Isolation and culture of human mesenchymal stem cells (MSCs) from the umbilical cord matrix (UCM)

Human umbilical cords were obtained after birth from healthy donors, with written informed consent of the parent(s) and the study was approved by the Ethics Committee of Maternidade de Bissaya Barreto – Centro Hospitalar de Coimbra (ref. 356/Sec). Samples were stored at room temperature (RT) in sterile 50 ml conical tubes (VWR International) for 12 to 48 h before tissue processing. The isolation procedure of MSCs was adapted from a protocol described by Reinisch et al. [27]. Each umbilical cord unit was manipulated under sterile conditions using a class-II biosafety cabinet (Heraeus HS-18) and cut into sections of about 5 cm. The pieces were washed 2 or 3 times using sterile PBS (Sigma-Aldrich), to remove the blood. The umbilical vein was washed with sterile PBS using a 10 ml sterile syringe to remove blood and blood clots. Subsequently, the vein and arteries were removed to avoid endothelial cell contamination. The Wharton's jelly (WJ) was then cut into fragments of 2–5 mm with the help of sterile scalpel and forceps (Fine Science Tools). Groups of approximately 24 fragments were transferred to each 55 cm² tissue culture (TC) plate (Corning) and left to dry for 5 min inside the biosafety cabinet to promote adhesion of the fragments to the polystyrene surface. Once the cord fragments were properly adherent to the plastic, MSC proliferation medium [Alpha-MEM without nucleosides (Life Technologies, cat. no. 22561-021) supplemented with 10% volume/volume (v/v) MSC-qualified Fetal Bovine Serum (FBS) (Hyclone), 10 U/ml of Penicillin, 10 µg/ml Streptomycin and 2.5 µg/ml Amphotericin B (all from Life Technologies)] was added to the culture plate, until all the fragments were completely covered. The fragments were then cultured for 10 days in an incubator (Shel Lab) at 37°C with 5% CO₂/95% air and 95% humidity, until MSCs started migrating out of the UCM pieces and forming well defined colonies. Then, UCM fragments were removed from the tissue culture plate and cells were passaged.

For the passaging, cells were detached and dissociated using Trypsin (500 µg/ml)-EDTA (200 µg/ml) solution (Life Technologies). Trypsin was inactivated using 10x the volume of MSC proliferation medium, centrifuged (300×g, for 5 min at RT), counted and seeded at a density of 3,000 cells/cm² in MSC proliferation medium and maintained in a CO₂ incubator (as above) until sub-confluence (80–90% confluence).

For cryopreservation, cells were treated in a similar way as described for the passaging procedure, except that cells were resuspended in cryopreservation solution (FBS supplemented with 10% DMSO – Sigma Aldrich) into cryotubes and transferred to a cell freezing container - Mr. Frosty - (both from Thermo Scientific), that was relocated inside a −80°C freezer to achieve a rate of cooling close to 1°C/minute. After 16 h, the cryotubes were transferred to a liquid nitrogen cryotank for long-term storage.

Proliferation kinetics of UCM-MSCs

MSCs isolated from 3 independent cord samples were continuously cultured from P2 to P8 and counted once they reached 80% confluence at each passage. The population doubling (PD) rate was determined at each passage using the equation N_H/N_I = 2 ^X, or [log₁₀(N_H) — log₁₀(N_I)]/log₁₀(2) = X, where N_I represents the number of cells plated at each passage, N_H the number of cells harvested at the end of each respective passage and X the population doubling (PD), as described [28].

The PD for each passage was calculated and added to the PD of the previous passages to generate data for cumulative population doublings (CPD). In addition, the generation time (GT) - average time between two cell doublings - was calculated from P2 to P8 using the following formula, as described [29]: X  =  [log₁₀(2) x Δt]/[log₁₀(N_H) — log₁₀(N_I)].

The total number of cells (TNC) was determined at each passage (P1-P8) by cumulative counting of the cells once they reached a confluence of 80%, using the formula: X  =  N_H x B/N_I, in which B represents the total number of cells in the previous passage. TNC accounts for the theoretical number of cells that could be obtained if no cells were discarded between each passage.

Colony-forming unit-fibroblast (CFU-F) assay

The colony-forming unit-fibroblast (CFU-F) assay of MSCs was determined for 3 independent cord samples at P2 and P8. Cells were seeded at 3 cells/cm² on 55 cm² TC dishes in MSC proliferation medium and cultured for 15 days. One third of the medium was replaced twice a week. Cells were fixed with 4% paraformaldehyde (PFA) (Sigma-Aldrich) for 20 min at RT and stained with Giemsa solution (Sigma-Aldrich). Individual colonies were counted manually.

Multilineage differentiation of UCM-MSCs

Chondrogenic, adipogenic and osteogenic differentiation capacity of isolated MSCs was assessed using early passage (P2-P3) MSCs. For osteogenic and adipogenic differentiation, cells were plated at 1,000 cells/cm² in the presence of Dulbecco's modified Eagle's medium (DMEM) (Life Technologies) supplemented with 10% MSC-qualified FBS (Hyclone) until reaching 80–100% confluence. Medium was then removed and StemPRO (Life Technologies) osteogenic or adipogenic differentiation medium was added and cells were cultured for 14 days. For chondrogenic differentiation, the cells were plated on low-attachment plates (Corning). A pellet of 5×10⁵ to 1×10⁶ cells was resuspended and droplets of this suspension were plated on the surface of each well. After plating, StemPRO chondrogenic differentiation medium was added and cells were cultured for 14 days.

For osteogenic differentiation assessment, alkaline phosphatase (ALP) and Von Kossa staining were performed. For ALP staining, the cells were washed with PBS and fixed in 10% cold neutral-buffered formalin (Sigma-Aldrich) for 15 min.

Cells were then washed and kept in distilled water for another 15 min and then stained with a 0.1 M Tris-HCl solution (Sigma-Aldrich) containing the substrate Naphtol AS MX-PO4 (0.1 mg/ml) (Sigma-Aldrich) for ALP in dimethylformamide (Fischer Scientific) and 0.6 mg/ml Red Violet LB salt (Sigma-Aldrich) for 45 min. The excess staining was removed by washing 3 times with distilled water. For the Von Kossa staining, the cells were washed with PBS and stained with 2.5% (w/w) silver nitrate (Sigma-Aldrich) for 30 min at RT and then washed 3 times with distilled water.

For adipogenic differentiation assessment, cells were washed with PBS and fixed with PFA using a 2.5% solution for 30 min at RT. Cells were washed once in distilled water and incubated with 0.3% Oil Red-O solution (Sigma-Aldrich) for 1 h at RT, and then washed twice with distilled water.

For chondrogenic differentiation assessment, cells were washed once with PBS and fixed with 2% PFA solution for 30 min at RT. The cells were then washed with PBS and stained with 1% Alcian Blue solution (Sigma-Aldrich) prepared in 0.1 M HCl for 30 min. The excess was removed by washing 3 times with PBS and the cells were then maintained in distilled water.

