A small molecule approach to engineering vascularized tissue

Biomaterials 34 (2013) 3053e3063

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Biomaterials journal homepage: www.elsevier.com/locate/biomaterials

A small molecule approach to engineering vascularized tissue Joyce Doorn a,1, Hugo A.M. Fernandes a,1, Bach Q. Le a, Jeroen van de Peppel b, Johannes P.T.M. van Leeuwen b, Margreet R. De Vries a, b, c, d, Zeen Aref c, Paul H.A. Quax c, Ola Myklebost d, Daniel B.F. Saris a, Clemens A. van Blitterswijk a, Jan de Boer a, * a

MIRA Institute for Biomedical Technology and Technical Medicine, Department of Tissue Regeneration, University of Twente, Drienerlolaan 5, 7522 NB Enschede, The Netherlands Erasmus MC, Department of Internal Medicine, Dr. Molenwaterplein 50, 3015 GE Rotterdam, The Netherlands c Einthoven Laboratory for Experimental Vascular Medicine, Department of Surgery, Leiden University Medical Centre, PO Box 9600, 2300 RC Leiden, The Netherlands d Oslo University Hospital, Department of Tumor Biology, The Norwegian Radium Hospital, Montebello, 0310 Oslo, Norway b

a r t i c l e i n f o

a b s t r a c t

Article history: Received 26 October 2012 Accepted 30 December 2012 Available online 29 January 2013

The repertoire of growth factors determines the biological engagement of human mesenchymal stromal cells (hMSCs) in processes such as immunomodulation and tissue repair. Hypoxia is a strong modulator of the secretome and well known stimuli to increase the secretion of pro-angiogenic molecules. In this manuscript, we employed a high throughput screening assay on an hMSCs cell line in order to identify small molecules that mimic hypoxia. Importantly, we show that the effect of these small molecules was cell type/species dependent, but we identified phenanthroline as a robust hit in several cell types. We show that phenanthroline induces high expression of hypoxia-target genes in hMSCs when compared with desferoxamine (DFO) (a known hypoxia mimic) and hypoxia incubator (2% O2). Interestingly, our microarray and proteomics analysis show that only phenanthroline induced high expression and secretion of another angiogenic cytokine, interleukin-8, suggesting that the mechanism of phenanthroline-induced hypoxia is distinct from DFO and hypoxia and involves the activation of other signaling pathways. We showed that phenanthroline alone was sufficient to induce blood vessel formation in a Matrigel plug assay in vivo paving the way to its application in ischeamic-related diseases. Ó 2013 Elsevier Ltd. All rights reserved.

Keywords: Angiogenesis Mesenchymal stem cells Stem cell Cell signaling

1. Introduction Human mesenchymal stromal cells (hMSCs) secrete a broad panel of factors that have trophic effects (e.g., anti-apoptotic, proangiogenic and anti-scarring) and immunomodulatory effects [1]. Several clinical trials have been completed or are currently on the way investigating the use of hMSCs for the treatment of, amongst others, autoimmune diseases [2e5], myocardial infarcts [reviewed in Ref. [6]], solid organ/graft transplantations [7,8] and ischemic wounds [9]. Debate exists on the mechanisms underlying the effects of infused MSCs. Although differentiation of MSCs into cells of the target tissue has been shown [10e12], low engraftment percentages, the short window in which effects are observed and the fact that conditioned medium alone also has effects, support a trophic effect [13e16]. Several studies have demonstrated that culture of hMSCs under hypoxic conditions, which closely resemble oxygen concentrations * Corresponding author. Fax: þ31 534 892 150. E-mail address: [email protected] (J. de Boer). 1 These authors contributed equally. 0142-9612/$ e see front matter Ó 2013 Elsevier Ltd. All rights reserved. http://dx.doi.org/10.1016/j.biomaterials.2012.12.037

in natural bone marrow (1e7%) [17], results in enhanced secretion of pro-angiogenic trophic factors e of which most importantly vascular endothelial growth factor (VEGF) e but also improves survival, engraftment and differentiation of implanted cells [18e 22]. Cellular responses to hypoxia are mainly regulated by hypoxia-inducible factors (HIFs)-heterodimers consisting of an a and b subunit [23,24]. The HIF-1a isoform is ubiquitously expressed in all cell types and, upon hydroxylation by prolyl hydroxylase domain proteins (PHDs), it is rapidly degraded by the proteasome [25]. PHD’s inactivation leads to accumulation and stabilization of HIF-1a and subsequent translocation into the nucleus where it binds to hypoxia responsive elements (HREs) and initiates transcriptional activation of HIF-target genes [23,24]. Subsets of genes containing an HRE have been identified, including angiogenic, endothelial and metabolism-related genes. Culture under hypoxia is expensive and, most importantly, restricted to an in vitro scenario. Alternatively, small molecules can be used that, similarly to hypoxia, activate HIF-1-target genes but are cheap and easy to use. In addition, controlled release of these molecules in an in vivo set-up is possible in a spatially and temporally controlled manner. Spatial control using antibodies

