First Characterization of Human Amniotic Fluid Stem Cell Extracellular Vesicles as a Powerful Paracrine Tool Endowed with Regenerative Potential

Carolina Balbi, Martina Piccoli, Lucio Barile, Andrea Papait, Andrea Armirotti, Elisa Principi, Daniele Reverberi, Luisa Pascucci, Pamela Becherini, Luigi Varesio, Massimo Mogni, Domenico Coviello, Tiziano Bandiera, Michela Pozzobon, Ranieri Cancedda, Sveva Bollini, Carolina Balbi, Martina Piccoli, Lucio Barile, Andrea Papait, Andrea Armirotti, Elisa Principi, Daniele Reverberi, Luisa Pascucci, Pamela Becherini, Luigi Varesio, Massimo Mogni, Domenico Coviello, Tiziano Bandiera, Michela Pozzobon, Ranieri Cancedda, Sveva Bollini

Abstract

Human amniotic fluid stem cells (hAFS) have shown a distinct secretory profile and significant regenerative potential in several preclinical models of disease. Nevertheless, little is known about the detailed characterization of their secretome. Herein we show for the first time that hAFS actively release extracellular vesicles (EV) endowed with significant paracrine potential and regenerative effect. c-KIT+ hAFS were isolated from leftover samples of amniotic fluid from prenatal screening and stimulated to enhance EV release (24 hours 20% O2 versus 1% O2 preconditioning). The capacity of the c-KIT+ hAFS-derived EV (hAFS-EV) to induce proliferation, survival, immunomodulation, and angiogenesis were investigated in vitro and in vivo. The hAFS-EV regenerative potential was also assessed in a model of skeletal muscle atrophy (HSA-Cre, SmnF7/F7 mice), in which mouse AFS transplantation was previously shown to enhance muscle strength and survival. hAFS secreted EV ranged from 50 up to 1,000 nm in size. In vitro analysis defined their role as biological mediators of regenerative, paracrine effects while their modulatory role in decreasing skeletal muscle inflammation in vivo was shown for the first time. Hypoxic preconditioning significantly induced the enrichment of exosomes endowed with regenerative microRNAs within the hAFS-EV. In conclusion, this is the first study showing that c-KIT+ hAFS dynamically release EV endowed with remarkable paracrine potential, thus representing an appealing tool for future regenerative therapy. Stem Cells Translational Medicine 2017;6:1340-1355.

Keywords: Apoptosis; Exosomes; Extracellular vesicles; Fetal stem cells; Paracrine communication; Proliferation; Tissue regeneration; miRNA.

© 2017 The Authors Stem Cells Translational Medicine published by Wiley Periodicals, Inc. on behalf of AlphaMed Press.

