Therapeutic potentials of mesenchymal stem cells (MSCs) depend largely on their ability to secrete cytokines or factors that modulate immune response, enhance cell survival, and induce neovascularization in the target tissues. We studied the secretome profile of gestational tissue-derived MSCs and their effects on functions of endothelial progenitor cells (EPCs), another angiogenic cell type that plays an important role during the neovascularization. MSCs derived from placental tissues (PL-MSCs) significantly enhanced EPC migration while BM-MSCs, which are the standard source of MSCs for various clinical applications, did not. By using protein fractionation and mass spectrometry analysis, we identified several novel candidates for EPC migration enhancing factor in PL-MSCs secretome that could be used to enhance neovascularization in the injured/ischemic tissues. We recommend that the strategy developed in our study could be used to systematically identify therapeutically useful molecules in the secretomes of other MSC sources for the clinical applications.
Mesenchymal stem cells (MSCs) are multipotent stem/progenitor cells which can differentiate to several mesodermal derivatives and possess an ability to secrete factors involved in neovascularization and immunomodulation [
For several decades, neovascularization is believed to be accomplished by proliferation of mature endothelial cells residing in the local vessels through the process of angiogenesis [
Apart from BM-MSCs which require highly invasive procedure for their isolation, MSCs can be easily obtained in large quantity from several gestational tissues using noninvasive procedure [
The present study aimed to investigate the effect of factors released from placental-derived MSCs (PL-MSCs) on EPC migration, which is a critical step of vasculogenesis, using an
This study was approved by the Siriraj Institutional Review Board, Faculty of Medicine Siriraj Hospital, Mahidol University, which was in accordance with the Declaration of Helsinki, the Belmont Report, CIOMS Guidelines, and ICH-GCP. Placental tissues and umbilical cord blood were obtained from healthy newborns after receiving signed informed consents from their mothers. Placental tissues were minced into small pieces and incubated with 0.25% trypsin-EDTA at 37°C for 30 minutes in shaking water bath. After incubation, the digested tissues were plated into 25 cm2 tissue culture flask containing complete medium (Dulbecco’s Modified Eagle’s Medium (DMEM; GIBCO, Invitrogen Corporation, USA) supplemented with 10% (v/v) Fetal Bovine Serum (FBS; Lonza, USA), 100 U/mL penicillin (General Drug House Co., Ltd., Thailand), and 100
Bone marrow samples (
The 3rd–5th passages of MSCs were harvested by trypsinization and incubated with 10
For adipogenic differentiation, 5 × 104 MSCs (3rd–5th passages) were cultured in NH AdipoDiff Medium (Miltenyi Biotec, Germany). The medium was replaced every 3 days according to the manufacturer’s instructions. After culture for 4 weeks, cells were stained with 0.5% (w/v) Oil Red O (Sigma Aldrich, USA) in isopropanol for 20 minutes at room temperature and were observed under phase-contrast microscope (Olympus, Japan). For osteogenic differentiation, 5 × 104 MSCs (3rd–5th passages) were cultured in NH OsteoDiff Medium (Miltenyi Biotec, Germany). The medium was replaced every 3 days according to the manufacturer’s instructions. After culture for 3 weeks, cells were stained with 40 mM Alizarin Red S (Sigma Aldrich, USA) for 20 minutes at room temperature and were observed under phase-contrast microscope (Olympus, Japan).
Twenty milliliters of heparinized umbilical cord blood was collected for EPC isolation. Mononuclear cell populations from umbilical cord blood were isolated using IsoPrep (Robbins Scientific Corporation, USA) density gradient centrifugation, washed twice with Phosphate Buffer Saline (PBS; GIBCO, Invitrogen Corporation, USA), resuspended in endothelial cell growth medium (endothelial basal medium-2 (LONZA, Germany), supplemented with EGM-2 single aliquots (LONZA, Germany) containing 2% (v/v) FBS, 5
Cells were characterized for EPC surface markers by incubating with the following mouse anti-human antibodies: anti-CD34-FITC (R&D Systems, USA), anti-VEGFR2-PE (R&D Systems, USA), anti-CD31-PE (BioLegend, USA), and anti-vWF-FITC (R&D Systems, USA) for 15 minutes at 4°C in the dark. After incubation, cells were washed twice with PBS and fixed with 2% (v/v) paraformaldehyde in PBS. Flow cytometry was performed using FACSCalibur flow cytometer (Becton Dickinson, USA) and CellQuest software. Cells labeled with FITC-conjugated mouse IgG1 (eBioscience, USA) and PE-conjugated mouse IgG1 (eBioscience, USA) served as negative controls.
