Comprehensive Profiling Of Secretome Formulations From Fetaland Perinatal Human Amniotic Fluid Stem Cells

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Furthermore, as from the bar charts in Figures 5B and 6B, the distinctive protein distribution according to the secreting cell hypoxic preconditioning is appreciable. In this case, for complete information, proteins that do not exceed the threshold of DAve and DCI set were also reported. Fetal has secretome results enriched with the 60 kDa heat Shock Protein (HSPD1, Figure 5B, left panel) in its hypoxic formulation, while the peri-natal counterpart highly expressed smooth muscle cell contractile myosin regulatory [52]light polypeptide 9(MYL9, Figure 5B, right panel). An important share of the difference between gestational stages seems to depend more on hypoxic preconditioning in hAFS. EVs; f-have-EVs obtained from hypoxic cell priming were found enriched with factors including Perlecan (HSPG2), Agrin (AGRN), Laminin Subunit α-5, and β-1(LAMA5 and LAMB1), Thrombospondin-1 (THBS1, Figure 6B, left panel). Hypoxic p-hAFS-EVs contained Ferritin Heavy Chain (FTH1), scaffolding proteins like Flotillin-1(FLOT1), Fascin (FSCN1), Annexin A6(ANXA6), Rab GDP dissociation inhibitor beta (GDI2), along with Thy-1 membrane glycoprotein (THY1), Neuropilin-1(NRP1) and Matrix Metalloprotein 14 (MMP14, Figure6B, right panel).

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Proteins were found with a frequency of at least 2 in each examined condition in f-hAFS. EVs and p-hAFS-EVs were further compared to the Vesciclepedia database [53]. As expected, the majority of the identified proteins (96%) have been previously described in EVs and exosomes in the reference database (Figure S3A). cistanche benefits In this respect, Gene Ontology (GO)enrichment analysis was performed by means of FunRich [54]. The abundance of GO terms in the dataset was compared against their natural amount in the reference database to find statistically over-represented groups of proteins, according to their involvement in biological processes, molecular function, and cellular components (for this last aspect data are not shown, but available on request). Regarding the analysis of the molecular functions associated with identified proteins, hAFS-CM fractions indicated enrichment in a structural constituent of extracellular matrix and cytoskeleton, cytoskeletal protein binding, and structural molecule activity (Figure S2), while hAFS-EVs were enriched with a structural constituent of cytoskeleton and ribosome, DNA and RNA binding and GTPase and chaperone binding factors (Figure S3C).

Biological processes enrichment analysis for both hAFS-CM and have-EVs indicated that the majority of proteins modulated in the fetal-and perinatal hAFS secretome fractions belong to cell growth/maintenance and protein metabolism (Figures 5C and 6C). Within hAFS-EVs we noticed that the term "extracellular matrix structural constituents" was exclusively associated with hypoxic f-hAF-EVs; the terms"calcium ion binding" and "structural molecular activity" were mainly enriched in hypoxic f-hAFS and p-hAFS samples (Figure S3B).

Figure 6. Comparative proteomics analysis of fetal- and perinatal hAFS-EVs. (A)Venn diagram illustrating the distribution of proteins identified with a frequency of at least 2 within f-hAFS-EVSnormo (dark yellow),f-hAFS-EVSHypo (red), p-hAFS-EVSnormo (light green), and p-hAFS-EVShypo (dark green). (B)Differentially expressed proteins were identified in fetal hAFS-EVs (left panel) and perinatal hAFS-EVs (right panel) by label-free quantification with MAProMa software. Left panel: histogram reporting the differential expression of proteins found upregulated between normoxic control (dark yellow bars and negative DAve values) and hypoxic preconditioning (red bars and positive DAve values) of f-hAFS-EVs over p-hAFS-EVs. Right panel: histogram reporting the differential expression of proteins found upregulated between control normoxic (light green bars and negative DAve values) and hypoxic preconditioning (dark green bars and positive DAve values) of p-hAFS-EVs over f-hAFS-EVs. Proteins with DAve (ratio of protein expression)> 10.4I and a DCI (confidence of differential expression) ≥I5I passed the filters and were considered differentially expressed; see Table S3 for the complete list and detailed parameters of the reported proteins. (C)Biological processes enrichment analysis of proteins identified with a frequency of at least 2 in f-hAFS-EVs (left panel) and p-hAFS-EVs(right panel) after hypoxic preconditioning. Based on the FunRich tool, gene ontology terms are shown in bar charts reporting the percentage of genes enriched for each category (dark yellow bars for f-hAFS-EVsnormo, red bars for f-hAFS-EVShypo, light green bars for p-hAFS-EVsnormo and dark green bars for p-hAFS-EVShypo). Only gene ontology terms with Bonferroni corrected*p<0.05 are reported.

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2.6.The Cytokine and Chemokine Profiling of Fetal vs.Perinatal has-CM and have-EVs Revealed Different Distribution Patterns

We have previously validated the regenerative capacity of f-hAFS-CMhypo on injured cardiovascular cells via paracrine effects [34,35,49]. Here we compared the cytokine and chemokine content of f-hAFS-CMHypo to the corresponding p-hAFS counterpart (Figure 7A, Figure S4A, and Table S4) and found some discriminating factors.

ANGIOGENIN, Extracellular Matrix Metalloproteinase Inducer (EMMPRIN), Interleukin 8 (IL-8), and Monocyte Chemoattractant Protein-1 (MCP-1) were found exclusively enriched inf-hAFS-CMNypo and were not detected in p-hAFS-CMhypo. Insulin-like Growth Factor Binding Protein 2 (IGFBP2) and Osteopontin (OPN) were significantly increased in f-hAFS-CMhypo over p-hAFS-CMHypo by 3.5-and 3.8-fold ("p<0.05 and **p<0.01 respectively, Figure 5A). Plasminogen Activator Inhibitor-1(PAI-1) was strongly expressed in both f-hAFS-CMhypo and p-hAFS-CMNypo (Figure7A). Other cytokines were detectable at low levels, namely Cystatin C(CST3), Fibroblast Growth Factor 19 (FGF-19)Interleukin-17a (IL-17a), Macrophage Migration Inhibitor Factor (MIF), Pentraxin 3 (PTX3).

