Dox-initiated senescence and apoptosis in response to the hAFS-CM. This work may lay the ground for the development of a stem cell-based paracrine therapy of chemotherapy-related cardiotoxicity.
2016-10-30 | GSE74513 | GEO
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.
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Project description:RNA-seq was performed on cultured human induced pluripotent cell derived cardiomyocytes (iPSC-CMs). There are four groups, with three samples per group: H1,H2,H3. Negative control. Healthy, normoxic iPSC-CMs D1,D2,D3. Positive control. iPSCs cultured under hypoxic (0-1% O2) for 48h with 2% v/v PBS as vehicle control EV1,EV2,EV3. Treatment 1. iPSCs cultured under hypoxic (0-1% O2) for 48h, with cardiac stromal cell derived extracellular vesicles, provided at a dose of 67ng/µl (2% v/v) EV21, EV23, EV24. Treatment 2 iPSCs cultured under hypoxic (0-1% O2) for 48h, with bone marrow mesenchymal stromal cell (BM-MSC) derived extracellular vesicles, provided at a dose of 67ng/µl (2% v/v) All EVs were isolated from cultured human cells using sequential centrifugation methods. Cells were cultured using commercial EV-depleted FBS to avoid contamination of bovine EVs. EVs were validated for CD81, CD9, ALIX positivity, and visualised by cryoEM. Particle to protein ratios were not different between cardiac and bone marrow EV isolates. Therefore EV doses were standardised so that the same dose by protein (67ng/µl) and EV number (~2,000 EVs per iPSC-CM) were added.
2024-10-02 | E-MTAB-13966 | biostudies-arrayexpress
Project description:<p><strong>BACKGROUND:</strong> Ischemia/reperfusion injury (IRI) is the leading cause of acute kidney injury (AKI). The current standard of care focuses on supporting kidney function, stating the need for more efficient and targeted therapies to enhance repair. Mesenchymal Stromal Cells (MSCs) and their secretome, either as conditioned medium (CM) or extracellular vesicles (EVs), have emerged as promising options for regenerative therapy, however, their full potential in treating AKI remains unknown.</p><p><strong>METHODS:</strong> In this study, we employed an in vitro model of chemically-induced ischemia using antimycin A combined with 2-deoxy-D-glucose to induce ischemic injury in proximal tubule epithelial cells. Afterwards, we evaluated the effects of MSC secretome, CM or EVs obtained from adipose tissue, bone marrow and umbilical cord, on ameliorating the detrimental effects of ischemia. To assess the damage and treatment outcomes, we analyzed cell morphology, mitochondrial health parameters (mitochondrial activity, ATP production, mass and membrane potential) and overall cell metabolism by metabolomics.</p><p><strong>RESULTS:</strong> Our findings show that ischemic injury caused cytoskeletal changes confirmed by disruption of the F-actin network, energetic imbalance as revealed by a 50% decrease in the oxygen consumption rate, increased oxidative stress, mitochondrial dysfunction and reduced cell metabolism. Upon treatment with MSC secretome, the morphological derangements were partly restored and ATP production increased by 40-50%, with umbilical cord-derived EVs being most effective. Furthermore, MSC treatment led to phenotype restoration as indicated by an increase in cell bioenergetics, including increased levels of glycolysis intermediates, as well as an accumulation of antioxidant metabolites.</p><p><strong>CONCLUSION:</strong> Our in vitro model effectively replicated the in vivo-like morphological and molecular changes observed during ischemic injury. Additionally, treatment with MSC secretome ameliorated proximal tubule damage, highlighting its potential as a viable therapeutic option for targeting AKI.</p>
2023-10-16 | MTBLS6279 | MetaboLights
Project description:Oligohydramnios is known to cause fetal pulmonary hypoplasia. Amniotic fluid stem cell extracellular vesicles (AFSC-EVs) have shown beneficial effects on hypoplastic lungs secondary to congenital diaphragmatic hernia. Herein, we investigated, whether AFSC-EVs also foster antenatal lung development in hypoplastic lungs secondary to oligohydramnion. We therefore induced surgically oligohydramnion in rat dams and harvested their fetuses. Controls underwent surgery without amniotic fluid drainage. The hypoplastic lungs were assigned to ex vivo to either medium alone or medium + AFSC-EV as a lung explant. Those samples were harvested 24h later and subjected to RNA extraction for subsequent RNA-seq.