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specific for certain tissues or cell types coupled to liposomes was pioneered in the early 80’s and has been broadly used ever since [26e28]. More recently, using phage display libraries, it was possible to identify small peptides possessing high-affinity for certain proteins and therefore use these instead of proteins [29]. Temporal control of drug release can be achieved using controlled hydrolysis of polymers, changes in polymer matrices leading to differences in diffusion rate, changes in cellular environment such as pH, temperature or enzymatic activity or even via external stimuli such as an electric field or ultrasounds and containing encapsulated drugs and therefore, tuning the release profile [29e35]. In the past, our lab has shown that by using a small molecule we can modulate the composition of trophic factors secreted by hMSCs and control their differentiation into the osteogenic lineage [36]. Nevertheless, the effects observed with hMSCs could not be extrapolated to different cell types or species, clearly demonstrating that the biological function attributed to some molecules in one species or cell type differ depending on the cellular context. For example, we have shown that the same molecule that induced osteogenesis in hMSCs (dibutyryl-cAMP) led to a distinct phenotype in MSCs from another species, i.e. adipogenesis in rat MSCs [37]. Based on this observation and on the fact that previous screens for activators of the HIF pathway have been performed mostly with cancer cells lines [21,38,39], we decided to develop a new screening strategy based on a more clinically relevant cell type. We generated a cell line using human immortalized MSCs containing an HRE-GFP reporter (HRE-GFP iMSCs) and used highthroughput screening (HTS) to identify compounds that activate the HIF-1 pathway. 2. Materials and methods 2.1. Ethics statement All animal experiments were performed in compliance with Dutch government guidelines and approved by the Institutional Committee for Animal Welfare of the Leiden University Medical Center (LUMC). 2.1.1. Cell culture Bone marrow aspirates (5e15 mL) were obtained from patients with written informed consent and isolated as previously described [40]. Human mesenchymal stromal cells (hMSCs) were expanded in proliferation medium consisting of alpha minimal essential medium (a-MEM; Gibco, Carlsbad, CA), 10% fetal bovine serum (Lonza, Verviers, Belgium), 0.2 mM ascorbic acid (Sigma Aldrich, St. Louis, MO), 2 mM L-Glutamine (Gibco), 100 U/mL of penicillin and 100 mg/mL of streptomycin (Invitrogen, Carlsbad, CA) and 1 ng/mL of basic fibroblast growth factor (bFGF, Instruchemie, Delfzijl, The Netherlands). Basic medium consisting of proliferation medium without bFGF was used during the experiments. Human umbilical vein endothelial cells (HUVECs) were commercially obtained from Lonza and cultured in Endothelial Growth Medium-2 (EGM-2) with addition of the microvascular bullet kit (MV, all from Clonetics, Lonza), containing hEGF, hydrocortisone, gentamicin, 5% FBS, VEGF, hFGF-B, R3-IGF-1 and ascorbic acid. Cells were kept at 37  C and 5% CO2. Medium was refreshed three times per week and cells were trypsinised when a confluency of 70e80% was reached. MG-63 cells were cultured in basic medium and ACL cells were cultured in DMEM High Glucose (PAA) containing 10% FBS and 0.2 mM ascorbic acid. 2.1.2. LOPAC screen To perform the screen, 4000 HRE-GFP iMSCs (see Supplementary information on preparation of the cell line) were seeded in black 96-well plates (BD Biosciences) and allowed to attach overnight. The next day, the LOPACÒ library, a library consisting of 1280 pharmacologically active compounds, was added. As negative and positive controls, basic medium and 100 mM desferoxamine (DFO, Sigma Aldrich) in basic medium were added respectively. After one day of incubation with the test compounds, an Alamar Blue assay was performed to assess cell numbers/metabolic activity [41] and GFP intensity was measured as readout for the HRE activity. Briefly, medium containing 10% (v/v) Alamar Blue solution (Biosource, Camarillo, CA) was added and incubated at 37  C for 4 h. Then, fluorescence was measured at 590 nm on a Victor plate reader (Perkin Elmer, Wellesley, MA). Upon removal of the Alamar Blue solution, cells were washed with phosphate buffered saline (PBS; Life Technologies) and GFP intensity was measured at 520 nm on a Victor plate reader.

