Grau-Vorster et al. Stem Cell Research & Therapy (2019) 10:164 https://doi.org/10.1186/s13287-019-1279-9 RESEARCH Open Access HLA-DR expression in clinical-grade bone marrow-derived multipotent mesenchymal stromal cells: a two-site study Marta Grau-Vorster1,2†, Anita Laitinen3†, Johanna Nystedt3* and Joaquim Vives1,4,5* Abstract Background: Contrary to the minimal criteria proposed by the International Society for Cell and Gene Therapy for defining multipotent mesenchymal stromal cells (MSC), human leukocyte antigen (HLA)-DR expression is largely unpredictable in ex vivo-expanded clinical-grade cultures. Although activation of MSC in culture does not appear to affect their functionality, a large study investigating the impact of HLA-DR expression on cell identity and potency is still missing in the literature. Methods: A retrospective analysis of HLA-DR expression in 130 clinical batches of bone marrow (BM)-MSC from two independent Good Manufacturing Practice-compliant production facilities was performed in order to identify the consequences on critical quality attributes as well as potential activation cues and dynamics of MSC activation in culture. Results: HLA-DR+ cells in culture were confirmed to maintain fibroblastic morphology, mesenchymal phenotype identity, multipotency in vitro, and immunomodulatory capacity. Interestingly, the use of either human sera or platelet lysate supplements resulted in similar results. Conclusions: HLA-DR expression should be considered informative rather than as a criterion to define MSC. Further work is still required to understand the impact of HLA-DR expression in the context of product specifications on BM-MSC qualities for clinical use in specific indications. Keywords: Multipotent mesenchymal stromal cell, Potency, Identity, HLA-DR, Cellular therapy, Cell culture, Quality compliance Introduction interest in the management of graft rejection and/or in- Multipotent mesenchymal stromal cells (MSCs) can be flammatory conditions, due to their capacity to modulate easily derived from bone marrow (BM) aspirates and the biological response of other cell types involved in further expanded ex vivo up to sufficient numbers for these processes [6]. In view of their increasing popularity clinical use [1, 2]. Indeed, several clinical applications of of MSC as a candidate drug for use in the treatment of a MSC are currently being explored in the fields of trans- range of pathologies, the International Society for Cell plantation and regenerative medicine, with a couple of and Gene Therapy (ISCT) established minimal criteria products already holding marketing approval [3–5]. In to define MSC identity in an attempt to standardize the addition to their potential to become specialized bone, product specifications and methods for characterization cartilage, and fat cells, the use of MSC is of particular of MSC regardless of their origin and derivation pro- tocols [7]. ISCT’s criteria are based on MSC’s adherence * Correspondence: johanna.nystedt@gmail.com; jvives@bst.cat to plastic surfaces, multipotency in vitro, and the ex- † Marta Grau-Vorster and Anita Laitinen contributed equally to this work. pression of a set of cell surface markers (positive for 3 Finnish Red Cross Blood Service, Advanced Cell Therapy Centre, Kivihaantie CD73, CD90, and CD105; negative for CD14, CD34, 7, FIN-00310 Helsinki, Finland 1 Servei de Teràpia Cel·lular, Banc de Sang i Teixits, Edifici Dr. Frederic Duran i CD45 or CD11b, CD79α or CD19, and HLA-DR). Intri- Jordà, Passeig Taulat, 116, 08005 Barcelona, Spain guingly, BM-MSC do express HLA-DR not only when 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. Grau-Vorster et al. Stem Cell Research & Therapy (2019) 10:164 Page 2 of 8 inflammatory molecules such as IFN-γ are present but Miltenyi Biotec), anti-human HLA-DR-FITC (L243; BD also under normal expansion culture conditions [8, 9]. Biosciences), anti-human CD90-PE (F15-42-1-5, Beckman In the present study, we re-analyzed HLA-DR ex- Coulter), mouse anti-human CD31-FITC (WM59; BD pression in 130 clinical batches of BM-MSC from two Pharmingen), and mouse anti-human CD73-PE (AD2, BD independent Good Manufacturing Practice (GMP)-com- Pharmingen). The Helsinki protocol used cell surface anti- pliant production facilities to study its potential effect on gens CD44 (CD44 –FITC, G44-26; BD Pharmingen), the attributes of MSC and the dynamics of activation in CD49e (CD49e-PE, IIA1; BD Pharmingen), CD13 (CD13- expansion cultures. APC, WM15; BD Pharmingen), CD90 (CD90-FITC, 5E10; Stemcell Technologies and BD Pharmingen), CD73 Methods (CD73-PE, AD2; BD Pharmingen), CD29 (CD29-APC, Cells and cell culture MAR4; BD Pharmingen), CD105 (CD105-PE, 266, BD In Barcelona, clinical-grade BM-MSC were produced Pharmingen; 43A3, Stem Cell Technologies; and MEM- within the context of four clinical trials (2009-016449- 229, Abcam), CD14, (CD14-PE, M5E2; BD Pharmingen), 24, 2010-023998-18, 2013-005025-23, 2010-023999-12) CD19 (CD19-PE, HIB19; BD Pharmingen), CD34 (CD34- with appropriate donor informed consent and approval PE, 8G12, BD Biosciences; AC136, Miltenyi Biotec), CD45 from a competent regulatory authority. When needed, (CD45-PE, H130; BD Pharmingen), and HLA-DR (HLA- cells were further expanded in vitro up to sufficient DR –PE, MEM-12, Abcam; L243, BD Biosciences). numbers (always under passage 4) using Dulbecco’s Cells were stained for 15 min at room temperature modified Eagle’s medium (DMEM; Gibco) supplemented (Barcelona) and for 30 min on ice (Helsinki), washed, with 10% human serum B (hSerB) containing 2 mM and resuspended in phosphate-buffered saline (DPBS; glutamine in T-flasks and CellSTACKS (Corning Incor- Gibco). Non-specific cell staining was ruled out by using porated Life Sciences) at 1 × 103 to 3.5 × 103 cells/cm2 mouse immunoglobulin isotype controls (BD Pharmin- seeding density [10]. All cultures were maintained at gen). The acquisition was performed on a FACSCalibur 37 °C and 5% CO2 in humidified incubators. Cell counting (Barcelona) or a Navios cytometer (Helsinki), and data and viability assessment of BM samples were performed were analyzed with the FlowJo software (TreeStar Inc., using a Guava EasyCyte Mini (Millipore). Ashland, OR, USA). In Helsinki, donor recruitment, eligibility assessment, In both laboratories, HLA-DR expression was analyzed and bone marrow aspiration protocol were approved by and used only for informative purposes but not for the local ethics committee, and the BM collection pro- product release. cedure was authorized by the Finnish Medicines Agency Re-analysis of HLA-DR for the standardization of data under a tissue establishment license. MSCs were pro- analysis from the two groups was performed using duced under a national advanced therapy medicinal FlowJo. The gating strategy with the use of IgG isotype products (ATMP) hospital exemption license authorized control for unspecific staining (below 1%) is shown in by the Finnish Medicines Agency (Fimea; national Additional file 1: Figure S1. ATMP manufacturing licenses #6322/20.10.01/2011 and #5103/20.30.01/2013). MSC were derived from BM aspi- Differentiation assays rates as reported elsewhere [11]. Briefly, primary cultures Specific differentiation media were used for the osteo- (P0) were initiated by plating isolated BM mononuclear genic, chondrogenic, and adipogenic induction of undif- cells (MNCs) at 4 × 105 cells/cm2. The MSCs were ex- ferentiated MSC cultures in vitro as reported previously panded in a medium consisting of Dulbecco’s modified [12–14]. Safranin O (Sigma), Oil Red O (Sigma), Sudan III Eagle’s medium (DMEM) low glucose (Life Technologies) (Sigma), and von Kossa (Sigma) stainings were per- supplemented with 10% human Platelet Lysate (hPL) and formed for the determination of the outcome of the 40 UI/mL heparin (Heparin LEO 5000 IE/KY/mL; Leo differentiation assays. Pharma) [12]. Culture medium was replaced twice a week, and the cells were detached with TrypLE Select CTS (Life Technologies). The cells were plated at a density of 1 × Cell