Comparison of different culture conditions for human

The Scandinavian Journal of Clinical & Laboratory Investigation, Vol. 68, No. 3, May 2008, 192–203

ORIGINAL ARTICLE

Comparison of different culture conditions for human mesenchymal stromal cells for clinical stem cell therapy M. Haack-Sorensen1, T. Friis1, L. Bindslev1, S. Mortensen1, H. E. Johnsen2 and J. Kastrup3*

Scand J Clin Lab Invest Downloaded from informahealthcare.com by University of Copenhagen North on 03/07/11 For personal use only.

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Cardiology Stem Cell Laboratory, The Heart Centre, University Hospital Rigshospitalet, Copenhagen, Denmark; 2Department of Haematology, Aalborg University Hospital, Aalborg, Denmark; 3Cardiac Catheterization Laboratory, The Heart Centre, University Hospital Rigshospitalet, Copenhagen, Denmark Objective. Mesenchymal stromal cells (MSCs) from adult bone marrow (BM) are considered potential candidates for therapeutic neovascularization in cardiovascular disease. When implementing results from animal trials in clinical treatment, it is essential to isolate and expand the MSCs under conditions following good manufacturing practice (GMP). The aims of the study were first to establish culture conditions following GMP quality demands for human MSC expansion and differentiation for use in clinical trials, and second to compare these MSCs with MSCs derived from culture in four media commonly used for MSC cultivation in animal studies simulating clinical stem cell therapy. Material and methods. Human mononuclear cells (MNCs) were isolated from BM aspirates by density gradient centrifugation and cultivated in a GMP-accepted medium (EMEA medium) or in one of four other media. Results. FACS analysis showed that the plastic-adherent MSCs cultured in EMEA medium or in the other four media were identically negative for the haematopoietic surface markers CD45 and CD34 and positive for CD105, CD73, CD90, CD166 and CD13, which in combined expression is characteristic of MSCs. MSC stimulation with vascular endothelial growth factor (VEGF) increased expression of the characteristic endothelial genes KDR and von Willebrand factor; the von Willebrand factor and CD31 at protein level as well as the capacity to develop capillary-like structures. Conclusions. We established culture conditions with a GMP compliant medium for MSC cultivation, expansion and differentiation. The expanded and differentiated MSCs can be used in autologous mesenchymal stromal cell therapy in patients with ischaemic heart disease. Keywords: Angiogenesis; bone marrow stem cells; cell cultures; endothelial differentiation; ischaemic heart disease; stromal cells

Introduction Cardiovascular disease is still the leading cause of morbidity and mortality around the world, and it is patients with end-stage ischaemic heart failure who carry the highest morbidity–mortality rate [1]. The clinical situation of a great number of patients with severe coronary artery disease, and who cannot be treated with conventional therapies, has led to extensive research to find new treatment regimes. In recent years, one focus has been on the use of stem cells as an experimental treatment to induce vasculogenesis or myogenesis in ischaemic heart disease [2– 4]. Transplantation of stem cells or progenitor cells into the injured site to replace the non-functional cells and enhancement of proliferation or differentiation of endogenous stem or progenitor cells are the two major cell-based strategies. Use of autologous human adult stem cells from the bone marrow (BM) in

transplantation is appealing because of its limited potential for immune rejection [5]. The adult BM harbours two different populations of stem cells with different biological properties: haematopoietic stem cells (HSCs) and mesenchymal stromal cells (MSCs) [6]. Both HSCs and MSCs have been used in animal trials to regenerate the heart [7– 11]. These studies have led to several small nonplacebo-controlled pilot studies and a few controlled studies using mononuclear cells (MNCs) isolated from BM aspirated from patients with acute myocardial infarction [2,12–17] or chronic myocardial ischaemia [18–23]. Granulocyte-colony stimulating factor (G-CSF), too, has been used to mobilize HSCs from the BM to the peripheral circulation in the treatment of patients with ischaemic heart disease [24–27]. Significant improvements in tissue perfusion and cardiac function were demonstrated in most of

*Correspondence author. Jens Kastrup, Cardiac Catheterization Laboratory 2014, The Heart Centre, University Hospital Rigshospitalet, Blegdamsvej 9, DK2100 Copenhagen Ø, Denmark. Tel: +45 3545 2819/2817. Fax: +45 3545 2705. Email: [email protected] and [email protected] (Received 8 June 2007; accepted 24 July 2007) ISSN 0036-5513 print/ISSN 1502-7686 online # 2008 Informa UK Ltd (Informa Healthcare, Taylor & Francis AS). DOI: 10.1080/00365510701601681 http://www.informaworld.com