While fetal-versus perinatal have-CM showed differential expression in their cytokine and chemokine profile, the corresponding fetal versus perinatal EV counterparts were more homogeneously distributed, although with lower expression profiles (Figure 7B, Figure S4B, and Table S5). Nonetheless, some differences could be appreciated: DiPeptidyl-Peptidase IV (DPPIV), Growth/differentiation factor 15 (GDF-15), and IL-8 were expressed only by f-hAFS-EVSHypo, although at low levels; ANGIOPOIETIN2, CD40 LIGAND and Vitamin D-binding Protein (VDBP) were found only in p-hAFS-EVShypo, despite once again detected in low amounts. Other cytokines such as Brain-derived Neurotrophic Factor (BDNF)ENDOGLIN, FGF-19, Insulin-like Growth Factor Binding Protein 3 (IGFBP3), IL-17a, MIF OPN, PTX3, Stromal Derived Factor-1 alpha (SDF-1a), were found in both f-hAFS-EVShypo and p-hAFS-EVSHvpo, with PAI-1 and EMMPRIN being more highly expressed (Figure 7B)

BDNF, ENDOGLIN, IGFBP3, and SDF-lo were exclusively enriched in all hypoxic have-EVs compared to the corresponding hAFS-CM, regardless of gestational stage. Moreover, while EMMPRIN was not detected within p-hAFS-CMhypo, it was found enriched in the EV corresponding fraction; conversely, OPN was more abundant in f-have-CMhypo than in f-have-EVShypo, while it was comparable among the corresponding p-hAFS secretome fractions. FGF-19,MIF, and PTX3 were similarly expressed in both fetal-and perinatal has-CM and in the corresponding hAFS-EVs. PAI-1 was highly enriched in hypoxic secretome fractions.

Figure 7. Cytokine and chemokine profiling within fetal- and perinatal hAFS secretome formulations. (A) Expression of cytokines and chemokines detected within the hypoxic fetal-versus perinatal have-CM (f-hAFS-CMHypo vs p-hAFS-CMHypo) are reported in pixel density by arbitrary unit [A.U.]. Values are expressed as mean ±s.e.m of independent experiments and reported in Table S4;*p=0.0485;**p=0.006.(B)Cytokine and chemokine content detected in the hypoxic fetal-versus perinatal hAFS-EVs (f-hAFS-EVSHypo vs p-hAFS-EVSHypo) and expressed by pixel density in arbitrary units [A.U.].cistanche cholesterol Values are expressed as mean±s.e.m of n=3 independent experiments and reported in Table S5. CST3: Cystatin C;EMMPRIN:Extracellular Matrix Metalloproteinase Inducer;FCFF-19: Fibroblast Growth Factor-19;IGFBP2:Insulin-like Growth Factor(IGF)Binding Protein 2;IL-8:Interleukin-8;IL-17a:Interleukin-17a; MCP-1: Monocyte Chemoattractant Protein-1;MIF:Macrophage migration Inhibitory Factor; PTX3: Pentraxin 3; PAI-1: Plasminogen Activator Inhibitor-1; BDNF: Brain-Derived Neurotrophic Factor; DPPIV:Dipeptidyl Peptidase IV; GDF-15:Growth Differentiation Factor-15;IGFBP3:Insulin-like Growth Factor(IGF)Binding Protein 3;SDF-1a: Stromal Derived Factor-1 alpha; VDBP: Vitamin D-Binding Protein.

2.7.Fetal-and Perinatal have-EVs Are Enriched with RNA Information in Their Cargo

Since small non-coding RNAs have been considered master regulators of EV paracrine influence on target cells [4,55], we mainly focused RNA sequencing analysis on microRNA (miRNA) content within f-hAFS-EVs and p-hAFS-EVs. Small RNA profiling showed enrichment of miRNA in both fetal- and perinatal hAFS-EVs (around 35-36%) when compared to the total small RNA amount (Figure 8A). The miRNA component was indeed among the two most represented RNA species in both EV formulations, together with rRNA(***p) p < 0.0001). The following miRNAs were the most highly enriched in the EV samples analysed: miR-31-5p; miR-196a-5p; miR-93-5p; miR-100-5p; miR-125a-5p; miR-27b-3p,let-7a-5p,let-7b-5p,let-7f-5p,let-7i-5p,miR-16-5p,miR-21-5p,miR-29a-3p,miR30a-5p, miR-125b-5p,miR-155-5p,miR-191-5p and miR-221-3p. Of note, fetal- and perinatal have-EVs shared the majority of such miRNAs (namely let-7a-5p,let-7b-5p,let-7f-5p, let-7i-5p, miR-16-5p, miR-21-5p, miR-29a-3p,miR30a-5p, miR-125b-5p, miR-155-5p, miR-191-5p and miR-221-3p, Figure7B).The 15 most enriched miRNAs species covered more than 60% of total miRNA content in each sample. On the other hand, about 100 miRNAs were found in the remaining 30% of the vesicular miRNA content (Figure 8C).

To further characterize the miRNA content within hAFS-EVs, we investigated whether the hAFS gestational stage or in vitro hypoxic cell preconditioning could influence the enrichment of specific miRNAs. The strongest modulation was found between gestational stages, where almost all modulated miRNAs were enriched in f-hAFS-EVs over the perinatal counterpart (Figure 9A and Table 1). Hypoxic preconditioning had a milder effect on miRNA cargo, whereas in this comparison modulation was in either direction, with some miRNA enriched in hypoxic-and others in normoxic control conditions (Figure 9A).

