Regenerative Medicine Has Recently Developed As An Emerging Field

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Abstract: We previously reported that c-KIT+htaman 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. have 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 Ⅱtrimester 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 the 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.

Keywords: amniotic fluid; stem cells; paracrine effects; extracellular vesicles; cell-conditioned medium; chemokine; cytokines; proteomics; RNA sequencing; microRNA

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1. Introduction

Regenerative medicine has recently developed as an emerging field to provide functional restoration of injured tissue by means of several strategies. As tissue engineering approaches have significantly advanced in recent years, the investigation of stem cell paracrine effects has concomitantly increasingly intensified. The therapeutic potential of transplanted stem cells has been broadly shown to be mostly mediated by their secreted soluble factors, which can orchestrate a pro-regenerative microenvironment in the host tissue while triggering the activation of endogenous mechanisms of functional recovery [1,2]. Therefore, the stem cell secretome the entirety of cell-released paracrine trophic molecules, as well as membrane-bound extracellular vesicles have been increasingly proposed as an innovative therapy medicinal product by multiple independent preclinical studies target-ing cardiovascular, neurological, and/or inflammatory disease. Accordingly, stem cells may be envisioned as biological factories for the exploitation of their therapeutic secretome by offering ready-to-use and off-the-shelf regenerative treatments. By applying such a cell-based, yet cell-free strategy, many limiting aspects associated with canonical cell therapy may be overcome, while still ensuring beneficial effects. what is a cistanche In this perspective, mesenchymal stromal cells (MSC) have been extensively tested as putative cell candidates? Indeed, MSC and stem/progenitor cells have been engineered and/or stimulated by different preconditioning strategies to enhance their regenerative capacity and secretory potential [3,4]with an explicit interest in the biological relevance of their secreted extracellular vesicles(EVs).

EVs are nano-sized particles delimited by a lipid bilayer and actively secreted by all cell types. EVs include very small (<200 nm) exosomes, medium-sized (200-500 nm)microvesicles or shedding vesicles, and larger-sized apoptotic bodies (>500 nm); they operate as critical biological conveyors of intercellular signaling by delivering their molecular cargo from a parental cell to a responder/target one [5,6]. Given that their peculiar paracrine potential in exerting beneficial effects is comparable to their cells of origin, stem cell EVs have arisen as appealing therapeutic options in preclinical models of diseases, such as ischemia, inflammation, or injury, as extensively reviewed in [7-9]. From a translational perspective, on top of cell modulatory potential, isolation feasibility and elevated self-renewal are key aspects of the ideal source of therapeutic EVs and soluble factors. In such a scenario, fetal- and perinatal MSC may offer an interesting option given their proliferative potential, and developmentally immature profile with intermediate features between embryonic and adult somatic progenitors [10,11]. Fetal MSC can be isolated from extra-embryonic annexes during gestation as left-over sampling obtained during prenatal screening (i.e., chorionic villi [12-14] and amniotic fluid [15,16]) or obtained as perinatal progenitors at birth, from clinical waste material (i.e, amniotic and placenta membranes [17-21], umbilical cord components [22-24] and term amniotic fluid [25,26]).

Cistanche can anti-aging

Notably, human amniotic fluid stem cells (hAFS) have been highlighted as promising therapeutic strategies in regenerative medicine. and have been shown to be broadly multipotent in vitro and in vivo [16,27,28], contribute to the hematopoietic lineage following in utero transplantation [29], and engraft in injured organs while exerting immunomodulatory effects [26,30] and activating endogenous reparative responses, as comprehensively described in [31]. Our team and others have further demonstrated that hAFS releases a secretome highly enriched with bioactive trophic molecules able to target different reparative mechanisms. hAFS paracrine factors have been reported to provide pro-survival stimuli with the quenching of inflammation [32], provide cardioprotection against pro-longed ischemia [33.34], and cardiotoxicity [35], and stimulate local angiogenesis with cardiomyocyte cell cycle re-entry [34,36]. Since most of these effects have been shown to be recapitulated by hAFS-EV administration alone, independent studies have focused on dissecting their regenerative profile against different pathological backgrounds, including skeletal and cardiac muscle injury, kidney disease, osteoarthritis, osteoporosis, necrotizing enterocolitis and neurodegenerative models [34,37-44].

