Mesenchymal stem cells have a high capacity for trans-differentiation toward many adult cell types, including endothelial cells. Feto-placental tissue, such as Wharton's jelly is a potential source of mesenchymal stem cells with low immunogenic capacity; make them an excellent source of progenitor cells with a potential use for tissue repair. We evaluated whether administration of endothelial cells derived from mesenchymal stem cells isolated from Wharton's jelly (hWMSCs) can accelerate tissue repair in vivo.
Mesenchymal stem cells were isolated from human Wharton's jelly by digestion with collagenase type I. Endothelial trans-differentiation was induced for 14 (hWMSC-End14d) and 30 (hWMSC-End30d) days. Cell phenotyping was performed using mesenchymal (CD90, CD73, CD105) and endothelial (Tie-2, KDR, eNOS, ICAM-1) markers. Endothelial trans-differentiation was demonstrated by the expression of endothelial markers and their ability to synthesize nitric oxide (NO).
hWMSCs can be differentiated into adipocytes, osteocytes, chondrocytes and endothelial cells. Moreover, these cells show high expression of CD73, CD90 and CD105 but low expression of endothelial markers prior to differentiation. hWMSCs-End express high levels of endothelial markers at 14 and 30 days of culture, and also they can synthesize NO. Injection of hWMSC-End30d in a mouse model of skin injury significantly accelerated wound healing compared with animals injected with undifferentiated hWMSC or injected with vehicle alone. These effects were also observed in animals that received conditioned media from hWMSC-End30d cultures.
These results demonstrate that mesenchymal stem cells isolated from Wharton's jelly can be cultured in vitro and trans-differentiated into endothelial cells. Differentiated hWMSC-End may promote neovascularization and tissue repair in vivo through the secretion of soluble pro-angiogenic factors.
Citation: Aguilera V, Briceño L, Contreras H, Lamperti L, Sepúlveda E, Díaz-Perez F, et al. (2014) Endothelium Trans Differentiated from Wharton's Jelly Mesenchymal Cells Promote Tissue Regeneration: Potential Role of Soluble Pro-Angiogenic Factors. PLoS ONE 9(11): e111025. https://doi.org/10.1371/journal.pone.0111025
Editor: Antonio Paolo Beltrami, University of Udine, Italy
Received: March 11, 2014; Accepted: September 26, 2014; Published: November 20, 2014
Copyright: © 2014 Aguilera et al. This is an open-access article distributed under the terms of the Creative Commons Attribution License, which permits unrestricted use, distribution, and reproduction in any medium, provided the original author and source are credited.
Funding: This study was supported by INNOVA CORFO (12IDL2-13351) and INNOVA BIOBIO (1245-EM.TES(12.21), Dirección de Investigación, Universidad de Concepción (DIUC 211.072.034-1.0), Chile and Convenio de desempeño, Universidad de Concepción, UCO1201.CE is supported by Fondecyt 1100684, DIUBB 122109 GI/EF. The funders had no role in study design, data collection and analysis, decision to publish, or preparation of the manuscript.
Competing interests: The authors have declared that no competing interests exist.
Mesenchymal cells (MSCs) are a heterogeneous group of adult stem cells able to proliferate and differentiate into osteocytes, chondrocytes or adipocytes , , as well as into non mesodermal precursors, from which they can be trans-differentiated towards hepatocytes , neurons  or endothelial cells , . In addition, MSCs are multipotent cells with a potential use in human regenerative medicine due to their ability to migrate into sites of injury and modulate immune response. MSCs have been proposed for therapeutic use in degenerative diseases of bone, muscle, nervous system and specially for cardiovascular and heart diseases – but one of the main limitations is the quantity of cells that can be isolated from sources such as bone marrow . However MSCs can also be obtained from other tissues, such as gingival tissue , skin , placenta , , amniotic fluid , whole blood or Wharton's jelly from umbilical cords , .
Mesenchymal stem cells isolated from Wharton's jelly (hWMSCs) have high telomerase activity , high proliferative capacity  and long- term culturing of expanded –. These cells cannot undergo tumor transformation – and have a low expression of histocompatibility complex class I molecules , , , while they do not express major histocompatibility complex class II , , . Therefore, these cells exhibit low immunogenicity and high immunosuppressive capacity that make them useful for therapeutic approaches , .
In vivo experiments showed that hWMSCs can repair ischemic tissue by promoting neovascularization  and re-endothelialization . The underlying mechanisms associated with pro-angiogenic effect of both MSCs and hWMSCs are under investigation. Indeed, MSCs can secrete angiogenic factors , and therefore, increase neovascularization in a mouse model of ischemia . Indeed, MSCs can be differentiated into endothelial cells and form capillary-like structures, an effect associated with the production of vascular endothelial growth factor (VEGF) by those cells –. Moreover, MSCs increased neovascularization and tissue perfusion through the secretion of VEGF and stromal cell-derived factor 1 (SDF-1) , . Despite these evidences there is limited information regarding potential pro-angiogenic activity of hWMSCs. Therefore we aimed to evaluate whether administration of endothelium derived from mesenchymal stem cells isolated from Wharton's jelly (hWMSCs) can accelerate tissue repair in vivo.
Materials and Methods
This investigation conforms to the principles outlined in the Declaration of Helsinki, and has received approval from the Ethics Committee of the Faculty of Pharmacy of Universidad de Concepción de Chile and Guillermo Grant Benavente Hospital (Concepción, Chile). Informed written consent was obtained from all patients.
Isolation and culture of hWMSC.
Umbilical cords were aseptically collected from healthy full-term pregnancies, at Guillermo Grant Benavente Hospital (Concepción, Chile). hWMSCs were isolated as described previously  with some modifications. Umbilical cords were cut into 2 to 3 mm3 pieces and vessels were stripped manually from those cord segments. Wharton's jelly was digested with collagenase 10 mg/mL (37°C for 4 hours). Mesenchymal cells were recovered, centrifuged (1000 g for 30 minutes) and suspended in fresh M-199 medium including 10% fetal bovine serum (FBS) and 5 ng/mL basic fibroblast growth factor, bFGF (Sigma, Aldrich). Cultures were maintained in a humidified atmosphere with 5% CO2 at 37°C for 10 days, until hWMSCs colonies were observed. Adherent cells were detached using a trypsin-EDTA solution .