Immunophenotypic characterization of UCM-MSCs

The immunophenotypic characterization of UCM-MSCs [30] was performed at passages 2 and 8. Cells were dissociated using StemPro Accutase (Life Technologies), labeled with antibodies against the indicated antigens and analyzed by flow cytometry (FACS Canto II, Becton-Dickinson). The following antibodies were used for the labeling: mouse PO anti-human CD45 IgG1, clone HI30 (5 µL/test) from Life Technologies; mouse APC anti-human CD90 IgG1, clone 5E10 (0.2 mg/ml) from BD Pharmingen; mouse PE anti-human CD105 IgM, clone ALB9 (10 µL/test) from Beckman Coulter; mouse PB anti-human CD11b IgG1, clone ICRF44 (0.2 mg/ml) from BD Pharmingen; mouse Pe-Cy7 anti-human CD13 IgG1, clone WM15 (2.5 µL/test) from BD Pharmingen; mouse PerCP-Cy5.5 anti-human CD34 IgG1, clone 8G12 (5 µL/test) from BD Pharmingen; mouse PE anti-human CD73 IgG1, clone AD2 (10 µL/test) from BD Pharmingen; mouse PE anti-human NFGR IgG1, clone C40-1457 (10 µL/test) from BD Pharmingen.

Neural-like induction of UCM-MSCs

Immunocytochemistry analysis and F-actin staining

Cells were fixed with 4% PFA for 20 min at RT, rinsed with PBS and permeabilized with PBS with 0.1% Triton X-100 for 20 min, except when using anti-human Golgi antibody, where permeabilization was performed using cold acetone for 15 seconds. Cells were incubated in blocking solution (PBS with 0.1% BSA) for 30 min at RT. Cells were incubated with primary antibodies diluted in blocking solution (see below), overnight at 4°C in humidified conditions. The cells were washed with PBS and then incubated with the appropriate fluorescently-labeled secondary antibodies (see below) in PBS with 0.1% BSA for 1 h at RT. Cells were washed and fixed with 4% PFA for 5 min, to crosslink antigen/antibody complexes, washed 3 times and then incubated for 4 min with 200 ng/ml of DAPI (Sigma-Aldrich). To stain cells for polymerized actin (F-actin), a solution of 4 µM FITC-phalloidin (Sigma-Aldrich) in PBS was incubated for 20 min at RT at the end of the immunocytochemistry protocol (after the incubation with the secondary antibodies), and then washed 3 times with PBS to remove unbound reagent.

The following primary antibodies were used: mouse anti-nestin, clone 10C2 (1∶400) from Millipore; mouse anti-O4, clone O4 (1∶400) from R&D Systems; mouse anti-A2B5, clone 105 (1∶500) from R&D Systems; rabbit anti-galactocerebroside (GalC) (1∶50) from Millipore; rabbit anti-MBP (1∶200) from Sigma-Aldrich; rabbit anti-GFAP (1∶500) from Dako Cytomation; rabbit anti-β-III-tubulin IgG1, clone TUJ1 1-15-79 (1∶2000) from Covance and mouse anti-human Golgi, clone 371-4 (1∶30) from Chemicon. The following secondary fluorescently-labeled antibodies were used according to the host species of each primary antibody: Alexa Fluor 488 donkey anti-mouse IgG (H+L) (1∶200) and Alexa Fluor 568 goat anti-rabbit IgG (H+L) (1∶200), both from Life Technologies.

Fluorescence microscopy was performed using a Zeiss Axiovert 200 M microscope using AxioVision Release 4.8 software (Zeiss) for image acquisition. Exposure time was the same for each marker analyzed and for each independent experiment.

Image analysis and fluorescence quantification

Mean fluorescence intensity (MFI) was quantified using ImageJ software (NIH) for each marker analyzed. In detail, for each marker and for each independent experiment (consisting of cells in all differentiation steps), the intensity of the background and the signal were obtained by determining the background and signal threshold levels (Threshold tool) of at least 3 different fields belonging to the differentiation step with the highest signal intensity. The average threshold levels were then calculated and applied (using the ‘Set threshold’ tool) to all images under analysis (for each marker in each independent experiment). After setting the thresholds, the MFI of the background and the signal were obtained using the Measure tool and the first was subtracted to the latter, analyzing at least 3 fields for each differentiation step. The resulting values were then averaged and subjected to statistical analysis.

RNA isolation and Real-Time RT-PCR

Total RNA was extracted from cells in culture in different stages of differentiation using the RNeasy Mini Kit (Qiagen) and treated with DNase-I (Qiagen). 250 ng of RNA were used in each duplicate per sample to synthetize cDNA using the SuperScript II Reverse Transcriptase (Life Technologies). Quantitative PCR analysis was done in each duplicate using the total amount of cDNA obtained in each reaction using Power Sybr Green PCR Master Mix (Life Technologies) using a real-time PCR system (7500 Fast real-time PCR System and 7500 Software V2.0.4, Applied Biosystems) and values were normalized to the levels of actin and undifferentiated MSCs were used as the control sample. The PCR cycling parameters were 94°C for 5 min; 30 cycles of 30 seconds at 94°C, 1 minute at 60°C, and 1 minute at 72°C; and final extension at 72°C for 10 min. The primers were as follows: actin forward, 5′-CAGAAGGATTCCTATGTGGGC-3′, reverse, 5′- GAGGGCATACCCCTCGTAGAT-3′; fibronectin, forward, 5′-GAGATCAGTGGGATAAGCAGCA-3′, reverse, 5′- CCTCTTCA-TGACGCTTGTGGA-3′; sox2, forward, 5′-CAGGAGAACCCCAAGATGC-3′, reverse, 5′-GCAGCCGCTTAGCCTCG-3′; nestin, forward, 5′-CAGCTGGCGCACCTCAAGATG-3′, reverse, 5′- AGGGAAGTTGGGCTCAGGACTGG-3′; mbp, forward, 5′-CTGGGCAGC-TGTTAGAGTCC-3′, reverse, 5′-TGGAGCAAAGGTTTGGTGTC-3′; gfap, forward, 5′-CTGTTGCCAGAGATGGAGGTT-3′, reverse, 5′-TCATCGCTCAGGAGGTCCTT-3′; neurofilament, forward, 5′-GAGCGCAAAGACTACCTGAAGA-3′, reverse, 5′-CAGC-GATTTCTATATCCAGAGCC-3′.