As a complementary analysis, we focused on the identification of investigated miRNAs with the lowest variability across the different donors and culture preconditioning, for EVs derived from both investigated gestational stages. miRNAs resulting from this analysis spanned from high to low expression levels (Figure 9B). Within the most stable miRNA core of hAFS-EVs cargo, some shared miRNA between f-hAFS-EVs and p-hAFS-EVs were identified (miR-21-5p, miR-29a-3p, miR-16-5p at high level; miR-221-3p,miR-221-5p and miR-22-3p at dim level, Table 2).

Figure 9. Analysis of differential enrichment of miRNAs in fetal- and perinatal hAFS-EVs. (A) Volcano plots for differential enrichment of hAFS-EV miRNA cargo according to the gestational stage (left panel) and in vitro hypoxic preconditioning of secreting cells (right panel). For miRNA details, please refer toTable1. (B)Scatter plot of the correlation between variability (X axis) and enrichment level (Y axis) of stable miRNAs within fetal hAFS-EVs (left panel) and perinatal hAFS-EVs(right panel) according to high (yellow dots), dim (green dots) and low (dark purple dots) enrichment. For miRNA details, please refer to Table 2.RPM: reads per million.

3. Discussion

Leftover discarded samples of human amniotic fluid have been identified as a valuable source of stromal cells with promising potential in regenerative medicine and tissue engineering. Ethical concerns associated with their isolation are minimal, since they can be obtained from either leftover samples of routine prenatal screening amniocentesis, during the II trimester of gestation (fetal hAFS), or from amniotic fluid discarded as clinical waste in III trimester scheduled C-section procedures (perinatal hAFS).In recent years, hAFS have been proposed as potential therapeutics for human tissue repair and regeneration given the encouraging evidence obtained from experimental disease models. Interestingly, they have been also proposed for in utero therapy of fetal-neonatal neurological diseases; indeed, preclinical studies suggested that hAFS administered prenatally via intra-amniotic delivery safeguarded the spinal cord during gestation via paracrine activity in a rat model of myelomeningocele [56-58], and reduced the damage of exposed bowel in experimental rodent gastroschisis[59]. From a translational perspective, in utero transplantation of hAFS could be replaced by the administration of the most suitable preparation of their secretome (hAFS-CM o hAFS-EVs). cistanche deserticola side effects This strategy would allow prompt and timely intervention during gestation by overcoming limitations of canonical cell therapy (i.e, time-consuming in vitro cell expansion) while providing off-the-shelf and ready-to-use pharmaceutical formulations.

The recent development of less invasive prenatal diagnostic techniques may result in a decrease in amniocentesis procedures in the near future, thus advocating perinatal hAFS as the more accessible option. Nevertheless, since fetal hAFS are more developmentally immature, they may harbor a more effective paracrine potential. Within this scenario, here we compared fetal- and perinatal c-KITt hAFS and we focused on profiling their secretome fractions. We highlighted relevant distinctions to be taken into consideration for the possible clinical translation of their paracrine capacity.

In agreement with previous independent studies, we show that the gestational stage did not influence the heterogeneous hAFS morphology and their mesenchymal antigen profile [25,26]. We then evaluated parameters more likely to impact cell secretory and paracrine activity beyond canonical stromal immunophenotype. Notably the presence of a CD146-positive, CD107a-high subpopulation within bone-marrow mesenchymal progenitors has been recently shown to correlate with remarkable modulatory and therapeutic paracrine activity [47]. Here we revealed that both fetal- and perinatal hAFS are strongly characterized by this molecular signature supporting their secretory potency with relevant translational implications. Moreover, fetal hAFS were characterized by inefficient aerobic metabolism, while more mature perinatal ones showed higher oxygen consumption rate and ATP synthesis. This may suggest a more immature metabolic profile of II trimester hAFS that resembles umbilical cord stromal cells of preterm newborns, which have shown the same trend [60].

In order to trigger paracrine potential, hAFS were exposed to 24 h serum-free hypoxic priming, a strategy we have previously successfully developed [34,35,37] for fetal cells and that here we investigated on their perinatal counterpart for the first time. Preconditioning fetal- and perinatal hAFS under hypoxia resulted in a positive trend in the increase of their secretome concentration and in the amount of EVs released, whereas the gestational stage did not exert any effect on the cell secretome yielc nor on EV morphology and size distribution.

Notably, characterization of the hAFS paracrine cargo revealed some specific differences, according to the different conditions we evaluated. The proteomic profiling of the fetal hAFS secretome revealed discernible factor distributions based on gestational stage and cell hypoxic preconditioning. This indicates that the hAFS paracrine potential can acquire a distinct identity during maturation from Ⅱ to Ⅲ gestation trimester that in turn can be modulated by stimulating the secreting cells in vitro. Biological processes enrichment analysis of hAFS-CM and hAFS-EVs suggested that most of the modulated proteins may concur to cell growth/maintenance and protein metabolism, thus supporting the cell beneficial paracrine effects reported far. In particular, the hypoxic fetal hAFS total secretome was found enriched with the heat shock protein HSPD1 (HSP60), which was demonstrated to support wound healing in a diabetic mouse skin injury model and to promote macrophage pro-resolving skewing into M2 phenotype [61]. Likewise, hypoxic fetal hAFS-EVs were enriched for factors promoting neurogenesis (HSPG2[62], cell self-renewal, and brain and cardiovascular development (LAMA5[63]and LAMB1[64]and migration (THBS1 [2]). The proteoglycan AGRN was also found in EVs following hypoxic priming of fetal hAFS. cistanche dosage reddit Our findings are in line with previous evidence of AGRN being upregulated in the proteome of mesenchymal stromal cells under inflammatory and hypoxic instructive stimuli[65]. AGRN has also been shown to be implicated in immune synapse signaling [66] and to concur with neonatal mouse heart regeneration [67], thus supporting a pronounced predisposition of developmentally young fetal hAFS towards regenerative paracrine effects. Moreover, fetal hAFS was confirmed to be more responsive to hypoxic preconditioning as shown by enrichment of predictors of vascular regenerative efficacy, such as ANGIOGENIN, EMMPRIM, IL-8, and MCP-1 cytokine[68], in their conditioned medium. This supports previous evidence of the paracrine potency of fetal hAFS-CM in boosting endogenous neo-arteriogenesis in preclinical rodent models of myocardial infarction, hind-limb ischemia, and ischemic fasciocutaneous flap [34,69-71]Furthermore, the fetal hAFS total secretome was found significantly more enriched with IGFBP2and OPN when compared to the perinatal one, thus suggesting a more pronounced pro-resolving and anti-aging modulatory profile[72-75]. The perinatal have-CM, while being less enhanced in paracrine factors, was similarly supplemented with neurotrophic and immunomodulatory factors, such as CST3[76,77] and MIF[78].