To confirm hWMSCs functionality, they were differentiated into osteocytes, adipocytes or chondrocytes. Cells were cultured in a 24-well plate at a density of 10×103 cell/cm2 and when they reached about 70% confluence, culture medium was replaced with M-199 medium containing 10% FBS and 2 mM glutamine, and supplemented as follows to induce either osteogenic differentiation (0,1 µM dexamethasone, 50 µg/ml ascorbic acid, and 10 mMβ-glycerophosphate), adipogenic differentiation (0,1 µM dexamethasone, 60 µM Indomethacin, 0,5 mM 3-isobutil-1- metilxantine and 10 µg/mL insulin) or chondrogenic differentiation (0,1 µM dexamethasone, 10 ng/mL transforming growth factor β1). Cells were incubated for 15 days and media was changed every 3 days. Osteogenic differentiation was analyzed by von Kossa staining, adipogenic differentiation was confirmed by neutral lipid vacuoles detected with Oil Red O staining  and chondrogenic differentiation was verified by extracellular matrix staining with Alcian blue , , .
Cell characterization by flow cytometry was carried out as previously described , , . In brief, hWMSCs in culture were trypsinized (0.25% trypsin/1 mM EDTA) washed and resuspended in phosphate-buffered saline at a density of 3×105 cells/100 µL. Cells were stained with an anti-human CD90-FITC monoclonal antibody (1∶100, Becton Dickinson, San Diego, CA, USA). Fifty thousand events were analyzed per sample in an argon laser FACS Calibur (BD Biosciences, Pharmingen, San Diego, CA, USA), at the Laboratory of Molecular Biology, Faculty of Medicine, Catholic University of Concepción.
Total RNA was isolated using the Trizol Reagent (Invitrogen, Carlsbad, CA, USA) according to the manufacturers instructions. RNA quality and integrity and quality were insured by gel visualization and spectrophotometric analysis (OD260/280). 1 µg of total RNA was reversed transcribed into cDNA for 1 hour at 37°C as described elsewhere. –.
mRNA for CD90, CD73, CD105 and Cyclophilin were assessed by quantitative real time PCR (qPCR) in a Rotorgene 2000 thermal cycler (Corbett Research). Thus, reactions in 25 µL were carried out using Brilliant II SYBR Green QPCR Master Mix (Agilent Technologies, USA) with 0.3 µM primers according to the manufacturer's instruction. Primers used are described in Table 1. Samples were incubated for 4 minutes at 95°C, followed by 25 cycles of 30 seconds at 95°C, 30 seconds at 54°C (for eNOS and Cyclofilin), 58°C (CD90, CD105) or 60°C (CD73), 30 seconds at 72°C, and finally 7 minutes at 72°C. A single melting point was observed for all samples. The gene expression was quantified using the 2−ΔΔCt (threshold cycle) method. Thus, a delta C(T) (ΔCt) value was obtained by subtracting the cyclophilin-CT value from the CT value of the studied gene. The ΔCt mean value obtained from the control group of each gene was used to calculate the 2(-Delta Delta C(t) (ΔΔCt) of the respective gene (2−ΔΔCt). Expected size products were separated by electrophoresis on 1.5% agarose gels and visualized with ethidium bromide under UV light.
Staining was performed on fixed cell monolayers (4% paraphormaldehyde, 10 min, room temperature), which were grown previously on coverslips. hWMSCs were permeabilized with PBS 0.25% Triton X-100 for 10 min and antibodies were added overnight at 4°C (anti-vimentin (1∶500, Abcam, Cambridge, UK), CD31-FITC (1∶200, BD Pharmingen, San Diego, CA, USA), and anti-KDR-FITC (1∶300, BD Pharmingen, San Diego, CA, USA). For vimentin labeling, a secondary Texas-Red conjugated antibody was used. Samples were placed on slides using mounting medium and DAPI was added for nuclei identification. Microscopic analysis of samples was performed by using an Olympus IX81 inverted microscope in conjunction with a DSU spinning disk confocal system.
Endothelial differentiation was performed as described previously with some modification , . hWMSCs were incubated in 100 mm2 plates up to 14 (hWMSC-End14d) or 30 days (hWMSC-End30d) in primary culture medium (PCM) composed of endothelial growth medium (GIBCO BRL Life Technologies, Bethesda, MD, USA) containing 5 mM D-glucose, 15% fetal calf serum, 10 mg/mL of human VEGF (GIBCO BRL Life Technologies, Bethesda, MD, USA) –. Endothelial phenotype was confirmed through presence of endothelial markers and nitric oxide (NO) production as described below.
Endothelial nitric oxide synthase (eNOS) activity.
L-[3H]-arginine conversion into L-[3H]-citruline was used to assess the endothelial nitric oxide synthase (eNOS) activity in hWMSCs trans-differentiated into endothelial cells. hWMSC, hWMSC-End14d or hWMSC-End30d were incubated with Na+-Krebs solution containing 100 µM L-arginine and L-[3H]arginine (4 µCi/mL) for 30min at 37°C. Cells were lysed and treated in a cation ion-exchange resin Dowex-50W (50X8-200, Na+ form), to separate L-[3H]-citrulline present in H2O eluates from L-[3H]-arginine retained in the column. When required, NG-nitro-L-arginine methyl ester (L-NAME, 100 µM), a NOS inhibitor, was added to cell cultures 30 min before experiments .
Intracellular NO formation.
Intracellular NO production was determined using the fluorescent probe 4-amino-5-methylamino-2′, 7′-difluorofluorescein diacetate (DAF-FM-DA, Calbiochem) as described previously , . hWMSCs, hWMSC-End14d or hWMSC-End30d were seeded in 96-well plates, incubated for 24 h under standard conditions. Before experiments, cells were incubated in serum-depleted medium for 4 h. DAF-FM-DA probe (5 µM) and L-arginine (100 µM) were added to cultures for 30 minutes and then fluorescence intensity was measured in a fluorimeter (Sinergy 2, Biotec). As a positive control, 1 mM histamine was added for the last 15 min. To assess the involvement of NOS activity in NO production, cells were pre-incubated with L-NAME (100 µM, 30 min).
eNOS and vimentin protein expression.