Co-culture of OPC/OL-like cells with mouse DRG neurons

Purified dorsal root ganglion (DRG) neurons were obtained by a culture system previously described [33] with minor modifications. Briefly, DRGs were dissected from E14-E16 C57BL/6 mice and explants were transferred to 22-mm glass coverslips previously coated with 0.1 mg/ml poly-D-lysine for 1 h at RT followed by 10 µg/ml laminin-1 (Sigma-Aldrich) for 1 h at 37°C. DRG maturation medium was composed by DMEM high glucose supplemented with 10% FBS, 50 ng/ml Nerve Growth Factor (NGF) and 10 U/ml of Penicillin/10 µg/ml Streptomycin (all from Life Technologies). After 2 to 3 days of culture, 0.5 µM Cytosine Arabinose (AraC; Sigma-Aldrich) was added to inhibit proliferating cells. After a 10 day period of DRG maturation, OPC-like cells derived from UCM-MSCs were plated on top of the neurons at a density of 20,000 cells/cm² and the culture remained in OL-like maturation medium supplemented with T3 [as described in the section Maturation of OPC-like cells into Oligodendrocyte (OL)-like cells]. Medium was changed every 3 or 4 days. By the end of 14 days, cells were fixed and immunocytochemistry analysis was performed.

Statistical analysis

Statistical analysis was performed by Kruskal-Wallis one-way ANOVA followed by Dunn's multiple comparison test or two-tailed Mann-Whitney test (when data was not normal or homoscedastic), or by repeated measures one-way ANOVA followed by Tukey's test (when a parametric analysis of paired data was appropriate), as indicated. Analysis was done using the software GraphPad Prism 5. Values represent mean ± SEM of at least 3 independent experiments (*P<0.05; **P<0.01 and ***P<0.001 for statistically significant differences).

Protein extraction and quantification, SDS-PAGE, and immunobloting

To obtain protein extracts from MSCs, cells from one TC plate with 21 cm² were detached using StemPro Accutase. The enzyme was inactivated with PBS and then cells were centrifuged (300×g, for 5 min at RT). The pellet was washed with PBS and centrifuged again. The supernatant was discarded and the pellet was frozen in liquid nitrogen for 20 seconds and stored at −80°C until further processing. To prepare protein extracts, frozen cell pellets were incubated with 200 µL of cold (4°C) extraction buffer [50 mM Tris-HCl pH 7.4 supplemented with cOmplete, EDTA-free protease inhibitor cocktail (Roche)]. Cells were disrupted using a 200 µl micropipette by repeatedly pipetting up and down on ice and then sonicated on ice, using an ultrasonic cell disrupter (VibraCell - model VCX 750, Sonics & Materials, Inc.) for 1 min (cycles of 1 s pulse interspaced by 1 s) with an amplitude of 40%, to fully disrupt the membrane structure. After centrifugation (at 20,000×g at 4°C for 30 min), the supernatants were retrieved and the protein was precipitated by adding 6 volumes of cold acetone (−20°C). Samples were then vortexed and centrifuged (at 20,000×g at 4°C for 30 min). The pellets were washed with 90% (v/v) cold acetone and then allowed to dry at RT for 15 min. Pellets were resuspended in 100 µl of 1x sample buffer [58.3 mM Tris/HCl, pH 6.8, 17 µg/ml SDS (BioRad), 50 µl/ml glycerol (GE Healthcare), 15.5 µg/ml DTT (Bioron), 20 µg/ml Bromophenol Blue (GE Healthcare)] and heated at 95°C for 5 min. Protein was quantified using the 2D quant kit (GE Healthcare), according to manufacturer's instructions.

To obtain protein extracts from brain cortex to be used as a positive control for MBP, the brain of one adult female Wistar rat was extracted and stored at −80°C until further processing (procedures were performed according to the European Union Directive 86/609/EEC and the legislation Portaria n. 1005/92, issued by the Portuguese Government for the protection of animals used for experimental and other scientific purposes). To obtain protein extracts, the brain was thawed with cold extraction buffer and then transferred to one centrifuge tube with 750 µL of extraction buffer. The tissue was homogenized using an ultrasonic cell disrupter microtip (VibraCell - model VCX 130, Sonics & Materials, Inc.) with an amplitude of 40–60% (cycles of 1 s pulse interspaced by 1 s) until homogenized. After centrifugation (at 5,000×g at 4°C for 5 min), the supernatant was collected. Next, protein was precipitated (200 µL of the total protein extract) and further processes as described to obtain protein extracts from MSCs.

Protein samples (15 µg/lane for rat brain cortex and 35 µg/lane for MSCs extracts) were separated by SDS-PAGE on SDS/discontinuous 4–12.5% (w/v) acrylamide–bisacrylamide (Bio-Rad) gels at constant voltage (80V while samples were in the stacking gel and 120V after entering the resolving gel, for about 1 h 30 min) using a Mini-Protean III (BioRad) apparatus, with running buffer (BioRad).

Blotting was done using polyvinylidene fluoride (PVDF) membranes (BioRad). Membranes were activated by rinsing in methanol, washed with deionized water and then kept in transfer buffer (BioRad). Protein transfer (western-blot) was performed using a Trans-Blot Turbo transfer system (BioRad) according to the manufacturer's instructions, using Trans-Blot Turbo transfer buffer (BioRad).

For immunoblotting, membranes were blocked with blocking solution [PBS-0.1% (v/v) Tween 20 (GE Healthcare) and 5 g/100 ml non-fat dried milk] for 1 h at RT. Incubation with anti-MBP antibody was performed with gentle agitation, overnight at 4°C, followed by 1 h at RT, while staining with anti-GAPDH antibody occurred for 2 h at RT. Membranes were washed with PBS-0.1% (v/v) Tween 20 and then incubated with the respective secondary antibody diluted in blocking solution (1 h at room temperature) and washed with PBS-0.1% (v/v) Tween 20.

Primary antibodies used were diluted in blocking solution and were rabbit monoclonal (clone EP1448Y) anti-MBP (Abcam, Ab53294) diluted 1∶1,000 and mouse monoclonal anti-GAPDH (Santa Cruz Biotechnology, SC-365062) diluted 1∶500. The respective secondary antibodies were also diluted in blocking solution and were goat anti-rabbit antibody conjugated with alkaline phosphatase (Jackson ImmunoResearch Laboratories, Inc.) diluted 1∶6,000 and donkey anti-mouse antibody conjugated with Alexa 488 (Life Technologies) diluted 1∶500.

An ECF (enhanced chemiflorescence) kit (GE Healthcare) was used for detection of the alkaline phosphatase-conjugated antibody, according to the manufacturer's instructions. Alkaline phosphatase activity (using ECF) and Alexa 488 fluorescence was visualized on a Molecular Imager FX system (BioRad) using the software Quantity One (BioRad). The integrated density of the western-blot bands was obtained using Image J software, using the ‘Threshold’ tool, followed by the command ‘Analyze Particles’.