2.1.3. Hit validation Obtained hits were further tested. Briefly, four different concentrations of each hit were tested for their capacity to induce HRE activity. In addition, twelve compounds previously identified in an HRE screen using a cancer cell line [38] were tested along with our hits. These experiments were performed as described above for the primary screen. Based on the HRE-GFP activity (high signal and low cytotoxicity) we selected a concentration for subsequent experiments with primary human mesenchymal stromal cells (hMSCs). 2.1.4. Gene expression analysis hMSCs (or when mentioned MG-63 and ACL cells) were seeded in triplicate in 6-well plates at 5000 cells/cm2 and allowed to attach for 10e15 h in basic medium. Upon reaching near confluency, cells were treated as described above. After 2 days of treatment and 2 subsequent days of incubation with fresh medium, cells were lysed immediately with TRIzol. RNA was isolated using a Bioke RNA II nucleospin RNA isolation kit (Machery Nagel) and RNA concentrations were measured using an ND100 spectrophotometer (Nanodrop technologies, USA). cDNA was synthesized from 100 ng of RNA, using iScript (BioRad) according to the manufacturer’s protocol. For qualitative PCR, a master mix, containing distilled water, forward primer, reverse primer (Sigma Genosys), BSA, and SYBR green I mix (all from Invitrogen) was prepared. Real-time qPCR was performed in a LightCycler (Roche). Light-Cycler data was analyzed using the fit points method of Light-Cycler software. The baseline was set at the lower log-linear part above baseline noise and the crossing temperature (Ct value) was determined. Ct values were normalized to the 18S housekeeping gene and DCt (Ct, control  Ct, sample) was used to calculate the upregulation in gene expression [42]. Primer sequences are listed in Table 1. 2.1.5. Protein expression analysis hMSCs were seeded at 5000 cells/cm2 in T25 flasks. Upon reaching nearconfluence, medium was changed for basic medium, basic medium with 150 mM DFO or 200 mM Phenanthroline (Phen, Sigma Aldrich) or cells were added to a hypoxia chamber (2% O2). After 2 days, cells were lysed with 250 mL RIPA buffer with addition of protease/phosphatase inhibitors (Roche). Total protein concentrations were determined using a BCA kit (Pierce) and 10 mg of total protein was used to determine concentrations of VEGF, IL-8, basic fibroblast growth factor (bFGF), growth-colony stimulating factor (G-CSF) and epidermal growth factor (EGF) using a Luminex assay (Invitrogen) according to the manufacturer’s protocol. Briefly, cells and standards were incubated with fluorescent beads, followed by incubation with a biotinylated detection antibody. After incubation with streptavidin-R-Phycoerythrin and washing, both the fluorescence of the coupled beads and the R-phycoerythrin were measured using a LuminexÒ FlexMapTM (Luminex). 2.1.6. Whole genome expression analysis hMSCs were seeded in T25 flasks at 5000 cells/cm2 and allowed to attach overnight in proliferation medium. The next day, medium was added with the following conditions; basic medium, basic medium supplemented with 150 mM DFO or basic medium supplemented with 200 mM Phen. After 48 h, RNA was isolated as described above. From 500 ng of RNA, cRNA was synthesized using the Illumina TotalPrep RNA amplification Kit (Ambion), according to the manufacturer’s protocol and the quality of RNA and cRNA was verified on a Bioanalyzer 2100 (Agilent). Microarrays were performed using Illumina HT-12 v4 expression Beadchips, according to the manufacturer’s protocol. 2.1.7. Proliferation hMSCs and HUVECs were seeded in triplicate in 6-well plates at 3000 cells/cm2 and allowed to attach overnight in culture medium as described above (see cell culture). Then, cells were washed and conditioned medium (CM, see Supplementary information for details on preparation) was added. After 3 days, proliferation of hMSCs was determined by measuring the metabolic activity using a 10% (v/v) Alamar Blue (Invitrogen) and for HUVECs, nuclei were stained with DAPI (Sigma Aldrich) and counted. 2.1.8. Scratch wound healing assay HUVECs were seeded in triplicate in 6-well plates at 10,000 cells/cm2 and allowed to attach for 10e15 h in culture medium as described above (see cell culture). When the cells reached near confluency, a wound was created by scratching the surface with a pipette tip, and the medium was changed to different types of conditioned medium. After 12 and 20 h pictures were taken to examine migration of cells into the wound. Table 1 Primer sequences. Gene

Forward primer

18S VEGF-A

CGGCTACCACATCCAAGGAA GCTGGAATTACCGCGGCT Commercially obtained from SA biosciences