While evidence may support the clinical translation of hAFS-EVs for future paracrine therapy, it is important to consider that most of these studies have mainly investigated the modulatory potential of fetal hAFS obtained during Ⅱ trimester prenatal screening Indeed, a complete profile of the secretome from the perinatal has counterpart (i.e, from III trimester c-sections) has not yet been explored in detail. Third-trimester perinatal hAFS have shown distinctive immune regulatory properties compared to me- and II-trimester ones [26], while maintaining relevant endothelial regenerative potential [25]. how much cistanche to take Of note, the recent report on the heterogeneous morphology of fetal hAFS[45] has provided new insights into their stemness and gene expression profile. bioflavonoids This has altogether shed new light on the regenerative value of the different cellular fractions of hAFS[46]. Hence, comprehensive characterization of the different subpopulations of hAFS is attracting mounting attention. We previously reported that a 24h hypoxic and serum-free stimulation represents an effective strategy to boost the paracrine potential of Ⅱ trimester fetal hAFS [34,35,37]. Since little is known about the composition of the secretome from III trimester hAFS, here we report the comprehensive comparison of Ⅱ versus Ⅲ trimester hAFS and their secretome fractions (including hats-EVs), in order to address the influence of gestational stage and hypoxic cell preconditioning on cell and secretome characteristics.

2. Results

2.1.Perinatal hAFS Present a Close Phenotypic Match to Fetal has

No statistically relevant difference was appreciated in donor age between fetal Il trimester and perinatal III trimester amniotic fluid samples. Fetal c-KIT* hAFS (f-hAFS from II trimester amniotic fluid samples) and perinatal c-KIT* hAFS (p-hAFS from III trimesters amniotic fluid clinical waste) confirmed similar features with fibroblast-like and oval-round morphology (Figure 1A) and mesenchymal stromal phenotype (data not shown), as previously reported [16,25]. Both f-hAFS and p-hAFS cultured in vitro up to passage 5 showed negligible levels of senescence from senescence-associated-β-galactosidase (SA-β-Gal) activation in about 4% of cells(Figure 1B). Both f-hAFS and p-hAFS presented a high level of co-expression of the mesenchymal markers CD107a and CD146, which have been recently reported to define a highly secretory phenotype[47]. CD107at CD146* cells represented the majority of the f-hAFS population (approximately 64%,*p<0.05), while p-hAFS showed a lower enrichment for this subpopulation, approximately 52% of total cells, yet this disparity was not statistically significant (Figure 1C).

Figure 1. Fetal has and perinatal have phenotypic evaluation. (A)Representative images of fetal hAFS (f-hAFS,left panel) and perinatal hAFS(p-hAFS, right panel) cultured in vitro in standard conditions; scale bar: 200 um. (B) Analysis of the senescent marker beta-galactosidase(SA-β-Gal, in blue) via cytochemistry staining on f-hAFS and p-hAFS after 5 passages in culture; representative images are reported in the left panel, scale bar: 200 um. The corresponding percentage of β-Gal-positive cells/field is reported in the graph in the right panel (f-hAFS:4.12±0.58% and p-hAFS:3.88±2.10%;p=0.1424,n=3 experiments).(C)Immunophenotype of hAFS expressing CD146 and CD107a mesenchymal markers. Representative flow cytometry plots of f-hAFS and p-hAFS (left panel) and corresponding values referred to double positive CD107a+ CD146+ cells; CD107a+CD146* f-hAFS:63.68±5.82%,*p=0.016compared to remaing36.32±5.82%f-hAFS (Other);CD107at CD146+p-hAFS:52.07±6.76% with remaining47.93±56.76% p-hAFS (Other);CD107a+CD146+ f-hAFS vs CD107a+ CD146+ p-hAFS p=0.2403,n=4 experiments. Other: total amount of remainingCD107a-CD146~hAFS,CD107a~CD146*hAFS and CD107at CD146-hAFS. All values are expressed as mean ± s.e.m of independent experiments. SA-β-Gal: Senescence-Associated-β-galactosidase.