Figures

Figure 1
Figure 1
Hypoxic preconditioning does not significantly affect hAFS stemness and phenotype. (A): Bright field, SSEA4 and NANOG immunostaining images of hAFS under: (a), (a′), and (a′′) 24 hours 20% O2 CTRL conditions; (b), (b′), and (b′′) 24 hours 20% O2 SF conditions; (c), (c′), and (c′′) 24 hours 1% O2 preconditioning in CTRL medium; (d), (d′), and (d′′) 24 hours 1% O2 preconditioning in SF condition; scale bar 50 µm. (B): Western Blot for HIF‐1α expression by hAFS after 24 hours of 20% O2 or 1% O2 preconditioning in complete (CTRL) or SF medium. (C): SSEA4+ hAFS under: (a) 24 hours 20% O2 CTRL condition: 87.8% ± 2.8%; (b) 24 hours 20%O2 SF condition: 89.9% ± 1.5%; (c) 24 hours 1% O2 preconditioning in CTRL medium: 83.0% ± 0.7%; and (d) 24 hours 1% O2 preconditioning in SF condition: 84.1% ± 2.2%; values expressed as mean ± SEM. (D): Real time qRT‐PCR and qualitative RT‐PCR analysis for NANOG and GAPDH expression of hAFS under 24 hours: (a) 20% O2 CTRL and (b) 20% O2 SF conditions, (c) 1% O2 CTRL and (d) 1% O2 SF conditions; +ve: positive control, human embryonic stem cells (****, p < .0001); −ve: negative control: HDF; n.d. (E): hAFS immunophenotype under 24 hours 20% O2 in CTRL (a), or SF conditions (b), or under 24 hours 1% O2 preconditioning, in either CTRL (c), or SF conditions (d). (F): Apoptosis analysis by Annexin V and PI of hAFS under 24 hours: (a) 20% O2 CTRL and (b) 20% O2 SF conditions, (c) 1% O2 CTRL and (d) 1% O2 SF conditions. Abbreviations: CD, cluster of differentiation; hAFS, human amniotic fluid stem cells; HIF‐1α, hypoxia inducible factor‐1 alpha; HLA, human leucocyte antigen; n.d., not determined; PI, propidium iodide; SF, serum‐free.
Figure 2
Figure 2
hAFS secrete EV containing microvesicles and exosomes. (A): Transmission electron microscopy (TEM) analysis of hAFS under 24 hours 20% O2 CTRL (a) or 20% O2 SF conditions (b) and 1% O2 CTRL (c) or 1% O2 SF (d) conditions; scale bars 500 nm. (B): TEM Analysis of 1% O2 hAFS‐EV (hAFS‐EVHypo); scale bar 500 nm in (d′); 200 nm in (d′′); and 100 nm (d′′′ and d′′′′). (C): Nanosight analysis measuring the amount of particles within hAFS‐EVNormo (283.3 ± 64.8 × 106 particle/ml) and hAFS‐EVHypo (283.8 ± 79.9 × 106 particle/ml) released by 106 cells (right panel); values are expressed as mean ± SEM. Representative images of the graphical output are reported in the left panel. (D): BCA assay of the protein concentration of hAFS‐EVHypo (4.2 ± 0.4 μg/106 cells, ***, p < .001, p = .0005) over hAFS‐EVNormo (2.4 ± 0.3 μg/106 cells) released by 106 cells. (E): Western Blot analysis of TSG101, ALIX, GRP94, and βACTIN by hAFS and hAFS‐EV, both under 20% O2 and 1% O2 SF conditions. (F): FACS analysis of hAFS‐EVNormo and hAFS‐EVHypo bound to the ExoCapTM Capture Beads compared to control empty beads (CTRL BEADS) for the exosomal markers CD81 (*, p < .05, p = .049; **, p < .01, p = .005; ***, p < .001, p = .0004), CD9 (*p < .05, p = .047; **, p < .01, p = .0015; ***, p < .001, p = .0002), and CD63 (*, p < .05, p = .02; **, p < .01, p = .004; ***, p < .001, p = .0002). Representative dot plots are illustrated in the left panel, the evaluation of the MFI ratio on the right panels. Abbreviations: CD, cluster of differentiation; EV, extracellular vesicles; hAFS, human amniotic fluid stem cells; Hypo, hypoxic; MFI, mean fluorescent intensity; Normo, normoxic; SF, serum‐free.
Figure 3
Figure 3