Reverse primer

J. Doorn et al. / Biomaterials 34 (2013) 3053e3063 2.1.9. In vivo murine matrigel plug assay In vivo angiogenesis analysis was performed using a Matrigel plug assay in male C57/BL6 mice (age 8 weeks) (Charles River). Growth factor reduced Matrigel (0.5 mL) (BD Biosciences) was injected into the subcutaneous space on the dorsal side of mice on both the left and right flank. Matrigel was mixed at 4  C with PBS, DFO (150 mM) or Phen (200 mM) (n ¼ 7 mice per group). Mice were sacrificed 7 days postimplantation. Matrigel plugs were excised and processed for histological analysis. Paraffin sections (5 mM) were stained with Hematoxylin, Phloxine and Saffron (HPS) or anti-CD31 (PECAM, Abcam, Cambridge, UK). Vascular ingrowth was scored by measuring the maximum ingrowth depth of capillary structures in 6 HPS stained sections per plug (one plug per mouse, 7 mice per group, expressed in mm). Quantification was performed in a double-blinded fashion by two individuals using morphometric image analysis methods (Qwin, Leica Imaging Systems). The endothelial nature of the infiltrating capillary structures was confirmed by the CD31 staining. 2.1.10. Statistics Data was analyzed using one-way ANOVA followed by Tukey’s multiple comparison’s test (P < 0.05). For the analysis of the ingrowth in the Matrigel plug statistical analysis was performed using ANOVA and unpaired t test.

3. Results 3.1. HRE activity in immortalized human mesenchymal stromal cells The first step to identify compounds capable of inducing HRE activity was to generate a reporter cell line in which the GFP gene is driven by a HRE element (reporter construct pCDNA3/HRE-GFP) (Fig. 1A). The uniqueness of our assay lies in the cellular background; human MSCs, a clinically relevant cell type for regenerative medicine as well as for cancer-related diseases [43]. Upon transfection of immortalized hMSCs and selection with G418 we selected 21 clones for further analysis. Using a known hypoxia mimic e DFO e we selected the clone with the highest HRE-GFP activity after 24 h exposure (Supplementary Fig. 1). To further confirm the ability of the selected clone to respond to hypoxia, we reduced the concentration of oxygen (O2) by increasing the number of cells per well and analyzed HRE-GFP activity [44]. As expected, this increase in cell density led to a dose-dependent increase in HRE-GFP activity (Fig. 1B). 3.1.1. Testing of known HRE activators To the best of our knowledge, this is the first HRE assay performed in hMSCs and as such, we decided to test compounds previously described as HRE activators in a cancer cell line (ME-180 cells from human cervical cancer cells) [38] in our model. Twelve compounds were tested at four different concentrations (1 nM, 10 nM, 1 mM and 100 mM), with DFO as a positive control, and HRE activity was measured after 24 h. Our results showed that exposure of HRE-GFP iMSCs to 100 mM DFO results in a 2.8-fold increase in

E1b 5xHRE VEGF min-P

HRE activity and a 38% reduction in metabolic activity relative to control (Fig. 2A). For the twelve compounds tested, concentrations that reduced metabolic activity more than 50% compared to 100 mM DFO were excluded (Fig. 2B). To our surprise, of these twelve compounds only 1,10-phenanthroline was able to induce HRE-GFP activity to the same level as 100 mM DFO (Fig. 2A). These results clearly highlight the importance of the cellular background on the regulation of HIF signaling. 3.1.2. Screening for new HRE activators: the LOPAC library In order to find new small molecules capable of activating the HRE we screened a library of pharmacological active compounds (LOPAC) using our HRE-GFP iMSCs cell line. Again, the well-known hypoxia mimic DFO was used as a positive control. Our results showed that DFO significantly decreased cell viability after 24 h exposure (24% reduction in viability: Bas ¼ 100  5 vs. DFO ¼ 76  3%; Supplementary Fig. 2A). In addition, HRE activity was significantly increased relative to the control when DFO was added (2.5-fold increase in HRE activity: Bas ¼ 100  11 vs. DFO ¼ 249  33%; Supplementary Fig. 2B). Given the fact that only one concentration per molecule was tested e thus reducing our chances to find hits, we decided to use a relaxed criteria to define a hit e any molecule that did not inhibit cell viability more than 24% (DFO reference value) and that showed an increase in HRE activity higher than 1.5-fold relative to the control. Based on these criteria we identified three hits: 1,10-phenanthroline monohydrate (Phen), quinacrine dihydrochloride (both in plate 13) and rottlerin (plate 14) (Fig. 3A). Interestingly, one of the hits (1,10-phenanthroline monohydrate) is the same as the compound previously identified as an HRE activator in the cancer cell line (1,10-phenanthroline). Selected hits were further tested using the same concentration range described above. As in the screening assay, 100 mM DFO was used as a positive control and, metabolic activity was again partially inhibited compared with the control (Supplementary Fig. 3A). Of the four different concentrations tested per compound, the ones that reduced metabolic activity more than 50% compared to 100 mM DFO were excluded from further analysis (Supplementary Fig. 3A). Moreover, only 1 mM quinacrine dihydrochloride and 100 mM Phen were able to induce HRE activity to a similar level as 100 mM DFO (Fig. 3B), but since quinacrine dihydrochloride did not affect VEGF gene expression, this compound was excluded from further analysis (Supplementary Fig. 3B). 3.2. Expression and secretion of angiogenic growth factors To analyze if different mechanisms are involved in DFO and Phen-induced hypoxia we performed a whole genome expression

