Cabrera-Pérez et al. Stem Cell Research & Therapy (2019) 10:356 https://doi.org/10.1186/s13287-019-1450-3 RESEARCH Open Access Osteogenic commitment of Wharton’s jelly mesenchymal stromal cells: mechanisms and implications for bioprocess development and clinical application Raquel Cabrera-Pérez1,2*, Marta Monguió-Tortajada3, Ana Gámez-Valero3, Raquel Rojas-Márquez1,4, Francesc Enric Borràs3,5, Santiago Roura6 and Joaquim Vives1,2,7* Abstract Background: Orthopaedic diseases are one of the major targets for regenerative medicine. In this context, Wharton’s jelly (WJ) is an alternative source to bone marrow (BM) for allogeneic transplantation since its isolation does not require an invasive procedure for cell collection and does not raise major ethical concerns. However, the osteogenic capacity of human WJ-derived multipotent mesenchymal stromal cells (MSC) remains unclear. Methods: Here, we compared the baseline osteogenic potential of MSC from WJ and BM cell sources by cytological staining, quantitative real-time PCR and proteomic analysis, and assessed chemical and biological strategies for priming undifferentiated WJ-MSC. Concretely, different inhibitors/activators of the TGFβ1-BMP2 signalling pathway as well as the secretome of differentiating BM-MSC were tested. Results: Cytochemical staining as well as gene expression and proteomic analysis revealed that osteogenic commitment was poor in WJ-MSC. However, stimulation of the BMP2 pathway with BMP2 plus tanshinone IIA and the addition of extracellular vesicles or protein-enriched preparations from differentiating BM-MSC enhanced WJ-MSC osteogenesis. Furthermore, greater outcome was obtained with the use of conditioned media from differentiating BM-MSC. Conclusions: Altogether, our results point to the use of master banks of WJ-MSC as a valuable alternative to BM-MSC for orthopaedic conditions. Keywords: Mesenchymal stromal cells, Bone marrow, Wharton’s jelly, Osteogenic differentiation, Bone regeneration Background off-the-shelf cell products derived from healthy and The development of new treatments for bone-related immune-compatible donors are very attractive since they diseases resulting from trauma or pathophysiological age-, are immediately available and provide a high number of sex- or infection-associated bone resorption has become a cells [14, 15]. priority in the field of regenerative medicine [1–4]. In this Multipotent mesenchymal stromal cells (MSC) consti- context, autologous cell-based therapy has been presented tute a heterogeneous population of non-haematopoietic as a promising approach to promote bone regeneration in multipotent cells which can be isolated from a variety of both pre-clinical and clinical settings [5–8]. However, their human body sources [3, 16, 17]. In particular, MSC have clinical translation needs the delivery of safe and efficacious a fibroblast-like appearance, plastic adherence, the ability products, which can be largely hampered by age and co- to differentiate into tissues of mesodermal lineages (adi- morbidities of the cell donor [9–13]. In contrast, allogeneic pocytes, chondrocytes and osteocytes) and a specific cell surface expression pattern, according to the minimal cri- * Correspondence: rcabrera@bst.cat; jvives@bst.cat teria established by the International Society for Cell and 1 Cell Therapy Service, Blood and Tissue Bank (BST), Barcelona, Catalonia, Gene Therapy (ISCT) [18]. Of note, a couple of MSC- Spain based products have already received marketing approval Full list of author information is available at the end of the article © The Author(s). 2019 Open Access This article is distributed under the terms of the Creative Commons Attribution 4.0 International License (http://creativecommons.org/licenses/by/4.0/), which permits unrestricted use, distribution, and reproduction in any medium, provided you give appropriate credit to the original author(s) and the source, provide a link to the Creative Commons license, and indicate if changes were made. The Creative Commons Public Domain Dedication waiver (http://creativecommons.org/publicdomain/zero/1.0/) applies to the data made available in this article, unless otherwise stated. Cabrera-Pérez et al. Stem Cell Research & Therapy (2019) 10:356 Page 2 of 11 [19], whereas most developments are still in clinical for Advanced Therapy Medicinal Products’ (GMP for evaluation, including