Total eNOS and vimentin protein expression was evaluated by western blot in hWMSCs, hWMSC-End14d or hWMSC-End30d. Total proteins (50 µg) were separated by a polyacrylamide gel (10%) electrophoresis and transferred to Immobilon-P polyvinylidenedifluoride membranes (BioRad Laboratories, Hertfordshire, UK). Membranes were probed with rabbit anti-eNOS (1∶1000), anti-vimentin (1∶500) or anti-β actin (1∶2000) (Santa Cruz Biotechnology, CA, USA). Membranes were incubated (1 h) with a horseradish peroxidase-conjugated goat anti-rabbit antibody. Proteins were detected by the enhanced chemiluminescence method, analyzed by densitometry and compared to β-actin expression (control).
Wound healing model and hWMSC transplantation.
Seventy- C57BL-6 mice (6 weeks old; female; body weight 20–23 g) were randomly divided into four groups, (n = 5 per group), and the excisional wound-splinting model was generated as described previously –. The experimental protocols were approved by Ethics Committee of the Universidad del Concepción and conform to the guidelines for the Care and Use of Laboratory Animals published by the US National Institute of Health. In brief, after anesthesia and hair removal from the dorsal surface, two 6-mm full thickness excisional skin wounds were created on each side of the midline. Each wound received 5×105cells (either, hWMSCs, hWMSC-End140d or hWMSC-End30d, in duplicate, each set derived from the same hWMSC line, with 5 hWMSC lines in total) in 60 µl of PBS injected intradermally around the wound at four injection sites; or 60 µl of conditioned media (see below), or PBS (control group). 20 µl of growth factor-reduced Matrigel (BD Biosciences) were applied onto the wound bed. A donut-shaped plastic splint was placed around the wound, with the area centered within the splint and the splinted hole represented the original wound size. An immediate-bonding adhesive was used to fix splints to the skin, followed by interrupted sutures to stabilize its position. Animals were housed individually. Digital photographs of wounds were taken at days 0, 3, 7 and 12 after cell injections and the percentage of wound closure was calculated  as: Wound closure (%) = (Areas of original would – Area of actual would)/Area of original would) ×100.
Time to wound closure was defined as the time at which the wound bed was completely re-epithelialized and filled with new tissue. Mice were sacrificed at day 12, and skin samples (including the wound and 4 mm of the surrounding skin) were harvested and placed on a tissue culture dish with the dermis side down, and photographed immediately.
Conditioned media (CM).
CM were obtained from confluent hWMSCs or MSC-End30d cultured for 48 h in M199 medium with 10% FBS. Cells and debris were removed by centrifugation (5 min, 500 g) and CM was stored at −80°C until use.
Tissue specimens were fixed (3% paraformaldehyde for 2 h) and embedded in paraffin. Six-micron-thick sections were stained with hematoxylin and eosin for light microscopy. Histological scoring was performed in a blinded fashion. The criteria used for histological scores of wound healing are summarized in Table 2, as described previously . In brief, each slide was given a histological score ranging from 1 to 10 according to the following parameters: re-epithelialization and regeneration, dermal cellularity, granulation tissue formation, and angiogenesis. Capillary density was assessed morphometrically by examining three fields per section of the wound between the edges in six successive sections after measuring by Image Pro plus software (Ipwin32, American, USA) , .
Microscopic analysis and immunohistochemistry.
Paraffin-embedded tissue sections were cut into 4-mm slices to use for vessel identification as previously described , . This approach, allowed us to measure the perimeter of every vessel and its corresponding total area of the reference field. These were expressed as mean vessel perimeter, in micrometers, divided by the respective total area. The blood vessels area in the pictures was analyzed using ImageJ software (National Institutes of Health, Bethesda, MD).
Presence of hWMSCs into the healing area.
hWMSCs were identified by human mitochondrial immunohistochemistry using same approach as described above. Sections were reacted with anti-human mitochondria (1∶1000, Abcam), and visualized with a peroxidase-conjugated secondary antibody (1∶100, Sigma Aldrich). Five fields belong to 5 tissue sections per animal were randomly selected. Positive cells in each field was evaluated as a dichotomic variable (i.e, present or absent). All analysis were carried out in a blinded way.
Values were expressed as mean ± standard deviation where n indicates number of different cell cultures (in duplicate). Statistical analysis of data was performed with a one-way ANOVA followed by a Tukey–Kramer. We used X2 test to analyze proportions. The statistical software GraphPad Prism 5.00 (GraphPad Software Inc., California, USA) and standard statistical software (SPSS 10.0) were used for data analysis. Differences were considered significant when the P-value was 0.05 or less.
Characterization of mesenchymal stem cells isolated from Wharton's jelly
Isolated cells showed a spindle-shaped fibroblast-like morphology (Figure 1A). After 15 days in culture in the presence of differentiating medium, hWMSCs exhibited positive staining for osteoblasts, adipocytes and chondrocytes markers (Figure 1A).
A) hWMSCs were cultured with basal medium (upper left panel), adipogenic (upper right panel), osteogenic (lower left panel) or chondrogenic (lower right panel) medium for 10 days. Cells were stained for lipid droplets (Oil Red O staining), calcium deposits (Von Kossa staining) or sulfated proteoglycans (toluidine blue staining), respectively. Magnification 20x. B) Mesenchymal gene expression (CD91, CD73, CD105, CD34) was assessed by RT-PCR in hWMSCs cultured in non-differentiated medium. Human umbilical vein endothelial cells (HUVEC) were used as control. Lower panel: qPCR shows RNA levels for Mesenchymal gene expression in hWMSC versus the corresponding controls. C) Surface CD90 expression was evaluated on hWMSCs by flow cytometry. Data are representative of 5 different isolations of MSC from Wharton's jelly.
hWMSCs cultured in non-differentiating media expressed the mesenchymal markers CD90, CD73 and CD105, as assessed by RT-PCR, but were negative for the hematopoietic marker CD34 (Figure 1B). On the contrary, CD34 mRNA level was significantly higher (7 fold) in human umbilical vein endothelial cells (HUVEC), as compared to hWMSCs (Figure 1B).
Flow cytometry analysis showed two subpopulations of hWMSC (i.e, P1, P2), which differ mainly in size. Moreover, both subpopulation were positive for CD90 (96.0% for P1 and 98.5% for P2) (Figure 1C). In addition, using flow cytometry, we also observed that hWMSCs were positive for vimentin (95%), but showed a low expression of the endothelial markers CD31 and KDR (data non show).