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Fig. 1. Generation of the HRE-reporter cell line. A, schematic representation of the reporter construct used (modified from Ref. [66]). B, GFP/Alamar represents HRE activity at different cell concentrations. Legend: 5  HRE ¼ 5 copies of the 35 bp fragment derived from the VEGF HRE; VEGF ¼ 385 bp of the 50 flanking region of the VEGF gene; E1b minP ¼ 32 bp of the adenovirus E1b minimal promoter.

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Fig. 2. Hypoxia mimics identified using a cancer cell line. A, twelve previously identified hypoxia mimics tested on our human HRE-GFP reporter cell line. Note that only one compound induces HRE activity. B, The capacity of the hypoxia mimics identified in a cancer cell line to activate the HIF pathway in hMSCs was tested at different concentrations. An arrow indicates a decrease in metabolic activity of more than 50% compared with DFO. An asterisk (*) denotes no statistical significant difference (One-way Anova and Tukey’s test, P < 0.05).

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Fig. 3. HTS assay on human HRE-GFP reporter cell line. A, HRE-GFP cells were seeded in sixteen 96-well plates (4000 cells/well) in basic medium. One day later the LOPAC library was added to the wells and two days later we measured GFP and Alamar Blue as readout for HRE activity and metabolic activity, respectively. Here are represented the wells with hits (top: GFP/Alamar and bottom Alamar Blue alone); Note: The same setup used in the top left plate for the controls was used in all cases. B, effect of different concentrations of the three identified hits in HRE activity. An asterisk (*) denotes no statistical significant difference (One-way Anova and Tukey’s test, P < 0.05).

analysis of primary hMSCs treated for two days with both compounds and compared this with cells grown under hypoxic conditions (2% O2) and normoxia. Heatmaps in Fig. 4 show the expression levels of previously identified HRE-containing genes [45], grouped by function. Both DFO and Phen increased expression of genes involved in metabolism, cell growth and survival as well as endothelial genes, but Phen markedly induced higher expression as compared with DFO. Gene Ontology (GO) analysis showed an enrichment of terms associated with cell death regardless of the treatment used, whereas terms associated with cell cycle, regulation of cell migration, blood vessel development and regulation of muscle contraction were mainly enriched when hMSCs where treated with Phen and DFO (Supplementary Fig. 4B). Interestingly, using our experimental setup, hMSCs exposure to 2% O2 does not seem to be sufficient to induce a characteristic hypoxia response. For example, considering a 1.5 < log2FC < 1.5 and an adjusted P value <0.05 we found, compared with control, 11 genes regulated by hypoxia (in contrast with 455 in DFO and 892 in Phen) for one donor, whereas for the other donor we found 9 genes regulated by hypoxia (in contrast with 369 in DFO and 911 in Phen) (see dataset 1). Remarkably, interleukin-8 (IL-8) was highly induced by Phen (top gene), whereas DFO and hypoxia did not affect its expression.

We then examined if two of the highly regulated genes (IL-8 and VEGF) were also highly expressed at a protein level when compared with the control. As shown in Fig. 5A, Phen indeed increased the secretion of IL-8 to 300e500 pg/mL, whereas in DFO and hypoxiatreated cells IL-8 levels were comparable to control (15e25 pg/mL). In contrast, although VEGF secretion was induced by all three treatments, it was higher in DFO-treated cells (140 pg/mL) than in Phen-treated cells (65 pg/mL), even though microarray data showed higher expression of VEGF after treatment with Phen. Secretion of growth-colony stimulating factor (G-CSF) and basic fibroblast growth factor (bFGF) was lower in the Phen-treatment group compared to DFO and hypoxia. To investigate the presence of trophic factors, conditioned medium (CM) was prepared from DFO-, Phen- or hypoxia-treated hMSCs, and used to culture HUVECs and MSCs. After 2 days of treatment and 2 more days of incubation with fresh medium, VEGF was still highly expressed in Phen-treated cells, whereas expression levels in DFO- or hypoxia-treated cells were comparable to non-treated cells (Fig. 5B). In MSCs, Phen-CM was the only medium to significantly increase proliferation compared to non-CM whereas in HUVECS both DFO-, Phen- and hypoxia-treated cells increased proliferation (Supplementary Fig. 9A and B). To investigate the effect of trophic