Comparison of culture conditions for MSCs for clinical therapy these small non-randomized trials using BM-aspirated mononuclear cells, but not in all the larger randomized placebo-controlled trials [28,29]. However, it is debatable whether any possible effect is caused by the stem cells or the cytokines secreted from the MNCs, since only 2 % of the injected cells were stem cells [21]. MSCs from the BM seem to have the potential to differentiate into different tissue lines, i.e. endothelial and muscle cells [6,30,31]. These cells may therefore constitute a new treatment regime for the development of new blood vessels in patients with myocardial ischaemia and in the establishment of new contractile myocardium in patients with scar tissue and dilated cardiomyopathy. It has been documented in a recent publication that injection of MSCs from the BM into chronic ischaemic dog myocardium improved cardiac function and that the MSC-derived cells could be detected in newly developed blood vessels and in myocytes [32]. Iwase et al. [33], who compared the angiogenetic potential of MNCs and MSCs in a rat hind limb ischaemic model, confirm these results in a study. MSC-treated animals had more improvement in blood flow and more newly developed capillaries than MNC-treated or placebo-treated animals. MSCs are rare in the BM and have to be isolated and expanded ex vivo before they can be used in clinical trials. The safety of the materials used is important and there is an increasing concern for transmission of infection particles from animal to humans; the use of MSC in cell therapy will require development of a GMP (good manufacturing practice) protocol for their isolation and cultivation. The aim of the study was to establish culture conditions following GMP quality demands for human MSC isolation, expansion and differentiation for use in clinical trials as well as to verify that these GMP cultured MSCs are identical to MSCs cultured in four media commonly used for MSC expansion in animal studies simulating clinical stem cell therapy.

Material and methods Isolation of mononuclear cells (MNCs) from the bone marrow There are several methods for isolating MSCs from the BM. In this study, the starting material consisted of BM obtained from patients undergoing elective hip replacement surgery. This procedure was approved by the local scientific ethics committee (KA 01053). The aspirated bone marrow was immediately combined with 25 mL RPMI 1640 medium containing GlutaMAX and 25 mM HEPES (GIBCO,

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Invitrogen, Taastrup, Denmark) plus 40 IE heparin/ mL (Hospital Pharmacy, Copenhagen, Denmark). The BM sample was diluted to a total of 1:5 with phosphate-buffered saline (PBS) (137 mM NaCl, 2.7 mM KCl, 1.4 mM NaH2PO4?H2O, 6.5 mM NA2HPO4?2H2O, pH 7.3–7.4) (Hospital Pharmacy, Copenhagen, Denmark) and portions of 5 mL were carefully transferred to 10 mL tubes each containing 5 mL Lymphoprep (Axis-Shield PoC AS, Oslo, Norway). After centrifugation at 1200g for 25 min, the mononuclear cells (MNCs) were collected from the interface, washed twice in PBS, and re-collected after centrifugation at 300g for 5 min. The cells were lysed by incubation with 3 mL Ortho-Mune lysing solution (Hospital Pharmacy, Copenhagen, Denmark) for 10 min in the dark. Afterwards, the cells were washed twice in PBS, counted and seeded at a density of 26107 cells per flask in T75 culture flasks (NUNC, Roskilde, Denmark) containing 15 mL of: (A) FBS medium composed of Dulbecco’s modified Eagle medium (DMEM) low glucose (1 g/L) with 25 mM HEPES and LGlutamine (PAA Laboratories, Pasching, Austria) plus 1 % penicillin/streptomycin (pen-strep) (GIBCO, Invitrogen, Taastrup, Denmark) and 10 % FBS (GIBCO, Invitrogen, Taastrup, Denmark) [30], which is a standard supplement in cell culture; (B) expansion medium (Exp-medium) composed of 60 % DMEM and 40 % MCDB-153 (Biochrom, Berlin, Germany) supplemented with ITS (insulin, transferrin, selerium), ascorbic acid 2-phosphate, hydroxycortisol, linoleic acid-BSA, epidermal growth factor (EGF) and platelet-derived growth factor-BB (PDGF-BB) (all purchased from Sigma-Aldrich, Brøndby, Denmark) [34]; (C) EBM-2-medium composed of EBM-2 (endothelial basal medium-2) (Cambrex Bio Science, Walkersville, Md., USA) plus 1 % pen-strep and 10 % FBS [35,36]; (D) HuSmedium composed of DMEM plus 1 % pen-strep and 10 % human serum (AB-HuS) (Sigma-Aldrich, Brøndby, Denmark) [12]; or (E) the GMP accepted MSC culture medium (EMEA medium) composed of DMEM plus 1 % pen-strep and 10 % EMEA-FBS (PAA Laboratories, Pasching, Austria) [37,38]. The EMEA-FBS, which we are using, is an Australian produced FBS tested by validated procedures to be free of BSE and FMD according to the European Agency for the Evaluation of Medicinal Products (EMEA) (the European counterpart of the FDA) and USDA (U.S. Drug Administration). It is treated by a validated irradiation process to inactivate potential contamination. The most common and best characterized method by which to isolate MSCs is adherence selection. After incubation for 5 days at 37 ˚C in humid air with