Compared to total hAFS-CM, the corresponding hypoxic fetal- and perinatal- EV counterparts showed lower expression of cytokines and chemokines, with the exception of the vascular remodeling mediator EMMPRIN [79], which was mostly enriched in the vesicle compartment. The previously reported cardio-active and pro-regenerative profile of fetal hAFS-EVs[34,37] has been confirmed herein by evidence of their exclusive expression of cardioprotective IL-8 [80] and GDF-15, a key paracrine factor triggering endogenous adult hippocampal neurogenesis[81,82], as well as counteracting anthracycline-induced cardiotoxicity [78]. Both fetal- and perinatal hAFS-EVs showed similar expression for the progenitor/stem cell trafficking regulator SDF-1α[83-85]. The proteomic analysis reported increased expression of proteins related to angiogenesis, like NRP1 [86] and MP14[87]in hypoxic perinatal hAFS-EVs; such stimulatory profile may explain previous results on the endothelial regenerative properties of Ⅲ trimester hAFS in a preclinical mouse model of skeletal muscle ischemic injury [25], despite the evidence of their hAFS-CM being less pro-angiogenic than the corresponding fetal one. Notably, the neural growth factor BDNF was found in both fetal- and perinatal EV cargo, although in low amounts, thus suggesting a putative neurotrophic activity for hAFS-EVs in neuronal survival and neurodevelopmental processes, as also observed for extracellular vesicles secreted by human bone marrow and umbilical cord blood-MSC[8889]. Both secretome formulations from fetal- and perinatal hAFS undergoing hypoxic stimulation showed to be enriched with PAI-1, a facilitator of endothelial activation [90] that has also been involved in the polarization of M2 macrophages in the heart and endowed with cardioprotective and anti-fibrotic potential [91].

MicroRNAs (miRNAs) have been broadly addressed as crucial regulators of stem cell- and mesenchymal stromal cell-EV paracrine activity [92,93]. Here we found that the 15 most enriched miRNA species within the hAFS-EVs cover more than 60% of the total miRNA content in each sample. These miRNAs have been reported to characterize the molecular cargo of mesenchymal stromal cell-EVs (let-7a-5p [4,95]),protect against myocardial ischemia by influencing vascular regeneration and inhibiting fibrosis (let-7b-5p, let-7f-5p, miR-21-5p and miR-155-5p [96,97]), promote wound healing by regulating keratinocyte function (miR-16-5p [98]) and counteract neuronal death after forebrain ischemia (miR-29a-3p [99,100]). On the other hand, about 1000 miRNAs were found in the remaining 30% of vesicular miRNA content. Such unbalanced distribution is consistent with previous studies [4] and highlights the mostly enriched miRNAs as the putative accountable ones for the main biological activity of hAFS-EVs. Interestingly, fetal- and perinatal hAFS-EVs shared the majority of 15 miRNAs. Of note, we also observed that both fetal hAFS-EVs and perinatal ones contained a set of very stable miRNAs across different enrichment levels. Such evidence may suggest a wide range of "housekeeping" candidates to be used as an internal reference control in qPCR experiments on hAFS-EVs. Moreover, a consistent subset of such stable miRNAs (miR-16-5p,miR-21-5p,miR-22-3p,miR-29a-3p,miR-221-3y miR-221-5p) is shared between the two gestational stages and overlaps with the 15 mostly enriched ones, suggesting an even more constant behavior and a reliable pre-resolving molecular signature([96,98-100]). Of note, a couple of candidates within such distinctive core have been recently reported as reference miRNAs within neuroprotective EVs obtained from II trimester amniotic fluid-derived mesenchymal stromal cells (miR-29a-3p and miR-221-3p[95]). Nevertheless, we also noticed that gestational age may modulate the miRNA cargo more than hypoxic preconditioning. Developmentally more juvenile EVs obtained from III trimester fetal hAFS were enriched with miRNAs previously shown to support the viability of embryonic stem cells (miR-302-3p [101]), cell proliferation, and osteogenic differentiation of bone marrow stromal cells (miR-217 [102]), while also harboring tumor suppressor potential (miR-302-3p[103,104]);miR-383-5p[105,106]).

Based on our results, here we confirm that fetal- and perinatal hAFS may represent attractive paracrine sources to be exploited for regenerative medicine. While their pheno-type and secretory activity were similar, we have highlighted some peculiar aspects in their secretome formulations as useful insights for their future therapeutic translation.