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Transcriptional cofactors

VEGFA VEGFA HMOX1 ADM TFRC LE P LE P VEGFB VEGFC VEGFB LTF EDN1 ADRBK1 HEBP2 HEBP1 FECH

HDAC3 ETS1 SMAD3 CITED4 MYC STAT3 MYC HDAC7A HDAC7 HBXIP CEBPa HDAC7 HDAC1 HDAC2 ARNT

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-3 Fig. 4. Phenanthroline induces strong expression of HRE-containing genes, compared to DFO and hypoxia culture. Primary hMSCs were cultured in the presence of 150 mM DFO, 200 mM Phen or in an hypoxia chamber (2% O2). After 2 days, whole genome expression analysis was performed and the expression of known HRE-containing genes was examined. Of the three culture methods, most genes were strongest induced using Phen. Heatmaps show the relative expression of denoted genes compared to cells in basic medium, with all genes statistically significant regulated compared to cells in basic medium (P < 0.05).

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DDIT4 BNIP3 RORA MCL1 IGFBP1 BCL12 BCL2L11 MCL1 RORA IGFBP1 Bcl2 NOXA1 Bclx Bclx BCL2L13 Bcl2 BCL2L12 BCL2L12 BAD BAD CTGF CTGF ENG PAWR CXCL12 NPM3 BIRC5 CXCL12 CXCL12

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Fig. 5. Phenanthroline induces secretion of IL-8, but DFO induces higher secretion of VEGF. A, hMSCs were cultured in the presence of 150 mM DFO, 200 mM Phen or in hypoxia (2% O2) for 2 days, after which cells were lysed. Protein concentrations of IL-8, VEGF, bFGF and G-CSF were determined in cell lysates and as shown, only Phen induced secretion of high levels of Il-8, which were not affected by DFO or hypoxia. In contrast, VEGF secretion was increased by all three culture methods, but highest by DFO. B, hMSCs were cultured in the presence of DFO, Phen or in a hypoxia chamber for 2 days. After refreshing the medium and 2 more days of culture in basic medium, expression of VEGF was examined. Only after treatment with Phen expression of these genes was still enhanced, whereas in DFO and hypoxia cultures expression levels had returned to basal levels. C, CM was prepared by culturing cells in the presence of 150 mM DFO, 200 mM Phen or in a hypoxia chamber (2% O2), after which the medium was changed and cells were kept in culture for 2 more days. As a control non-treated cells were incubated with medium. CM was then used to culture HUVECs for a scratch wound healing assay. Migration of cells was increased in CM, but no differences between the treatments was observed although the remaining open area was smaller after culture of the cells in Phen-CM, suggesting a higher migration rate of the cells in this type of medium. D, response of different cell types to Phen. Note that regardless of the cell type used, Phen induces high expression of VEGF compared to the control. D231 and D142; donor number as specified in the laboratory database, (*) denotes P < 0.05, (**) denotes P < 0.01, (##) denotes P < 0.01 compared to basic, DFO and hypoxia (one-way Anova and Tukey’s test).

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factors on gene expression of HUVECs, cells were seeded on Matrigel in different types of CM. As shown in Supplementary Fig. 9C, whereas expression of endothelial genes was slightly decreased in basic-CM, DFO- and Phen-CM increased expression of these genes and demonstrated similar effects as hypoxia-CM. Similarly, a scratch wound healing assay demonstrated that migration of HUVECs was increased by CM, but no differences were observed between these groups (Fig. 5C). The observed effects were not restricted to hMSCs. When human osteoblasts (MG-63 cells) or human anterior cruciate ligament derived cells (ACL) were exposed to Phen for two days a clear up-regulation of VEGF was observed compared to the control (Fig. 5D). A synergistic effect of DFO and hypoxia (two days exposure) was not observed in any of the donors tested whereas one of the donors showed a statistically significant synergistic effect of Phen and hypoxia (Supplementary Fig. 8). 3.3. In vivo blood vessel formation Having shown that exposure of hMSCs to DFO and Phen can induce the secretion of trophic factors we decided to test if both compounds can act in vivo in a similar manner. Ultimately, we tested the efficacy of DFO and Phen as factors that can stimulate the hypoxia driven angiogenic response in vivo by incorporating DFO (150 mM) or Phen (200 mM) in Matrigel plugs (without incorporation of any cells) that are injected subcutaneously in mice. After 7 days a significantly increased capillary ingrowth could be observed in the DFO-containing as well as the Phen-containing matrigel plugs that were implanted into mice (Fig. 6). The capillary ingrowth after injecting DFO and Phen did not differ significantly, however it should be noted that in the Phen-treated group two mice showed excessive capillary ingrowth that resulted in the presence of capillary structures throughout the total plug (Fig. 6C). Those structures were functional as demonstrated by the presence of erythrocytes in the lumen (Fig. 6D). 4. Discussion Pre-conditioning or “training” of hMSCs prior to in vivo delivery can alter their differentiation status and the secretion of trophic factors, which might increase their therapeutic value [18e22,46]. Hypoxia pre-conditioning increases the secretion of pro-angiogenic and pro-survival factors improving engraftment of infused MSCs. Treatment of MSCs with growth factors, such as epidermal growth factor or sonic hedgehog, has similar effects [47e49], but relies on the use of recombinant proteins, which are expensive and pose challenges to incorporate into drug delivery systems while maintaining their bioactivity. Although some of the core components of the hypoxia pathway are conserved between cell types, variations occur between different cell types and species, which can result in a distinct activation pattern of HIF target genes [21,50,51]. To the best of our knowledge, this is the first screen for activators of the HIF-1 pathway using hMSCs. Due to their heterogeneity, primary hMSCs pose additional issues for screening assays and therefore, we used a clonally expanded immortalized cell line reasoning that the cellular components of the hypoxia pathway will closely resemble those in primary hMSCs [52]. Surprisingly, of the 12 previously identified hypoxia mimics [38], only 1 was able to activate the HRE reporter in HRE-GFP iMSCs, thus clearly demonstrating the cell type specificity for mechanisms regulating hypoxia. Phen can act as a chelator and form a complex with iron [53], but even though most molecules have a described molecular target, the lack of specificity should not be ignored [54]. Treatment of hMSCs with Phen results in enhanced secretion of VEGF and IL-8, both involved in proliferation, survival, sprouting and angiogenesis [55]. The effects of Phen-CM on proliferation and