MSC-based tissue engineering ATMPs, European Commission Guidelines of 2017.11.22) products (EudraCT Nos. 2010-024041-78, 2010-023998- and further expanded in Dulbecco’s modified Eagle’s 18, 2010-023999-12 and 2013-005025-23) under devel- medium (DMEM) (31885-023; Gibco) containing 2 mM opment in our laboratory [20]. glutamine and supplemented with 10% human serum B Bone marrow (BM) has become the most used source of (hSerB)—‘expansion medium’ [37, 38]. All cell cultures MSC in the orthopaedic field because of its intrinsic osteo- were maintained at 37 °C and 5% CO2 in humidified incu- genic differentiation potential, but alternative sources are bators, and media were changed every 3–4 days. Cell num- garnering attraction. These include Wharton’s jelly (WJ), ber and viability were determined by the haemocytometer- which is the connective tissue surrounding the human um- based trypan blue dye exclusion assay. bilical cord and is advantageous for cell collection since its isolation is not painful, does not require invasive procedures Phenotype assessment and does not raise major ethical concerns [21]. Accordingly, Immunophenotypic characterisation of MSC was per- we reported the feasibility of expanding clinical-grade WJ- formed using the following antibodies: mouse anti-human MSC from samples typically discarded from public cord CD45-fluorescein isothiocyanate (CD45-FITC) (Clone blood banking programmes [22]. Moreover, in contrast to HI30; 555482; BD Pharmingen), anti-human CD105- adult BM-MSC, WJ-MSC are expected to be more primi- phycoerythrin (CD105-PE) (Clone 43A4E1; 130-117-696; tive, proliferative and immunosuppressive cells, particularly Miltenyi Biotec), anti-human HLA-DR-FITC (Clone L243; for the lack of HLA-DR antigens [23–26]. Nevertheless, the 347363; BD Biosciences), anti-human CD90-PE (Clone osteogenic capacity of WJ-MSC remains under scrutiny. F15-42-1-5; IM1840U; Beckman Coulter), anti-human Osteogenic differentiation of mesenchymal precursors CD31-FITC (Clone WM59; 555445; BD Pharmingen) and and bone regeneration are extremely complex processes anti-human CD73-PE (Clone AD2; 550257; BD Pharmin- regulated by the interaction of different signalling pathways gen). Cells were stained for 15 min at room temperature including TGFβ/BMP, MAPK, Wnt, Hedgehog, Notch and (RT), washed and re-suspended in phosphate-buffered sa- AKT/mTOR [27]. Among them, the TGFβ/BMP pathway line (PBS) (14190-094; Gibco) as described elsewhere [38]. plays the major role in the regulation of osteoblast lineage- Acquisition and data analysis were performed using a specific differentiation, bone induction, maintenance and FACSCalibur cytometer and the CellQuest Pro software repair and constitutes a promising target for the treatment (Becton Dickinson), respectively. of bone diseases [28–30]. Up to date, about 60 TGFβ family proteins have been identified so far, being TGFβ1 and Osteogenic differentiation assays BMP2 ligands the most widely investigated due to their BM-MSC and WJ-MSC (passage 3–5) were seeded until positive role in bone formation in vivo [31, 32]. However, 70–80% confluence (104 and 2 × 104 cells/cm2, respect- while BMP proteins have been demonstrated to induce the ively). ‘Differentiation media’ composed of the StemPro expression of MSC differentiation factors (such as DLX5 osteogenesis differentiation kit (A1007201; Gibco) sup- and RUNX2) [33, 34], several in vitro studies have described plemented with 100 units/mL of penicillin and 100 μg/ a negative impact of TGFβ1 on the terminal differentiation mL streptomycin (Penicillin-Streptomycin; P4458; of osteoblast precursors [34–36]. Sigma-Aldrich) was used for the osteogenic induction Despite several reports investigating the osteogenic cap- in vitro. Alkaline phosphatase (ALP) (B5655; Sigma- abilities of BM- and WJ-MSC have been published to date, Aldrich) and alizarin red (AR) (2003999; Merck Milli- it is still difficult to integrate existing data due to heterogen- pore) staining were finally carried out to assess cell eity in MSC isolation and culture procedures. In the differentiation. present study, we aimed to provide homogeneous and com- parative data regarding the ability of BM- and WJ-MSC to Gene expression assays differentiate towards the osteogenic lineage by means of Total RNA was purified from cell cultures using the cytological staining and molecular and proteomic