hAFS‐EV mediate paracrine effects on target cells. (A): PKH67+ hAFS‐EV uptake by HDF, C2C12, and hPBMC by FACS (values normalized to mode on the y‐axis) and immunostaining (scale bar: 20 µm). PKH67+ cells after treatment with hAFS‐EVNormo and hAFS‐EVHypo were: 65.4% ± 4.4% and 63.5% ± 5.6% HDF (**, p < .01, p = .0033 and p = .0036, respectively); 75.9% ± 3.4% and 83.7% ± 1.1% C2C12 (****, p < .0001); 9.3% ± 2.6% and 12.5% ± 2.5% hPBMC (*, p < .05, p = .017). (B): BrdU Enzyme‐Linked ImmunoSorbent Assay (ELISA) on HDF treated with 1 µg/104, 2 µg/104, and 4 µg/104 cells of hAFS‐EVNormo and hAFS‐EVHypo versus untreated cells (CTRL); ****, p < .0001; #, p referring to hAFS‐EVHypo versus hAFS‐EVNormo: #, p < .05 (p = .033); ##, p < .01 (p = .0077); ###, p < .001 (p = .0002). Values expressed as the fold change of sample absorbance read at 370 nm (reference wavelength approximately 492 nm). (C): MTT assay on C2C12 primed with 1 µg/104, 2 µg/104, and 4 µg/104 cells of hAFS‐EVNormo or hAFS‐EVHypo and exposed to oxidative stress versus untreated cells (CTRL) or damaged cells without hAFS‐EV stimulation (H2O2). ****, p < .0001; #, p referring to hAFS‐EVHypo versus hAFS‐EVNormo: ####, p < .0001. Values expressed as the fold change of the sample absorbance read at 560 and 670 nm. (D): hPBMC proliferation under unstimulated (Naive) and activating (PHA or PWM) conditions with 1 μg/105 cells of hAFS‐EVNormo and hAFS‐EVHypo versus untreated cells (CTRL hPBMC); CPS. (E): FACS analysis of mature CD19+CD27+ B cells treated with 1 μg/105 cells of hAFS‐EVNormo or hAFS‐EVHypo. Naive hPBMC: 3.48% ± 0.86%; CTRL hPBMC + PWM: 8.48% ± 2.63%; hPBMC + PWM + hAFS‐EVNormo: 2.32% ± 0.96%; hPBMC + PWM + hAFS‐EVHypo: 2.36% ± 0.62%. *, p < .05 (p = .045 and p = .047). Abbreviations: CD, cluster of differentiation; CPS, counts per second; DAPI, 4′,6‐diamidino‐2‐phenylindole; EV, extracellular vesicles; hAFS, human amniotic fluid stem cells; HDF, human dermal fibroblast; hPBMC, human peripheral blood mononuclear cell; Hypo, hypoxic; MVB, multivesicular body; Normo, normoxic; PHA, phytohemagglutinin; PWM, pokeweed mitogen; SF, serum‐free.
Figure 4
Figure 4
hAFS‐EV exert a certain degree of angiogenic potential in a mouse Matrigel plug assay. (A): Representative images of Matrigel plugs loaded with 10 μg of hAFS‐EVNormo or hAFS‐EVHypo and recovered after 3 weeks from C57Bl/6J mice; scale bar 5 mm. (B): Evaluation of blood hemoglobin content on the Matrigel plugs loaded with 10 μg of hAFS‐EVNormo or hAFS‐EVHypo versus the PBS control *, p < .05 (p = .04). (C): Real time qRT‐PCR analysis on the recovered plugs for Cdh5, VegfA, and Pecam1 expression as normalized to B2M expression. (D): Representative images of histological evaluation of vessel formation within the Matrigel plug by infiltrating host cells via hematoxylin and eosin and CD31 staining, scale bar: 50 µm. CTRL: vehicle loaded (PBS) Matrigel plug; hAFS‐EVNormo: hAFS‐EVNormo loaded Matrigel plug; hAFS‐EVHypo: hAFS‐EVHypo loaded Matrigel plug. Abbreviations: CD, cluster of differentiation; EV, extracellular vesicles; hAFS, human amniotic fluid stem cells; Hypo, hypoxic; Normo, normoxic.
Figure 5
Figure 5