migration of HUVECs were comparable to those of hypoxia-CM, even though hMSCs numbers were significantly decreased after treatment with Phen while preparing CM. In our experimental setup we observed that the effects of both DFO and Phen regarding metabolic activity are donor- and dose-dependent and in either case showed cytotoxic effects compared with the control (Supplementary Figs. 5 and 6). Besides optimizing exposure and concentration one can envision a strategy where toxic effects are minimized and cell survival is optimized. For example, a screening similar to the one performed in this study can be devised in an attempt to find compounds that increase cell proliferation and survival while the cells are exposed to the hypoxia mimic. Interestingly, only Phen was able to induce expression of Il-8, suggesting mechanistic differences between Phen and other ways of hypoxia induction. Whole genome analysis showed that, although DFO and the 2% O2 increase expression of several hypoxia target genes, only hMSCs exposed to Phen showed a dramatic increase in IL-8 expression. Although known to be regulated by hypoxia [56,57] IL-8 can also be increased by hypoxia-independent mechanisms. One hypothesis for the increase of IL-8 solely in Phentreated cells could be that Phen activates another signaling pathway that in turn activates IL-8 transcription. A candidate pathway is the NF-kB pathway, which activation leads to IL-8 expression [58,59] and indeed, our microarray data shows that expression of RelA e a NF-kB target gene e is higher in the presence of Phen than in hypoxia or DFO-treated cells. Alternatively, increased stability of the heterodimeric complex in the nuclei can explain the stronger activation of HIF target genes, as we also observed a higher expression of P300, a known co-activator of HIF, in Phen-treated cells [21,60]. IL-8 is also a known chemo-attractant for inflammatory cells [61,62]. Although some controversy persists on the role of inflammatory cells on tissue regeneration e it seems that under certain conditions this can be highly beneficial e co-cultures of hMSCs with peripheral mononuclear cells enhances expression of bone related markers and inflammation during bone repair leads to local recruitment of osteoprogenitors [63,64]. Furthermore, we showed for the first time that in vivo delivery of Phen to the matrigel as a single factor is sufficient to enhance vessel formation in vivo. Compared with DFO, the stronger activation of HIF target genes by Phen suggests this compound is more potent, which was partially reflected in the Matrigel plug assay, where Phen induced extensive capillary growth in 2 out of 7 mice compared to moderate growth in DFO-plugs (although overall, the differences to DFO did not differ significantly). Our in vitro data suggests that the mechanism by which Phen enhances vessels formation in vivo can involve differentiation of progenitor cells such as MSCs into cells from the endothelial lineage. Gene expression analysis of iMSCs treated with Phen showed an increase in endothelial-related genes (Supplementary Fig. 7B). This observation was corroborated by the tube formation assay using iMSCs (iMSCs were used because they are clonally expanded therefore ruling out different responses to the treatments by subpopulations of cells) where we showed that tube-like structures were only presented after 14 d in iMSCs pre-treated with Phen (Supplementary Fig. 7A). The involvement of matrix degrading enzymes in blood vessel formation is well known [65]. However, our microarray data does not show differential expression of matrix degrading enzymes upon treatment with DFO or Phen, indicating that under our experimental conditions other mechanisms are involved in in vivo vessel formation. Nevertheless, in vivo immunocytochemistry for matrix degrading enzymes as well as their respective inhibitors should be performed in the future to have a temporal and spatially understanding of their expression levels. Local release of DFO and Phen can increase hypoxia levels therefore triggering a cascade of events leading to the formation of blood