4. Materials and Methods

4.1.Human Amniotic Fluid Stem Cell Isolation and In Vitro Culture

Human amniotic fluid stem cells (hAFS) were isolated from leftover samples of amniotic fluid (AF) collected by routine prenatal screening via Ⅱ trimester amniocentesis (fetal hAFS, f-hAFS), or as clinical waste during scheduled cesarean-section delivery during Ⅲ trimester (perinatal hAFS,p-hAFS) at the Prenatal Diagnosis and Perinatal Medicine Unit, IRCCS San Martino Hospital, at the Fetal- and perinatal Medical and Surgery Unit and Human Genetics Laboratory at IRCCS Istituto Gaslini hospital (Genova, Italy). Informed written consent was obtained from all donors according to local ethical committee authorization (protocol P.R.428REG2015)and in compliance with Helsinki Declaration guidelines. II trimester fetal AF samples were obtained from female donors with an average age of about 37.42±0.32 years old (n=15 ranging from 36-up to 41 years old);Ⅲ trimester perinatal AF samples were obtained from female donors with an average age of 34.25±1.31 years old (n=10 ranging from 26- up to 42 years old). Fetal- and perinatal hAFS were obtained from samples validated for normal karyotype and isolated by immunomagnetic sorting for c-KIT expression (CD117 MicroBead Kit, Miltenyi Biotechnology, Bologna, Italy)from adherent AF mesenchymal stromal cells[16]. cistanche extract benefits c-KIT* hAFS were cultured in Minimal Essential Medium (MEM)-alpha with 15% FBS (Fetal Bovine Serum, Gibco-Thermo Fisher Scientific, Monza, Italy),18%Chang B, and 2% Chang C Medium (Irvine Scientific, Santa Ana, CA, USA) with 1%L-glutamine and 1% penicillin/streptomycin (Gibco-Thermo Fisher Scientific,Monza, Italy), in an incubator at 37°C with 5% CO2 and 20% Oz atmosphere and cultured up to 5 passages in vitro before being used to isolate their secretome.

4.2.Biochemical Evaluation of hAFS Metabolism

Cell aerobic metabolism was evaluated in terms of oxygen consumption and ATP synthesis through the FI-Fo ATP synthase. Oxygen consumption rate (OCR) was measured at 37 ℃ in a closed chamber magnetically stirred using an amperometric electrode (Unisense-Microrespiration, Unisense A/S, Denmark). One hundred thousand (10>) cells were used for each experiment. To evaluate basal respiration, hAFS were permeabilized with 0.03 mg/mL digitonin for 10 min and suspended in phosphate buffer saline (PBS).10 mM pyruvate plus 5 mM malate or 20 mM succinate were added to stimulate the path-ways composed by Complexes I, II, and IV or Complexes Ⅱ and IV, respectively [60].

To evaluate the relative contributions to respiration of glutamine, long-chain fatty acid oxidation, and glucose, after the digitonin permeabilization, cells were suspended in a growth medium and 4 uM of BPTES,4 uM Etomoxir, and 4 uM UK5099 were added to inhibit glutaminase, carnitine palmitoyl-transferase 1A(CPT1A), or the mitochondrial pyruvate carrier (MPC), respectively. Fi-F。ATP synthase(ATP synthase) activity was detected by measuring ATP production by the highly sensitive luciferin/luciferase method. The assays were conducted at 37℃, for 2 min, and data were collected every 30 s. In a first set of experiments,10°cells were incubated for 10 min in medium containing 50mM KCl,1mMEGTA,2mMEDTA,5mMKH2PO4,2mMMgC12,0.6mMouabain,1mM P1P5-Di(adenosine-5')penta-phosphate,0.040 mg/mL ampicillin, and 10mM Tris-HCl pH7.4. Afterward, ATP synthesis was induced by the addition of the respiratory substrates (10mM pyruvate +5 mM malate or 20 mM succinate) and 0.1 mM ADP. The reaction was measured using the luciferin/luciferase ATP bioluminescence assay kit CLSII(Roche, Basel, Switzerland) in a Luminometer (GloMax 20/20 Luminometer, Promega,Milan, Italy)ATP standard solutions (Roche, Basel, Switzerland) ranging 10-10-10-/M were used for calibration. In the second set of experiments, ATP synthesis was evaluated in the presence of 4 uM BPTES,4 uM Etomoxir, or 4 uM UK5099.In this case,10 cells were incubated for 10 min in a growth medium in the absence or presence of a metabolism inhibitor, and ATP synthesis was induced with 0.1 mM ADP. OxPhos (oxidative phosphorylation) efficiency (P/O ratio) was calculated as the ratio between the concentration of produced ATP and the amount of consumed oxygen in the presence of respiratory substrate and ADP. When oxygen consumption is completely devoted to energy production, the P/O ratio should be approximately 2.5 and 1.5 after pyruvate + malate or succinate addition, respectively [48]To evaluate the contribution of anaerobic glycolysis to hAFS metabolism, glucose and lactate concentrations were evaluated in the growth medium. Glucose consumption was evaluated by the hexokinase (HK) and glucose-6-phosphate dehydrogenase(G6PD)coupling system, following the reduction of NADP at 340 nm. The assay medium contained 100mM Tris-HCl,pH7.4,2mM ATP,10mMNADP,2mMMgC12,2IU of hexokinase,and 2 IU of glucose-6-phosphate dehydrogenase. Lactate release was assayed following the reduction of NAD+ at340 nm. The assay medium contained 100 mM Tris-HCl (pH8),5 mM NAD+,and 1 IU/mL of lactate dehydrogenase. Samples were analyzed before and after the addition of 4 ug of purified lactate dehydrogenase. In both cases, data was normalized to the cell number and expressed as mM glucose/10° cells or mM lactate released/10° cells, respectively[107].