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5 % CO2, the medium was changed and almost all of the haematopoietic stem cells (non-adherent cells) were washed away. Subsequently, the medium was changed every 3 or 4 days. When the adherent cells in the flasks were confluent, cells were harvested by incubation for 10 min at 37 ˚C with 3 mL Tryple Select (animal origin free) (GIBCO, Invitrogen, Taastrup, Denmark). To inactivate Tryple Select, 7 mL of the respective culture medium was added and the cell suspension was centrifuged for 5 min at 300g. Cells were re-suspended, counted and re-plated for further experiments. During cultivation, morphological changes were followed visually by phasecontrast microscopy. Proliferation capacity Cell proliferation was measured using the PKH26GL cell linker kit (Sigma-Aldrich, Brøndby, Denmark). Following the manufacturer’s instructions, MSCs were marked with PKH26, which is a membrane-labelling agent that incorporates a fluorescent dye with long aliphatic tails (PKH26) into lipid regions of the cell membrane. The PKH-stained cells were plated in 24 well plates at a density of 10,000 cells/well in 1 mL culture medium. Up to 7 days after PKH staining, the fluorescent intensity on the cells was measured in duplicate/triplicate by flow cytometry. The half-lifetime of fluorescent intensity on the cells was proportional to the doubling time for the MSCs. Differentiation towards endothelial cells MSCs that were approximately 80 % confluent were induced to differentiate towards endothelial cells; the cells were cultured for 7 days in a modified EMEA medium containing only 2 % serum and 50 ng/mL human recombinant vascular endothelial growth factor (rhVEGF-A165) (R&D Systems, Minneapolis, Minn., USA). The medium was changed every 2 or 3 days [30,37,38]. Flow cytometric analysis of BM cells MSCs were harvested using cell scrapers (NUNC, Roskilde, Denmark). The cells were washed in 3 mL FACS-PBS mixture (445 mL FACS-PBS; Hospital Pharmacy, Copenhagen, Denmark), 5 mL 1 mM/L EDTA (Hospital Pharmacy, Copenhagen, Denmark) and 50 mL newborn calf serum (NCS) (GIBCO, Invitrogen, Taastrup, Denmark)), and centrifuged for 5 min at 300g. Cell pellet was re-suspended in a suitable volume FACS-PBS mixture and distributed to FACS tubes with antibodies.

The MSCs were incubated for 30 min at 4 ˚C with the following panel of monoclonal antibodies conjugated with fluorescein isothiocyanate (FITC), phycoerythrin (PE), peridinin chlorophyll-a protein (PerCP) or allophycocyanin (APC): anti-CD45 (HI30 clone, IgG1; Becton Dickinson (BD), San Jose, Calif., USA) and anti-CD34 (8G12 clone, IgG1; BD, San Jose, Calif., USA) to exclude haematopoietic cells as well as anti-CD105 (NI-3A1, clone, IgG1; Ancell Corporation, Bayport, Minn., USA), anti-CD73 (AD2 clone, IgG1; BD, San Jose, Calif., USA), anti-CD166 (3A6 clone, IgG1; BD, San Jose, Calif., USA), anti-CD13 (F0831 clone, IgG1; Dako Cytomation, Glostrup, Denmark), or anti-CD90 (Immunotech, Brussels, Belgium). IgG1 FITC, IgG1 PE, IgG1 PerCP and IgG1 APC (BD, San Jose, Calif., USA) were used as negative controls. Afterwards, the cells were washed twice in FACSPBS solution and cell fluorescence was measured by flow cytometry on 10,000/15,000 cells per sample using a FACS Calibur instrument (BectonDickinson, San Jose, Calif., USA). Cell analysis was done using Cell Quest PRO software (Becton Dickinson, San Jose, Calif., USA). The forward scatter/side scatter (FSC/SSC) was divided into two gates and the data were analysed individually for each gate. RNA extraction and gene expression Total RNA was extracted from unstimulated MSCs (control, cultivated in EMEA medium) and VEGFstimulated MSCs (cultivated 7 days in EMEA medium with 50 ng/mL VEGF and only 2 % EMEA-FBS) using the micro-kit (Qiagen, Hilden, Germany) in accordance with the manufacturer’s protocol. For reverse transcription (RT), 1 mg RNA was used in 20 mL reactions (1 mL oligo(dT); primer (from 50 mM stock, mix of oligo 12T, 15T and 18T; Invitrogen, Taastrup, Denmark) was added to 1 mg RNA in 13 mL DEPC-treated water and heated to 65 ˚C for 10 min. The reaction was then placed on ice and added to 2 mL 106RT buffer (200 mM TrisHCL, pH 8.4, 500 mM KCl, 25 mM MgCl2, and 1 mg/mL BSA), 2 mL 0.1 M DTT, 1 mL 10 mM dNTP mix, and 1 mL RT enzyme (M-MLV, 200 U/m; GIBCO, Invitrogen, Taastrup, Denmark). Synthesis of cDNA was performed at 37 ˚C for 60 min, followed by 5 min at 95 ˚C, as suggested by the manufacturer. The gene expression levels of VEGF receptor 2 (KDR) and von Willebrand factor (vWF) were determined by real-time PCR and related to expression of the housekeeping gene b-actin. b-actin: Forward: CCT TTT TGT CCC CCA ACT TGA, Reverse: TGG CTG CCT CCA CCC A, Probe: ATG