Isolation, expansion and characterization of human umbilical cord matrix mesenchymal stem cells (UCM-MSCs)

Human MSCs were isolated from 12 umbilical cords, as described in the ‘Materials and Methods’ section, with a success rate of 100% (in agreement to what has been previously reported [34]). At the end of 5 to 10 days in culture, several Wharton's jelly fragments were still attached to the tissue culture (TC) dishes and showed cells migrating from the tissue. Colonies of cells displaying an MSC-like phenotype, with spindle-shaped morphology could be readily identified by phase-contrast microscopy (Figure S1). Cells proliferated rapidly and formed compact colonies by the end of 10-14 days in vitro. Then, the cells were detached and dissociated and by the end of Passage 1 (P1) constituted a homogeneous monolayer with MSC-like morphology (adherent, spindle-shaped fibroblastoid-like cells). The cells were then either cryopreserved or expanded until passage 8 (P8) and further characterized.

UCM-MSCs have been described to proliferate readily in vitro [34], [35], hence we sought to thoroughly characterize their proliferation kinetics, namely the total number of cells - TNC (Figures 1A, B), generation time - GT (Figure 1C), population doubling - PD (Figure 1D) and cumulative population doubling - CPD (Figure 1E) of cells isolated from 3 randomly selected independent samples (CM#2, CM#3 and CM#7) between passages 2 and 8 (P2-P8). Notably, by the end of P4 (17 to 21 days after the initial isolation of the UCM explants), the total number of cells obtained from each sample (i.e., if no cells had been discarded until that point) had surpassed 1×10⁹ cells (Figures 1A, B), well above what is considered relevant for clinical applications [29] (about 1–2 million cells per kg of body weight) and at passage 8 the TNC was 5.98×10¹³ (±4.76×10¹³). On average, between P2 and P8, the generation time ranged from 1.02±0.071 (SEM) to 1.55±0.316 (SEM) days (Figure 1C). Moreover, the colony-forming unit-fibroblast (CFU-F) capacity of the cells (Figure 1F) was maintained throughout P2 to P8 [48.5±7.52 (SEM) and 43.8±16.20 (SEM)]. For both types of assays (GT and CFU-F), no statistically significant differences were found between the time points, indicating that neither the generation time nor the CFU-F capacity were significantly affected by passaging the cells until P8. Hence, the cells showed high proliferative capacity typical of bona-fide MSCs, maintained a short generation time from passages 2 to 8 and reached a clinically relevant number (superior to 1×10⁹ cells) within a time-frame of 17 to 21 days.

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Figure 1. Proliferation and colony-forming capacity of human umbilical cord mesenchymal stem cells (hUCM-MSCs) in vitro.

Total number of cells (hUCM-MSCs) along time (days) and passage number (A, B), generation time (C), population doubling (D), cumulative population doubling (E) from passage 1 (P1) or P2 until P8, as indicated, and colony forming unit-fibroblast (CFU-F) number at P2 and P8 (F). Cells were isolated from 3 distinct human umbilical cord matrix samples (CM#2, #3 and #7). From P2 onwards (inclusively), cells were plated at a fixed density of 3,000 cells/cm², allowed to proliferate until sub-confluence and re-plated in the same way (A-E), or plated at 3 cells/cm² at P2 and P8 and cultured for 15 days for the CFU-F study (F). The total number of cells (TNC) was determined at each passage (P1-P8) by cumulative counting of the cells once they reached a confluence of 80% (A, B). The TNC designates the theoretical number of cells that could be obtained if no cells were discarded between each passage. The observed mean generation time (GT) was between 1.02 (±0.071) and 1.55 (±0.316) days (C), and no statistically significant differences were found in GT from passages 2 to 8 (Kruskal-Wallis one-way ANOVA followed by Dunn's Multiple Comparison test). The CFU-F capacity was maintained from P2 to P8 (F) and no statistically significant differences were found (two-tailed Mann-Whitney test). Bars represent mean ± SEM (B-F).

https://doi.org/10.1371/journal.pone.0111059.g001

Multilineage differentiation capacity and immunophenotypic characterization of UCM-MSCs

MSCs are multipotent stem cells that can differentiate in vitro into chondrocytes, osteocytes and adipocytes [2]. The multilineage differentiation potential of UCM-MSCs was demonstrated in culture (Figure 2A-C), under conditions that favor osteogenic, adipogenic or chondrogenic differentiation (Materials and Methods section). Chondrogenic induction was observed by Alcian Blue staining (Figure 2A), while osteogenic differentiation was evident by an increase in alkaline phosphatase (ALP) activity (reddish areas) and enhanced mineralization showed by von Kossa staining (dark areas), as shown in Figure 2B. Adipogenic induction, although less efficient, was visible by the cellular accumulation of lipid-rich vacuoles that stained with Oil Red-O (Figure 2C). These results indicate that UCM-MSCs possess the multilineage differentiation capacity characteristic of MSCs [2].

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Figure 2. Multilineage differentiation potential and immunophenotypic characterization of hUCM-MSCs.

The multilineage differentiation potential of hUCM-MSCs was demonstrated after 14 days in culture, under conditions that favour chondrogenic, osteogenic or adipogenic differentiation. Phase contrast images of cells stained with Alcian Blue for chondrogenesis (A), ALP and von Kossa for osteogenesis (B) or Oil Red-O for adipogenesis (C). Scale bar represents 100 µm. For immunophenotypic characterization, hUCM-MSCs at passage 2 were dissociated using Accutase (Life Technologies), labelled with antibodies against the indicated antigens and analysed by flow cytometry. Cells were positive for CD105 (D), CD73 (E), CD90 (F) and CD13 (G) and negative for CD34 (H), NGFR (I), CD11b (J) and CD45 (K) (pink lines) when compared with unlabelled MSCs (green lines), as depicted in the histograms. Histograms were obtained from one sample (UCM#2) at passage 2 and are representative of 3 independent samples at P2 and P8.

https://doi.org/10.1371/journal.pone.0111059.g002

Flow cytometry analysis showed that UCM-MSCs were positive for CD13, CD73, CD90 and CD105, while cells did not express CD34 and CD45 (hematopoietic lineage markers), CD11b and NGFR (Figure 2D-K and Table 1 represent the analysis of one sample in passage 2, while data from 3 independent samples at passage 2 and 8 is presented in Figure S2), showing that this phenotypic profile was consistent between different donors and with the MSC phenotype previously described by us [36] and others [30], [37], [38].