4.3.Flow Cytometry Characterization of hAFS

One hundred thousand (105)fetal-and p-hAFS cells were detached and incubated with mouse anti-human-CD107a-Alexa Fluor 647-and anti-human CD146-FITC-conjugated antibodies (eBioscience, Thermo Fisher Scientific, Monza, Italy). Cell apoptosis was assessed using a FITC Annexin V Apoptosis Detection Kit (BD Pharmingen, Becton Dickinson,Mi-lan, Italy) following the manufacturer's instructions. Events were acquired on a BD Bioscience FACS Aria II sorter and analyzer, equipped with FACS Diva software (BD Bioscience, Bec-ton Dickinson, Milan, Italy).Data were analyzed using FlowJo V9.0 software (BD Bioscience, Becton Dickinson, Milan, Italy). 4.4.Senescence Staining

The senescence phenotype of hAFS cultured up to passage 5 in standard in vitro conditions was evaluated with Senescence β-Galactosidase Staining Kit (Cell Signaling Technology, Danvers,MA, USA):f-hand p-hAFS were fixed with 1x Fixative solution at 70% confluency and stained for SA-β-gal at 37 °C overnight, according to the manufacturer's instructions. Senescent events were acquired on a Leica DMil microscope (equipped with Leica Acquire software V3.4.4, Leica Microsystems,Milan, Italy) and evaluated as a percentage of SA-β-gal-positive cells over total cells per field.

4.5. Separation and Concentration of the hAFS Secretome Fractions

f-hAFS and p-hAFS were cultured for 24 h in serum-free medium (SF) in 1% O2 hypoxia versus 20% O2 normoxia (control), the latter of which was used as the baseline reference. This preconditioning strategy was used to enhance the release of bio-active paracrine factors, as we previously reported [34,35,37,49]. hAFS were cultured for24h in serum-free (SF) medium (high glucose Dulbecco's Modified Eagle's Medium, DMEM, with 1% L-glutamine and 1% penicillin/streptomycin, all from Gibco-Thermo Fisher Scientific, Monza, Italy), under normoxic(20% O2 and 5% CO2 at 37°C) or hypoxic(1% O2 and 5%CO2 at 37°C in CellXpert C170i and Galaxy 48R CO2 incubators, from Eppendorf, Milan, Italy)conditions.

f-hAFS-CM and p-hAFS-CM were collected and centrifuged at 4℃at 300x g for 10 min and 2000× g for 20 min to remove cell debris; hAFS-CM was concentrated using ultrafiltration membranes with a 3kDa selective cut-off(Amicon Ultra-15, Merck Millipore Darmstadt, Germany)at 4℃ at 3000× g for90 min and then further concentrated at 4℃at 3000× g for 30 min. hAFS-EVs were separated and concentrated by serial ultracentrifugation from hAFS-CM. Briefly, hAFS-CM was collected and centrifuged at 4℃at 300× g for 10 min, and 2000x g for 20 min to remove cell debris. The supernatant was then processed at 10,000× g for 40 min. The pellet was discarded and the supernatant was further processed by ultracentrifugation in an Optima L-90K (Beckmann Coulter,Milan, Italy) at 10,000×g for 120 min using Beckman Coulter's swinging-bucket SW55Ti centrifuge rotors. The pellet containing heterogenous hAFS-EVs was washed in PBS with final centrifugation at 100,000× g for 120 min and then resuspended in PBS filtered with a 0,22 um pore filter membrane. Protein concentrations in hAFS-CM and on the surface of hAFS-EVs were measured using the BiCinchoninic Acid (BCA) assay(Thermo Fisher Scientific, Monza, Italy). Samples were acquired on a Gen5 Microplate Reader at 570 nm to evaluate hAFS-CM and hAFS-EVs yield in terms of μg of solution/10°producing cells.

4.6. Characterization of hAFS-EVs by Transmission Electron Microscopy and Nanoparticle Tracking Analysis

Transmission electron microscopy (TEM) analysis was performed on a Hitachi TEM microscope (HT7800 series, Hitachi High Technologies, Monza, Italy). Digital images were taken with a Mega view 3 camera and Radius software (EMSIS, Muenster, Germany). f-hAFS and p-hAFS were fixed in 3.7% paraformaldehyde (PA)solution diluted 1:1 with hAFS complete medium, washed in 0.1 M cacodylate buffer, and then immediately incubated for 1 h at room temperature in 0.1 M cacodylate buffer containing 2.5% glutaraldehyde (Electron Microscopy Science, Hatfield,PA, USA). Cells pellets were post-fixed in osmium tetroxide for lh and in a 1% uranyl acetate solution for 1h. Samples were dehydrated for 24 h at 42℃and 48 h at 60℃ through a graded ethanol series and embedded in epoxy resin (Poly-Bed; Polysciences Europe GmbH, Minneapolis, Germany). Ultrathin sections(50 nm) were cut with Leica Ultracut microtome(Leica Microsystems,Milan, Italy)and counterstained with a 5% uranyl acetate in 50% ethanol solution. f-hAFS-EVs and p-hAFS-EVs were resuspended in 20 uL PBS solution and fixed by adding an equal volume of 2% paraformaldehyde in 0.1 M phosphate buffer solution (pH7.4). EVs were then adsorbed for 10 min onto formvar-carbon coated copper grids by floating the grids on 5 μL drops on parafilm. Subsequently， grids with adhering EVs were rinsed in PBS and negatively stained by 2% uranyl acetate solution for 5 min at room temperature. Stained grids were embedded in 2.5% methylcellulose for improved preservation and air dried before the examination. Morphometry analysis of hAFS-EVs was measured on 10 randomly taken micrographs at 40.000× g magnification. The size was calculated using the arbitrary line function embedded in the measurement dialog box of Radius software (EMSIS, Muenster Germany). To visualize hAFS-EVs size distribution, results were plotted as scatter dot plot and as frequency distribution in which each size is represented as a point along with lines for the median value and the range.

