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Proteomic Analysis of the Secretome and Exosomes of Feline Adipose-Derived Mesenchymal Stem Cells

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Proteomic Analysis of the Secretome and Exosomes of Feline Adipose-Derived Mesenchymal Stem Cells animals Article Proteomic Analysis of the Secretome and Exosomes of Feline Adipose-Derived Mesenchymal Stem Cells Antonio J. Villatoro 1,2,† , María del Carmen Martín-Astorga 1,† , Cristina Alcoholado 1 , María del Mar Sánchez-Martín 1 and José Becerra 1,3,4,* 1 Laboratory of Bioengineering and Tissue Regeneration, Department of Cell Biology, Genetics and Physiology, University of Málaga, IBIMA, 29071 Málaga, Spain; [email protected] (A.J.V.); [email protected] (M.d.C.M.-A.); [email protected] (C.A.); [email protected] (M.d.M.S.-M.) 2 Instituto de Immunología Clínica y Terapia Celular (IMMUNESTEM), 29071 Málaga, Spain 3 Biomedical Research Networking Center in Bioengineering, Biomaterials, and Nanomedicine (CIBER-BBN), 28029 Madrid, Spain 4 Andalusian Centre for Nanomedicine and Biotechnology, University of Málaga (BIONAND), 29590 Málaga, Spain * Correspondence: [email protected]; Tel.: +34-952-131966 † These authors contributed equally to the work. Simple Summary: The enormous advances in stem cell research have generated high expectations in the development of new therapies to repair or regenerate damaged tissues. For this reason, laboratory studies of stem cells enable scientists to learn about cells’ essential properties. Specifically, in recent years, therapies based on mesenchymal stem cells have become an interesting alternative for the treatment of different complex pathologies in veterinary medicine. Mesenchymal stem cells secrete a wide variety of therapeutic elements such as bioactive molecules and extracellular vesicles Citation: Villatoro, A.J.; (e.g., exosomes). Thus, it is essential to characterize them before future use as biotechnological Martín-Astorga, M.d.C.; Alcoholado, products. Therefore, the objective of this study was to determine and compare their protein profile C.; Sánchez-Martín, M.d.M.; Becerra, to understand better the mechanisms of action of these components and facilitate their possible J. Proteomic Analysis of the use in future therapies. The data demonstrate the existence of different proteins responsible for the Secretome and Exosomes of Feline biological effects of cells. In addition, these approaches and techniques can contribute to the better Adipose-Derived Mesenchymal Stem prediction of clinical outcomes of mesenchymal stem cell treatment. Cells. Animals 2021, 11, 295. https:// doi.org/10.3390/ani11020295 Abstract: Mesenchymal stem cells (MSCs) have been shown to have therapeutic efficacy in different complex pathologies in feline species. This effect is attributed to the secretion of a wide variety of Academic Editor: Alessio Soggiu bioactive molecules and extracellular vesicles, such as exosomes, with significant paracrine activity, Received: 4 December 2020 Accepted: 21 January 2021 encompassed under the concept of the secretome. However, at present, the exosomes from feline Published: 24 January 2021 MSCs have not yet been studied in detail. The objective of this study is to analyze and compare the protein profiles of the secretome as a whole and its exosomal fraction from feline adipose-derived Publisher’s Note: MDPI stays neutral MSCs (fAd-MSCs). For this, Gene Ontology (GO), Kyoto Encyclopedia of Genes and Genomes with regard to jurisdictional claims in (KEGG) and Protein–Protein Interaction Networks Functional Enrichment Analysis (STRING) were published maps and institutional affil- utilized. A total of 239 proteins were identified in the secretome, and 228 proteins specific to exosomes iations. were identified, with a total of 133 common proteins. The proteins identified in the secretome were located in the extracellular regions and in the cytoplasm, while the exosomal proteins were located mainly in the membrane, cytoplasm and cytosol. Regarding function, in the secretome, proteins involved in different metabolic pathways, in pathways related to the immune system and the Copyright: © 2021 by the authors. endocrine system and in the processing of proteins in the endoplasmic reticulum predominated. Licensee MDPI, Basel, Switzerland. In contrast, proteins specific to exosomes were predominantly associated with endocytosis, cell This article is an open access article junctions, platelet activation and other cell signaling pathways. The possible future use of the distributed under the terms and secretome, or some of its components, such as exosomes, would provide a non-cell-based therapeutic conditions of the Creative Commons strategy for the treatment of different diseases that would avoid the drawbacks of cell therapy. Attribution (CC BY) license (https:// creativecommons.org/licenses/by/ 4.0/). Animals 2021, 11, 295. https://doi.org/10.3390/ani11020295 