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Table 1. Summary of the flow cytometry analysis of UCM-MSCs.

https://doi.org/10.1371/journal.pone.0111059.t001

Differentiation of UCM-MSCs into an oligodendrocyte-like lineage

After assuring the fidelity of the UCM-MSCs obtained, we focused on the differentiation of these cells into oligodendrocyte-like cells using a stepwise protocol, based on the literature for neural- and oligodendroglial-like induction of distinct types of stem cells, including embryonic stem cells [24], [25], [39] and MSCs [7], [9], [22]. MSCs were subjected to distinct soluble factors, culture surfaces and coating conditions during the differentiation process (Figure S3). Briefly, undifferentiated MSCs (Figures 3A and 4A-C) were treated with epidermal growth factor (EGF) for 3 days for generation of neuroectodermal-induced MSCs (niMSCs) (Figure 3B). Next, niMSCs were cultured on non-treated polystyrene in the presence of EGF and basic fibroblast growth factor (bFGF) for induction into neural stem-like (NSC-like) cells (Figures 3C, D, F and 4D-F). The latter were then cultured on tissue culture polystyrene using different coating conditions (Figure S3) for the generation of oligodendrocyte precursor-like (OPC-like) cells (Figure 4G-O). Finally, these progenitor cells were seeded on poly-D-lysine (PDL) and laminin-2/merosin (MN) double-coated surfaces for the generation of oligodendrocyte-like (OL-like) cells (Figures 5 and 6).

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Figure 3. Cell morphology and expression of nestin in hUCM-MSC and during the first steps of differentiation.

Phase contrast images of undifferentiated hUCM-MSCs (A), neuroectodermal-like induced MSCs – niMSCs (B) and NSC-like cells after 3 (C) and 18 days (D) in culture in NSC induction medium. Immunofluorescence microscopy images for the neural precursor marker nestin (in green) in undifferentiated MSCs (E) and NSC-like cells (F). Counterstaining of nuclei was performed with DAPI (in blue). In (A-D) the scale bar represents 200 µm and in (E-F) represents 50 µm. Images are representative of at least 3 independent experiments.

https://doi.org/10.1371/journal.pone.0111059.g003

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Figure 4. Expression of neural- and oligodendroglial-precursor markers in hUCM-MSCs, NSC- and OPC-like cells.

The expression of nestin (neural precursor marker), A2B5 (OPC marker) and O4 (OPC/OL marker) in undifferentiated hUCM-MSCs (A-C), after neural-like induction (D-F) and OPC-like induction using non-coated (G-I), laminin-2 (merosin-MN)-coated (J-L) and fibronectin (FN)-coated (M-O) tissue culture polystyrene was determined by immunofluorescence microscopy (in green). (P) and (Q) are inserts of (C) and (O) images, respectively, to highlight the presence of the typical OPC punctate and perinuclear distribution of A2B5 in OPC-like cells (Q), in contrast with control hUCM-MSCs (P). Counterstaining of nuclei was performed with DAPI (in blue) and the scale bars represent 50 µm. Images are representative of at least 3 independent experiments.

https://doi.org/10.1371/journal.pone.0111059.g004

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Figure 5. Expression of neural precursor, astrocytic and neuronal markers by OL-like cells.

The expression of the astrocytic marker GFAP (A-D and I-J), the neuronal marker beta-III-tubulin (E-H and K-L) and the neural precursor marker nestin (M-P and Q-S) in OL-like cells derived from OPC-like cells cultured on fibronectin (A-H and M-P) was determined by immunofluorescence microscopy. OL-like cells were differentiated for 10 days on laminin-2 (merosin)-coated wells, in the presence of T3 (thyroid hormone), F3 (contactin), T3+F3 or 7 days T3 followed by 3 days T3+F3 (T3+3d F3), as indicated. It was evident that the neural precursor marker nestin decreased in the OL-like stage of differentiation (M-P), as compared to NSC- or OPC-like cells (R and S, respectively), indicating maturation of the OL-like cells. It was also clear that the astrocytic marker GFAP was present in rat astrocytes as expected (I), but essentially absent in OL-like cells (A-D) or hUCM-MSCs (J). Similarly, the neuronal marker beta-III-tubulin was detected in the neuronal cell line SH-SY5Y (K) but undetected in OL-like cells (E-H) or hUCM-MSCs (L). Counterstaining of nuclei was performed with DAPI (in blue) and the scale bars represent 50 µm - scale bar in image (P) is representative for images (A-P) and scale bar in image (S) is representative for images (Q-S). Images are representative of at least 3 independent experiments.

https://doi.org/10.1371/journal.pone.0111059.g005

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Figure 6. Expression of oligodendroglial markers by OL-like cells.

The expression of the OPC marker A2B5 (A-D), the OPC/OL marker O4 (E-H) and the OL markers GalC (I-L) and MBP (M-P) in OL-like cells derived from OPC-like cells cultured on fibronectin was determined by immunofluorescence microscopy. OL-like cells were differentiated for 10 days on laminin 2 (merosin)-coated wells, in the presence of T3 (thyroid hormone), F3 (contactin), T3+F3 or 7 days T3 followed by 3 days T3+F3 (T3+3d F3), as indicated. It could be observed that the OPC marker A2B5 greatly decreased in the last stage of differentiation (A-D), as compared to OPC-like cells (Q), indicating maturation of the OL-like cells. The decrease was also more evident when OL-like cells were derived from OPC-like cells cultured on fibronectin (FN)-coated wells (U) than in laminin-2/merosin (LM/MN)-coated wells (V). The OPC/OL marker O4 was expressed at high levels in OL-like cells (E-H), in contrast to the low levels in hUCM-MSCs (R). The OL marker GalC was more highly expressed in OL-like cells (I-L) as compared to MSCs (S), while MBP seemed to be expressed (M-P) at similar levels (T). Counterstaining of nuclei was performed with DAPI (in blue) and the scale bars represent 50 µm - scale bar in image (T) is representative for images (A-T) and scale bar in image (V) is representative for images (U-V). Images are representative of at least 3 independent experiments.

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Induction of MSCs into neural-like precursor cells

MSCs used for differentiation experiments were taken from cryopreserved cell batches between passages 3 and 7 obtained from 3 independent donors, characterized as described above (Figures 1 and 2). After 3 days under neuroectodermal-inducing conditions (in the presence of N2 and EGF) the niMSCs kept the spindle-shape morphology (Figure 3B) characteristic of MSCs (Figure 3A) and seemed to maintain proliferation.

In order to induce the differentiation of niMSCs into a NSC-like phenotype, cells were seeded on non-treated polystyrene plates (sterile, non-tissue culture ‘bacterial dishes’) and cultured up to 18 days (Figure 3C,D). The induction medium contained the soluble factors bFGF and EGF, described to favor neuroectodermal-like fate of MSCs [7], [9]. After 3 days in culture (Figure 3C), cells had formed patches or small colonies that were maintained until 18 days in NSC-induction medium (Figure 3D). Although undifferentiated MSCs already expressed nestin [11] to a certain extent (Figures 3E and 4A), there was an increase in the expression of this NSC marker after the NSC-like induction phase, as addressed by immunocytochemistry (Figures 3F and 4D). The quantification of the mean fluorescence intensity (MFI) of immunofluorescence images (see Materials and Methods) was addressed for distinct neural markers at the different stages of differentiation under study (Figure 7A-D). The MFI of nestin in cells after NSC-like induction (NSC-like) showed a significant increase compared to undifferentiated MSCs (Figure 7A).