analysis. RNeasy Plus Mini Kit (74134; Qiagen) according to the Moreover, we evaluated a variety of strategies based on the manufacturer’s instructions, quantified using NanoDrop modulation of the TGFβ/BMP pathway and the use of the Lite (Thermo Scientific), and electrophoresed in 1% BM-MSC secretome to enhance osteogenesis in WJ-MSC agarose gels to confirm integrity and purity. cDNA and emulate BM-MSC osteogenic commitment. synthesis was then performed by reverse-transcription PCR (RT-PCR) using the High-Capacity cDNA Reverse Methods Transcription Kit (4368814; Thermo Fisher Scientific) Cell culture according to the manufacturer’s instructions. cDNA was BM-MSC (n = 3) and WJ-MSC (n = 3) (passage 3–5) finally amplified by quantitative real-time PCR (qRT- were isolated according to ‘Good Manufacturing Practice PCR) using the TaqMan gene expression assays listed in Cabrera-Pérez et al. Stem Cell Research & Therapy (2019) 10:356 Page 3 of 11 Table 1. In all cases, target gene expression was referred aldehyde/sulphate-latex microspheres (A37304; Invitrogen) to GAPDH expression by using the 2−ΔCt method. for 15 min at RT and blocked in BCB buffer (PBS supple- mented with 0.1% BSA (A4503) and 0.01% NaN3 (S8032); Inhibition/activation of TGFβ/BMP2 signalling pathways Sigma-Aldrich) on overnight rotation. EV-coated beads WJ-MSC (passage 4) were seeded at a cell density of 2 × were spun down at 2000 × g for 10 min, washed with BCB 104 cells/cm2. Inhibition of the TGFβ signalling pathway buffer and re-suspended in PBS. EV-coated beads were la- was carried out by addition of galunisertib (LY2157299) belled with the primary antibodies anti-CD9 (Clone VJ1/ (sc-391123; Santa Cruz Biotechnology) to the osteogenic 20) and anti-CD63 (Clone TEA3/18) (kindly provided by differentiation media at a final concentration of 10 μM. M. Yáñez-Mó (CBM-SO, IIS-IP, UAM, Madrid, Spain) and For BMP2 pathway stimulation, human recombinant F. Sánchez-Madrid (Hospital Universitario de la Princesa, BMP2 (SRP6155; Sigma-Aldrich) and/or tanshinone IIA IIS-IP, UAM, CNIC, Madrid, Spain)) or the IgG isotype (sc-200932; Santa Cruz Biotechnology) were added to a control (a637355; Abcam) and secondary antibody FITC- final concentration of 100 ng/mL and 5 μM, respectively. conjugated Goat F(ab′)2 Anti-Mouse IgG (1032-02; All compounds were added immediately after every Bionova). EV-coupled beads were washed after each step media change after 1 week of in vitro osteogenic with BCB buffer and centrifuged at 2000 × g for 10 min. induction with differentiation media. Data was acquired in a FACSLyric flow cytometer (BD) and analysed by FlowJo v.X software (Tree Star). Extracellular vesicle isolation and characterisation SEC-EV-containing fractions were examined for EV size To avoid sample contamination with exogenous extracel- and morphology by cryo-electron microscopy (cryo-EM). lular vesicles (EVs), cells were cultured in EV-depleted Vitrified specimens were prepared by placing 3 μL of a sam- media. For EV depletion, 2× differentiation media was ple on a Quantifoil® 1.2/1.3 TEM grid, blotted to a thin film ultra-centrifuged at 100,000 × g for ≥16 h and diluted with and plunged into liquid ethane-N2(l) in the Leica EM CPC StemPro Basal Media (A10069-01; Gibco) to 1× working cryoworkstation (Leica). The grids were transferred to a concentration. 626 Gatan cryoholder and maintained at −179 °C. Samples Supernatants derived from undifferentiated BM-MSC were analysed with a Jeol JEM-2011 transmission electron and WJ-MSC (passage 3–5) or from BM-MSC under microscope (Jeol) operating at an accelerating voltage of osteogenic differentiation (passage 3–5) were collected 200 kV. Images were recorded on a Gatan UltraScan 2000 at weeks 0, 1, 2 and 3, and were sequentially centrifuged cooled charge-coupled device (CCD) camera with the at 400 × g for 5 min and at 2000 × g for 10 min to ex- DigitalMicrograph software package (Gatan). clude cells and cell debris. Conditioned medium (CM) was then concentrated by 100-kDa ultrafiltration using Proteomic analysis Amicon Ultra (UFC910024; Millipore) at 2000 × g for 35 The protein content of EV-enriched fractions was analysed min, obtaining typically 250 μL of concentrated CM by liquid chromatography followed by mass spectrometry (CCM). EVs were isolated by size exclusion chromatog- (LC-MS/MS) on Orbitrap XL (Thermo Fisher) for three in- raphy (SEC) as