REGENERATIVE MEDICINE Islet-1 Overexpression in Human Mesenchymal Stem Cells Promotes Vascularization Through Monocyte Chemoattractant Protein-3 JIA LIU,a,b WEIQIANG LI,c,d YINFEN WANG,a WENDONG FAN,a PANLONG LI,c WANYI LIN,c DAYA YANG,a RONG FANG,a MINGZHE FENG,a CHENGHENG HU,a ZHIMIN DU,a GUIFU WU,a,b,e ANDY PENG XIANGc,d a

Department of Cardiology, The First Affiliated Hospital of Sun Yat-sen University, Guangzhou, Guangdong, People’s Republic of China; bHeart Center, The Affiliated Futian Hospital of Guangdong Medical College, Shenzhen, People’s Republic of China; cCenter for Stem Cell Biology and Tissue Engineering, Key Laboratory for Stem Cells and Tissue Engineering, Ministry of Education; dDepartment of Biochemistry, Zhongshan Medical School, Sun Yat-sen University, Guangzhou, Guangdong, People’s Republic of China; eKey Laboratory on Assisted Circulation, Ministry of Health, Guangzhou, Guangdong, People’s Republic of China Correspondence: Andy Peng Xiang, Ph.D., Center for Stem Cell Biology and Tissue Engineering, Key Laboratory for Stem Cells and Tissue Engineering, Ministry of Education, Sun Yat-Sen University, Guangzhou, Guangdong 510080, People’s Republic of China. Telephone: 186-20-87335822; Fax: 186-20-87335858; e-mail: [email protected]; or Guifu Wu, M.D., Ph.D., FACC, Department of Cardiology, The First Affiliated Hospital of Sun Yat-sen University, Guangzhou, Guangdong 510080, People’s Republic of China. Telephone: 186-75583982222; Fax: 186-75583980805; e-mail: wuguifu@mail. sysu. edu.cn Received August 23, 2013; accepted for publication February 11, 2014; first published online in STEM CELLS EXPRESS February 27, 2014. C AlphaMed Press V

1066-5099/2014/$30.00/0 http://dx.doi.org/ 10.1002/stem.1682

Key Words. Islet 1 • Human mesenchymal stem cells • Vascularization • Angiogenesis

ABSTRACT The LIM-homeobox transcription factor islet-1 (ISL1) has been proposed to mark a cardiovascular progenitor cell lineage that gives rise to cardiomyocytes, endothelial cells, and smooth muscle cells. The aim of this study was to investigate whether forced expression of ISL1 in human mesenchymal stem cells (hMSCs) influenced the differentiation capacity and angiogenic properties of hMSCs. The lentiviral vector, EF1a-ISL1, was constructed using the Multisite Gateway System and used to transduce hMSCs. We found that ISL1 overexpression did not alter the proliferation, migration, or survival of hMSCs or affect their ability to differentiate into osteoblasts, adipocytes, cardiomyocytes, or endotheliocytes. However, ISL1-hMSCs differentiated into smooth muscle cells more efficiently than control hMSCs. Furthermore, conditioned medium from ISL1-hMSCs greatly enhanced the survival, migration, and tube-formation ability of human umbilical vein endothelial cells (HUVECs) in vitro. In vivo angiogenesis assays also showed much more vascular-like structures in the group cotransplanted with ISL1-hMSCs and HUVECs than in the group cotransplanted with control hMSCs and HUVECs. Quantitative RT-PCR and antibody arrays detected monocyte chemoattractant protein-3 (MCP3) at a higher level in conditioned medium from ISL1-hMSCs cultures than in conditioned medium from control hMSCs. Neutralization assays showed that addition of an anti-MCP3 antibody to ISL1-hMSCs-conditioned medium efficiently abolished the angiogenesispromoting effect of ISL1-hMSCs. Our data suggest that overexpression of ISL1 in hMSCs promotes angiogenesis in vitro and in vivo through increasing secretion of paracrine factors, smooth muscle differentiation ability, and enhancing the survival of HUVECs. STEM CELLS 2014;32:1843–1854