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Figure 7. Quantification of mean fluorescence intensity (MFI) of immunofluorescence images and mRNA expression of differentiation stage markers.

MFI values for nestin (A), O4 (B), GalC (C) and MBP (D) of immnunofluorescence images (representative images on Figures 4 to 6) were quantified using Image J software. The graphics represent results regarding cells cultured during the OPC-like differentiation stage on fibronectin-coated TC polystyrene. OL-like cells were differentiated for 10 days on laminin 2 (merosin)-coated wells, cultured in the presence of T3, F3, T3F3 or 7dT3+3dT3F3, as indicated. Bars represent mean ± SEM of at least 3 independent experiments. Statistical analysis was performed by repeated measures one-way ANOVA followed by Tukey's Multiple Comparison Test. Statistically significant differences for each marker between MSCs and cells at other differentiation stages were indicated on top of the corresponding graphic bars, while differences between other conditions were represented using connectors (*P<0,05; **P<0,01 and ***P<0,001). Real time RT-PCR analysis (E) was performed for genes representative of neural progenitors (sox2 and nestin), neurons (neurofilament), astrocytes (gfap) and oligodendrocytes (mbp), while fibronectin was used as a mesenchymal marker. Bars represent mean ± SEM of at least 3 independent experiments and are expressed as fold change of 2^−ΔΔCt using actin as a reference gene and undifferentiated MSCs as the control condition. Statistical analysis was performed by Kruskal-Wallis one-way ANOVA followed by Dunn's multiple comparison test (*P<0,05, **P<0,01 and ***P<0,001).

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No substantial expression of the oligodendrocyte precursor markers A2B5 or O4 (the latter being also present in mature oligodendrocytes) could be detected at the NSC-like stage or in undifferentiated MSCs (Figures 4 E/F and B/C, respectively), and the faint staining visible in the immunocytochemistry images seem to represent background staining, as will become evident when comparing MSCs with cells at more mature differentiation stages (Figures 4, 6 and 7B). Interestingly, galactocerebroside (GalC, a marker of mature oligodendrocytes) seems to be already expressed by undifferentiated MSCs (Figure 6S), confirming observations made by others [7]. However, its expression was significantly increased after NSC-like induction (Figure 7C). The expression of myelin basic protein (MBP), a protein expressed by mature oligodendrocytes, also showed a tendency to increase during the NSC-like differentiation stage (Figure 7D). Nevertheless, MBP could already be detected in low amounts in undifferentiated MSCs, as determined by immunocytochemistry (Figures 6T and 7D) and semi-quantitative RT-PCR (not shown), in agreement with results by others [7].

Oligodendrocyte precursor cell (OPC)-like induction

At the end of the NSC-like induction protocol, cells were detached and seeded on 6-well plates coated with fibronectin (FN), described to play an essential role in survival and proliferation signaling during the OPC differentiation stage [40]. In parallel, cells were also seeded on laminin-2/merosin (MN) coated wells, as previously described by Zhang and colleagues [22] for the differentiation of MSC into OPC-like cells. As control, cells were also plated on non-coated (NC) tissue culture wells.

By the end of the OPC-like induction stage, the cells cultured on FN- or MN-coated plates survived well and maintained the spindle shape morphology, when compared with cells cultured on uncoated wells, which did not attach well to the surface and only some small adherent clusters and single cells survived until the end of this stage (Figure 4G-O).

Figure 4 shows that there seemed to be a tendency for increased expression of Nestin, O4 and A2B5 in OPC-like cells (regardless of the coating conditions used) when compared with undifferentiated MSCs. The quantification of the MFI of Nestin and O4 (Figure 7A,B) showed that despite not being statistically significant, there was a slight increase of these markers in OPC-like cells cultured in FN when compared with MSCs. RT-PCR analysis showed that nestin was in fact upregulated in terms of gene expression in OPC-like cells when compared to undifferentiated MSC (Figure 7E). The presence of Nestin in OPC-like cells is in agreement with the literature, since it has been reported that OPCs may express nestin [41], [42]. Nonetheless, there seems to be a peak of Nestin expression during the NSC-like stage, and then a significant decrease at the OPC-like stage (Figure 7A), which is consistent with a NSC-like stage followed by an OPC-like stage of differentiation. Although MFI analysis of the levels of O4 between OPC-like cells and undifferentiated MSCs showed that these differences were not statistically significant (Figure 7B), there was a trend of increased expression of O4 on OPC-like cells when comparing with MSCs (Figure 4B and N), indicating at least some degree of oligodendroglial-like commitment of the cells. Moreover, the presence of A2B5 was visible in OPC-like cells (especially when cultured on FN or MN-coated surfaces) when compared to MSCs (Figure 4C, I, L, O, P and Q). Although it was not possible to quantify the MFI of this marker (due to its punctate distribution), it can be readily perceived in Figures 4P and Q (which show a higher magnification of the images depicted in Figures 4C and O, respectively) that the punctate and perinuclear distribution of A2B5 similar to that reported in bona-fide OPCs [42] was only visible in OPC-like cells and not in MSCs.

Overall, the protocol tested using FN or MN-coated tissue culture polystyrene (TCPS) seemed to favor a neural- and oligodendroglial precursor-like fate of MSCs.

Oligodendrocyte (OL)-like induction

OPC-like cells were detached and dissociated from the non-coated, fibronectin- or laminin-coated TCPS and re-seeded on PDL and MN double-coated 96 well plates, in distinct OL differentiation media (see Materials and Methods). The presence of merosin is known to favor oligodendrocyte differentiation [43], hence this extracellular matrix (ECM) protein was chosen as a coating element for the final stage of the protocol.

In order to induce the differentiation of OPC-like cells into OL-like cells, the thyroid hormone T3 (known to induce the differentiation of oligodendrocyte progenitor into mature OLs [44]), F3/Contactin (a ligand known to induce the maturation of oligodendrocytes [21], [22], [45]), or T3 combined with F3 (T3F3) were added to the basal differentiation media and incubated for 10 days. It was also tested the effect of T3 in the first 7 days of culture and T3 together with F3 during the last 3 days of differentiation (7dT3+3dT3F3).

Immunocytochemistry analysis of nestin in OL-like cells previously cultured on FN-coated TCPS during the OPC-like stage (the preceding differentiation step) showed that the expression of this neural stem/progenitor marker decreased significantly during the final stage of differentiation in all the conditions tested in the final differentiation stage when compared with NSC-like cells, or when compared with OPC-like cells when differentiated terminally in the presence of 7dT3+3dT3F3 or T3 (Figures 5M-S and 7A). Nestin levels peaked at the NSC-like stage and then decrease during the final differentiation step (OL-like stage), to levels similar to those of undifferentiated MSCs (Figures 5M-S and 7A). These results support the idea that the cells underwent a neural progenitor-like state during the NSC- and OPC-like stages of the differentiation protocol and then differentiated into a more mature phenotype at the OL-like stage (similar to what has been described for bona-fide OLs [46]).

