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The Protein Content of Extracellular Vesicles Derived from Expanded Human Umbilical Cord Blood-Derived CD133+ and Human Bone Marrow-Derived Mesenchymal Stem Cells Partially Explains Why both Sources are Advantageous for Regenerative Medicine

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Abstract

Adult stem cells have beneficial effects when exposed to damaged tissue due, at least in part, to their paracrine activity, which includes soluble factors and extracellular vesicles (EVs). Given the multiplicity of signals carried by these vesicles through the horizontal transfer of functional molecules, human mesenchymal stem cell (hMSCs) and CD133+ cell-derived EVs have been tested in various disease models and shown to recover damaged tissues. In this study, we profiled the protein content of EVs derived from expanded human CD133+ cells and bone marrow-derived hMSCs with the intention of better understanding the functions performed by these vesicles/cells and delineating the most appropriate use of each EV in future therapeutic procedures. Using LC-MS/MS analysis, we identified 623 proteins for expanded CD133+-EVs and 797 proteins for hMSCs-EVs. Although the EVs from both origins were qualitatively similar, when protein abundance was considered, hMSCs-EVs and CD133+-EVs were different. Gene Ontology (GO) enrichment analysis in CD133+-EVs revealed proteins involved in a variety of angiogenesis-related functions as well proteins related to the cytoskeleton and highly implicated in cell motility and cellular activation. In contrast, when overrepresented proteins in hMSCs-EVs were analyzed, a GO cluster of immune response-related genes involved with immune response-regulating factors acting on phagocytosis and innate immunity was identified. Together our data demonstrate that from the point of view of protein content, expanded CD133+-EVs and hMSCs-EVs are in part similar but also sufficiently different to reflect the main beneficial paracrine effects widely reported in pre-clinical studies using expanded CD133+ cells and/or hBM-MSCs.

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Acknowledgements

We would like to thank all the staff of the Instituto Carlos Chagas (Fiocruz-PR) for the laboratory and administrative support; the staff of the Núcleo de Tecnologia Celular (PUCPR) for tissue collection and the staff of the Electron Microscopy facilities from UFPR and UFSC for image acquisition and help. We thank the Program for Technological Development in Tools for Health-PDTIS-FIOCRUZ for use of its facilities, specifically the flow cytometry core facility and mass spectrometry facility. We thank Paulo Moro for the flow cytometry half off set histogram design. This study was supported by Fundação Araucária (Grant number 1005/2013) and MCTI/CNPq/MS (Grant number 404656/2012-9). We thank Capes (Ministério da Educação) for Addeli B.B. Angulski’s fellowship.

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  1. Instituto Carlos Chagas, Fiocruz-Paraná, Rua Professor Algacyr Munhoz Mader, 3775, Curitiba, PR, 81350-010, Brazil

    Addeli B. B. Angulski, Michel Batista, Bruna H. Marcon, Marco A. Stimamiglio & Alejandro Correa

  2. Núcleo de Tecnologia Celular, Pontifícia Universidade Católica do Paraná, Rua Imaculada Conceição, 1155, Curitiba, PR, 80215-901, Brazil

    Luiz G. Capriglione & Alexandra C. Senegaglia

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  1. Addeli B. B. Angulski
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Figure S1

Immunophenotypic characterization by flow cytometry of hMSCs. Representative histograms of human bone marrow-derived mesenchymal stem cells with immunophenotypes assessed by flow cytometry. Cells were labeled with fluorescent human antibodies against CD34, CD45, CD19, CD11b, HLA-DR, CD31, CD90, CD105 and CD73. Red histograms indicate the percentage of positive cell populations for each antibody, whereas black histograms indicate isotype-control antibodies. (GIF 66 kb)

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Figure S2

Immunophenotypic characterization by flow cytometry of expanded CD133 + cells. Representative histograms of expanded CD133+ cells and immunophenotype assessed by flow cytometry. Cells were labeled with fluorescent antibodies against CD34, CD45, CD14, CD133, CD146, CD31, CD309, CD105 and von Willebrand factor (vWF). Red histograms indicate the percentage of positive cell populations for each antibody, whereas black histograms indicate isotype-control antibodies. (GIF 72 kb)

High Resolution Image (TIFF 1285 kb)

Figure S3

Apoptosis and necrosis assay with hMSCs and expanded CD133 + cells. (A) (B) (C) Dot plots of three different donors of hMSCs labeled with the PE Annexin V apoptosis detection kit with 7-AAD. (D) (E) (F) Dot plots of three different donors of expanded CD133+ cells labeled with the PE Annexin V apoptosis detection kit with 7-AAD. The dot plots in the lower left corner indicate the live cell population, the dot plots on top indicate the necrotic population (Annexin V and 7-AAD positive), and the dot plots in the lower right corner indicate the apoptotic population. (GIF 44 kb)

High Resolution Image (TIFF 1184 kb)

Figure S4

Venn diagrams showing an overlap in proteins identified in the four LC-MS/MS analysis. A total of 467 proteins were identified in the two replicates of CD133+-EVs, and 464 proteins were identified in the two replicates of hMSC-EVs. A total of 427 proteins were identified in all MS runs. (GIF 18 kb)

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Figure S5

Multi-scatter correlation plot of different replicate runs of EVs derived from expanded CD133 + and hMSC cells. Scatter plots demonstrating the degree of correlation between the 12 different replicate runs (6 replicate runs for CD133+-EVs and 6 replicate runs for hMSC-EVs). The number at the top of each graphic represents the correlation (R square) between the conditions. The correlations are uniformly high and vary only between r = 0.780 and 0.804 among the replicates of the same EV sample and are uniformly low, varying only between r = 0.396 and 0.558 among the replicates of different EV samples. Axis values represents the log 2 (x) value. (GIF 54 kb)

High Resolution Image (TIFF 3368 kb)

Figure S6

TreeMap visualization of the summary of exclusive GO Biological process for each type of EV obtained by REVIGO analysis. The size of each box is correlated with the frequencies of the occurrence of the GO term. Boxes with the same color are grouped by semantic similarity. (A) and (B) Exclusive GO Biological process for expanded CD133+-EVs and hMSC-EVs, respectively. (GIF 107 kb)

High Resolution Image (TIFF 946 kb)

Figure S7

Venn diagram showing the intersections of proteins identified in hMSC-EV samples and the study by Kim and coworkers. A total of 450 proteins overlapped between hMSC-EVs and Kim’s study. (GIF 10 kb)

High Resolution Image (TIFF 298 kb)

Figure S8

Comparison of functional enrichment analysis of the hMSC-EV proteome with the study by Kim and coworkers. (A) and (B) Gene ontology enrichment analysis of total proteins identified in hMSC-EVs and Kim and coworkers’ study, showing the most enriched terms for biological processes and molecular function. Pie chart shows some selected significantly enriched categories (p < 0.05). GO analysis was conducted using Funrich software. (GIF 57 kb)

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Angulski, A.B.B., Capriglione, L.G., Batista, M. et al. The Protein Content of Extracellular Vesicles Derived from Expanded Human Umbilical Cord Blood-Derived CD133+ and Human Bone Marrow-Derived Mesenchymal Stem Cells Partially Explains Why both Sources are Advantageous for Regenerative Medicine. Stem Cell Rev and Rep 13, 244–257 (2017). https://doi.org/10.1007/s12015-016-9715-z

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