Characterization of hWMSCs trans-differentiated to endothelial cells.
Using both inmmunocytochemistry (Figure 2A) and western blot analysis (Figure 2B), it was observed that after 15 days of culture without differentiating medium hWMSCs expressed high protein levels of vimentin protein, but little eNOS, CD31 and KDR levels. On the contrary, in the presence of culture medium for endothelial differentiation (see Methods), hWMSC-End14d and hWMSC-End30d showed low levels of vimentin, but high levels of eNOS, CD31 and KDR. In addition, expression of eNOS protein (an endothelial marker) was higher (4 fold) in hWMSC-End30d compared with MSC-End14d (Figure 2B).
A) Confocal microscopy for vimentin (mesenchymal cell marker), KDR and CD31 (endothelial markers) by immunofluorescent staining. Normal mouse IgG was used as a negative control. Nuclei were counter-stained with DAPI (blue). Magnification 40X (CD31 and KDR), 20X (vimentin). Bars 50 µm. B) Vimentin and eNOS protein abundance in hWMSCs (line 1) or hWMSC transdifferentiated into endothelial cells for 14 days (hWMSC-End14d, line 2) or 30 days (hWMSC-End30d, line 3) with endothelial-differentiating medium. Upper panel: Representative western blot for vimentin, eNOS and β-actin (internal reference). Lower panel: densitometric analysis of protein abundance, expressed as ratios for Vimentin/β-actin (▪) or eNOS/β-actin (□). Data are representative of 5 different MSC isolations from Wharton's jelly.
Conversion of L-arginine to L-citrulline (i.e., eNOS activity) and intracellular NO formation were elevated (5 and 3 fold, respectively) in trans-differentiated endothelial cells, when compared with non-differentiated cells. Indeed, NOS activity was higher (2 fold) in hWMSC-End30d than hWMSC-End14d (Figure 3A); whereas NO intracellular formation reached the same level in both stages of differentiation. Both L-citrulline formation and NO formation were inhibited by L-NAME in all conditions (Figure 3).
NO synthesis was estimated in hWMSCs or hWMSC transdifferentiated into endothelial cells for 14 days (hWMSC-End14d) or 30 days (hWMSC-End30d). Cells were incubated in the absence (control □) or presence of L-nitroarginine methyl ester (L-NAME, 100 mM, ▪) for 30 minutes. A) L-citrulline formation assay; B), NO detection with the 4,5-diaminofluorescein diacetate probe (DAF-FM-DA, 1 mM, 30 minutes). Values are mean±S.E.M (n = 3), **P<0.01 v/s MSCs; *P<0.05 v/s without L-NAME. Data are representative of 5 different MSC isolations from Wharton's jelly.
Wound healing capacity of endothelial-trans differentiated hWMSCs.
From hWMSC was possible to obtain trans-differentiated endothelial cells of 14 (MSC-End14d) and 30 day (MSC-End30d). Thus, three cell populations were obtained from the same hWMSC isolation, each one was injected in two animals, for wound healing experiments (n = 5 experiments). Mice treated with hWMSCs exhibited accelerated wound closure as compared to control animals (Figure 4A). Among hWMSC, those cells that were trans differentiated toward an endothelial phenotype showed faster wound closure than non-differentiated hWMSC. Thus, at day 7, the extent of wound closure was higher in mice treated with hWMSC-End14d and hWMSC-End30d (90±3%) compared to those mice that received either hWMSCs (55±4%, p<0.05) or vehicle (39±1%, n = 5, p<0.05). Similarly, complete wound closure was observed at day 12 in the hWMSC group, but at this time wound closure remain incomplete (97±2%) in mice treated with vehicle (Figure 4B).
A) Representative images of wounds at day 1 (top panels) and 12 (lower panels) after injury and subcutaneous injection of hWMSCs, hWMSC trans-differentiated into endothelial cells for 14 days (hWMSC-End14d) or 30 days (hWMSC-End30d), or control (PBS). B) Wound healing quantified in PBS (○), hWMSC (•), hWMSC-End14d (□) or hWMSC-End30d (▪) treated mice (n = 5 independent experiments, in duplicate). Values are expressed as mean±S.E.M, +P<0.05 in hWMSC-End30d v/s hWMSC, hWMSC-End14d, at the corresponding time; **P<0.03 in hWMSC-End30d v/s PBS; *P<0.001 in hWMSC-End30d v/s PBS; # P<0.01 in hWMSC-End30d v/s PBS.
Histological analysis of the healed tissue at day 12 showed enhanced re-epithelialization in mice treated with hWMSC-End14d and hWMSC-End30d compared with those treated with either hWMSCs or vehicle (Figure 5A). Indeed, in animals treated with hWMSCs, hWMSC-End14d or hWMSC-End30d, wounds exhibited augmented thickness of the epidermis, newly formed granular, tissue, and a more organized extracellular matrix (Figure 5A), as well as increased area and number of vessels (Figure 5B), but the effect only reached statistical significance in endothelial-trans differentiated hWMSC (14d or 30d) compared with the vehicle-treated group.
A) Representative photographs of wounds (hematoxilin/eosin staining) 12 days after injury and subcutaneous injection of PBS, hWMSCs, hWMSC-End14d or hWMSC-End30d. Quantification of histological images, for blood vessels area (B) and histological score (C) for each group of mice. Values are mean ± S.E.M (n = 5 independent experiments, in duplicate), *P<0.001 in hWMSC-End30d or hWMSC-End14d v/s MSC; +P<0.05 in hWMSC-End30d or hWMSC-End14d v/s hWMSC. Magnification x40 (-). Ep, epidermis; D, dermis; H, hypodermis.
Consistent with these findings, the histological score, an index of repairing capacity, was significantly higher in the groups treated with hWMSC-End14d and hWMSC-End30d compared with hWMSC or vehicle (Figure 5C). There was no significant difference among histological parameters when comparing histological scores in the hWMSC-End14d and hWMSC-End30d groups.
Engraftment of endothelial-trans differentiated hWMSCs into wounded skin.