2.2.Fetal hAFS Show a Different Metabolism from Perinatal hAFS

To evaluate whether the gestational stage may influence mitochondrial metabolism, f-hAFS and p-hAFS were analyzed in standard in vitro culture conditions by biochemical analyses. Evaluation of aerobic metabolism showed that the oxygen consumption rate (OCR) and ATP synthesis were lower in f-hAFS with respect to p-hAFS, both when stimulated with pyruvate + malate (P/M;***p<0.001 for OCR, and *****p<0001, for ATP synthesis), and with succinate(**p<0.01 for OCR and ATP synthesis, Figure 2A). Moreover, f-has displayed a lower oxidative phosphorylation efficiency when compared to p-hAFS, as shown by thep/O values(***p<0.001 for P/M and *****p<0001 for succinate). Values for f-hAFS were lower than those reported in the literature[48], and suggest uncoupling between oxygen consumption and ATP production (Figure 2A). By evaluating the relative contributions of glutamine, long-chain fatty acid oxidation, and glucose in oxidative phosphorylation (OxPhos)metabolism, we noticed that f-hAFS were sensitive to the addition of BPTES (glutaminase inhibitor,** p< 0.01) and etomoxir(carnitine palmitoyl-transferase 1A inhibitor, **p<0.01), but not to UK5099 (mitochondrial pyruvate carrier inhibitor). By contrast,BPTES(*****p<0.0001)and UK5099(****p<0.001),but not etomoxir,inhibited the metabolism of p-hAFS (Figure 2B, upper panel). This observation was confirmed by the inhibition percentage of the single inhibitor (Figure 2B, lower panel). Therefore, both cell types similarly rely on glutamine as a respiratory substrate; yet, f-hAFS prefer fatty acids as a second substrate, while p-hAFS are sustained by glucose. Interestingly, f-hAFS showed a higher increment of glucose consumption and lactate release when compared to p-hAFS (*p<0.05 and***p<0.001 respectively, Figure 2C), which indicates the attempt to balance inefficient aerobic metabolism by lactate fermentation. This difference could also explain the reaction of f-hAFS to the addition of etomoxir and UK5099. Since f-has favor the use of glucose during anaerobic glycolysis(* p<0.05), they are likely forced to use fatty acids and glutamine to supply the aerobic metabolism.

2.3.Hypoxic Preconditioning Does Not Affect Fetal- and Perinatal has Viability and Sustains Their Secretory Activity

In order to define hAFS secretome formulations, cells were cultured in serum-free conditions to avoid any contamination from FBS. We previously showed that 24 h serum-free (SF) and 1% O2 hypoxic culture conditions did not significantly alter the viability of Ⅱtrimester fetal hAFS (f-hope), while they supported the release of regenerative paracrine factors in their cell-conditioned medium (hAFS-CM) and in extracellular vesicles (hAFS. EVs)[34,35,37,49]. Herein, in addition to profiling the p-hAFS secretome fractions for the first time, we evaluated whether p-have presented similar behavior under the same preconditioning regime, using the normoxic culture condition as the control. f-hAFS and p-hAFS viability were analyzed after 24 h in the following settings: normoxic (20% O2) condition in complete control(Ctrl) culture medium(Ctrlf-hAFSnormo and Ctrl p-hAFSnormo), a normoxic condition in SF medium (SF f-hAFSnormo and SF p-hAFSnormo), hypoxic (1% O2) condition in complete control medium (Ctrlf-has hypo and Ctrl p-hAFSnypo), and hypoxic condition in SF medium (SF f-has hypo and SF p-has hypo, Figure 3A). We confirmed that f-has viability was unaltered in both Ctrl and SF conditions and under hypoxic stimulation, with more than 80% (up to almost 88%) of total cells being unaffected. Early and late apoptotic cells ranged from ca. 13% to 18% in SF conditions, without any statistically significant relevance. Likewise, perinatal have viability was in the range of 80-92%, and early and late apoptotic cells represented up to 18% in SF conditions. p-hAFS were marginally influenced only under the combined hypoxic and SF conditions; indeed, while preconditioning did not influence cell survival when p-hAFS were cultured in a complete medium, the corresponding SF condition showed an increase by ca. 4-fold (* p<0.05) of late apoptotic cells(Figure 3B).

Figure 2. Metabolic characterization of fetal- and perinatalhAFS. (A) Oxygen consumption rate (OCR), ATP synthesis through F1-F。ATP synthase, and P/O ratio in f-have and-have in the presence of pyruvate plus malate (P/M) or succinate(Succ);***p=0.0005,**p=0.0012,****p<0.0001,**p=0.0013,****p=0.0002,*****p<0.0001.(B)OCR and ATP synthesis in presence of BPTES, Etomoxir, and UK5099(upper panel) was sequentially added during the experiments to evaluate the relative contributions of glutamine, long-chain fatty acid oxidation and glucose in OxPhos metabolism in f-hAFS and p-hAFS.For OCR experiments: f-hAFS+BPTES**p=0.0014;for f-hAFS + Etomoxir**p=0.0088;for p-hAFS ＋BPTES***p<0.0001;for p-hAFS＋UK5099**p<0.0001. For ATP experiments: f-hAFS + BPTES**** p<0.0001; for f-hAFS＋Etomoxir**p=0.0013;for p-hAFS＋BPTES****p<0.0001;for p-hAFS + UK5099 ** p<0.0001). cistanche Australia The comparison of the percentage of inhibition of OCR and ATP synthesis in f-and-hAFS due to the inhibitors indicated above is reported in the lower panel B. For OCR experiments: hAFS + Etomoxir**** p<0.0001; for hAFS + UK5099 **** p<0.0001. For ATP experiments: hAFS+Etomoxir*****p<0.0001;for hAFS +UK5099 **** p<0.0001).(C) Glucose consumption,lactate release and anaerobic glycolysis yield, used as markers of the anaerobic glycolysis, in f-hAFS and p-hAFS. All values are expressed as mean ± use.m of n=4 independent experiments;*p=0.016,**p=0008,*p=0.0416,respectively.