previously reported [39]. Protein elution dependent undifferentiated cultures for each MSC type. was checked by reading absorbance at 280 nm using Data was searched against the Swiss-Prot human database NanoDrop (Thermo Scientific). (downloaded in August 2016), using the search algorithm The presence of EVs in the SEC fractions was determined Mascot v2.5.1. Only peptides showing a false discovery rate according to the presence of tetraspanins by bead-based (FDR) lower than 5% were retained. Proteins identified with flow cytometry [39]. Briefly, EVs were coupled to 4-μm at least two unique peptides and found in all three samples were considered for further analysis. Table 1 TaqMan gene expression assay ID The obtained proteomic profile for our samples was com- Gene Assay ID pared with previous studies compiled in EV-specific data- MSX2 Hs00741177_m1 bases EVpedia [40], ExoCarta [41] and Vesiclepedia [42]. DLX5 Hs01573641_mH RUNX2 Hs01047973_m1 Data analysis SP7 Hs05049492_s1 Statistical analysis was performed with the GraphPad ALPL Hs01029144_m1 Prism 6 software (GraphPad Software, Inc.). Descriptive TFGβ1 Hs00998133_m1 data were expressed as mean ± standard deviation (SD). BGLAP Hs01587814_g1 Multiple t tests were used for investigating differences COL1A2 Hs01028970_m1 between BM- and WJ-MSC at different time points along the osteogenic differentiation. Statistical signifi- GAPDH Hs02786624_g1 cance was set at *p < 0.05 and **p < 0.01. Cabrera-Pérez et al. Stem Cell Research & Therapy (2019) 10:356 Page 4 of 11 Results BM-MSC show increased expression of osteogenic WJ-MSC exhibit delayed osteogenic induction compared markers compared to WJ-MSC with BM-MSC We further analysed the delayed osteogenesis in WJ-MSC BM-MSC and WJ-MSC were highly positive for CD73, cultures. For that purpose, we comparatively assessed the CD90 and CD105 and negative for CD31, CD45 and time-course expression of key osteogenic genes implicated HLA-DR expression according to the ISCT criteria in MSC differentiation. Interestingly, we found some sig- (Additional file 1). nificant differences along both BM- and WJ-MSC osteo- The osteogenic differentiation of BM-MSC and WJ- genic differentiation. Regarding osteogenic transcription MSC cultures were assessed by alkaline phosphatase factors (Fig. 2a), BM-MSC showed a progressive increase (ALP) and alizarin red (AR) staining. All BM-MSC and in DLX5, RUNX2 and SP7 expression. In contrast, in WJ- WJ-MSC cell lines showed osteogenic potential in vitro MSC, DLX5 expression was gradually decreased and SP7 after osteogenic induction with specific differentiation levels exhibited an increment from week 2 to 5. Additional media. However, a delay in osteogenesis was observed in differences were observed in MSX2. Remarkably, a steady WJ-MSC compared to BM-MSC. In particular, as shown increase of MSX2 expression was observed up to week 5 in Fig. 1a, most of the cells in BM-MSC cultures in WJ-MSC, whereas no changes in MSX2 expression displayed a marked baseline activity of the osteogenic were detected in BM-MSC. marker ALP. In contrast, in WJ-MSC cultures, only few Moreover, the expression patterns of main early and cells exhibited ALP activity even at week 5. Regarding late osteogenic markers were comparatively assessed the results obtained for AR staining, calcium depositions (Fig. 2b). In this sense, the early marker ALPL reached were clearly visible in BM-MSC cultures at week 3. its maximum expression level during the first week in However, 5 weeks were required in order to obtain simi- BM-MSC. However, in WJ-MSC, ALPL expression was lar results in WJ-MSC (Fig. 1b). reduced at this time compared to week 0 and started to Fig. 1 Differences in timing for osteogenic differentiation in BM-MSC and WJ-MSC cultures. Alkaline phosphatase (a) and alizarin red (b) staining at indicated times. N = 3 for both MSC types. Scale bars: 100 μm Cabrera-Pérez et al. Stem Cell Research & Therapy (2019) 10:356 Page 5 of 11 Fig. 2 Gene expression profiles of the main markers involved in osteogenic differentiation. Expression levels of osteogenic transcription factors (a) and early/late osteogenic markers (b). Bars represent mean ± SD. *p < 0.05 and **p < 0.01 (multiple t tests). N = 3 for each MSC type Cabrera-Pérez et al. Stem Cell Research & Therapy (2019) 10:356 Page 6 of 11 increase again from the third week. The expression patterns As