Positive staining for human mitochondria was found in scar tissues in the three groups of animals (hWMSC, hWMSC-14d and hWMSC-30d treated mice), being the hWMSC-30d group the most evident. Positive staining was localized in epidermis, dermis, hair follicles and glandular structures in all grafted animals, in contrast, PBS-treated mice were completely negative for human mitochondrial staining (Figure 6A). The number of positive cells observed in vessels is restricted and there are no significant differences in different types of cells implanted. (Figure 6B)
A. Immunohistochemical staining of human mitochondria was performed in permeabilized tissue sections obtained after 12 days of subcutaneous injection of PBS, hWMSCs, hWMSC-End14d or hWMSC-End30d in mice. Cell nuclei were stained with hematoxyline. In B. Number of positive cells per vessel. Representative images of 5 independent experiments, in duplicate. Magnification x40 and insert 100x. Bars 50 µm.
Wound healing with conditioned medium.
Conditioned medium (CM) recovered from hWMSCs or hWMSC-End30d cultures increased the formation of capillary-like structures in HUVEC cultured on Matrigel. Thus, when compared with medium derived from hWMSCs, the conditioned medium from hWMSC-End30d significantly increased length, number of branch points and relative area of capillary-like structures in HUVEC cultured on Matrigel (data not show).
When injecting CM directly on wounds in the animal model of injury, there was a significant increase in the percentage of wound closure, from day 7 to day 9, in mice treated with hWMSC-30d medium compared with hWMSCs medium or PBS (p<0.05) (Figure 7).
Wound closure was quantified in injured mice that received an injection of non-conditioned medium (○), conditioned medium derived from hWMSC (•) or hWMSC-End30d (□) (n = 5 experiments in duplicate). Values are expressed as mean±S.E.M, *p<0.04 vs non-conditioned and conditioned medium derived from hWMSC.
Mesenchymal stem cells isolated from Wharton's jelly of human umbilical cords (hWMSCs) have been proposed as an alternative source of progenitor cells for use in regenerative medicine , –. On the other hand, loss of endothelial cells or endothelial dysfunction is associated to several chronic metabolic diseases. Endothelial repair using adult cells is extremely difficult, since their isolation from large vessels is challenging, and once isolated they display a low proliferation rate in culture . In this regard, our results show that: 1) hWMSCs can be differentiated into adipocytes, osteocytes and chondrocytes. 2) hWMSCs express classic surface mesenchymal markers (CD90, CD73 and CD105), but not hematopoietic markers (CD34). 3) These cells can be trans-differentiated into endothelial cells (hWMSC-End), which in turn show expression of endothelial markers, such as CD31, KDR and eNOS, and importantly, they produce NO. 4) In vivo experiments showed that hWMSCs trans-differentiated into endothelial cells have greater regenerative capacity than non-differentiated hWMSC. 5) Conditioned medium derived from hWMSC-End, also enhanced the percentage of wound healing and vascularization in the scar, suggesting that these cells may release pro-angiogenic factors. Therefore, trans-differentiation of hWMSC into endothelial cells enhances wound healing and potentially exhibits a broad range of pre and clinical applications.
hWMSCs characterization and endothelial differentiation
Our phenotypic characterization of hWMSCs agrees with previous literature –, where primary cultures include at least two subpopulations of cells that differ in size: a larger (P1) and a smaller population (P2). Both cell types were positive for the mesenchymal marker CD90 (96 and 98.5%, respectively) (Figure 1C), but not for the hematopoietic marker CD34, as reported previously , , . Functional studies showed that hWMSCs can be differentiated into chondrocytes, osteocytes, adipocytes, as well as they can be trans-differentiated into endothelial cells. The International Society for Cellular Therapy (ISCT) propose three criteria to define MSC include: plastic adherence in conjunction with a fibroblastoid phenotype; cell surface expression of CD105, CD73 and CD90 and lack of expression of CD45, CD34, CD14 (or CD11b), CD79α (or CD19) and human leukocyte antigen (HLA)-DR molecules; and in vitro differentiation capacity toward chondrocyte, adipocyte and osteocyte lineages. Our results agree with these criteria proposed by the ATCC. In addition, Lui et al., (2014) showed that the human umbilical cord Wharton's jelly is an potential alternative source of highly pure mesenchymal cells, since contrary to bone marrow or adipose tissue, the Wharton's jelly preparations contained reduced amount of stem and hemogenic cells .
In addition, when hWMSCs were exposed to an endothelial-differentiating medium, they showed morphological changes (polygonal-elongated morphology, enlarged nuclei and prominent nucleoli), which have been associated with hWMSCs trans-differentiation , . Furthermore, endothelial marker expression (CD31, KDR and eNOS) increased during differentiation and was significantly higher in hWMSC-End30d than in hWMSCs or hWMSC -End14d, supporting cell differentiation towards an endothelial phenotype as demonstrated by others , , . Therefore, our results further support the capacity for trans-differentiation of hWMSCs.
Alamino et al., (2010) demonstrated that specific genes related to endothelial cell phenotype and function were up-regulated in hWMSCs stimulated by endothelial differentiating medium . However, despite this and the phenotypical differentiation highlighted in other studies , ,  relevant endothelial-related functions, such as NO synthesis, had not been analyzed. Bone marrow mesenchymal stem cells can also be differentiated into endothelial-like cells , , , . However, bone marrow harvesting is a highly invasive procedure to the donors, and proliferation efficiency, multipotent differentiation potential, and maximal lifespan of these cells decline with aging . This is one of the main reasons for seeking other sources of stem cells. In this context, it has been shown that mesenchymal stem cells derived from human placenta were able to differentiate into endothelial-like cells , whereas differentiation capacity toward osteocytes, adipopocytes or chondrocytes was not tested. This last limitation was overcome in our study, showing that hWMSCs can be differentiated in osteocytes, adipopocytes, chondrocytes and even endothelial cells.
Mature endothelial cells are characterized by the synthesis and release of vasoactive substances, such as NO. We find that eNOS protein expression was significantly increased only in hWMSC-End30d, and effect associated with high L-arginine conversion into L-citrulline (i.e., an index of NOS activity) and intracellular formation of NO, confirming that hWMSC-End30d displays an endothelial phenotype. In this regard, the effect of NO on tissue repair is mediated by at least two of the three isoforms described, inducible nitric oxide synthase (iNOS) and eNOS, since mice deficient to those enzymes exhibited an impaired healing processes , . Moreover, eNOS gene-transduction in mice restored the attenuated NOS production from the injured endothelium, resulting in vasodilation and regeneration –. The underlying mechanisms triggered by NO during tissue repair remain largely unknown. However, it has been suggested that NO regulates the expression of an increasing number of genes, including VEGF, which in turn plays a crucial role in tissue repair processes , . In agreement with this last idea, our results suggest that hWMSCs, and particularly hWMSCs-End30d release a proangiogenenic factor(s), since the use of conditioned medium from those cells, also accelerated tissue repair compared to animals treated with vehicle or non-conditioned medium. However, to determine the precise mechanism involved, identification of these factors is required, as well as characterization of morphological and/or histochemical changes in the repaired tissue.