hAFS‐EV decrease muscle inflammation in a mouse model of muscle atrophy. (A): In vivo procedure. (B): IgG (red), CD68 (red), and LAMININ (green) expression in the Tibialis Anterior muscle of untreated (CTRL) and 1 μg hAFS‐EVNormo‐ and hAFS‐EVHypo‐treated mice at 1 and 7 days post injection (p.i.); scale bar: 100 μm and 50 μm, respectively. (C): IgG‐infiltrated fibers at 1 and 7 days p.i. (CTRL 1 Day: 14.9% ± 3.9%; hAFS‐EVNormo 1 Day: 2.9% ± 1.4%; hAFS‐EVHypo 1 Day: 1.6% ± 0.7%; *, p < .05 p = .031 and p = .016; CTRL 7 Days: 16.1% ± 4.3%; hAFS‐EVNormo 7 Days: 3.8% ± 1.0%; hAFS‐EVHypo 7 Days: 2.0% ± 0.7%; *, p < .05, p = .035) and corresponding mean fluorescent area (CTRL 1 Day: 8.33% ± 3.0%; hAFS‐EVNormo 1 Day: 0.71% ± 0.11%; hAFS‐EVHypo 1 Day: 0.42% ± 0.1%; **, p < .01, p = .002; CTRL 7 Days: 8.90% ± 3.2%; hAFS‐EVNormo 7 Days: 1.64% ± 0.44%; hAFS‐EVHypo 7 Days: 0.80% ± 0.15%; **, p < .01, p = .006 and p = .003). (D): Central nucleated fibers at 1 and 7 days p.i.: CTRL 1 Day: 9.4% ± 1.6%; hAFS‐EVNormo 1 Day: 4.0% ± 1.3%; hAFS‐EVHypo 1 Day: 2.6% ± 1.3%, *, p < .05, p = .015; CTRL 7 Days: 9.8% ± 1.7%; hAFS‐EVNormo 7 Days: 13.1% ± 3.5%; hAFS‐EVHypo 7 Days: 3.7% ± 0.9%; #, p < .05, p = .033, as hAFS‐EVHypo versus hAFS‐EVNormo. (E): Ratio of Nos2 over Arg2 by real time qRT‐PCR; *, p < .05, p = .045 and p = .036 for hAFS‐EVNormo and hAFS‐EVHypo;. A.U. (F): Real time qRT‐PCR for Il‐1α (***, p < .001, p = .0002 and p = .001; *, p < .05, p = .046 and p = .0163, for hAFS‐EVNormo and hAFS‐EVHypo) and Il‐10 (****, p < .0001, **, p < .01, p = .004). Abbreviations: A.U. arbitrary units; CD, cluster of differentiation; DAPI, 4′,6‐diamidino‐2‐phenylindole; EV, extracellular vesicles; hAFS, human amniotic fluid stem cells; Hypo, hypoxic; Normo, normoxic; SF, serum‐free.
Figure 6
Figure 6
hAFS‐EV act as biological carrier of regenerative miRNAs which are significantly increased in the target cells following direct uptake. (A): Analysis of hAFS‐EV small non‐coding RNA content by Agilent technology. (B): Real time qRT‐PCR of hAFS‐EVHypo versus hAFS‐EVNormo showing significant enrichment of the following regenerative miRNAs: miR‐223 (***, p < .001, p = .0001), miR‐146a (**, p < .01, p = .009), miR‐let7c (***, p < .001, p = .0002), miR‐21 (***, p < .001, p = .0003), miR‐199a‐3p (**, p < .01, p = .002), miR‐126, miR‐146b, and miR‐210 (****, p < .0001). (C): Uptake analysis of hAFS‐EV fluorescently labeled RNA by HDF (upper panel) and C2C12 (lower panel) via FACS (on the left representative histogram graphs with corresponding values in the graphs on the right). 88.67% ± 4.21% and 86.73% ± 4.46% of HDF (****, p < .0001) and 91.5% ± 3.38% and 85.97% ± 5.51% of C2C12 cells (****, p < .0001) became fluorescent following incubation with 1 µg/104 cells of SYTO®RNASelect+ hAFS‐EVNormo and hAFS‐EVHypo, showing direct uptake of the marked RNA cargo. (D): Real time qRT‐PCR analysis for the enrichment of specific hAFS‐EV‐delivered miRNAs in the target proliferative HDF: miRNA‐146a, **, p < .01, p = .006; miRNA‐126 and miR‐199a‐3p (*, p < .05, p = .03), miR‐210 (*, p < .05, p = .04). (E): Real time qRT‐PCR analysis for the enrichment of specific hAFS‐EV‐delivered miRNAs in the target C2C12 with or without H2O2 treatment: miR‐let7c (**, p < .01, p = .005 and *, p < .05, p = .04); miR‐21 (**, p < .01, p = .002 and p = .001, respectively); miRNA‐146b (*, p < .05, p = .020 and p = .012); miRNA‐199a‐3p (*, p < .05, p = .013 and **, p < .01 p = .008); and miRNA‐210 (**, p < .01, p = .006 and *, p < .05 p = .02). Gene expression was normalized to human GAPDH and to mouse B2M as internal controls in the HDF and C2C12 cells, respectively. Abbreviations: EV, extracellular vesicles; FITC, fluorescein isothiocyanate; hAFS, human amniotic fluid stem cells; HDF, human dermal fibroblast; Hypo, hypoxic; Normo, normoxic.

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