https://www.mdpi.com/journal/animals Animals 2021, 11, 295 2 of 15 Keywords: mesenchymal stem cells; feline; exosomes; secretome; ultrahigh-performance liquid chromatography-high-resolution mass spectrometry (UHPLC–HRMS) 1. Introduction In recent years, mesenchymal stem cells (MSCs) therapy has become an interesting alternative in different complex pathologies in feline medicine [1–3]. Feline MSCs were first isolated from different sources such as bone marrow [4], fat [5], peripheral blood [6], and fetal fluids and amniotic membranes [7]. Currently, adipose-derived MSCs are the most used in clinical applications in feline species due to their ease of obtaining, affordability, and abundance [3,5–13]. The therapeutic effect of MSCs is attributed to the secretion of a wide variety of bioac- tive molecules and extracellular vesicles with important paracrine activity, encompassed under the concept of the secretome [14–16]. Among extracellular vesicles, exosomes are of great interest as therapeutic elements. Exosomes are nanovesicles of endocytic origin with a bilipid membrane that transport different types of proteins, lipids, and microRNAs, among other molecules. Their role is influenced by the cellular origin and different phys- iological and pathological conditions, and they play a fundamental role in intercellular communication [17–19]. Future use of the secretome or some of its components, such as exosomes, as biotech- nological products in regenerative medicine, provides the basis for a cell-free therapeutic strategy that avoids some drawbacks of cell therapy [20–22]. They have shown their effi- cacy in a wide variety of pathologies, mainly in the human species [23–26] but studies in veterinary medicine are still scarce [22,27–29]. Therefore, it is essential to characterize the secretome and its components before their possible clinical use. Although exosomes have been characterized in different types of MSCs, both in human species and in some domestic animals [17,30–32], to date, exosomes in MSCs of feline species have not been described in detail. Considering that proteins mediate most intracellular and intercellular activities, ap- proaches in proteomics are of great value to elucidate different cellular functions and possible patterns of cellular communication [33–35]. Therefore, the characterization of the proteomic profile of the MSCs secretome under standard culture conditions, as well as that of MSCs exosomes, will allow a better understanding of the mechanisms of action of these components and facilitate their possible use in future therapies. Ongoing research on feline MSCs confirms that they also have an immunomodulatory potency similar to that most recently defined for human MSCs [2,16]. Since the cat is an interesting preclinical model that naturally suffers from certain human-like pathologies, the translation of its results with these new therapies should be very useful in human medicine [36]. Therefore, the objective of this study was to determine and compare the protein profile of the secretome with that of exosomes contained therein that were previously isolated, quantified and characterized from feline adipose-derived MSCs (fAd-MSCs) under standard culture conditions. 2. Materials and Methods All protocols were approved by the Institutional Committee for the Care and Use of Animals of BIONAND (Centro Andaluz de Nanomedicina y Biotecnología), Malaga, Spain with the project identification code 07/2017. The procedures with animals were performed by veterinarians after obtaining consent from the owners for the transfer of samples. 2.1. Isolation, Expansion and Characterization of fAd-MSCs fAd-MSCs were isolated from subcutaneous fat of the inguinal region of three healthy, European breed female cats, aged between 3 and 6 months; the fat samples were obtained Animals 2021, 11, 295 3 of 15 at the time of ovariectomy. The adipose tissue was digested with collagenase type I (Merck) to separate the adipose fraction from the vascular stroma. The isolated fraction was seeded in Dulbecco’s modified Eagle’s medium (DMEM) supplemented with 10% fetal bovine serum (FBS), 2.5 mM L-glutamine, 100 U/mL penicillin, 100 µg/mL streptomycin, and 1.25 µg/mL fungizone (Merck) in T-175 flasks. The fAd-MSCs were maintained under standard culture conditions until reaching a confluence of 80%, at which time they were subcultured. All experiments were performed in cells of culture passage 2. fAd-MSCs were characterized by flow cytometry and the multipotential differentiation capacity into adipogenic, osteogenic and chondrogenic lineages, according to the criteria of the International Society of Cellular Therapy (ISCT) [37,38]. The detailed procedure was published previously by our group [3]. 2.2. Production of the fAd-MSC Secretome Each donor was processed individually. fAd-MSCs (5 × 105) were seeded in DMEM supplemented with 10%
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