INTRODUCTION Vasculogenesis and angiogenesis [1, 2]—processes of blood vessel formation—are vital for development and tissue repair and are important contributors to disease progression. Poor blood circulation caused by inadequate blood vessel growth and/or impaired functionality of blood vessels may lead to ischemic diseases, such as myocardial ischemia, stroke, and delayed wound healing. Considerable efforts have been devoted to the development of new therapeutic strategies for generating an accessory vasculature in ischemic tissues. Angiogenic factors, gene therapy, and cellreplacement therapy represent important therapeutic options for ischemia diseases [3]. To date, however, most clinical studies that have focused on gene therapy or small molecule approaches for therapeutic vascular regeneration have an acceptable safety profile but received controversial results [4]. Nevertheless, results from various animal models indicate

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that cell therapy and gene therapy effectively promote neovascularization [5]. Among them, stem cell-based approaches using human mesenchymal stem cells (hMSCs) have been intensively investigated as treatments for ischemic disease because these cells have the ability to differentiate into smooth muscle cells, endothelial cells, and cardiomyocytes or secrete soluble factors that promote angiogenesis [6]. However, one of the major obstacles in using stem cell for therapeutic angiogenesis is the poor survival rate of transplanted stem cells in ischemic tissues due to ischemic environment [4]. Interestingly, Gnecchi et al. [7] provided evidence that the combination of MSCs and AKT (protein kinase B)-overexpression-based gene therapy enhances cell survival, therapeutic neovascularization, myocardial protection, and functional improvement through paracrine mechanisms. Two other reports also provided evidence that gene modification such as prolyl hydroxylase domain protein 2 silencing [8] or

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granulocyte chemotactic protein 2 overexpression [9] can enhance the survival and paracrine function of transplanted adipose-derived stem cells. The LIM-homeobox transcription factor islet-1 (ISL1) plays an important role in the development of cardiovascular cells during embryogenesis. Pfaff et al. [10] found that ISL1-knockout mouse embryos arrest begin on embryonic day 9.5 (E9.5) and are necrotic at E11.5 because of impaired development of the vasculature and its surrounding mesenchyme. Moreover, another study [11] showed that ISL1-null mice completely lack the outflow tract, right ventricle, and much of the atria. Using lineage-tracing studies, the authors of this study further demonstrated that ISL1 is a marker for a distinct population of undifferentiated cardiac progenitors in the secondary heart field. Moretti et al. [12] used genetic fate-mapping studies to document that ISL1-positive precursors from the secondary heart field can generate each of these diverse cardiovascular cell types in vivo and have the potential for self-renewal and the ability to differentiate into endothelial cells, cardiomyocytes, and smooth muscle cells in vitro. More recently, Fonoudi et al. [13] found that transduction of ISL1 promotes cardiomyocyte differentiation from human embryonic stem cells. However, as one of the key transcription factors in cardiovascular development, whether ISL1 influences the biological properties of hMSCs remains largely unknown. In this study, we constructed a lentiviral vector for ISL1 overexpression under the control of the human elongation factor 1a (EF1a) promoter. Using this construct to transduce hMSCs, we investigated whether ISL1 overexpression enhances the cardiovascular-differentiation capacity, angiogenesis properties, and paracrine function of hMSCs.

MATERIALS

AND

METHODS

Cell Culture hMSCs at passage 2 were obtained from Cyagen Biosciences Inc. (Guangzhou, China, http://www.cyagen.com/), and the MSCs were isolated from heparinized bone marrow of healthy human donors after informed consent. The culture medium was 90% Dulbecco’s modified Eagle medium (DMEM; Hyclone, Logan, UT, http://www.hyclone.com) supplemented with 10% fetal bovine serum (FBS; Invitrogen, Carlsbad, CA, http://www. invitrogen.com), 100 IU/ml penicillin (Hyclone), and 100 lg/ml streptomycin (Hyclone). The medium was changed every 2 days. After reaching 90% confluence, cells were detached by incubating with 0.125% trypsin for 1–2 minutes at 37 C and replated for continuous passage. Control hMSCs and ISL1-hMSCs between 5 and 10 cell passages were used for all experiments. Human umbilical vein endothelial cells (HUVECs) were purchased from American Type Culture Collection (ATCC; Rockville, MD, http://www.atcc.org) and cultured in endothelial growth medium-2 (EGM-2; Lonza, Balitmore, MD, http://www.lonza. com/). Medium was changed every 2 days, and cells were passaged every 2–3 days. All experiments with HUVECs were performed at no more than 3–5 cell passages.