count, viability, and apoptosis 103 cells/cm2 from P1 onwards. Cells were counted either by Trypan blue dye exclusion methods or with Perfect-Count Microspheres (Cytognos) Phenotype assessment in a FACSCalibur cytometer (Becton Dickinson). Via- Immunophenotypic characterization of BM-MSC was bility was determined using the 7-amino-actinomycin D performed using the following antibodies. The Barcelona (7-AAD, BD Biosciences) exclusion method and expressed protocol used mouse anti-human CD45-fluorescein as a percentage (%) of total cells. Data were analyzed with isothiocyanate (CD45-FITC, HI30, BD Pharmingen), the CellQuest Pro (Becton Dickinson) software. Alter- anti-human CD105-phycoerythrin (CD105-PE, 43A4E1; natively, cell numbers and viability were determined using Grau-Vorster et al. Stem Cell Research & Therapy (2019) 10:164 Page 3 of 8 the NucleoCounter NC-100 device (ChemoMetec A/S, (average 19.8 ± 15.6%, from patients enrolled in clinical Denmark). trials for autologous use), whereas batches in Helsinki ranged from < 1 to 60.5% (average 19.2 ± 17.4%, from Lymphocyte proliferation assays healthy donors for allogeneic use) (Table 1). In order to The immunomodulation potential of BM-MSC was bring consistency to the comparability of analyses per- tested in the presence of peripheral blood mononuclear formed in different laboratories following different tech- cells (PBMC) at a ratio of 1:5 (Barcelona procedure) and niques, we reanalyzed a fraction of data from both 1:10, 1:20, and 1:50 (Helsinki procedure) as described laboratories (n = 65 and n = 37 from Barcelona and elsewhere [12, 15]. The proliferation of carboxyfluores- Helsinki, respectively; Additional file 1: Figure S2) BM- cein diacetate succinimidyl ester (CFSE)-labeled PBMC MSC batches from both centers complied with the specifi- after polyclonal stimulation was determined by measur- cations approved by their own respective regulatory ing the reduction of fluorescence intensity at day 5 by authority in terms of phenotype, adherence to culture- flow cytometry. treated plastic surfaces, fibroblastic morphology, differen- tiation potential, and immunomodulation capacity in vitro Effect of different medium supplements (Additional file 1: Table S1, Fig. 1). The effect of different medium supplements on the In order to determine the potential triggers of cell acti- variation of HLA-DR expression was investigated after vation in culture, standard parameters were analyzed, in- incubation of the same BM-MSC lines with different cluding density, medium supplements, and forced batches of sera. For this purpose, three independent activation with IFN-γ and its recovery after removal of BM-MSC lines were expanded under the same condi- the stimulus. tions for one passage and subsequently incubated with either one of two different conditions for an additional Effect of cell density and passaging on HLA-DR expression passage: one condition that was previously found experi- Regarding cell density and HLA-DR expression, no cor- mentally related to activation of BM-MSC (14.3 ± 12.8% relation was found (Fig. 2); dispersion without statistical HLA-DR+ cells, n = 7) and another one related to low correlation was observed in both cases (p value = 0.2287 HLA-DR+ cell number in culture (3.3 ± 3.3% HLA-DR+ and 0.2272, in Barcelona and Helsinki datasets, respec- cells, n = 2). tively). However, culture passaging was found to change the HLA-DR+ cell percentage. We collected HLA-DR Activation of MSC values from the two passages required in the production For cell activation experiments, IFN-γ (R&D Systems, process, which typically take a total of 3 weeks and cor- Minneapolis, MN, USA) was used as a stimulus at 10, respond to 24.6 ± 1.5 cumulative population doublings 100, and 200 UI/mL, based on the concentrations used (CPD). We observed a decrement in HLA-DR positivity in previously reported experiments [16]. of at least 25% in 71.9% of the cultures, whereas only For the recovery after stimulation experiments, the 5.3% increased HLA-DR more than 25% of