International Journal of Molecular Sciences Article Comparative Proteomic Analysis of the Mesenchymal Stem Cells Secretome from Adipose, Bone Marrow, Placenta and Wharton’s Jelly Sungho Shin 1,2,†, Jeongmin Lee 3,4,† , Yumi Kwon 1, Kang-Sik Park 2,5, Jae-Hoon Jeong 6, Suk-Joo Choi 7, Sa Ik Bang 8, Jong Wook Chang 3,4,* and Cheolju Lee 1,2,9,* 1 Center for Theragnosis, Korea Institute of Science and Technology, Seoul 02792, Korea; [email protected] (S.S.); [email protected] (Y.K.) 2 KHU-KIST Department of Converging Science and Technology, Kyung Hee University, Seoul 02447, Korea; [email protected] 3 Stem Cell & Regenerative Medicine Institute, Samsung Medical Center, Seoul 06351, Korea; [email protected] 4 R&D Center, ENCell Co., Ltd., Seoul 06351, Korea 5 Department of Physiology, School of Medicine, Kyung Hee University, Seoul 02447, Korea 6 Division of Radiation Cancer Research, Korea Institute of Radiological and Medical Sciences, Seoul 01812, Korea; [email protected] 7 Department of Obstetrics and Gynecology, Samsung Medical Center, Sungkyunkwan University School of Medicine, Seoul 06351, Korea; [email protected] 8 Department of Plastic Surgery, Samsung Medical Center, Sungkyunkwan University School of Medicine, Seoul 06351, Korea; [email protected] 9 Division of Bio-Medical Science & Technology, KIST School, Korea University of Science and Technology, Seoul 02792, Korea Citation: Shin, S.; Lee, J.; Kwon, Y.; * Correspondence: [email protected] (J.W.C.); [email protected] (C.L.) Park, K.-S.; Jeong, J.-H.; Choi, S.-J.; † These authors contributed equally to this work. Bang, S.I.; Chang, J.W.; Lee, C. Comparative Proteomic Analysis of Abstract: Mesenchymal stem cells (MSCs) have the potential to be a viable therapy against vari- the Mesenchymal Stem Cells ous diseases due to their paracrine effects, such as secretion of immunomodulatory, trophic and Secretome from Adipose, Bone protective factors. These cells are known to be distributed within various organs and tissues. Al- Marrow, Placenta and Wharton’s Jelly. though they possess the same characteristics, MSCs from different sources are believed to have Int. J. Mol. Sci. 2021, 22, 845. different secretion potentials and patterns, which may influence their therapeutic effects in disease https://doi.org/10.3390/ijms22020845 environments. We characterized the protein secretome of adipose (AD), bone marrow (BM), placenta Academic Editor: (PL), and Wharton’s jelly (WJ)-derived human MSCs by using conditioned media and analyzing the Bogusław Machali´nski secretome by mass spectrometry and follow-up bioinformatics. Each MSC secretome profile had Received: 28 December 2020 distinct characteristics depending on the source. However, the functional analyses of the secretome Accepted: 13 January 2021 from different sources showed that they share similar characteristics, such as cell migration and Published: 15 January 2021 negative regulation of programmed cell death, even though differences in the composition of the secretome exist. This study shows that the secretome of fetal-derived MSCs, such as PL and WJ, Publisher’s Note: MDPI stays neutral had a more diverse composition than that of AD and BM-derived MSCs, and it was assumed that with regard to jurisdictional claims in their therapeutic potential was greater because of these properties. published maps and institutional affil- iations. Keywords: mesenchymal stem cells; secretome; adipose; bone marrow; placenta; Wharton’s jelly; mass spectrometry Copyright: © 2021 by the authors. Licensee MDPI, Basel, Switzerland. 1. Introduction This article is an open access article In early studies, mesenchymal stem cells (MSCs) [1] were first associated with the distributed under the terms and potential for differentiation into specific cells [2–6]. While many studies have described conditions of the Creative Commons Attribution (CC BY) license (https:// the self-renewal and differentiation properties of MSCs as progenitors, recent studies are creativecommons.org/licenses/by/ more focused on the non-progenitor functions of MSCs, such as immune modulation 4.0/). and the secretion of trophic and protective factors (paracrine actions) [7,8]. Based on the Int. J. Mol. Sci. 2021, 22, 845. https://doi.org/10.3390/ijms22020845 https://www.mdpi.com/journal/ijms Int. J. Mol. Sci. 2021, 22, x FOR PEER REVIEW 2 of 17 Int. J. Mol. Sci. 2021, 22, 845 2 of 16 the secretion of trophic and protective factors (paracrine actions) [7,8]. Based on the clini- cal safety of the immunomodulatory feature, the applications of MSCs in clinical trials are clinicaldiverse: safety bone of and the cartilage immunomodulatory diseases, neurological feature, the diseases, applications cardiovascular of MSCs in diseases, clinical trialsgraft- areversus