Comparison of culture conditions for MSCs for clinical therapy TAT GAA GGC TTT TGG TCT CCC TGG GA; KDR: Forward: CAG CAG GAT GGC AAA GAC TAC A, Reverse: GGC AGA GAG AGT CCA GAA TCC TC, Probe: TGT TCT TCC GAT ATC AGA GAC TTT GAG CAT GG, and vWF: Forward: ATG TAT GAA GGC TTT TGG TCT CCC TGG GA, Reverse: CCA TCC TGG AGC GTC TCA TC, Probe: AAG AGG AGG ACA GAT CAT GAC ACT GAA GCG. All probes were labelled with FAM (6-carboxy-fluorescein) as reporter at the 59 end and the quencher TAMRA (6-carboxy-tetramethyl-rhodamine) at the 39 end. All Primer sets spanned introns to reduce the risk of amplifying genomic DNA. Primers were purchased from Invitrogen (Taastrup, Denmark) and probes from MWG-Biotech (Ebersberg, Germany). All the reactions were 30 mL (reaction mixture: Platium QPCR supermix-UDG + Rox reference dye, MgCl2 (concentration optimized for each primer set) (Invitrogen, Taastrup, Denmark), primer forward and reverse (10 mM), probe (5 mM), H2O and cDNA), and each sample was run in duplicate. Two ‘‘no-templatecontrol’’ samples were included for each run. The thermal cycling conditions included 2 min at 50 ˚C and 10 min at 95 ˚C, followed by 45 cycles of 95 ˚C for 15 s and 58 ˚C for 1 min. All reactions were performed on an Mx3000 (Stratagene, AHDiagnostics, Aarhus, Denmark). Data collection and analysis was done with the Mx3000 version 2.0 software for Windows (Stratagene, AH-Diagnostics, Aarhus, Denmark). All assays were tested in logarithmic dilutions of template to verify that their affinity is comparable at high and low expression levels. Immunocytochemical analysis To detect the endothelial-specific proteins, the monoclonal mouse anti-human CD34 antibody (1:40) (Becton Dickinson, San Jose, Calif., USA), the CD31 antibody (1:20) (Dako Cytomation, Glostrup, Denmark) and polyclonal rabbit antihuman vWF antibody (1:400) (Dako Cytomation, Glostrup, Denmark) were used. All antibodies were diluted in 16buffer II+III solution (8.8 g/L disodiumhydrogen phosphate, 14 g/L sodium dihydrogen phosphate, 88 g/L sodium chloride, 5.0 mL/L Tween 20, pH 7.0) +2 % human serum albumin (HSA) (Hospital Pharmacy, Copenhagen, Denmark). The secondary antibodies used were Dako REAL Envision/horseradish peroxidase (HRP) and goad anti-rabbit/mouse IgG (K5007) (Dako Cytomation, Glostrup, Denmark). MSCs grown on round sterile, thermanox plastic coverslips (NUNC, New York, USA), either in the

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presence or absence of VEGF, were fixed in 4 % formalin (Hospital Pharmacy, Copenhagen, Denmark) for 5 min, then washed 262 min in 16buffer II+III solution; incubated with premier antibody for 1 h, washed 363 min with 16buffer II+III solution and then incubated with peroxidasemarked second antibody for 30 min. The coverslips were then washed 463 min in 16buffer II + III solution and incubated for 15 min with Dako REAL Envision detection system (DAB+ + chromogen diluted in substrate buffer) (K5007) (Dako Cytomation, Glostrup, Denmark). An enzymatic reaction is formed because DAB acts as a substrate that precipitates in the presence of peroxidase, and the reaction becomes visible. The nuclei were then stained with Mayer’s Hematoxylin (Hospital Pharmacy, Copenhagen, Denmark). In vitro tube formation assay Endothelial cells have the capacity to form networks of capillary-like ring structures when cultivated on ECMatrix. The in vitro matrigel angiogenesis kit (Chemicon International Inc., Millipore, Billerica, Mass., USA) was used (in accordance with the manufacturer’s instructions) to study the angiogenic capacity of unstimulated and VEGF-stimulated MSCs. Briefly, unstimulated and VEGF-stimulated MSCs were harvested, and 5,000–10,000 cells were added on top of the solid ECMatrix in 150 mL EMEA medium containing either 10 % serum (control) or 2 % serum and 50 ng/mL VEGF. The cells were maintained at 37 ˚C in humid air with 5 % CO2 for 8 h. The MSC capillary-like network formation was inspected and digitally photographed (Nikon Coolpix 4500, Copenhagen, Denmark) under a phase contrast microscope (Leica DMIL, Herlev, Denmark) at magnification 6100.