Lentiviral Vector Construction and Transduction Entry vectors were generated by flanking the human EF1a promoter [14] and human ISL1 gene with attB4/B1r and attB1/B2 sites, respectively, by polymerase chain reaction C AlphaMed Press 2014 V

(PCR) (Supporting Information Table S1 and Fig. S1A). The promoter PCR product was cloned into pDONR P4-P1r (Invitrogen) using the Gateway BP recombination method, following the manufacturer’s instructions. The att-flanked ISL1 fragment was cloned into pDONR 221 (Invitrogen) using the same method (Supporting Information Fig. S1B). The resulting vectors, termed pUp-EF1a and pDown-ISL1, were next recombined into the pDest-puro vector [15] using a recognized LR recombination reaction protocol described in the Gateway LR kit and a clonase enzyme mix (Invitrogen; Supporting Information Fig. S1C). The final lentiviral expression vector was designated pLV/puro-EF1a-ISL1 (EF1a-ISL1). Lentiviruses were prepared by transient cotransfection of 293FT cells with the EF1a-ISL1 construct together with the ViraPower Lentiviral packaging mix (Invitrogen) using Lipofectamine 2000 (Invitrogen). Three days after transfection, supernatants containing viral particles were harvested, filtered through polyethersulfone membranes (pore size, 0.45 lm), and titered. For lentiviral transduction, hMSCs were dissociated into single-cell suspensions using 0.125% TrypLE Select (Invitrogen) and then replated with lentiviral particles and 5 lg/ml polybrene (Sigma-Aldrich, St Louis, MO, http://www.sigmaaldrich. com). The medium was replaced with fresh culture medium 12–24 hours after infection. Four days after transduction, puromycin (Sigma) was added to the culture medium at a concentration of 1–5 lg/ml and cells were maintained in this medium for 5 days. HUVECs were also labeled with EF1ahrGFP [15] by lentivirus transduction as described above and then purified by fluorescence activated cell sorting (FACS).

Characterization of ISL1-hMSCs For flow cytometry analyses, ISL1-hMSCs were trypsinized, washed with phosphate-buffered saline (PBS), and incubated with primary fluorescein isothiocyanate-conjugated (anti-CD29, anti-CD34, anti-CD45) or phycoerythrin-conjugated (anti-CD73, anti-CD105, anti-CD90, anti-CD166) antibodies, all from BD Pharmingen (Palo Alto, CA, http://www.bdbiosciences.com/ index_us.shtml). Irrelevant isotype-matched antibodies (BD Pharmingen) were used as negative controls. Flow cytometry analyses were performed with an Influ cell sorter (BD Biosciences, San Diego, CA, http://www.bdbiosciences.com). The data were analyzed using FlowJo software. Cell proliferation assay was carried out to determine the population doubling time (PDT) of ISL1-hMSCs and control cells by direct cell counting or the fluorescence-based Cell Counting Kit-8 (CCK8) Cell Proliferation Assay (Dojindo, Kumamoto, Japan, http://www.dojindo.com/), following the manufacturer’s instructions. In brief, 2 ml of cell suspension was plated onto a six-well plate at 20,000 cells per well and cultured for 7–10 days. Viable cells (as determined by trypan blue exclusion) were counted by Cellometer Auto T4 automated cell counters (Nexcelom Bioscience, Lawrence, MA, http://www.nexcelom.com/) everyday. PDT were calculated according to the following formula: (t 2 t0) log2/log(N 2 N0), where t 2 t0 is culture time (hours), N the number of harvested cells, and N0 is the number of cells in the initial. For CCK8 assay, 100 ll of cell suspension was plated onto a 96-well plate at 5,000 cells per well and cultured for 24 hours, after which 10 ll of CCK-8 solution was added to each well and the plate was incubated for 1–4 hours. Cell proliferation was assessed by measuring absorbance at 450 nm using STEM CELLS