diverse:-host bonedisease and (GVHD), cartilage diabetes, diseases, hematological neurological diseases, diseases, cardiovascular inflammatory diseases,diseases, graft-versus-hostand diseases in liver disease and (GVHD), kidneys diabetes,[9,10]. hematological diseases, inflammatory diseases, and diseasesOne of inthe liver unique and characteristics kidneys [9,10]. of MSCs is that they can be isolated from various originsOne [11,12]. of the MSCs unique are characteristics known to exist of in MSCs numerous is that perivascular they can be tissues isolated (including from various bone originsmarrow [11 (BM),,12]. MSCsadipose are tissue known (AD), to exist peripheral in numerous blood, perivascularteeth, skeleta tissuesl muscle, (including placenta bone (PL), marrowumbilical (BM), cord, adipose amniotic tissue fluid (AD), and peripheral cord blood), blood, while teeth, other skeletal stem muscle, cells are placenta distributed (PL), umbilicalmainly in cord,the tissues amniotic of their fluid defined and cord lineage. blood), Many while comparison other stem studies cells of are source distributed-specific mainlyMSCs [13 in the–16 tissues] have shown of their that defined these lineage. cells share Many common comparison characteristics, studies of source-specificand these have MSCsbeen proposed [13–16] have by the shown International that these Society cells share for Cell common Therapy characteristics, [17]. and these have been proposedThe expression by the of International genes and proteins Society forare Cellknown Therapy to have [17 an]. important role in tissue specificityThe expression [18,19]. Thus, of genes the origin and proteins of MSCs are may known contribute to have to an the important protein expression role in tissue, in- specificitycluding those [18, 19found]. Thus, in the the secretome. origin of In MSCs this study, may contribute we compared to the the protein secreted expression, proteins in includingthe culture those media found of human in the secretome.MSCs derived In this from study, four we different compared sources: the secreted AD, BM, proteins PL, and in the culture media of human MSCs derived from four different sources: AD, BM, PL, Wharton’s jelly (WJ) (Figure 1). For this purpose, we used label-free quantification (LFQ) and Wharton’s jelly (WJ) (Figure1). For this purpose, we used label-free quantification mass spectrometry (MS) because it allows the relative comparison of proteomes between (LFQ) mass spectrometry (MS) because it allows the relative comparison of proteomes samples, showing the proteins that are released more often from certain cell types [20]. between samples, showing the proteins that are released more often from certain cell Our results confirmed a significant difference in the secretome profile between MSCs de- types [20]. Our results confirmed a significant difference in the secretome profile between pending on each source. However, the functional analyses of the secretome from different MSCs depending on each source. However, the functional analyses of the secretome from MSC sources were similar despite variation in the composition of the secretome. different MSC sources were similar despite variation in the composition of the secretome. FigureFigure 1. 1. Analysis of the the secretome secretome from from mesenchymal mesenchymal stem stem cells cells (MSCs). (MSCs). MSCs MSCs isolated isolated from from adiposeadipose tissuetissue ((nn == 3), placenta ( (nn == 3) 3) and and Wharton’s Wharton’s jelly jelly (n (=n 3)= were 3) were incubated incubated in serum in serum-free-free conditioned media for 24 h. The conditioned media were collected, concentrated and analyzed by conditioned media for 24 h. The conditioned media were collected, concentrated and analyzed LC–MS/MS. by LC–MS/MS. Int. J. Mol. Sci. 2021, 22, 845 3 of 16 Int. J. Mol. Sci. 2021, 22, x FOR PEER REVIEW 3 of 17 2. Results 2.1. Characterization2. Results of MSCs from Different Sources 2.1. Characterization of MSCs from Different Sources Human MSCs that were isolated from four different sources (AD, BM, PL, and WJ) Human MSCs that were isolated from four different sources (AD, BM, PL, and WJ) adhered to plastic cell culture plates and exhibited a spindle-shape (Figure2A). The MSCs adhered to plastic cell culture plates and exhibited a spindle-shape (Figure 2a). The MSCs revealedrevealed the the potential potential to to differentiate differentiate into a a mesodermal mesodermal lineage. lineage. Mineralization Mineralization of the of the extracellularextracellular matrix matrix and and accumulation accumulation of of lipid lipid-rich-rich vacuoles vacuoles inside inside the cells the cellswere weredetected detected as a resultas a result of osteogenesis of osteogenesis and and adipogenesis.