Results Isolation and cultivation of bone-marrow-derived mesenchymal stromal cells (MSCs) Cultivation of lymphoprep gradient isolated MNCs (Figure 1A), which contained approximately 1 MSC for every 100,000 nucleated marrow cells (MNC) [39], resulted in an evolving population of plastic-adherent cells, which constituted the primary ex vivo source of MSCs. After 5 days of cultivation, the non-adherent haematopoietic cells were washed away by medium change, with only the adherent MSCs left in the culture flask (Figure 1B). These cells displayed a fairly homogeneous population of spindle-shaped


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Figure 1. Cell morphology – from seeding of MNCs to harvest of MSCs. Phase-contrast microscopy photographs at magnification 6100 of A: lymphoprep gradient isolated MNCs, B: plastic-adherent MSCs after one week of cultivation in EMEA medium, and C: confluent MSCs after approximately 3 weeks of cultivation in EMEA medium.

and fibroblast-like cells. After approximately 3–4 weeks of cultivation, large areas in the culture flasks were confluent (Figure 1C) and the MSCs were harvested for further use. The forward scatter/side scatter (FSC/SSC) analysis on the FASC calibur showed that the MSCs were a heterogeneous population of cells of very different size and granulation (Figure 2). For analysis of the raw data, we divided the MSCs into two regions (R1 and R2); the data presented in this article represent the entire population (R1+R2). Characterization of MNC and MSC Flow cytometry was performed to characterize the expression patterns of characteristic surface proteins on lymphoprep gradient isolated MNCs and the MSCs after ex vivo cultivation.

Figure 2. Forward side-scatter plot of MSCs in culture. The plot shows that the cells have a wide range in size (FSC-H) and granulation (SSC-H). The data shown have been analysed separately for gate R1+R2, but are presented as one data set.

MNCs were positive for the characteristic haematopoietic markers CD45 and CD34, whereas the plastic-adherent MSCs were almost negative for both CD45 and CD34 (Figure 3). Expression of the surface markers CD105 (SH2), CD73 (SH3/SH4), CD90 (Thy-1), CD166 (ALCAM) and CD13, which in combined expression is characteristic of mesenchymal stromal cells, was much higher on MSCs than on MNCs (Figure 3). This indicates that by culturing the MNCs it is possible to separate the MSCs from the haematopoietic cells based on their selective adherence to plastic surfaces. Comparison of different culture media Morphology of the MSCs in different culture media To compare the GMP-accepted MSC culture medium (EMEA medium) with four media commonly used for MSC cultivation in animal studies, BM aspirates were obtained from 10 donors and the freshly isolated MNCs from each donor were divided into 5 aliquots and seeded at a density of 26107 per culture flask in 15 mL of A: FBS medium; B: Exp medium; C: EBM-2 medium; D: HuS medium or E: the GMP accepted

Figure 3. FACS analysis of MNCs and MSCs. MNCs contained haematopoietic cells expressing CD45, whereas MSCs were predominantly CD452/CD342. The expression of CD105, CD73, CD90, CD166 and CD13 was higher on MSCs than on MNCs (n510).

Comparison of culture conditions for MSCs for clinical therapy

Expression of surface markers on MSCs cultivated in the different media To investigate whether cultivation in the different culture media influenced the expression of surface markers we performed flow cytometry on MSCs cultured in the five different media. As shown in Figure 4F, there were no differences in the presence of surface markers CD105, CD73, CD90, CD166 and CD13 on MSCs cultured in any of the five media, although the expression level of some of the surface proteins was lower on cells in FBS medium, EBM-2


EMEA medium. After one week of cultivation, singlecell derived colonies were visible in all five culture media (Figure 3A–E). When inspecting the cells, we found only small differences in their morphology. The colonies in FBS medium (Figure 4A), EBM-2 medium (Figure 4C) and EMEA medium (Figure 4E) contained both spindle-shaped cells and large flat cells; in Exp medium, the cells mostly had spindle-shaped morphology (Figure 4B) and the colonies in HuS medium contained both spindle-shaped cells and small flat cells (Figure 4D).