its initial same line was used for all three conditions. Cells were value (n = 57). Furthermore, one MSC line was cultured previously stimulated 48 h with 100 UI/mL of IFN-γ. up to passage 5 confirming the decrease in HLA-DR+ After 48 h incubation, cells were left without IFN-γ to cell number (Additional file 1: Figure S3). see the decrease in HLA-DR. Controls without stimu- lation at any point and with maintained stimulation with Effect of cell culture medium supplement on HLA-DR 100 UI/mL of IFN-γ were performed. expression Different batches of sera experimentally related with Data analysis high and low HLA-DR values were tested in three Descriptive data were expressed as mean ± standard BM-MSC lines derived from three different donors. deviation. Pearson correlation p values were calculated to evaluate the significance of the correlation of cell Table 1 Summary of HLA-DR expression in 130 batches of density and HLA-DR expression in BM-MSC. clinical-grade BM-MSC from two independent GMP-compliant facilities. Batches of BM-MSC were distributed according to the production site Results Group Number Mean (%) Standard Minimum Maximum BM-MSC express variable levels of HLA-DR deviation (%) (%) Despite compliance with the ISCT criteria, BM-MSC in (%) culture showed variable percentages of HLA-DR+ cells in Barcelona 91 19.8 15.6 <1 77.7 130 batches produced in two manufacturing sites (namely, Helsinki 39 19.2 17.4 <1 60.5 Barcelona, n = 91, and Helsinki, n = 39). HLA-DR ex- Total 130 pression in Barcelona batches ranged from < 1 to 77.7% Grau-Vorster et al. Stem Cell Research & Therapy (2019) 10:164 Page 4 of 8 Fig. 1 Differentiation and immunomodulation potential of BM-MSC. Selected examples of characterization assays performed on BM-MSC at Barcelona’s and Helsinki’s facilities. a Differentiation potential was confirmed by von Kossa staining after 4-week culture in osteogenic medium (black depositions), Oil Red O/Sudan III staining after 3-week culture in adipogenic medium (lipid droplets in the cytoplasm of adipocytes stained red), and Safranin O staining after 3-week culture in chondrogenic medium (glycosaminoglycans stained red). b Immunomodulatory properties of MSC were tested by means of the lymphocyte proliferation assay, in which MSC demonstrated their capacity to suppress proliferation of polyclonally stimulated lymphocytes. Barcelona scale bars = 200 μm. Helsinki von Kossa scale bars = 500 μm and Oil Red O scale bars = 100 μm Despite starting with similar HLA-DR values (17.1%, Activation of HLA-DR expression 19.4%, and 15.0%; corresponding to an average of We took advantage of IFN-γ being a strong activator of 17.2% ± 2.2%), the percentage of activation after incu- MSC in vitro and performed a series of experiments to bation varied independently of the cell line or supple- study the effect of its addition to determine the kinetics ment. Remarkably, none of the other mesenchymal of BM-MSC activation upon stimulation with increasing surface markers depended on HLA-DR expression doses of IFN-γ. We observed that the maximum acti- (Table 2). Additionally, the potentiality of the three vation was reached at 48 h after induction, resulting in cell lines was not altered in either case, showing a high levels of activation when using 100 and 200 UI/mL similar capacity to differentiate in vitro into the doses (Fig. 3a). Interestingly, when IFN-γ stimulus was osteogenic and chondrogenic lineages (Additional file 1: removed, the number of HLA-DR+ cells decreased Figure S4). gradually over time (Fig. 3b). Grau-Vorster et al. Stem Cell Research & Therapy (2019) 10:164 Page 5 of 8 Fig. 2 Analysis of cell culture parameters. Effect of cell density on HLA-DR expression was studied in batches released from both manufacturing sites, namely Barcelona (a) and Helsinki (b). However, high dispersion without statistical correlation was observed in both cases (p value = 0.2287 and 0.2272, respectively) Discussion number of HLA-DR+ cells gradually decreased over time. Variability of HLA-DR expression