To further examine the characteristics of EPCs, an
7 × 105 PL-MSCs (3rd–5th passages) were plated into 75 cm2 flask containing complete medium and incubated in a humidified atmosphere containing 5% CO2 for 24 hours. After incubation, cells were washed twice with 5 mL sterile PBS and incubated with 15 mL serum-free medium (SFM) (DMEM supplemented with 100 U/mL penicillin and 100
The 100 kDa fraction of PL-MSCs conditioned medium was further fractionated into several subfractions according to the hydrophobicity of their protein composition by reverse-phase chromatography. Firstly, Sep-Pak C18 Vac cartridge (Waters Associates, USA) was washed twice with 35 mL 100% acetonitrile (ACN) followed by equilibration with 35 mL 0.1% trifluoroacetic acid (TFA). The solutions were allowed to drain at the rate of 1 mL/min by connecting to Perista pump AC2110 (ATTO, Japan). After equilibration, the 100 kDa fraction was transferred into the preequilibrated Sep-Pak C18 cartridge followed by the addition of 35 mL 0.1% TFA. The fluid was allowed to drain and the hydrophobic proteins presented in the 100 kDa fraction were sequentially eluted from the column by increasing ACN concentration from 10% to 100%. The eluted solutions were collected and separated into distinct subfractions and the amount of proteins presented in each subfraction were determined by measuring an absorbance at 280 nm and 220 nm by nanodrop 2000 spectrophotometer (Thermo Scientific, USA). Finally, acetonitrile and TFA remaining in each subfraction were eliminated by vacuum centrifugation.
To investigate the paracrine effect of BM-MSCs and PL-MSCs on EPC migration, EPCs were cocultured with MSCs through 8
The effect of fractionated PL-MSCs conditioned medium on EPC migration was studied using an
Mass spectrometry analysis was performed at the National Center for Genetic Engineering and Biotechnology (BIOTEC), Thailand. The proteins presented in each PL-MSCs conditioned medium fraction were digested by incubation with trypsin and analyzed by ESI ion trap mass spectrometry. Identification and quantification of each protein was determined by DeCyder MS differential analysis software 2.0 (GE Healthcare, USA) and MASCOT search engine software (Matrix Science, UK) based on NCBInr human protein databases. The identified proteins were then categorized into nonsecretory, classical secretory, and nonclassical secretory proteins by SignalP and SecretomeP software (Center for Biological Sequence Analysis (CBS), Denmark). Finally, the secretory proteins were further categorized by PANTHER and UniProt software into separated groups according to their functions.
Data were presented as mean ± standard error of the mean (SEM). The Mann-Whitney test and nonparametric Kruskal-Wallis test were used to assess the significance of differences between observed data.