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Figure 4. Morphology and surface protein expression of MSCs cultured in different media. MSCs cultivated in A: FBS medium were spindle-shaped or large flat cells; B: Exp medium were predominantly spindle-shaped; C: EBM-2 medium were spindle-shaped or large flat cells; D: HuS medium were spindle-shaped or small flat cells; and E: EMEA medium were spindleshaped or large flat cells. Magnification 6200. F: MSCs cultivated in different culture media all expressed CD105, CD73, CD90, CD166 and CD13 (n510).

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Stimulation with VEGF

Figure 5. Cell proliferation. MSCs cultured in five different media conditions. The average doubling time varied only slightly among the five media (n510).

medium and HuS medium than on cells in EMEA medium and especially Exp medium, in which MSCs had a markedly higher expression of CD105, CD73, CD90 and CD13 than in any of the other four media. Proliferation of MSCs cultured in the different media The proliferation capacity of MSCs cultured in the five different media was investigated. The cells were labelled with the fluorescent membrane-labelling agent PKH26 and their PHK26 fluorescence intensity was analysed by flow cytometry up to 7 days after cell labelling. The doubling time of the cells, which was proportional to the half-life period for the PKH26labelling intensity of the cells, varied only slightly among the five media (Figure 5). The average doubling time ¡ SEM was found to be 29.4¡5.2 h in FBS medium, 28.2¡3.6 h in Exp medium and EBM-2 medium, 31.8¡4.1 h in HuS medium and 35.4¡4.5 h in EMEA medium.

MSCs were stimulated to differentiate towards endothelial cells by culturing 80 % confluent cells for 7 days in EMEA medium containing 50 ng/mL rhVEGF and only 2 % EMEA-FBS, whereas control cells were kept in EMEA medium. To monitor the differentiation pattern, changes in the expression of the characteristic endothelial genes KDR/VEGFR-2 and vWF were detected by real-time PCR measurements. The gene expression of both KDR and vWF was increased in VEGF-stimulated MSCs (Figure 6A, B). To further confirm endothelial differentiation of MSCs, cells were examined immunocytochemically with monoclonal anti-CD34, CD31 and polyclonal anti-vWF antibodies. Consistent with our previous real-time PCR results, expression of vWF was much higher in VEGFtreated cells compared to control cells. Moreover, some of the treated cells also expressed CD31, but not the control cells. As positive control, human cardiac microvascular endothelial cells (HCMECs) were used. HCMECs were small cells compared to MSCs and were positive for vWF; CD31 and some cells were also positive for CD34, as shown in Figure 7A. Furthermore, we investigated whether MSCs have the capacity to form capillary-like ring structures on ECMatrix. Unstimulated MSCs cultured in EMEA medium and VEGF-stimulated MSCs were incubated for 8 h on ECMatrix, and the ability to form capillary-like structures was evaluated. VEGF-stimulated MSCs formed networks of capillary-like ring structures, whereas unstimulated MSCs had a more limited capacity to form networks of capillary-like ring structures (Figure 7B). Together, these results indicate that the MSCs can be induced to

Figure 6. Real-time PCR gene expression of KDR and vWF in unstimulated MSCs and VEGF-stimulated MSCs. Expression of the characteristic endothelial cell genes, A: KDR (VEGFR2) and B: vWF were increased in VEGF-stimulated MSCs. The expression levels are presented relative to a-actin (n510).



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Figure 7. Functional characterization of MSCs differentiated towards endothelial cells. A: Immunocytochemical staining of MSCs un-stimulated (control) and stimulated with VEGF. MSCs were stained for CD34, endothelial marker vWF and CD31. The control cells were stained negative for CD34 and the endothelial markers, but VEGF-stimulated MSCs stained positive for vWF and few cells stained positive for CD31. HCMEC were used as positive control. The phase-contrast microscopy photographs were taken at magnification 6200. B: VEGF-stimulated MSCs had the capacity to form capillary-like ring structures when cultivated for 8 h on ECMatrix, whereas C: unstimulated MSCs had a more limited capacity to form the capillary-like ring structures. The phase-contrast microscopy photographss were taken at magnification 6100.

differentiate towards endothelial cells when cultured for 7 days in EMEA medium with 50 ng/mL VEGF and low serum content.