shown in Fig. 3b, the inhibition of TGFβ1 signalling of late osteogenic markers TGFβ1 and BGLAP were also had no effect on WJ-MSC osteogenesis. Similarly, negative different. In BM-MSC, TGFβ1 achieved the highest expres- results in AR staining experiments were seen when BMP2 sion level at week 2 and BGLAP expression increased pro- and tanshinone IIA were present alone or in combination gressively. On the contrary, no changes were observed for with galunisertib. In contrast, when BMP2 and tanshinone these genes in WJ-MSC. In regard to COL1A2, similar IIA were added together, calcium depositions were clearly expression patterns were obtained for both cell types, visible in WJ-MSC cultures after 3-week induction, emu- although expression in BM-MSC was twofold higher than lating the behaviour of BM-MSC under standard osteo- that in WJ-MSC. Remarkably, the expression of RUNX2, genic inducing conditions. This suggests the promotion of DLX5, ALPL, TGFβ1, BGLAP and COL1A2 was promoted WJ-MSC osteogenic differentiation following stimulation in BM-MSC even when they were in an undifferentiated of BMP2 signalling. stage. Taken together, these findings indicate a higher osteogenic differentiation commitment in BM-MSC. BM-MSC-conditioned media strongly enhances WJ-MSC osteogenic differentiation The results obtained after the characterisation of the gene Promotion of BMP2 signalling primes osteogenic expression profiles of some of the main osteogenic markers differentiation of WJ-MSC explained, in part, the superior osteogenic potential pre- Subsequently, the role of TGFβ1 and BMP2 signalling sented by BM-MSC. However, with the aim of deepening pathways in the promotion of osteogenic differentiation in the elucidation of the increased osteogenic capacity of WJ-MSC was investigated. To prevent TGFβ activa- shown by BM-MSC, the proteomic content associated with tion, differentiation media was supplemented from week the isolated extracellular vesicles (EVs) from the super- 1 to 3 with galunisertib. On the other hand, in order to natant of undifferentiated BM-MSC and WJ-MSC cultures stimulate osteogenic differentiation through the BMP2 was analysed. Proteomic analysis by LC-MS/MS allowed signalling pathway, human recombinant BMP2 and/or the identification of several EV markers, such as annexin the BMP activator tanshinone IIA were also added to A2, A5 and A6, glyceraldehyde-3-phosphate dehydrogen- the differentiation media (Fig. 3a). ase and CD5L. As depicted in Fig. 4, 99 proteins were Fig. 3 Modulation of the TGFβ/BMP signalling pathway to stimulate WJ-MSC osteogenic differentiation. a Scheme of the experimental design. From day 0 to week 1, cells were cultured in osteogenic differentiation media, which was supplemented from week 1 to 3 with galunisertib, BMP2 and/or tanshinone IIA. b Representative AR staining results obtained in passage 4 WJ-MSC after 2 weeks of culture in osteogenic media supplemented with galunisertib 10 μM, BMP2 100 ng/mL and tanshinone IIa 5 μM in different combinations. Scale bars: 100 μm Cabrera-Pérez et al. Stem Cell Research & Therapy (2019) 10:356 Page 7 of 11 Fig. 4 Osteogenic markers found in undifferentiated BM-MSC- and WJ-MSC-secreted EVs. Number of proteins found in extracellular vesicles (EVs) isolated by size exclusion chromatography (n = 3 different cell lines for BM-MSC and WJ-MSC). The osteogenic markers differentially overrepresented in BM-MSC EV samples are indicated. Results were obtained by LC-MS/MS, two peptides restricted, FDR<5% found in common between both types of cells. However, (Fig. 5b). As shown in Fig. 5b, the addition of CM from five osteogenic markers (namely COL6A1, COL6A2, PCO differentiating BM-MSC to WJ-MSC cultures resulted in LCE, COL12A1 and COL6A3) were differentially overrep- higher osteogenesis than the addition of purified EVs or resented in BM-derived EVs compared to WJ-MSC EVs. protein fractions separately. Furthermore, the fact that This finding could contribute to the explanation of the calcium depositions were clearly identified from the very higher osteogenic commitment observed in BM-MSC and first week indicated a powerful synergistic positive effect suggested the possibility of using the BM-MSC secretome between EV and proteins secreted by BM-MSC in the to prime osteogenesis in WJ-MSC. progression along the osteogenic linage differentiation. To test the feasibility of this