Placenta-derived mesenchymal stem cells (PL-MSCs) and bone-marrow-derived mesenchymal stem cells (BM-MSCs) established in this study displayed fibroblast-like morphology (Figure
Characteristics of PL-MSCs and BM-MSCs. (a) Fibroblast-like morphology of PL-MSCs and BM-MSCs (scale bar = 500
Characteristic of endothelial progenitor cells (EPCs). (a) Cobblestone-like morphology of EPC colony appeared after 7 days of culture (scale bar = 500
Effects of soluble factors released from BM-MSCs and PL-MSCs on EPC migration. (a) Diagram of the transwell culture system for an
To investigate the effect of MSC-derived soluble factors on EPC migration, EPCs were cocultured with BM-MSCs and PL-MSCs using the transwell culture system (Figure
To identify PL-MSC-derived factors which are able to induce EPC migration, PL-MSCs conditioned medium was fractionated into 5 distinct fractions according to the molecular weight of their protein compositions. The effect of each PL-MSCs conditioned medium fraction on EPC migration was then determined by
Effect of PL-MSCs conditioned medium fractions on EPC migration. (a) Diagram of the transwell culture system for an
To identify EPC migratory enhancing factors presented in the 100 kDa of PL-MSCs conditioned medium, proteins presented in the 100 kDa fraction of PL-MSCs conditioned medium were further fractionated into 11 subfractions according to their hydrophobicity (see Supplementary Figure 1 of the Supplementary Material available online at
Effect of the 100 kDa subfractions on EPC migration. (a) Hematoxylin stained EPCs which migrated to the other side of membrane in response to soluble factors presented in 11 distinct subfractions (PF) of the 100 kDa fraction of PL-MSCs conditioned medium (scale bar = 200
EPC migratory enhancing factors presented in PL-MSCs secretome were identified by mass spectrometry. Proteins presented in the 100 kDa fraction, subfraction 5, and subfraction 6 were identified by DeCyder differential analysis software based on their signal intensities. The 100 kDa fraction contained 251 proteins while subfraction 5 and subfraction 6 contained 258 and 239 proteins, respectively (Figure
Characterization of PL-MSCs secretome. (a) Venn diagram illustrates the proteins presented in 100 kDa fraction of PL-MSCs conditioned medium as well as those presented in subfraction 5 and subfraction 6 of the 100 kDa fraction. Number of proteins presented in the 100 kDa fraction which were subsequently fractionated into subfraction 5 and/or subfraction 6 (labeled with white color) were regarded as candidate proteins. (b) Pie chart illustrates 239 candidate proteins which were further categorized into nonsecreted protein, classically secreted protein, and nonclassically secreted protein, by SignalP 4.1 and SecretomeP 2.0 software. (c) Graph demonstrates functions of 77 secretory proteins based on PANTHER classification and UniProt.
Those 239 (183 + 38 + 18) candidate proteins were further categorized into nonsecreted protein, classically secreted protein, and nonclassically secreted protein by SignalP 4.1 and SecretomeP 2.0 software. Of 239 proteins, 131 were nonsecreted proteins, 85 were nonclassically secreted proteins, and 23 were classically secreted proteins (Figure
Among those 77 secreted proteins, we further identified the possible candidates for EPC migratory enhancing factor using the following criteria: (A) the candidates must be classical secretory proteins, (B) the candidates must not belong to an apoptotic pathway, and (C) the candidates must be previously reported to be involved in cell migration and/or neovascularization process. According to those criteria, 12 proteins were considered to be possible candidates for EPC migratory enhancing factors. These proteins include astrotactin-1, ADAMTS1, plexin-B1, heparin cofactor 2, Sushi domain-containing protein 2, plasminogen (Angiostatin), PILR alpha-associated neural protein, lymphocyte antigen 75, type IV collagen, laminin, semaphorin receptor
List of EPC migratory enhancing factor candidates in PL-MSCs secretome.
Protein name | Accession |
Secretory pathway prediction | Biological function |
UniProtKB |
---|---|---|---|---|
Astrotactin-1 | gi|46488923 | Classical | Neuron migration and neuronal adhesion | O14525 |
ADAMTS1 | gi|119631213 | Classical | Metalloprotease involve in extracellular matrix remodeling | Q9UHI8 |
Plexin-B1 | gi|6010211 | Classical | Axon guidance and cell migration | O43157 |
Heparin cofactor 2 (SERPIND1) | gi|23273330 | Classical | Chemotactic activity for monocyte and neutrophil | P05546 |
Sushi domain-containing protein 2 | gi|10092665 | Classical | Immune response | Q9UGT4 |
Plasminogen (angiostatin) | gi|38051823 | Classical | Blood coagulation |
P00747 |
PILR alpha-associated neural protein | gi|24308547 | Classical | Immune regulation | Q8IYJ0 |
Lymphocyte antigen 75 | gi|32307817 | Classical | Inflammation and immune process | O60449 |