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Fig. 6. In vivo capillary ingrowth in matrigel containing DFO or Phen. Matrigel plugs (0.5 mL) containing either PBS, DFO (150 mM) or Phen (200 mM) were injected subcutaneously and analyzed for capillary ingrowth after 7 days. A, vessel ingrowth was scored morphometrically and expressed as mean  SEM per group (n ¼ 7) in mm ingrowth. B, the endothelial nature of the ingrowing cells structures is confirmed by CD31 staining. C, in two plugs in the Phen treated group extraordinary ingrowth of capillary like structures throughout the total plug could be observed, demonstrated a clear lumen surrounded by CD31 positive cells. D, the functionality of the vessels was confirmed by the presence of erythrocytes in the lumen of the vessels.

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vessels. Recruitment of different cell types to the vicinity of the plug should be analyzed in detail as some of the secreted factors play a role in recruitment of inflammatory cells that can enhance vessel formation or even the recruitment of fully differentiated endothelial cells that will eventually form new blood vessels. Our data suggest that Phen offers a cheap and easy alternative for hypoxia cultures and in addition, Phen could be applied directly in an in vivo setup. 5. Conclusions Our results showed that small molecules could be used to control the secretome of hMSCs. Moreover, we showed that the activity of previously identified hypoxia mimics is dependent on the cell type. By identified phenanthroline as a potent hypoxia mimic in several cell types we paved the way to its broad application in ischemic-related diseases. Our in vivo data clearly showed the potential of phenanthroline to induce blood vessel formation. Conflict of interest The authors declare no conflict of interests. Acknowledgments We would like to thank Roderick Beijersbergen and Pasi Halonen for technical support and thoughtful discussions, and Bram Koopman and Lorenzo Moroni for technical support. Furthermore, the authors gratefully acknowledge the support of the TeRM Smart Mix Program of the Netherlands Ministry of Economic Affairs and the Netherlands Ministry of Education, Culture and Science (JD, JdB and CvB), the STW program (HF, JdB and CvB), the BMM PENT project (PQ, MV and ZA), the OM at the Norwegian Stem Cell Centre from the Norwegian Research Council (OM) and Erasmus MC (JvP and HvL). Appendix A. Supplementary information Supplementary information related to this article can be found at http://dx.doi.org/10.1016/j.biomaterials.2012.12.037. References [1] Haynesworth SE, Baber MA, Caplan AI. Cytokine expression by human marrow-derived mesenchymal progenitor cells in vitro: effects of dexamethasone and IL-1 alpha. J Cell Physiol 1996;166(3):585e92. [2] Le Blanc K, Frassoni F, Ball L, Locatelli F, Roelofs H, Lewis I, et al. Mesenchymal stem cells for treatment of steroid-resistant, severe, acute graft-versus-host disease: a phase II study. The Lancet 2008;371(9624):1579e86. [3] Caplan AI. Why are MSCs therapeutic? New data: new insight. J Pathol 2009; 217(2):318e24. [4] Baker M. Stem-cell drug fails crucial trials. Nat News 2009. [5] Garcia-Olmo D, Herreros D, Pascual I, Pascual JA, Del-Valle E, Zorrilla J, et al. Expanded adipose-derived stem cells for the treatment of complex perianal fistula: a phase II clinical trial. Dis Colon Rectum 2009;52:79e86. [6] Menasche P. Cardiac cell therapy: lessons from clinical trials. J Mol Cell Cardiol 2010;50(2):258e65. [7] Bartholomew A, Sturgeon C, Siatskas M, Ferrer K, McIntosh K, Patil S, et al. Mesenchymal stem cells suppress lymphocyte proliferation in vitro and prolong skin graft survival in vivo. Exp Hematol 2002;30(1):42e8. [8] Chabannes D, Hill M, Merieau E, Rossignol J, Brion R, Soulillou JP, et al. A role for heme oxygenase-1 in the immunosuppressive effect of adult rat and human mesenchymal stem cells. Blood 2007;110(10):3691e4. [9] Falanga V, Iwamoto S, Chartier M, Yufit T, Butmarc J, Kouttab N, et al. Autologous bone marrow-derived cultured mesenchymal stem cells delivered in a fibrin spray accelerate healing in murine and human cutaneous wounds. Tissue Eng 2007;13:1299e312. [10] Orlic D, Kajstura J, Chimenti S, Bodine DM, Leri A, Anversa P. Bone marrow stem cells regenerate infarcted myocardium. Pediatr Transplant 2003;7(Suppl. 3):86e8.

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