Reinforcing this idea was the behavior of the OPC marker A2B5 [46], which decreased during the final stage of differentiation (OL-like cells) when compared with OPC-like cells (Figure 6 A-D, Q, U, V). This effect was more evident in oligodendrocyte-like cells that had been previously cultured on FN-coated dishes during the OPC-like differentiation stage than in cells that had previously been cultured on MN-coated TCPS (Figure 6U, V). The loss of this OPC marker indicates that OL-like cells derived from OPC-like cells cultured on FN-coated wells might be more mature than those obtained from OPC-like cells cultured on MN-coated or non-coated wells, in agreement with reports highlighting the important role of fibronectin during the OPC stage of differentiation [40].

To further characterize the OL-like cells obtained, we screened for the presence of neural maturation markers for astrocytic (GFAP), neuronal (beta-III-tubulin) and oligodendroglial (O4, GalC and MBP) lineages. The images of OL-like cells presented in Figures 5 and 6 were obtained from cells that had previously been cultured on FN-coated TCPS during the OPC-like state, and from this point onwards will be considered as the main condition under analysis during the final stage of differentiation, unless stated otherwise.

The expression of GFAP was negative in OL-like cells in all differentiation conditions (Figure 5 A-D), similar to what had been observed in undifferentiated MSCs (Figure 5J) and in contrast to what was shown in Figure 5I, where primary rat astrocytes were used as positive control for this marker. Cells that were cultured on laminin-2 or non-coated wells during the OPC-like stage showed some expression of this astrocytic marker in the final differentiation stage (OL-like stage) - data not shown. These results further supported the idea that the oligodendroglial-like fate of MSCs seems to be favored by the presence of FN during the OPC-like stage.

In Figure 5E-H, it can be observed that the expression of beta-III-tubulin was essentially absent in OL-like cells obtained in the presence of any of the distinct differentiation media, although there was some expression in cells that were cultured on non-coated wells during the OPC-like stage (data not shown). In Figure 5K, the SH-SY5Y cell line was used as a positive control for this neuronal marker, which was absent in undifferentiated MSCs (Figure 5L).

The expression of O4, an OPC marker which is maintained in mature oligodendrocytes [46], increased during the differentiation of MSCs into OL-like cells (Figure 6E-H). For cells cultured on fibronectin during the OPC-like stage, the quantification of the MFI of O4 showed a significant increase between MSCs, NSC-like or OPC-like cells and the cells obtained during the final stage of differentiation (OL-like cells) cultured in the presence of any combination of differentiation factors (Figure 7B). Similar results were obtained for cells previously cultured on non-coated TCPS, or for cells maintained on MN-coated TCPS during the OPC-like stage when finally differentiated in presence of F3 only (data not shown).

GalC seemed to be already expressed at some extent by undifferentiated MSCs (Figure 6S). However, the expression of this oligodendrocyte marker increased in oligodendrocyte-like cells differentiated under all tested conditions (Figure 6I-L). The MFI of GalC showed a tendency to increase between MSCs and OL-like cells in all conditions. Compared to MSCs, the level of GalC expressed by OL-like cells was significantly increased only in OL-like cells cultured in the presence of T3 or F3 (Figure 7C). Unexpectedly, the presence of GalC was also higher in NSC-like cells, when compared with undifferentiated MSCs. Nevertheless, only during the final stage of differentiation (in OL-like cells) it was possible to observe cells with a branched morphology similar to oligodendrocytes and strongly positive for GalC, like those terminally differentiated in the presence of T3 or F3 (asterisk in Figures 6I and J, respectively). For cells cultured on non-coated wells during the OPC-like stage, there was a significant increase in the expression of GalC between OL-like cells (cultured in the presence of any of the final differentiation factors) and MSCs, but no significant differences were found between OL-like cells derived from OPC-like cells cultured on laminin-coated wells and MSCs (data not shown).

Immunocytochemistry analysis of MBP showed that this mature oligodendrocyte marker was present in OL-like cells (Figure 6M-P) after the final differentiation step, however, the levels of MBP were similar to those observed in undifferentiated MSCs, as illustrated in Figure 6T (the presence of MBP in MSCs was confirmed by western-blot analysis – Figure S4). The quantification of the MFI throughout the distinct stages of the differentiation protocol showed that the expression of MBP had a tendency to increase (although the differences were not statistically significant) during the NSC- and OPC-like stages of differentiation (Figure 7D). In fact, there was a significant decrease in the expression of MBP in the final step of differentiation, between NSC-like and OL-like cells cultured in the presence of F3 or T3F3, but not T3 or 7dT3+3dT3F3 (Figure 7D). Also, for cells cultured on uncoated surfaces during the OPC-like stage, there was a significant decrease in the expression of MBP between NSC-like cells and oligodendrocyte-like cells cultured in the presence of F3 and between OPC-like cells and OL-like cells cultured in the presence of T3 or F3 (not shown).

The expression of the oligodendrocyte markers MBP, GalC and O4, together with the downregulation of nestin and A2B5 in the OL-like differentiated cells, supports the idea that MSCs could be induced to differentiate into OL-like cells, sharing the behavior of several markers expressed by bona-fide oligodendrocytes and its precursors [46].

The more evident downregulation of A2B5 and the absence of neuronal (beta-III-tubulin) and astrocytic (GFAP) markers observed in OL-like cells derived from OPC-like cells cultured on fibronectin, suggests that these coating conditions (during the OPC-like stage) are suitable for the differentiation of MSCs into oligodendrocyte-like cells. Hence, UCM-MSCs could be differentiated into NSC-like cells, OPC-like cells and finally into oligodendrocyte-like cells, with expression of specific markers for each stage of differentiation. Figure 6 I and J (staining for GalC) illustrates terminally differentiated OL-like cells displaying a branched morphology (asterisks within images), resembling oligodendrocytes. Despite several cells with this morphology could be observed after the final differentiation step, they represented a minority of the cells.

Pattern of expression of MBP

Although we could not observe an increase of MBP expression in OL-like cells when compared with undifferentiated MSCs, it could be observed that there were distinct patterns of expression of MBP in the differentiated cells. One of the patterns was common to all stages of differentiation, including undifferentiated MSCs (Figure 6T), which was a strong staining in the nuclear area and more diffuse in cytosolic areas surrounding the nucleus (e.g.: Figure 8A, MBP panel, arrows). The other patterns suggested a structured distribution of MBP, resembling cytosolic filaments organized in a parallel manner, or at the periphery of the cells (e.g.: Figure 8A, MBP panel, arrow heads) and were present only in OL-like cells.

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Figure 8. Co-localization of MBP and F-actin in oligodendrocyte-like cells.