We then evaluated the yield of secretome fractions obtained from f-hAFS versus p-hAFS based on protein enrichment. The total has secretome, as the entirety of the cell-secreted paracrine factors, is here represented by the hAFS-CM. The protein concentration of f-hAFS-CM and p-hAFS-CM in SF medium following hypoxic cell preconditioning vs control normoxic condition as baseline (namely, f-hAFS-CMnormo, f-hAFS-CMNypo, P has-CMnormo, and p-hAFS-CMHypo,) was evaluated by BCA assay and measured as per 10§ cells. cistanch The results acquired suggested that f-has-CM and p-hAFS-CM showed an equal positive trend in protein enrichment following hypoxic priming (f-hAFS-CMnypo'166.10±22.13 μg/10°cells；p-hAFS-CMNypo∶182.30±29.71 μg/10°cells） over their normoxic counterparts (f-hAFS-CMnormo:105.50±19.89 ug/10° cells; p-hAFS-CMhypoi 91.12±24.39 μg/10° cells）. Likewise， the surface protein concentration of hAFS-EVs was measured in f-have-EVSnormo, f-hAFS-EVShypo, p-hAFS-EVSnormo, and p-hAFS-EVShypo EVs showed comparable yield when obtained from f-hAFS or p-hAFS. As for hAFS-CM formulations, a positive trend in the increase of protein content on f-hAFS-EVs and p-hAFS. EVs were appreciated after hypoxic stimulation over the corresponding normoxic condition （f-hAFS-EVSHypo∶2.03±0.67 ug/10°cells and p-hAFS-EVSHypo∶1.85±0.47 μg/10°cells；f-have-EVsnormo∶1.28±0.36μg/10°cells and p-hAFS-EVsnormo∶1.19±0.31 μg/10°cells， Figure 3C).

2.4.Fetal-and Perinatal hAFS Release EVs with Analogous Morphology and Size Distribution

Morphological analysis by transmission electron microscopy (TEM) heightened the high EV-secretory prolife of both f-hAFS and p-hAFS (Figure 4). We further investigated the size and area of f-hAFS-EVs and p-hAFS-EVs (Figure 4B) following hypoxic preconditioning compared to normoxic baseline. Fetal- and perinatal have released EVs heterogeneous in size, in the range of 40-250 nm, hence including both exosomes/small EVs and microvesicles/shedding vesicles. The average size of EVs/field in the different groups was comparable, fetal hAFS-EVs measured 90-100 nm (f-hAFS-EVsnormo:104.00 ±3.00 nm;f-have-EVSHvpo:97.10±10.10 nm)and perinatal ones measured 70-114 nm (p-hAFS-EVsnormo:94.60±19.53 nm; p-hAFS-EVShypo:76.43±4.86 nm, Figure 4B, left panel). As for the yield, hAFS stimulated under hypoxia showed a positive trend in the increase of the amount of small EVs, although this increase was not statistically significant. f-hAFS-EVStypo measured 40-70 nm which was almost twice that compared to their normoxic counterpart. Perinatal-has-EVSHypo that measured 40-70 nm,70-100 nm and 100-130 nm were almost triple the amount of those obtained in normoxic culture (Figure 4B).

Nanoparticle tracking analysis (NTA)showed an elevated number of particles in both f-hAFS-EV and p-hAFS-EV preparations, and confirmed the increase of EVs in the hypoxic samples, as also observed from the previous analyses (f-hAFS-EVsnormo:182±0.10'particles/10° cells; f-have-EVshypo: 3.30±0.22×10°particles/10°cells;p-hAFS-EVsnormo:2.43±0.80×10°particles/10°cells;p-hAFS-EVshypo:3.05±0.62×10°particles/10°cells, Figure 4C).