Type IV collagen | gi|15991848 | Classical | Extracellular matrix/cell adhesion | Q14031 |
Laminin | gi|119613854 | Classical | Cell adhesion and cell migration | Q13751 |
Semaphorin receptor | gi|6010211 | Classical | Axon guidance and cell migration | O43157 |
Small inducible cytokine subfamily E, member 1 (endothelial monocyte-activating) | gi|119626608 | Nonclassical | Angiogenesis | Q12904 |
MSCs have been regarded as promising sources for cell therapy. The therapeutic potentials of MSCs depend on their multilineage differentiation capacity and their ability to secrete broad range of cytokines and growth factors which modulate immune response, enhance cell survival, and induce neovascularization in the target tissues [
We showed that PL-MSCs derived soluble factors significantly enhanced EPC migration and their migration enhancing effect was even greater than that of BM-MSCs. Despite the fact that there are several reports describing the positive effect of BM-MSCs on the function of mature endothelial cells (ECs), there have been no previous reports on the effects of BM-MSCs on the properties of endothelial progenitor cells (EPCs). Previous studies demonstrated the enhancing effect of BM-MSCs on EC migration; however our results showed that BM-MSCs did not affect EPC migration. It is possible that the EC and EPC migration requires different proangiogenic factors. Previous studies showed that VEGF released from BM-MSCs and chorionic blood vessel-derived MSCs (bv-MSCs) enhances EC migration [
To further identify EPC migration enhancing factors presented in PL-MSCs secretome, PL-MSCs conditioned medium was separated into several distinct fractions according to the molecular weight and hydrophobicity of their protein components. EPC migration enhancing factors were enriched in subfractions 5 and 6 of the 100 kDa fraction of PL-MSCs secretome. Mass spectrometry analysis revealed that PL-MSCs secreted hundreds of different proteins through classical and nonclassical secretory pathway. The nonsecretory intracellular proteins leaked from dead cells during culture were also identified in the PL-MSCs conditioned medium and excluded from subsequent analysis [
Using this strategy, 77 PL-MSC-derived secretory proteins presented in subfraction 5 and/or 6 of the 100 kDa fraction of PL-MSCs secretome were identified. Among those secreted proteins, 12 proteins previously reported to be involved in cell migration and/or neovascularization process were identified as candidates for EPC migration enhancing factors. Those include (A) laminin and collagen IV which are extracellular matrix proteins involved in cell adhesion and migration of endothelial and tumor cells [
The positive effect of PL-MSCs on EPC migration described in our study is in agreement with a previous report demonstrating that several gestational tissue-derived MSCs, including chorionic blood vessel-derived MSCs (bv-MSCs), amniotic membrane-derived MSCs (hAMCs), and umbilical cord-derived MSCs (UC-MSCs) released proangiogenic factors which enhance migration and vessel-forming capacity of mature endothelial cell [
We herein report for the first time that PL-MSCs secreted unique combination of factors which enhance EPC migration and their effect is greater than that of BM-MSCs. We also identified several novel candidates for EPC migration enhancing factor in PL-MSCs secretome which have been reported to enhance proliferation, migration, and vessel-forming capacity of mature endothelial cells. The factors discovered in this study could be used to implement the therapeutic effect of MSCs by enhancing neovascularization in the injured/ischemic tissues. Moreover, the strategy developed in our study could be used to systematically identify other therapeutically useful molecules in the secretomes of other MSC sources. However, the therapeutic effects of EPC migration enhancing factors identified in this study should be further confirmed by
The authors declare no conflict of interests. There were no commercial organizations or funding bodies associated with data collection and analysis, or with this study paper.
Witchayaporn Kamprom performed the experiments and drafted the paper. Pakpoom Kheolamai designed the experiments, analyzed the data, supervised the study, wrote and finalized the paper. Aungkura Supokawej, Yaowalak U-Pratya, Methichit Wattanapanitch, Chuti Laowtammathron, and Sittiruk Roytrakul analyzed the data. Surapol Issaragrisil conceived and supervised the study and wrote and finalized the paper. All authors read and approved the final paper.
This research project was funded by grants from Thailand Research Fund (Grant no. RTA 488-0007), the Commission on Higher Education (Grant no. CHE-RES-RG-49). S. Issaragrisil is a Senior Research Scholar of Thailand Research Fund. Witchayaporn Kamprom was supported by the Royal Golden Jubilee Ph.D. Program of the Thailand Research Fund.