in MSC cultures is a This observation is relevant since HLA-DR expression on nuisance for developers adhering to the criteria estab- cells during expansion cultures is variable over time; how- lished by the ISCT, so it tends to be overlooked provided ever, in-process and final product quality controls are per- that quality control (QC) panels are adapted to robust formed only at specific time points during the production product specifications in order to bring consistency from process, typically after 8 to 10 days from seeding, there- batch to batch using reliable markers [17]. This makes it fore showing only a snapshot of the status of the cells difficult to fully realize how different cell preparations and as a result could be potentially masking a dy- from different production sites can be. In this context, namic pattern of expression of HLA-DR. Regardless of standardization of QC criteria and methodology is a HLA-DR, the identity of MSC is clearly defined by other pressing need as new MSC-based therapies reach the parameters, such as the presence of mesenchymal cell sur- clinical setting and comparability of studies is required face markers and absence of hematopoietic and endothe- to define the efficacy of such new drug candidates [4, 5]. lial markers, fibroblastic cell morphology on plastic In the present study, we focused on the analysis of HLA surfaces, and immunomodulation and differentiation po- class II molecule expression in cultured MSC which was tential [7]. Indeed, we also showed that HLA-DR expres- demonstrated to be unpredictable and dynamic over time sion had no effect on immunomodulation and thus confirming previous studies [8]. Expression of multilineage differentiation potential of MSC [8]. HLA-DR has been proved, both in the present work In one of the first reports on this topic, Le Blanc and and reported by others, that it can be triggered in collaborators analyzed the intracellular protein levels of vitro by IFN-γ [9, 18] and other pro-inflammatory stimuli HLA-DR in MSC in parallel with extracellular HLA-DR [19]. Interestingly, when IFN-γ stimulus was removed, the phenotype by flow cytometry [18]. They found that MSC Table 2 Result summary after expanding cells in different medium formulations associated with “activating” or “neutral” cues. Activation of cells occurred randomly regardless of the supplement used. Nonetheless, differentiation potential was not altered (please refer to Additional file 1: Figure S2) Cell line Passage number supplement %CD31 %CD45 %HLA-DR %CD73 %CD105 %CD90 1 P0 3.6 1.9 17.1 99.8 99.9 99.83 P1 Activating 2.0 0.3 14.2 99.7 99.9 99.6 P1 Neutral 7.7 2.0 29.6 99.3 99.4 99.4 2 P0 6.4 1.1 19.4 100 98.7 99.9 P1 Activating 8.4 3.0 37.7 99.7 99.6 99.9 P1 Neutral 7.6 3.3 40.9 99.8 99.5 99.9 3 P0 7.2 1.0 15.0 99.7 99.7 99.84 P1 Activating 4.7 3.3 23.9 99.4 99.4 99.5 P1 Neutral 4.3 2.9 25.4 99.1 99.3 99.3 P passage number Grau-Vorster et al. Stem Cell Research & Therapy (2019) 10:164 Page 6 of 8 Fig. 3 Dynamics of BM-MSC activation in vitro. In culture, BM-MSC are activated by the addition of IFN-γ, which is illustrated by increasing concentration up to 200 UI/mL, reaching a peak 48 h after stimulation (a). Interestingly, the reversal of the process for the recovery of HLA-DR expression levels using IFN-γ-activated cells takes longer than the activation time (b) expressed first HLA-DR at the protein level intracellu- its expression is known to be indicative of inflammatory larly, and only later HLA-DR molecules were detected in microenvironments and maybe also differentiation com- the cellular membrane upon activation. The intracellular mitment [20]. In any case, the major concern regarding synthesis of HLA-DR was initiated after 2 days of expo- HLA-DR expression in MSC would be the potential rejec- sure to IFN-γ, but required up to 7 days for cell surface tion of these cells. Lately, new hypotheses about the MSC expression. Nevertheless, we detected MSC activation mechanisms of action involved are being proposed, and extracellularly