Fluorescence microscopy images of OL-like cells (A) stained with fluorescent phalloidin (in green) to assess the presence of polymerized actin (F-actin) and an antibody against MBP (in red) and both channels merged, as indicated. Cells were derived from OPC-like cells cultured on FN-coated wells. Cells showing structured MBP and with partial co-localization with F-actin (arrow heads) or with diffuse MBP and no co-localization with F-actin (arrows) were visible after OL-like differentiation in the presence of T3, F3, T3+F3 or 7 days T3 followed by 3 days T3+F3 (T3+3d F3), as indicated. The bottom panel is a magnification of the T3 condition. Images are representative of at least 3 independent experiments and scale bar corresponds to 50 µm. The percentage of cells expressing structured MBP was calculated (B) under two categories: cells expressing structured cytoplasmic (left) or peripheral (right) MBP. Values were expressed as percentage of total cells and bars represent mean ± SEM percentage of cells present in at least 14 fields, belonging to 3 independent experiments. No statistically significant differences were found between the distinct differentiation conditions (Kruskal-Wallis one-way ANOVA followed by Dunn's multiple comparison test).

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The quantification of the percentage of OL-like cells expressing structured MBP showed that the parallel distribution pattern of MBP could be observed in between approximately 25% and 45% of the cells (Figure 8B, left) and the occurrence of structured MBP at the periphery of the cells (Figure 8B, right) was present in between about 18% and 34% of the cells, depending on the differentiation medium used. Although the combined presence of T3 and F3 seemed to favor the appearance of patterned MBP, we could not find statistically significant differences between the distinct differentiation conditions.

The structured distribution of MBP resembled the pattern of polymerized actin filaments (F-actin). It is known that, in mature oligodendrocytes, MBP associates with F-actin and that this interaction seems to be important for the myelinating activity of OLs [47], [48].

In order to test whether the structured MBP was coincident with polymerized actin filaments, the cells were analyzed for the presence of F-actin using fluorescently-labeled phalloidin. The immunocytochemistry results showed that whenever MBP could be detected in a structured manner (either with the parallel or the peripheral distribution), it always co-localized with F-actin (Figure 8A, merge panel, arrow heads), in contrast to the diffuse MBP, which did not (e.g., Figure 8A, merge panel, arrows). Cells with oligodendrocyte-like morphology could also be observed, displaying co-localization of MBP with F-actin (e.g.: Figure 8A, asterisk).

Real-time RT-PCR analysis

In order to further characterize the cells obtained at each distinct phase of the differentiation process, cells were analyzed at the transcript level by real-time RT-PCR. Actin was used as a house-keeping gene, fibronectin as a mesenchymal marker and several neural genes, representative of neural progenitors (sox2 and nestin), neurons (neurofilament), astrocytes (gfap) and oligodendrocytes (mbp), were analyzed.

Data presented in Figure 7E shows that comparing to undifferentiated MSCs, the mesenchymal marker fibronectin remained essentially unchanged throughout the differentiation process, while the neural progenitor genes sox2 and nestin were upregulated during the OPC-like stage of differentiation, then returning to lower levels after the OL-like stage. The presence of nestin during the OPC differentiation stage of oligodendroglial lineages has been known for some time [41], [42], whereas the expression of sox2 has only been reported more recently to occur in bona-fide OPCs, but not by mature oligodendrocytes [49]–[51]. All other markers did not show any statistically significant difference comparing with MSCs, except for neurofilament, that exhibited a peak at the NSC-like stage and then, in subsequent differentiation steps, decreased to levels similar to those already expressed by MSCs, suggesting that an eventual neuronal-like phenotypic tendency was lost during the later differentiation steps.

Overall, the RT-PCR data seems to be in agreement with the immunocytochemistry results, suggesting that the NSC/OPC-like differentiation stages are associated with the increased expression of neural progenitor markers, while the oligodendroglial maturation marker MBP seemed to be expressed by OL-like cells at levels similar to those already present in undifferentiated MSCs.

Co-culture experiments

In order to address the interaction between the obtained OL-like cells and neuronal cells, a co-culture experiment was set up [33]. Mouse dorsal root ganglion (DRG) explants were cultured for 10 days until the axons were well developed, and then MSC-derived cells at the end of the OPC-like stage were added to the DRG culture and allowed to be in co-culture for further 14 days in OL-like maturation medium (Materials and Methods).

By the end of the co-culture period, it could be observed that OL-like cells typically tended to be present in the vicinity of neurons (Figure 9A - neurons stained in red for beta-III-tubulin and OL-like cells stained in green for human Golgi). This observation suggests that there might occur a positive crosstalk between both types of cells (neurons and OL-like cells).

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Figure 9. Immunofluorescence microscopy images of OL-like cells derived from hUCM-MSCs in co-culture with mouse dorsal root ganglion (DRG) neurons.

The co-cultures were stained using an anti-human Golgi antibody (green) to specifically identify the human cells (OL-like cells derived form MSCs) and an anti-beta-III-tubulin antibody (red) to label the mouse DRG neurons. The images acquired in the green channel (anti-human Golgi antibody) were deliberately slightly overexposed to allow for a better understanding of the cellular morphology, which did not affect the identification of human versus mouse cells, as evidenced by the lack of green signal in the mouse DRG neurons (in red). Counterstaining of the nuclei was performed using DAPI (blue). It was apparent that the OL-like cells and DRG neurons tend to cluster together, as illustrated in a lower magnification image (A). Higher magnification images (B-D) suggest the existence of contact points (arrows) between branches of OL-like cells displaying an immature oligodendrocyte-like morphology (asterisks) and neurites. Scale bars correspond to 50 µm (A) or 20 µm (B-D).

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Occasionally, OL-like cells with branched morphology could be observed (Figure 9B-D, asterisk). Although there was no evidence of robust structures resembling myelin sheaths wrapping neurons, in contrast to those described for bona-fide oligodendrocytes [33], in some cases there were apparent contact points between branches of OL-like cells and axons of DRGs (Figure 9C, D – arrows). These results suggest that although there was no evidence for robust axonal ensheathment, there might be physical interactions between OL-like cells and neuronal axons.

Overall, the results indicate that MSCs are prone to differentiate into neural-like cells, namely oligodendrocyte-like cells, expressing some of the typical oligodendroglial lineage markers along the distinct differentiation steps. The OL-like cells obtained expressed some of the mature oligodendrocyte markers (e.g.: MBP, GalC), but seemed to be immature comparing to actual bona-fide oligodendrocytes in terms of morphology and function. Although some cells did present an OL-like morphology and seemed to establish contacts with axons of neuronal cells, there was no evidence for robust myelination in OL-like/DRG co-culture experiments. Nevertheless, MSCs seem to have the potential to differentiate into oligodendroglial-like lineages and with improvements to the protocol, a more mature phenotype might be attained, with a more conclusive functional phenotype.