approach, conditioned media (CM) from BM-MSC cultures were obtained Discussion weekly up to week 3 of the osteogenic differentiation and The use of allogeneic MSC is promising for the treatment processed by size exclusion chromatography to separate of bone-related conditions. In this context, some studies and purify fractions enriched in EVs or soluble proteins have suggested that master cell banks of WJ-MSC may (Additional file 2). Once isolated, the EV or protein frac- offer advantages over the use of BM-MSC [20, 22]. How- tions were added to WJ-MSC differentiating cultures. ever, to guarantee the success of WJ-MSC in situations Monitoring of the osteogenic differentiation was per- where bone repair or bone regeneration is required, it is formed by AR staining. Negative results were obtained essential to demonstrate that osteogenic commitment is in all cases after 2 weeks of media supplementation with equally driven in both BM- and WJ-MSC. either EVs or protein fractions (data not shown). How- In the present study, we have extensively characterised ever, after 3 weeks, calcium depositions were observed in the osteogenic differentiation process of BM- and WJ-MSC all the WJ-MSC cultures supplemented with BM- in order to provide homogeneous data because, despite secreted EVs and protein fractions purified from BM- there are many reports in the field describing osteogenic MSC differentiating cultures at weeks 1, 2 and 3, while properties of both cell lines, data sets are often incomplete the secretome from undifferentiated BM-MSC (week 0) and non-comparable due to heterogeneity in isolation and did not (Fig. 5a). Although both EVs and protein frac- culture procedures. tions positively stimulate WJ-MSC osteogenesis, the ef- In concordance with previous observations reported fect produced by the soluble protein fraction resulted in by other authors in the field [43, 44], our findings evi- a greater outcome than that produced by EVs collected denced that WJ-MSC are less prone to differentiate into at the same differentiation stage. Based on this observa- bone cells than BM-MSC. We thus aimed to compara- tion, the full CM collected from BM-MSC cultures after tively analyse the molecular machinery associated with 1 week of in vitro osteogenic induction was also tested osteogenic differentiation in BM- and WJ-MSC since we Cabrera-Pérez et al. Stem Cell Research & Therapy (2019) 10:356 Page 8 of 11 could be critical in MSC, resulting in either a proliferat- ing or differentiating outcome. Trying to revert the scenario found in WJ-MSC, we then attempted to modulate TGFβ- and BMP-mediated signal- ling pathways by adding specific activators or inhibitors in order to repress MSX2 and/or to stimulate DLX5 expres- sion. To this end, we used different combinations of chem- ical factors including (i) galunisertib, a receptor antagonist that specifically targets and binds TGFβRI [49]; (ii) BMP2, which has been reported to induce osteoblast differenti- ation by promoting DLX5 expression [33, 50] and (iii) tanshinone IIA, a phytochemical compound reported to enhance BMP signalling stimulation [51]. Neither the use of each molecule separately nor the combination of a repressing and activating agent resulted in an osteogenic output in WJ-MSC. However, agreeing with the results published by Heo and collaborators in umbilical cord blood MSC [52], the addition of tanshinone IIA to BMP2- induced cultures significantly increased calcium depositions in WJ-MSC after 3 weeks, thus matching WJ-MSC osteo- genic behaviour to that of their BM-MSC counterparts. Although systemic infusion of MSC has been shown to increase bone growth and repair in clinical trials [53–55], administered cells engraft poorly. In this line, previous in vivo studies carried out by our group in which ovine eGFP+ BM-MSC were infused in an ovine model of osteo- necrosis of the femoral head demonstrated the presence of non-stained eGFP osteocytes in newly formed bone matrix, suggesting that contribution of MSC lies also in paracrine signalling that activate and recruit host osteoblasts to the bone repair areas [56]. Increasing evidence have shown that nanosized, membrane-encapsulated EVs are one of the most active MSCs’ secreted factors [25]. Indeed, EVs can serve as powerful tools for cell-free therapy due to pre- cise multifunctional molecular cargoes [57, 58]. However, Fig. 5 Effect of