f-hAFS-EVs and p-hAFS-EVs were also analyzed by Nanoparticle Tracking Analysis (NTA) to assess particles released by 10° cells. hAFS-EVs were diluted 1:100 in PBS solution and acquired on a NanoSight LM10 (Malvern Instruments, Malvern, UK) that recorded at least 3 different frames of 60 s each. Three different acquisitions of each sample were analyzed using the Batch Process option in the software. 4.7.LC-MS/MS Analysis of hAFS-CM and hAFS-EVs 4.7.1.In-Solution Digestion

Proteomic analysis was performed on 3 biological replicates of hAFS-CM and hAFS. EVs from f-hAFS and p-hAFS after normoxic or hypoxic preconditioning (n=24 different conditions).hAFS-CM and hAFS-EVs samples were suspended in 0.1M NHCO3 pH 7.9 and treated with RapigestIM SF reagent (Waters Co, Milford,MA, USA) at the final concentration of 0.25% (w/v). The resulting suspensions were incubated while stirring at 100°C for 20 min. The digestion was carried out on each sample by adding Sequencing Grade Modified Trypsin (Promega Inc, Madison, WI, USA) at an enzyme/substrate ratio of 1:50(w/w) overnight at 37°C in 0.1 M NH4HCO3 pH7.9 buffer with 10% CHSCN.An additional aliquot of trypsin (1:100 w/w) was added in the morning, and the digestion continued for 4h. Moreover, the addition of 0.5% Trifluoroacetic acid (TFA) Gigma-Aldrich Inc., St Louis, MO, USA)stopped the enzymatic reaction, and a subsequent incubation at 37 ℃ for 45 min completed the RapiGest acid hydrolysis[108]. The water-immiscible degradation products were removed by centrifugation at 13.000 rpm for 10 min. Finally, the tryptic digest mixtures were desalted using PierceTM C-18 spin columns (Thermo Fisher Scientific, Monza, Italy), according to the manufacturer protocol, and were resuspended in 0.1% formic acid (Sigma-Aldrich Inc., St. Louis, MO, USA) in water (LC-MS Ultra CHROMASOLVTM, Honeywell Riedel-de HaenTM, Muskegon,MI, USA) at a concentration of 0.1 μg/μL.

4.7.2.Liquid Chromatography

Trypsin digested mixtures were analyzed by means of a platform consisting of a nano-liquid chromatographic system, Eksigent nanoLC-Ultra[2]2D System (Eksigent, part of AB SCIEX Dublin, Dublin, CA, USA) configured in trap-elute mode, coupled with a high-resolution mass spectrometer. Briefly, samples (0.8 ug injected) were first loaded on a peptide trap (200 um × 500 um ChromXP C18-CL,3 um,120 A)and washed with the loading pump running in isocratic mode with 0.1% formic acid in water for 10 min at a flow of 3 μL/min. The automatic switching of a ten-port valve then eluted the trapped mixture on a nano-reversed phase column (75 um × 15 cm ChromXP C18-CL,3 um,120 A)through a 150 min gradient of eluent B(eluent A,0.1% formic acid in water; eluent B,0.1%formic acid in acetonitrile) at a flow rate of 300 nL/min. In-depth, the gradient was: from 5-10%Bin 3min,10-40% Bin 130min,40-95% Bin 10 min and holding at 95% B for7min. 4.7.3.Mass Spectrometry

MS/MS analyses were performed on an LTQ-OrbitrapXL mass spectrometer (Thermo Fisher Scientific, Monza, Italy) equipped with a nanospray ion source. The spray capillary voltage was set at 1.7 kV and the ion transfer capillary temperature was held at 220℃. Full MS spectra were recorded over a 400-1600 m/z range in positive ion mode, with a resolving power of 60000 (full width at half-maximum) and a scan rate of 2 spectra/s. This step was followed by five low-resolution MS/MS events that were sequentially generated in a data-dependent manner on the top five ions selected from the full MS spectrum (at 35% collision energy), using the dynamic exclusion of 0.5 min for MS/MS analysis. Mass spectrometer scan functions and high-performance liquid chromatography solvent gradients were controlled by the Xcalibur data system version 1.4(Thermo Fisher Scientific, Monza, Italy).

4.7.4.Proteomic Data Processing and Data Mining

All generated data were searched using the Sequest HT search engine contained in the Thermo Scientific Proteome Discoverer software, version 2.1. The experimental MS/MS spectra were correlated to tryptic peptide sequences by comparison with the theoretical mass spectra obtained by in silico digestion of the Uniprot Homo Sapiens proteome database (74600 entries), downloaded in January 2020 (www.uniprot.org, accessed on 10 March 2021). The following criteria were used for the identification of peptide sequences and related proteins: trypsin as an enzyme, three missed cleavages per peptide, mass tolerances of ±50 ppm for precursor ions, and ±0.8 Da for-fragment ions. Percolator node was used with a target-decoy strategy to give a final false discovery rate (FDR) at Peptide Spectrum Match (PSM) level of 0.01(strict) based on q-values, considering a maximum deltaCN of 0.05 [109]. Only peptides with a minimum peptide length of six amino acids and rank1 were considered. Protein grouping and strict parsimony principles were applied. The MS data have been deposited to the ProteomeXchange Consortium via the PRIDE [10]partner repository (ftp://massive.ucsd.edu/MSV000087013/,acessed on 10 March 2021)The 48 proteins obtained from the SEQUEST algorithm were aligned, normalized, and label-free compared. An in-house algorithm, namely, the Multidimensional Algorithm Protein Map (MAProMa) was employed for this aim, using the average peptide spectrum matches (aPSM) [111,112] that correspond to the average of all the spectra identified for a protein and, consequently, to its relative abundance, in each analyzed condition. In-depth, to select differentially expressed proteins, subgroups (for both fetal- vs perinatal-hAFS-CM and hAFS-EVs, considering also hypoxic cell preconditioning stimulation), were pairwise compared by applying a threshold of 0.4 and 5 on the two MAProMa indexes DAve (Differential Average) and DCI (Differential Confidence Index), respectively. DAve, which evaluates changes in protein expression, was defined as(X-Y)/(X＋Y)/0.5, while DCI which evaluates the confidence of differential expression, was defined as (X+Y)×(X-Y)/2 The X and Y terms represent the PSM of a given protein in two compared samples. In addition, the average protein lists, obtained from each examined condition, were subjected to linear discriminant analysis (LDA), and proteins with the largest F ratio (≥4.5) and smallest p-value(≤0.001) were retained and processed by hierarchical clustering, applying Ward's method and the Euclidean's distance metric using JMP 15.2 software. Specifically, the F ratio represented the model mean square divided by the error mean square, whereas the p-value indicated the probability of obtaining an F value greater than that calculated if, in reality, there was no difference between the population group means. 4.8. Cytokine and Chemokine Profiling of hAFS-CM and have-EVs