Wound healing with endothelial trans-differentiated hWMSCs
Mesenchymal stem cells accelerate wound healing in animal models , –. Our results agree with these evidences, and indicate that implantation of hWMSCs differentiated into endothelial cells and cultured during 14 or 30 days showed faster wound healing than hWMSCs. The mechanisms are unclear; however, as described above we found some differences in the capacity for NO synthesis, which may impact the healing capacity of these cells. In addition, the capacity and functionality of differentiated mesenchymal stem cell might differs in vivo depending on the source from where they were isolated . For instance, hWMSC-End, or mesenchymal stem cells isolated from amnion  but not bone marrow-derived cells  improve wound repair. More studies are required in order to confirm higher capacity of feto-placental derived mesenchymal cells compared those isolated from bone marrow or adipose tissue.
Histological analysis of scar tissue obtained from wounds showed higher capacity regeneration of hWMSC-End compared to hWMSCs. Thus, hWMSC-End treated animals showed the highest regeneration capacity, associated with better organization of epidermis and dermis compared to animals treated with hWMSC, as indicated by the high histological score observed in those animals. Interestingly, tissue recovery in the group of animals treated with hWMSC-End was associated with an elevated number of blood vessels than hWMSCs or PBS-treated animals. In this regard, neovascularization is a crucial step in the wound healing process , , , since this process is necessary to sustain the newly formed granulation tissue and the survival of keratinocytes.
We conduct additional experiments in order to answer whether the presence of hWMSCs or release of pro-angiogenic factors may be taken part in the tissue recovery. Firstly, we demonstrated that hWMSCs are incorporated into the scar tissue (epidermis, dermis, hair follicles and glandular structures), a finding more evident when hWMSC-End30d were used. These results are consistent with previous studies where bone marrow derived mesenchymal stem cells were incorporated into hair follicles and sebaceous glands in a mouse model of injury , . However, the number of human cells identified in the lesion area was reduced, as previously reported using mesenchymal stem cells isolated from mouse bone marrow . In addition, a recent study showed a dramatic decline of engrafted bone marrow mesenchymal stem cells in the acutely infarcted myocardium after intramyocardial injection . The mechanisms involved with the decline of implanted cells are not fully understood. It is likely that with progression of wound healing process, cytokines and extracellular matrix molecules favor both mesencymal stem cells survival and engraftment decrease. In this scenario, we further investigate whether hWMSCs and hWMSC-End may promote wound healing via soluble factors.
In order to test the last hypothesis, we recovered conditioned media from hWMSC or hWMSC-End30d cultures, which were injected into mice wounds. Interestingly, conditioned medium derived from hWMSC-End accelerated wound closure, in a similar pattern that observed with hWMSC itself (figures 4B and 7, respectively). These results, suggest that wound healing is mainly carried out by soluble factors (i.e. cytokines or growth factors) produced by hWMSC-End. Indeed, it is well known that hWMSCs may secrete angiopoietin-1, angiogenin, interleukin 8 (IL-8), monocyte chemotactic protein-1 (MCP-1), serpin E1 (plasminogen activator inhibitor 1), tissue inhibitor of metalloproteinase-1 (TIMP-1) and trombospondrina-1 . A combination of those factors (and perhaps many others) may participate in the healing effect of conditioned medium derived from hWMSC-End . In our study we also found that CM recovered from hWMSCs or hWMSC-End30d cultures increased the formation of capillary-like structures in HUVEC cultured on Matrigel, supporting that pro-angiogenic factor(s) might taken part in the promotion of wound healing. Alternatively, other group has suggested that mesenchymal stem cells may recruit mature endothelial cells and/or release pro-angiogenic molecules , which then may contribute to the healing process . Clearly, more studies are required in this field.
In conclusion, our results represent the first evidence showing that endothelial-trans differentiated hWMSCs have the capacity to promote neovascularization and wound healing in vivo through the secretion of pro-angiogenic factors. However, further studies are required to identify whether NO or other secreted angiogenic factor(s) are responsible for tissue regeneration in vivo, and evaluate their future use in regenerative therapy.
We thank midwives of Hospital Clínico Guillermo Grant Benavente and Clínica Sanatorio Alemán, Concepción labor ward for the supply of umbilical cords. We also thanks to Dr. Helen Jones, Assistant Professor, of the Center for Molecular Fetal Therapy (MLC 11025), Division of General and Thoracic Surgery, Cincinnati Children's Hospital Medical Center for editing this manuscript.
Conceived and designed the experiments: VA PF LL FAZ JRT CR CE CA. Performed the experiments: VA AC LB HC PF ES FDP FAZ ML CV RM CA. Analyzed the data: VA HC ES LL FAZ CR CE CA. Contributed reagents/materials/analysis tools: FAZ CR CE CA. Wrote the paper: VA FAZ CE CR CA.
- 1. Pittenger MF, Mackay AM, Beck SC, Jaiswal RK, Douglas R, et al. (1999) Multilineage potential of adult human mesenchymal stem cells. Science 284: 143–147.
- 2. Dominici M, Le Blanc K, Mueller I, Slaper-Cortenbach I, Marini F, et al. (2006) Minimal criteria for defining multipotent mesenchymal stromal cells. The International Society for Cellular Therapy position statement. Cytotherapy 8: 315–317.
- 3. Yoon HH, Jung BY, Seo YK, Song KY, Park JK (2010) In vitro hepatic differentiation of umbilical cord-derived mesenchymal stem cell. Process Biochemistry 45: 1857–1864.
- 4. Yu YL, Chou RH, Chen LT, Shyu WC, Hsieh SC, et al. (2011) EZH2 regulates neuronal differentiation of mesenchymal stem cells through PIP5K1C-dependent calcium signaling. J Biol Chem 286: 9657–9667.