BM-MSC-derived products on osteogenic significant differences have been described in the content commitment of WJ-MSC. a Representative images of AR staining in of EV purified from MSC cultures of different origins. In passage 4 WJ-MSC after 3 weeks of culture with ODM alone (top) or supplemented with either EV or protein fractions collected from terms of miRNA profiles, BM-MSC-derived EVs have been undifferentiated BM-MSC (week 0) or BM-MSC cultures at week 1, 2 described to present a miRNA cargo that is tightly related or 3 of osteogenic differentiation (bottom). b Representative images to MSC differentiation [59]. Furthermore, different profiles of AR staining in passage 4 WJ-MSC after 1, 2 or 3 weeks of culture of miRNAs have also been reported depending on the dif- with CM obtained from differentiating BM-MSC cultures at week 1. ferentiation stage of the secretory cell [60]. Here, we stud- CM conditioned media, EV extracellular vesicles, ODM osteogenic differentiation media, SN supernatant. In all cases, scale bars: 100 μm ied the differences in the protein content of EVs obtained from undifferentiated BM- and WJ-MSC. As a result, COL6A1, COL6A2, PCOLCE, COL12A1 and COL6A3 found a marked delay in the osteogenic progression of osteogenic markers were differentially overrepresented in WJ-MSC compared to BM-MSC. Results obtained by BM-derived EVs compared to WJ-MSC EVs. Interestingly, qRT-PCR confirmed that BM-MSC exhibit an osteo- COL6 and COL12 interactions have been reported to con- genic gene profile more similar to that of osteoblast and trol and promote bone formation in early phases due to evidenced that MSX2, a TGFβ1-induced factor reported their role in the establishment of matrix bridges between to promote cell proliferation and supress osteogenic adjacent cells when pre-osteoblasts establish cell-cell com- differentiation by inhibiting DLX5-driven effects [36, munication [61]. These observations prompted us to evalu- 45–48], was overexpressed in WJ-MSC. This suggests ate the effect of BM-MSC-derived EVs in the osteogenic that the balance between MSX2 and DLX5 expression differentiation of WJ-MSC. Additionally, the impact of the Cabrera-Pérez et al. Stem Cell Research & Therapy (2019) 10:356 Page 9 of 11 soluble protein fractions (eluted in the latter size exclusion Supplementary information chromatography (SEC) fractions) purified from BM-CM Supplementary information accompanies this paper at https://doi.org/10. 1186/s13287-019-1450-3. was also determined. Both EV-rich and soluble protein fractions from differ- Additional file 1 Immunophenotypic characterization of BM-MSC and WJ- entiating BM-MSC cultures promote bone differen- MSC. Boxes represent median and 5-95 percentiles. N = 3 for each cell type. tiation in WJ-MSC. On the contrary, the secretome of Additional file 2. EV and protein fractions isolation and characterization. undifferentiated BM-MSC does not have an osteogenic (A) Scheme of the methodological procedure followed for EV and protein isolation from BM-MSC and WJ-MSC conditioned media. CCM, effect. This fact matches the differences reported in the concentrated conditioned media; CM, conditioned media; EV, extracellular miRNA content of MSC-EVs depending on differenti- vesicles; SEC, size exclusion chromatography; SN, cell culture supernatant. ation stages and explains, in part, the little differences (B) Example of representative elution profile obtained for CD9 and CD63 EV markers quantification by bead-based flow cytometry (left axis) and found in the protein content of undifferentiated BM- for protein elution monitoring by absorption at 280 nm (right axis) in the MSC and WJ-MSC EVs. Remarkably, the use of unpro- different SEC fractions. MFI, mean fluorescence intensity. (C) Cryo-EM cessed CM obtained from differentiating BM-MSC has a images confirming EVs presence in pooled EV fractions. Scale bar: 200 nm. greater impact on osteogenic induction and produces not only an exacerbation on the mineralisation of the Abbreviations culture but also a shortening in the differentiation time. ALP: Alkaline phosphatase; AR: Alizarin red; BM: Bone marrow; CM: Conditioned media; EV: Extracellular vesicle; FDR: False discovery rate; This suggests that EV- and protein-rich fractions from GMP: Good manufacturing practice; ISCT: International Society for Cell and differentiating BM-MSC act by different and synergistic