after 48 h after IFN-γ exposure and pro- among them, efferocytosis is gaining strength. This hypoth- vided data of slow HLA-DR downregulation. With this, esis supports that MSC releases soluble molecules and it seems clear from these studies that the expression of extracellular macrovesicles that signal to the target environ- HLA-DR can come from a previous exposure since ment and then disappear [6]. Without considering HLA- clinical-grade manufacture bioprocesses are relatively DR expression, MSCs have not been reported to cause short (that is, two passages only) and we provided evi- rejection, but on the contrary, MSCs are being used in the dence of variability of HLA-DR expression even in the management of graft-versus-host disease (GvHD) [21, 22]. very first passage. One might think that the condition of The mechanisms by which these cells induce tolerance the donor could determine MSC activation in culture; were reported by Galleu and collaborators in vivo in an however, our study collected data from both healthy animal model and were apoptosis of MSC resulted in the donors and patients, and in both cases, the occurrence induction of immunosuppression [23]. Interestingly, IFN-γ- of HLA-DR expression appeared to be random. activated MSCs are being used for the treatment of GvHD With the removal of HLA-DR expression of MSC to induce tolerance [9]. Moreover, Zachar and collaborators from the QC panels, and the acceptance that HLA-DR illustrated that MSCs have to be activated in order to pro- expression in cultured MSC does not follow a predic- vide their therapeutic effects in the regenerative medicine table pattern under standard in vitro growth conditions, field [19]. Therefore, the presence of HLA-DR molecules it would be interesting to further investigate its triggers as on the cell surface does not compromise MSC identity, Grau-Vorster et al. Stem Cell Research & Therapy (2019) 10:164 Page 7 of 8 neither their function and application in the clinical setting Ethics approval and consent to participate for immunomodulation and differentiation potential appli- Cells were sourced from Banc de Sang i Teixits’ Biobank (Barcelona, Spain) and had appropriate donor informed consent for use in research. An cations. Further clinical studies could discard the secondary authorization is issued by Hospital de la Vall d’Hebron’s Ethics Committee effects of activated MSC as well as their therapeutic effect. (Barcelona, Spain) to JV. Consent for publication Conclusions Not applicable. We concluded that the use of HLA-DR as a negative marker of BM-MSC does not add any additional value to Competing interests QC panels and its expression does not affect other attri- The authors declare that they have no competing interests. butes, such as phenotype and functionality in vitro. Further- Author details more, we showed for the first time that the expression of 1 Servei de Teràpia Cel·lular, Banc de Sang i Teixits, Edifici Dr. Frederic Duran i HLA-DR is dynamic in MSC cultures. Finally, we believe Jordà, Passeig Taulat, 116, 08005 Barcelona, Spain. 2Transfusion Medicine that we can only reliably describe the full clinical potential Group, Vall d’Hebron Research Institute (VHIR), Universitat Autònoma de Barcelona, Passeig de la Vall d’Hebron 129-139, 08035 Barcelona, Spain. of MSC if robust QC panels directly related to MSC’s func- 3 Finnish Red Cross Blood Service, Advanced Cell Therapy Centre, Kivihaantie tionality are characterized, so the definition and broad 7, FIN-00310 Helsinki, Finland. 4Musculoskeletal Tissue Engineering Group, adoption of meaningful potency assays are urgently needed. Vall d’Hebron Research Institute (VHIR), Universitat Autònoma de Barcelona, Passeig de la Vall d’Hebron 129-139, 08035 Barcelona, Spain. 5Departament de Medicina, Universitat Autònoma de Barcelona, Passeig de la Vall d’Hebron Additional file 129-139, 08035 Barcelona, Spain. Received: 5 April 2019 Revised: 13 May 2019 Additional file 1: Table S1. 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