Discussion Transfer of treatment regimes from animal studies to human clinical trials is often difficult because of safety issues which have to be taken into account if severe side effects related to the treatment are to be avoided. The present study evaluated a GMPapproved cell expansion technique which can be used when implementing autologous mesenchymal stromal cell treatment in clinical settings and compared this with the non-GMP culture conditions normally used in animal studies simulating clinical stem cell treatment. MSCs isolated from the BM have the potential to differentiate into several cell types, i.e. tri-lineage

mesenchymal stromal differentiation: osteoblasts, adipocytes, condrocytes, and other cell types such as endothelial cells and myocytes [6,31,40], which can all potentially be used for regenerative stem cell treatment in different human diseases. In the present study, our focus was on culture conditions and the cells’ potential to differentiate in an endothelial direction, which can be used in clinical treatment of patients with coronary artery disease. In animal studies, it has been demonstrated that treatment with MSCs improves cardiac perfusion and function in a chronic myocardial ischaemia model [32] and also perfusion in an ischaemic hind leg [33]. The improvement seems to result from increased numbers of MSC-derived endothelial cells and myocytes. In order to be able to implement MSC treatment in clinical practice, it is necessary to expand the number of MSCs ex vivo. Despite the fact that MSCs can readily be isolated and expanded ex vivo [41,42],


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many of the applied culture media are unsuitable for use in clinical trials, since they are based on animal products and consequently involve the risk of transference of infective particles from animal to human. Variations in the isolation techniques and culture media used for cultivation of MSCs in different laboratories have led to varying findings regarding characterization of these cells [43–45]. Therefore, we consider it important that a simple and uniform GMP-approved culture medium be developed for MSC expansion. We tested a culture medium (EMEA ) approved for human cell therapy and compared it with four media often used in ex vivo and animal studies to verify that these GMP cultured MSCs were identical to MSCs cultured in media used for MSC expansion in animal studies. When using lymphoprep-gradient isolated MNC from the BM as initial cell source, we found that the adherent cells cultured in the GMPapproved EMEA medium had the same surface characteristics as previously described for MSCs [30,31,45,46]. Moreover, when comparing cells cultured in EMEA medium with cells cultured in four media commonly used in animal studies, we found some small differences in expression level of surface markers between cells in the different media. However, the expression patterns of the surface markers were identical among the five media. Our results show that about 20 % of the cells were CD105 positive, whereas the literature describes a much higher expression of CD105 on MSCs. However, MSCs are a heterogeneous population and, when selected from bone marrow by their plastic adherence property, there probably remains some non-MSC contamination in the culture and not all cells are in the same cell cycle proliferation phase when the culturing period is terminated. To have a purer MSC population, it is necessary to use methods such as multiparametric fluorescence activated cell sorting (FACS) or magnetic bead separation with selected antibodies. Many variables and parameters must be considered when expanding MSCs for clinical purposes. They thrive very well in Exp medium, but it is important to keep culture conditions as simple as possible, and Exp medium contains several different growth factors and supplements not all of which are useable in clinical settings [34,42]. All synthetic media will require growth factor supplementation, and serum is a crucial ingredient in media owing to its supply of nutrients. For expansion of cells for clinical use, it is not possible to use standard (nonirradiated) FBS because of the potential risk of transmitting infection particles from animals to

humans. Several data on the use of human serum for MSC cultivation have been reported [12,16,23,47,48]. Although Stute et al. described MSC expansion being identical in medium containing either 10 % autologous serum or 10 % FBS, they found it difficult to obtain enough autologous serum to expand MSC on a clinical scale [48]. Our results show that MSCs cultured in EMEA medium or EXP medium were CD45 and CD34 negative and had almost identical expression levels of the surface markers CD105, CD73, CD90, CD166 and CD13, which was higher compared to HuS medium or standard FBS medium that in combined expression is characteristic of MSCs. In EMEA medium, some of the cell surface markers were superior to those in standard FBS medium. It is possible that different batches of standard FBS can have some influence on cell culture outcome. For the clinical trials, however, standard FBS is not approved, so we did not consider it important to optimize the different standard FBS batches. Difficulties in collection, output and quality of the autologous serum can vary from patient to patient and lead to difficulties in standardization of culture conditions. On the other hand, different risks are associated with the use of materials of animal origin, including bovine serum, which is an essential ingredient of the cell culture media. Indeed, the nature and quality of bovine serum can profoundly influence the quality of the manufacturing process and of the finished product. We therefore conclude that the GMP-accepted EMEA medium is useful in ex vivo expansion of MSCs for use in clinical settings. The materials and methods used within this research have been evaluated and controlled by the Danish Medicines Agency for use in clinical trials. The method has been approved in two ongoing clinical trials in patients with coronary artery disease. Regarding isolation and expansion of MSCs, unfortunately serum-free media have not yet been defined. Serum-free media have been reported [49–51] but have not been extensively used by independent laboratories, and development of new serum-free media for isolating and expanding MSCs is an area of investigation within the biotechnology industry for clinical use. We tried different serum-free media in our laboratory, but our cells died (data not shown). To evaluate whether the isolated MSC could differentiate toward endothelial cells, we stimulated the expanded MSC with VEGF at low serum content and found, as demonstrated in several previous MSC studies, that VEGF stimulation of MSC increased gene expression of the endothelial cell markers vWF and VEGF receptor 2 (KDR) [30,34,37,38].