- 5. Oswald J, Boxberger S, Jorgensen B, Feldmann S, Ehninger G, et al. (2004) Mesenchymal stem cells can be differentiated into endothelial cells in vitro. Stem Cells 22: 377–384.
- 6. Harichandan A, Buhring HJ (2011) Prospective isolation of human MSC. Best Pract Res Clin Haematol 24: 25–36.
- 7. Tang YL, Zhao Q, Zhang YC, Cheng L, Liu M, et al. (2004) Autologous mesenchymal stem cell transplantation induce VEGF and neovascularization in ischemic myocardium. Regul Pept 117: 3–10.
- 8. Tang J, Wang J, Yang J, Kong X, Zheng F, et al. (2009) Mesenchymal stem cells over-expressing SDF-1 promote angiogenesis and improve heart function in experimental myocardial infarction in rats. Eur J Cardiothorac Surg 36: 644–650.
- 9. Li Z, Guo J, Chang Q, Zhang A (2009) Paracrine role for mesenchymal stem cells in acute myocardial infarction. Biol Pharm Bull 32: 1343–1346.
- 10. Mitrano TI, Grob MS, Carrion F, Nova-Lamperti E, Luz PA, et al. (2010) Culture and characterization of mesenchymal stem cells from human gingival tissue. J Periodontol 81: 917–925.
- 11. Salvolini E, Lucarini G, Zizzi A, Orciani M, Di Benedetto G, et al. (2010) Human skin-derived mesenchymal stem cells as a source of VEGF and nitric oxide. Arch Dermatol Res 302: 367–374.
- 12. Battula VL, Bareiss PM, Treml S, Conrad S, Albert I, et al. (2007) Human placenta and bone marrow derived MSC cultured in serum-free, b-FGF-containing medium express cell surface frizzled-9 and SSEA-4 and give rise to multilineage differentiation. Differentiation 75: 279–291.
- 13. Parolini O, Alviano F, Bagnara GP, Bilic G, Buhring HJ, et al. (2008) Concise review: isolation and characterization of cells from human term placenta: outcome of the first international Workshop on Placenta Derived Stem Cells. Stem Cells 26: 300–311.
- 14. Alviano F, Fossati V, Marchionni C, Arpinati M, Bonsi L, et al. (2007) Term amniotic membrane is a high throughput source for multipotent mesenchymal stem cells with the ability to differentiate into endothelial cells in vitro. BMC Dev Biol 7.
- 15. La Rocca G, Anzalone R, Corrao S, Magno F, Loria T, et al. (2009) Isolation and characterization of Oct-4+/HLA-G+ mesenchymal stem cells from human umbilical cord matrix: differentiation potential and detection of new markers. Histochem Cell Biol 131: 267–282.
- 16. Rocha V, Labopin M, Sanz G, Arcese W, Schwerdtfeger R, et al. (2004) Transplants of umbilical-cord blood or bone marrow from unrelated donors in adults with acute leukemia. N Engl J Med 351: 2276–2285.
- 17. Mitchell KE, Weiss ML, Mitchell BM, Martin P, Davis D, et al. (2003) Matrix cells from Wharton's jelly form neurons and glia. Stem Cells 21: 50–60.
- 18. Sarugaser R, Hanoun L, Keating A, Stanford WL, Davies JE (2009) Human mesenchymal stem cells self-renew and differentiate according to a deterministic hierarchy. PLoS One 4: e6498.
- 19. Lu LL, Liu YJ, Yang SG, Zhao QJ, Wang X, et al. (2006) Isolation and characterization of human umbilical cord mesenchymal stem cells with hematopoiesis-supportive function and other potentials. Haematologica 91: 1017–1026.
- 20. Karahuseyinoglu S, Cinar O, Kilic E, Kara F, Akay GG, et al. (2007) Biology of stem cells in human umbilical cord stroma: In situ and in vitro surveys. Stem Cells 25: 319–331.
- 21. Weiss ML, Medicetty S, Bledsoe AR, Rachakatla RS, Choi M, et al. (2006) Human umbilical cord matrix stem cells: preliminary characterization and effect of transplantation in a rodent model of Parkinson's disease. Stem Cells 24: 781–792.
- 22. Lund RD, Wang S, Lu B, Girman S, Holmes T, et al. (2007) Cells isolated from umbilical cord tissue rescue photoreceptors and visual functions in a rodent model of retinal disease. Stem Cells 25: 602–611.
- 23. Rachakatla RS, Marini F, Weiss ML, Tamura M, Troyer D (2007) Development of human umbilical cord matrix stem cell-based gene therapy for experimental lung tumors. Cancer Gene Ther 14: 828–835.
- 24. Conconi MT, Burra P, Di Liddo R, Calore C, Turetta M, et al. (2006) CD105(+) cells from Wharton's jelly show in vitro and in vivo myogenic differentiative potential. Int J Mol Med 18: 1089–1096.
- 25. Liu Y, Wu J, Zhu Y, Han J (2012) Therapeutic application of mesenchymal stem cells in bone and joint diseases. Clin Exp Med.
- 26. Crisostomo PR, Wang Y, Markel TA, Wang M, Lahm T, et al. (2008) Human mesenchymal stem cells stimulated by TNF-alpha, LPS, or hypoxia produce growth factors by an NF kappa B- but not JNK-dependent mechanism. American Journal of Physiology-Cell Physiology 294: C675–C682.
- 27. Choi M, Lee HS, Naidansaren P, Kim HK, O E, et al. (2013) Proangiogenic features of Wharton's jelly-derived mesenchymal stromal/stem cells and their ability to form functional vessels. Int J Biochem Cell Biol 45: 560–570.
- 28. Miranville A, Heeschen C, Sengenes C, Curat CA, Busse R, et al. (2004) Improvement of postnatal neovascularization by human adipose tissue-derived stem cells. Circulation 110: 349–355.
- 29. De Francesco F, Tirino V, Desiderio V, Ferraro G, D'Andrea F, et al. (2009) Human CD34(+)/CD90(+) ASCs Are Capable of Growing as Sphere Clusters, Producing High Levels of VEGF and Forming Capillaries. PLoS One 4.
- 30. Fu X, Li H (2009) Mesenchymal stem cells and skin wound repair and regeneration: possibilities and questions. Cell Tissue Res 335: 317–321.