Cytokine and chemokine profiling of hAFS-CM and have-EVs obtained by f-hAFS and p-hAFS after hypoxic preconditioning was assessed by means of the Proteome ProfilerTM Human XL Cytokine Array kit (R&D System, Minneapolis, MN, USA) according to the manufacturer's instructions. Twenty ug of the hAFS-CM and hAFS-EVs samples were used. Membranes images were acquired by a Chemidoc Mini HD9 Auto (Uvitec Cambridge, UK)Specific cytokine/chemokine content was evaluated by the quantification of positive pixel intensity (by means of the arbitrary unit) for each detectable cytokine using ImageJ software (available at https://imagej.nih.gov/ij/,accessed on 10 March 2021 [13]). 4.9.RNA Extraction from hAFS-EVs and Next

Generation Sequencing

RNA was isolated from f-hAFS-EVs and p-have-EVs with miRNeasy Micro Kit (Qiagen, Milan, Italy) according to the manufacturer's instructions. RNA integrity and size distribution were evaluated using the Agilent Small RNA Kit with the small noncoding RNA chip in order to assess the content of small RNAs ranging from 6 to 150 nucleotides (nt). The Qubit microRNA Assay Kit (Thermo Fisher Scientific, Monza, Italy) was used to quantify microRNA (miRNAs) content, following the manufacturer's instructions. miRNA sequencing libraries were prepared and amplified using the QIAseq miRNA Library kit (Qiagen, Milan, Italy)using 18.5ng of isolated miRNAs as input and following the manufacturer's instructions. Libraries were pooled after a quality check and quantification by TapeStation (Agilent Technologies, Foster City, CA, USA) was performed using Agilent High Sensitivity D1000 ScreenTape. Pooled libraries were assessed for quality control by real-time qPCR following the "Sequencing Library qPCR Quantification" Guide (Illumina Inc, San Diego, CA, USA)and sequenced by Illumina NextSeq platform using High Output Hit v2.5(75 cycles) (Illumina Inc, San Diego, CA, USA). Base calling was performed with the default Illumina NextSeq500 workflow.

4.10.Bioinformatic Data Analysis of miRNA Sequencing

Fastq files were first processed by trimming off the 3' adapter and low-quality bases using Cutadapt [114]. Following trimming, the insert sequences and UMI sequences were identified. Reads with no adapter sequence, reads with less than 16 bp insert sequences, and Reads with less than 10 bp UMI sequences were discarded. To annotate the insert sequences, reads were aligned to GRCh38 human genome assembly using Bowtie [15]For each sample all reads assigned to a particular miRNA were counted, and the associated UMIs were aggregated to count unique molecules. Secondary analysis was performed by custom R scripts available upon reasonable request. Differential enrichment analysis was performed using Limma [116] and EdgeR Bioconductor packages [117].

4.11. Statistical Analyses

Results are presented as mean ±s.e.m of at least three(n=3)independent experiments. Comparisons were drawn by one-way ANOVA followed by posthoc Tukey's multiple comparisons test or by Student's t-test. Analyses were performed using Graph-Pad Prism Version 8.0.2 (GraphPad Software, https://www.graphpad.com, accessed on 10 March 2021) with statistical significance set at* p<0.05. For proteomics analysis, the distribution of proteins in the examined conditions, functional enrichment analysis, and comparison of data versus the Vesiclepedia database (http:/microvesicles.org, accessed on 10 March 2021) were achieved using FunRich (version 3.1.3,http://www.funrich.org,accessed on 10 March 2021[54]), that uses hypergeometric test and Bonferroni for statistics and allows the graphical visualization of data with Venn and bar charts [118]. 5. Conclusions

In conclusion, fetal- and perinatal hAFS were found phenotypically equivalent with comparable secretory potency and EV enrichment in size and distribution; yet some distinctions in their secretome profile could be appreciated. Specifically, the developmentally immature profile of fetal hAFS may be recapitulated by their secretome formulations endowed with a more pronounced pro-vasculogenic, pro-regenerative and rejuvenating secretome. However, perinatal hAFS still retain a relevant paracrine profile via the expression of factors related to endothelial cell migration, immune-modulatory, anti-inflammatory, and neurotrophic potential similar to fetal hAFS. These findings may provide useful insights supporting a future paracrine therapy of injury-related and inflammatory/ischemic-based disease. Therefore, the selection of either fetal or perinatal hAFS as the most ideal cell source should be evaluated considering the specific clinical scenario.

This article is extracted from Int. J. Mol. Sci. 2021, 22, 3713. https://doi.org/10.3390/ijms22073713 https://www.mdpi.com/journal/ijms