Comparison of culture conditions for MSCs for clinical therapy Moreover, stimulation with VEGF increased expression of the endothelial proteins vWF and CD31 verified by immunocytochemical staining, and increased the capacity of MSCs to form capillarylike ring structures when incubated on ECMatrix. Many patients still suffer from the severe clinical consequences of coronary artery disease with angina and end-stage ischaemic heart failure despite improved medical and revascularization therapies. Based on the impressive results in studies in both small and larger animals, stem cell therapy can be an option in cardiac patients. Many laboratories are investigating how progenitor/stem cells can be used to treat regenerative diseases. To date, most of the published work on transplantation of stem cells, especially MNCs, has been done in animal models. Some clinical trials within cardiology have shown that intra-coronary injection of autologous MNCs from the bone marrow can improve the perfusion and contractile function of the heart, while other trials cannot find any improvement [15,28–30]. However, cell-based therapy seemed both safe and feasible, but the precise mechanism underlying the apparent improvement in tissue perfusion is unclear. MSC transplantation has been shown to induce neovascularization in animal models [2,34,36,52–55], and in some studies MSCs have been superior to MNCs in the development of new capillaries and improvement of left ventricular function in animals with myocardial ischaemia [32,33]. These findings suggest that MSCs will have at least the same beneficial effects in the treatment of patients with myocardial ischaemia as MNCs. The present method for isolation and expansion of MSCs from the bone marrow meets the recommendations for a GMP protocol. Therefore, it is now possible to take the next step and test whether clinical autologous MSC therapy has the potential to induce vasculogenesis in ischaemic tissues. There have been no clinical studies on the therapeutic potency of human ex vivo cultureexpanded MSCs compared to MNCs in ischaemic heart disease, but there has been one study where injected intra-coronary autologous MSCs expanded in FCS-containing media together with endothelial progenitor cells in patients after an acute myocardial infarction [56,57]. An improved recovery of left ventricular function was found and no ventricular arrhythmia was detected. In addition, ex vivo expanded MSCs have been administrated safely and without adverse reaction in patients with osteoarthritis [58], in haematological malignancy that was in complete remission [59] and

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co-transplanted with HSCs in haematologic malignancy patients [60] and in stroke patients [61]. Conclusions Using flow cytometry, we have demonstrated that MSCs obtained from the bone marrow and cultured in GMP-approved EMEA medium express the same surface proteins as MSCs cultured in four media commonly used for MSC expansion in animal studies using non-GMP approved media. We found that the MSC proliferation rate was similar in the five culture media. VEGF stimulation of the MSCs increased expression of the characteristic endothelial genes KDR and vWF as well as vWF and CD31 at protein level and the capacity to form capillary-like ring structures. We conclude that the use of fetal bovine serum is potentially hazardous, but until a suitable alternative is found this remains the only choice in initiating clinical MSC trials. Our GMP-accepted EMEA medium can be used for expansion and differentiation of MSCs to endothelial cells for autologous stem cell therapy in clinical trials in patients with ischaemic heart disease. Acknowledgement The Lundbeck Foundation and the Research Foundation at Rigshospitalet supported the study. References [1] Graham RM, Bishopric NH, Webster KA. Gene and cell therapy for heart disease. IUBMB Life 2002;54:59–66. [2] Hamano K, Li TS, Kobayashi T, Tanaka N, Kobayashi S, Matsuzaki M, et al. The induction of angiogenesis by the implantation of autologous bone marrow cells: a novel and simple therapeutic method. Surgery 2001;130:44–54. [3] Siepe M, Heilmann C, von Samson P, Menasche P, Beyersdorf F. Stem cell research and cell transplantation for myocardial regeneration. Eur J Cardiothorac Surg 2005. [4] Yongzhong W, Johnsen HE, Jorgensen E, Kastrup J. The clinical impact of vascular growth factors and endothelial progenitor cells in the acute coronary syndrome. Scand Cardiovasc J 2003;37:18–22. [5] Lee MS, Lill M, Makkar RR. Stem cell transplantation in myocardial infarction. Rev Cardiovasc Med 2004;5:82–98. [6] Minguell JJ, Erices A, Conget P. Mesenchymal stromal cells. Exp Biol Med (Maywood) 2001;226:507–20. [7] Fuchs S, Baffour R, Zhou YF, Shou M, Pierre A, Tio FO, et al. Transendocardial delivery of autologous bone marrow enhances collateral perfusion and regional function in pigs with chronic experimental myocardial ischemia. J Am Coll Cardiol 2001;37:1726–32. [8] Kamihata H, Matsubara H, Nishiue T, Fujiyama S, Tsutsumi Y, Ozono R, et al. Implantation of bone marrow mononuclear cells into ischemic myocardium enhances collateral perfusion and regional function via side supply of angioblasts, angiogenic ligands, and cytokines. Circulation 2001;104:1046–52.


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