- 31. Wang SH, Lin SJ, Chen YH, Lin FY, Shih JC, et al. (2009) Late outgrowth endothelial cells derived from Wharton jelly in human umbilical cord reduce neointimal formation after vascular injury: involvement of pigment epithelium-derived factor. Arterioscler Thromb Vasc Biol 29: 816–822.
- 32. Kinnaird T, Stabile E, Burnett MS, Shou M, Lee CW, et al. (2004) Local delivery of marrow-derived stromal cells augments collateral perfusion through paracrine mechanisms. Circulation 109: 1543–1549.
- 33. Kondo K, Shintani S, Shibata R, Murakami H, Murakami R, et al. (2009) Implantation of adipose-derived regenerative cells enhances ischemia-induced angiogenesis. Arterioscler Thromb Vasc Biol 29: 61–66.
- 34. Meyer T, Pfeifroth A, Hocht B (2008) Isolation and characterisation of mesenchymal stem cells in Wharton's jelly of the human umbilical cord: potent cells for cell- based therapies in paediatric surgery? European Surgery-Acta Chirurgica Austriaca 40: 239–244.
- 35. Guzman-Gutierrez E, Sandoval C, Nova E, Castillo JL, Vera JC, et al. (2010) Differential expression of functional nucleoside transporters in non-differentiated and differentiated human endothelial progenitor cells. Placenta 31: 928–936.
- 36. Diaz-Perez F, Radojkovic C, Aguilera V, Veas C, Gonzalez M, et al. (2012) L-arginine transport and nitric oxide synthesis in human endothelial progenitor cells. J Cardiovasc Pharmacol 60: 439–449.
- 37. Fernandez P, Jara C, Aguilera V, Caviedes L, Diaz F, et al. (2012) Adenosine A(2)A and A(3) receptors are involved in the human endothelial progenitor cells migration. J Cardiovasc Pharmacol 59: 397–404.
- 38. Kim SW, Zhang HZ, Guo L, Kim JM, Kim MH (2012) Amniotic mesenchymal stem cells enhance wound healing in diabetic NOD/SCID mice through high angiogenic and engraftment capabilities. PLoS One 7: e41105.
- 39. Wu YJ, Chen L, Scott PG, Tredget EE (2007) Mesenchymal stem cells enhance wound healing through differentiation and angiogenesis. Stem Cells 25: 2648–2659.
- 40. Galiano RD, Michaels J5th, Dobryansky M, Levine JP, Gurtner GC (2004) Quantitative and reproducible murine model of excisional wound healing. Wound Repair Regen 12: 485–492.
- 41. Kim SW, Zhang HZ, Guo L, Kim JM, Kim MH (2012) Amniotic mesenchymal stem cells with robust chemotactic properties are effective in the treatment of a myocardial infarction model. Int J Cardiol.
- 42. Panepucci RA, Siufi JLC, Silva WA, Proto-Siquiera R, Neder L, et al. (2004) Comparison of gene expression of umbilical cord vein and bone marrow-derived mesenchymal stem cells. Stem Cells 22: 1263–1278.
- 43. Chen MY, Lie PC, Li ZL, Wei X (2009) Endothelial differentiation of Wharton's jelly-derived mesenchymal stem cells in comparison with bone marrow-derived mesenchymal stem cells. Exp Hematol 37: 629–640.
- 44. Wang HS, Hung SC, Peng ST, Huang CC, Wei HM, et al. (2004) Mesenchymal stem cells in the Wharton's jelly of the human umbilical cord. Stem Cells 22: 1330–1337.
- 45. Orbay H, Tobita M, Mizuno H (2012) Mesenchymal stem cells isolated from adipose and other tissues: basic biological properties and clinical applications. Stem Cells Int 2012: 461718.
- 46. Petsa A, Gargani S, Felesakis A, Grigoriadis N, Grigoriadis I (2009) Effectiveness of protocol for the isolation of Wharton's Jelly stem cells in large-scale applications. In Vitro Cell Dev Biol Anim 45: 573–576.
- 47. Alaminos M, Perez-Kohler B, Garzon I, Garcia-Honduvilla N, Romer B, et al. (2010) Transdifferentiation Potentiality of Human Wharton's Jelly Stem Cells Towards Vascular Endothelial Cells. J Cell Physiol 223: 640–647.
- 48. Gang EJ, Jeong JA, Han S, Yan Q, Jeon CJ, et al. (2006) In vitro endothelial potential of human UC blood-derived mesenchymal stem cells. Cytotherapy 8: 215–227.
- 49. Oswald J, Boxberger S, Jorgensen B, Feldmann S, Ehninger G, et al. (2004) Mesenchymal stem cells can be differentiated into endothelial cells in vitro. Stem Cells. 22: 377–384.
- 50. Liu JW, Dunoyer-Geindre S, Serre-Beinier V, Mai G, Lambert JF, et al. (2007) Characterization of endothelial-like cells derived from human mesenchymal stem cells. J Thromb Haemost. 5: 826–834.
- 51. Stenderup K, Justesen J, Clausen C, Kassem M (2003) Aging is associated with decreased maximal life span and accelerated senescence of bone marrow stromal cells. Bone. 33: 919–926.
- 52. Miao Z, Jin J, Chen L, Zhu J, Huang W, et al. (2006) Isolation of mesenchymal stem cells from human placenta: comparison with human bone marrow mesenchymal stem cells. Cell Biol Int 30: 681–687.
- 53. Lee PC, Salyapongse AN, Bragdon GA, Shears LLII, Watkins SC, et al. (1999) Impaired wound healing and angiogenesis in eNOS-deficient mice. Am J Physiol 277: H1600–H1608.
- 54. Li HH, Fu XB, Ouyang YS, Cai CL, Wang J, et al. (2006) Adult bone-marrow-derived mesenchymal stem cells contribute to wound healing of skin appendages. Cell Tissue Res 326: 725–736.
- 55. Noiseux N, Gnecchi M, Lopez-Ilasaca M, Zhang L, Solomon SD, et al. (2006) Mesenchymal stem cells overexpressing Akt dramatically repair infarcted myocardium and improve cardiac function despite infrequent cellular fusion or differentiation. Mol Ther 14: 840–850.
- 56. Chen L, Tredget EE, Wu PY, Wu Y (2008) Paracrine factors of mesenchymal stem cells recruit macrophages and endothelial lineage cells and enhance wound healing. PLoS One 3: e1886.