Gene Therapy; MSC: Mesenchymal stromal cell; ODM: Osteogenic pathways, thus pointing out that BM-MSC and WJ-MSC differentiation media; qRT-PCR: Quantitative real-time PCR; RT: Room temperature; SEC: Size exclusion chromatography; WJ: Wharton’s jelly therapeutic efficacy could be equivalent when adminis- tered within the bone microenvironment, where BM- Acknowledgements MSC are present, and mitigating the need to overstimu- The authors would like to acknowledge Clémentine Mirabel and Blanca late WJ-MSC osteogenesis ex vivo. This is particularly Reyes for the technical support and advice, and Dr. Kristel van Landuyt for critically reviewing the manuscript and providing helpful comments. relevant due to the implications of WJ-MSC osteogenic priming in manufacturing development and clinical ap- Authors’ contributions plications which include (i) prolonged cell cultures for RC-P, FEB, SR and JV conceived the study. RC-P, MM-T, AG-V and RR-M per- ex vivo stimulation; (ii) difficulties of cell trypsinisation formed the experiments and analysed the data. RC-P wrote the manuscript. once the differentiation process is started; (iii) higher All authors revised the manuscript. All authors approved the final version of the manuscript. costs associated with the increment in time for cell cul- ture and the use of additional GMP grade products; and Funding (iv) possible clinical complications surrounding the use Work in our laboratory is supported by the Spanish Cell Therapy Network of priming compounds. Furthermore, the effect associ- (TerCel, expedient No. RD16/0011/0028 and RD16/00111/0006), Ministerio de Ciencia Innovación y Universidades de España (Instituto de Salud Carlos III, ated with the use of CM could be enhanced in vivo due expedient No. PI19/01788), Fundació la Marató de TV3 (expedient number: to the immune response produced under pathophysio- 122831), and ISCIII-REDinREN (RD16/0009 Feder Funds), and developed in the logical conditions, which positively contributes to bone context of AdvanceCat with the support of ACCIÓ (Catalonia Trade & Invest- ment; Generalitat de Catalunya) under the Catalonian ERDF operational regeneration. programme (European Regional Development Fund) 2014-2020. FEB is a re- searcher from Fundació Institut de Recerca en Ciències de la Salut Germans Conclusions Trias i Pujol, supported by the Health Department of the Catalan Govern- ment (Generalitat de Catalunya). Our laboratory is awarded by the Generalitat Despite their multipotentiality, the intrinsic molecular de Catalunya as Consolidated Research Groups (ref. 2017-SGR-719, 2017-SGR- signature of WJ-MSC described here highly counteracts 301 and 2017-SGR-483). their osteogenic differentiation and thus their future ap- plication in cell-based therapies against orthopaedic con- Availability of data and materials The datasets used and/or analysed during the current study are available ditions. However, our findings demonstrate that secreted from the corresponding authors on request. factors in the CM from differentiating BM-MSC cultures greatly enhance WJ-MSC osteogenesis and suggest that Ethics approval and consent to participate intra-bony environment could be enough to guarantee Clinical grade BM-MSC were produced within the context of four clinical tri- als (EudraCT Nos. 2010-023998-18, 2010-023999-12, 2013-005025-23 and WJ-MSC-promoted bone regeneration. This fact avoids 2015005786-23). WJ-MSC were isolated from umbilical cord tissue collected the need either to overstimulate WJ-MSC osteogenesis within the Concordia Program (EudraCT No. 2015-005786-23). Appropriate ex vivo or to use genetically modified WJ-MSC. There- donor informed consent for use in research was obtained in all cases. fore, although further research is required in order to evaluate the therapeutic benefit of WJ-MSC in the con- Consent for publication Not applicable. text of orthopaedic diseases, the use of GMP-grade master cell banks of WJ-MSC may be a valuable alternative Competing interests to those of BM-MSC. The authors declare that they have no competing interests. Cabrera-Pérez et al. Stem Cell Research & Therapy (2019) 10:356 Page 10 of 11 Author details The International Society for Cellular Therapy position statement. 1 Cell Therapy Service, Blood and Tissue Bank (BST), Barcelona, Catalonia, Cytotherapy. 2006;8(4):315–7. 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