Figure 4. Morphological characterization of fetal- and perinatal have-EVs. (A) Representative images of transmission electron microscopy (TEM) of f-hAFS and p-hAFS (upper and lower left panel, respectively, with black arrows indicating intracytoplasmic multi-vesicular bodies with small EVs/exosomes within them), and of f-hAFS-EVs and p-hAFS-EVs (upper and lower right panel, respectively) released in serum-free conditions and under normoxic versus hypoxic preconditioning (f-hAFS-EVSnormo; f-have-EVSHypoi p-hAFS-EVSnormo; and f-hAFS-EVshypo, respectively), scale bars: 200 nm. (B)Left panel: TEM analysis of hAFS-EVs size distribution; right panel: distribution of the number f-hAFS-EVs and p-hAFS-EVs per field size intervals from 40 nm up to 250 nm were considered; values are expressed as mean±s.e.m of n=3 independent experiments. (C)Nanoparticle tracking analysis for hAFS-size and distribution. Left panel: representative image of the graphical output; right panel: hAFS-EVs concentration measured as 10° particles per 10° secreting cells; nm: nanometer; mL: milliliter.

2.5.Proteomic Characterization of Fetal vs. Perinatal hAFS Highlights Differences in Their Secretome Composition According to Gestational Age and Hypoxic Preconditioning

Proteomic characterization of both f-hAFS and p-hAFS secretome formulations was performed by means of a shotgun label-free platform, based on the coupling of nano liquid chromatography and high-resolution mass spectrometry (nLC-HRMS). Forty-eight proteomic profiles were acquired by the duplicate analysis of three biological replicates of hAFS-CM and have-EVs from f-hAFS and p-hAFS undergoing hypoxic cell precondition-ing compared to the normoxic condition as a control. A total of 4179 distinct protein groups were identified with at least one unique peptide and with molecular weights ranging from 2 to 3900 kDa and isoelectric points from 3.6 to 13. A higher average protein expression in hAFS-EVs was observed when compared to hAFS-CM. The alignment of all protein lists obtained was carried out on the basis of identified proteins. For each experimental condition, a unique list was created normalizing and averaging[50] the peptide spectrum match values (PSMs) attributed to the proteins, which represent the number of mass spectra assigned to each and indirectly represent their abundance in the samples. The complete list of proteins identified in hAFS-CM and have-EV formulations is reported in Table S1.

The application of linear discriminant analysis (LDA[51]) on this master list allowed the extraction of statistically significant proteins (Fratio ≥4.5 and** p <0.001)to be processed by hierarchical clustering. Figure SLA shows a clear separation and different behavior between hAFS-CM and have-EV fractions generating two main branches, as highlighted by the heatmap color code. A further subgrouping was also observed according to the gestational age and the hypoxic preconditioning adopted. The fact that each analyzed condition presented a unique identity is confirmed by the Venn diagrams (Figures 5A and 6A, Tables S1-S3) that report the distribution of proteins identified with a frequency >1 in hAFS-CM and have-EV formulations considered separately. While about 69.5% and 69.9% of proteins were shared among hAFS-EVs and hAFS-CM conditions respectively, the remaining content appeared exclusive in different proportions, ranging from 3.7% to 13.4%, among the formulations.

To quantitatively examine the proteomic changes, a label-free differential analysis was performed by using the home-made MAProMa software and applying two algorithms, DAve (Differential Average) and DCI (Differential Confidence Index, representing the ratio and the confidence in differential expression, respectively), on the PSMs of every single protein between the two compared terms. Using stringent filters for DAve and DCI to maximize the confidence of identification and to consider proteins with a variation greater than a fold change of 1.5, pairwise comparisons of f-hAFS-CM versus p-hAFS-CM and of f-hAFS-EVs versus p-hAFS-EVs were made according to cell gestational stage. A total of 58 and 109 proteins were found differentially expressed in the above has-CM and have-EV compartments, respectively, (Figure S1B, C for selected details and Tables S2-S3 in extended form). Among these, 30 proteins resulted up-regulated in f-hAFS-CM and 28 were up-regulated in p-hAFS-CM (Figure S1B); likewise,44 distinct proteins resulted in upregulated in f-have-EVs and 65 were up-regulated in p-hAFS-EVs (Figure S1C). Notably, proteins that resulted in up-regulated in f-have are to be considered down-regulated in p-has and vice versa.

values are reported; see Table S2 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 fetal hAFS-CM (left panel) and perinatal have-CM (right panel) according to cell hypoxic preconditioning. Based on the FunRich tool, gene ontology terms are shown in bar charts reporting the percentage of genes enriched for each category (pink bars for-have-CMnormo, purple bars for f-hAFS-CMhypor light blue bars for p-hAFS-CMnormo, and blue balls for p-hAFS-CMhypo). buy cistanche Only gene ontology terms with Bonferroni corrected with*p<0.05 are reported.

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