2024-10-17 | GSE247978 | GEO
Project description:Background: Advanced Renal cell carcinoma (RCC) is therapeutically challenging. RCC progression is facilitated by mesenchymal stem/stromal cells (MSCs) that exert remarkable tumor tropism. The specific mechanisms mediating MSCs’ migration to RCC remain unknown. Here, we comprehensively analyzed RCC secretome to identify MSCs attractants. Methods: Conditioned media (CM) were collected from five RCC-derived cell lines (Caki-1, 786-O, A498, KIJ265T, KIJ308T) and non-tumorous control cell line (RPTEC/TERT1) and analyzed using cytokine arrays targeting 274 cytokines in addition to global CM proteomics. MSCs were isolated from bone marrow of patients undergoing standard orthopedic surgeries. RCC CM and the selected recombinant cytokines were used to analyze their influence on MSCs migration and microarray-targeted gene expression. The expression of genes encoding cytokines was evaluated in 100 matched-paired control-RCC tumor samples. Results: When compared with normal cells, CM from advanced RCC cell lines (Caki-1, KIJ265T) were the strongest stimulators of MSCs migration. Targeted analysis of 274 cytokines and global proteomics of RCC CM revealed decreased DPP4 and EGF, as well as increased AREG, FN1, and MMP1, with consistently altered gene expression in RCC cell lines and tumors. AREG and FN1 stimulated, while DPP4 attenuated MSCs migration. RCC CM induced MSCs’ transcriptional reprogramming, stimulating the expression of CD44, PTX3, and RAB27B. RCC cells secreted hyaluronic acid (HA), a CD44 ligand mediating MSCs’ homing to the kidney. AREG emerged as an upregulator of MSCs’ transcription. Conclusions: advanced RCC cells secrete AREG, FN1 and HA to induce MSCs migration, while DPP4 loss prevents its inhibitory effect on MSCs homing. RCC secretome induces MSCs’ transcriptional reprograming to facilitate their migration. The identified components of RCC secretome represent potential therapeutic targets. We used microarrays to determine the effect of the conditioned media (CM) collected from two RCC-derived cell lines (Caki-1, KIJ265T) and non-tumorous control cell line (RPTEC/TERT1) on the transcriptome change in mesenchymal stem/stromal cells (MSCs).
2023-07-18 | GSE232951 | GEO
Project description:Hypoxic pre-conditioning has been demonstrated to increase the resistance of neural stem cells (NSCs) to hypoxic conditions, as well as to improve their capacity for differentiation and neuro-genesis. Extracellular vesicles (EVs) have recently emerged as critical mediators of cell-cell com-munication, but their role in this hypoxic conditioning is presently unknown. Here, we demon-strated that three hours of hypoxic pre-conditioning triggers significant neural stem cell EV release. Proteomic profiling of EVs from normal and hypoxic preconditioned neural stem cells identified 20 proteins that were up-regulated and 22 proteins that were down-regulated after hypoxic precon-ditioning. We also found an upregulation of some of these proteins by qPCR, thus indicating dif-ferences also at the transcript level within the EVs. Among the up-regulated proteins are CNP, Cyfip1, CASK, and TUBB5, which are well known to exhibit significant beneficial effects to neural stem cells. Thus, our results not only show a significant difference of protein cargo in EVs conse-quent to hypoxic exposure, but identify several candidate proteins that might play a pivotal role in the cell-to-cell mediated communication underlying neuronal differentiation, protection, matura-tion and survival following exposure to hypoxic conditions.
2022-11-07 | PXD037966 |
Project description:Patient-derived prostate fibroblast primary cultures PCF-54 and PCF-55 were established from two specimens of PC tissues. Urinary EVs were isolated from urine samples of 3 patients with PC and 2 healthy males and used for the treatment of prostate fibroblast primary cultures and normal foreskin fibroblasts. Normoxic and hypoxic EVs were isolated from cell culture medium of PC3 and LNCaP prostate cancer cell lines, cultivated in normoxic and hypoxic conditions respectively. The EV-treated fibroblasts were subjected to RNA sequencing analysis.
2023-04-01 | E-MTAB-11317 | biostudies-arrayexpress
Project description:The anthracycline doxorubicin (Dox) is widely used in oncology, but it may cause a cardiomyopathy with bleak prognosis that cannot be effectively prevented. The secretome of human amniotic fluid-derived stem cells (hAFS) has previously been demonstrated to significantly reduce ischemic cardiac damage. Here it is shown that, following hypoxic preconditioning, hAFS conditioned medium (hAFS-CM) antagonizes senescence and apoptosis of cardiomyocytes and cardiac progenitor cells, two major features of Dox cardiotoxicity. Mechanistic studies with mouse neonatal ventricular cardiomyocytes (mNVCM) reveal that hAFS-CM inhibition of Dox-elicited senescence and apoptosis is associated with decreased DNA damage, nuclear translocation of NF-kB, and upregulation of the NF-kB controlled genes, Il6 and Cxcl1, promoting mNVCM survival. Furthermore, hAFS-CM induces expression of the efflux transporter, Abcb1b, and Dox extrusion from mNVCM. The PI3K/Akt signaling cascade, upstream of NF-kB, is potently activated by hAFS-CM and pre-treatment with a PI3K inhibitor abrogates NF-kB accumulation into the nucleus, modulation of Il6, Cxcl1 and Abcb1b, and prevention of Dox-initiated senescence and apoptosis in response to hAFS-CM. These results support the concept that hAFS are a valuable source of cardioprotective factors and lay the foundations for the development of a stem cell-based paracrine treatment of chemotherapy-related cardiotoxicity.
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