Liu, Li, Wang et al. a microplate reader (Tecan Trading AG, Switzerland, http:// www.tecan.com/). Untransduced hMSCs at the same passage served as controls. For osteogenic differentiation [16], cells were incubated in osteogenic medium containing 1027 M dexamethasone, 0.2 mM ascorbic acid, and 10 mM b-glycerophosphate (all from Sigma-Aldrich). The medium was replaced twice weekly. After 14 days in differentiation medium, cells displayed bone-like nodular aggregates of matrix mineralization. Mineral deposition was visualized by Alizarin Red S staining for calcium. Quantitative reverse transcription-PCR (qRT-PCR) and Western blot assay were used to analyze the expression of osteogenic related genes including transcription factor Runt-related transcription factor 2 (RUNX2) at day 7 and other markers for osteoblast differentiation, including osteopontin (OPN) and type I collagen (COL1A1) at day 14. Osteocalcin secretion by differentiated cells was also determined using enzyme-linked immunosorbent assay (ELISA) kit according to manufacturer’s instruction (Invitrogen). For adipogenesis [16], induction media and maintenance media were used alternately for 3 days. The induction media consisted of DMEM (high glucose), 10% FBS, 1% antibiotic solution, 0.5 mM 1-methyl-3-isobutylxanthine, 200 mM indomethacin, 10 mg/ml insulin, and 0.1 mM dexamethasone (all from Sigma-Aldrich). The maintenance media consisted of DMEM, 10% FBS, 1% antibiotic solution, and 10 mg/ml insulin. Adipogenic differentiation was analyzed for the expression of adipogenesis marker peroxisome proliferator-activated receptor c (PPARc) at day 7, lipoprotein lipase (LPL), and fatty acid binding protein 4 (FABP4) at day 21 by qRT-PCR or Western blot. The lipid droplet morphology was detected by oil red O staining (Sigma-Aldrich) after induction for 3 weeks. For neural differentiation, cells were cultured in medium consisted of DMEM/F12 supplemented with 1% N2, 2% B27, 50 ng/ml brain-derived neurotrophic factor, 50 ng/ml nerve growth factor, and 10 ng/ml neurotrophin-3 (all from Invitrogen) for 2 weeks [17]. The expression of neural lineage related genes including PAX6, ISL2, LHX3, LHX4, HB9, CHAT, TUBB3, MAP2, neurofilament light chain protein (NFL), and GFAP were then analyzed by immunostaining or qRT-PCR.

Cell Survival and Migration Assay The influence of ISL1 transduction on hMSCs survival was determined by seeding 1 3 105 ISL1-hMSCs or control cells per well of a six-well plate. After incubation overnight, media were replaced with serum-free DMEM and cells were cultured for an additional 48 hours [18]. The impact of conditioned medium from ISL1-hMSCs on HUVECs survival was also investigated. For these experiments, equal numbers of ISL1-hMSCs or control (untransduced) hMSCs were cultured in serum-free medium for 24 hours, and then the medium was collected as conditioned medium. HUVECs at 100% confluence were trypsinized to yield a single-cell suspension and replated on sixwell plates at a density of 1 3 104 cells per cm2 in EGM-2 medium. After culturing for 12 hours, the culture medium was aspirated and conditioned medium from ISL1-hMSCs or control hMSC cultures was added, and cells were cultured for an additional 24 hours. Cell survival was determined using an Alexa Fluor 488 Annexin V/PI Kit (Invitrogen) according to the manufacturer’s instructions [19]. Green and red fluorescence indicate dead cells, whereas unlabeled cells represent live

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cells. Cell death or cell survival rates were obtained from five randomly selected fields for each treated/untreated group. The migration ability of hMSCs and HUVECs was determined using Transwell inserts with 8-lm-pore membrane filters (Chemicon, Temecula, CA, http://www.chemicon.com), as described by the manufacturer [20]. Cells were grown to 70% confluence prior to harvesting by trypsinization. After rehydration of the extracellular matrix layer of the inserts by incubating in serum-free media for 1–2 hours, a 300-ll cell suspension (1 3 105 cells per ml) in serum-free medium was loaded into the upper chamber. Then, 500 ll of culture media (for hMSCs/ISL1-hMSCs assays) or conditioned medium from hMSCs/ISL1-hMSCs (for HUVECs assays) were loaded into the lower chamber. After incubating for 6 hours (for hMSCs) or 24 hours (for HUVECs), the upper sides of the filters were carefully washed with cold PBS, and any remaining nonmigrating cells were gently removed with a cotton-tipped swab. It was important to repeat the procedure with a second clean cotton-tipped swab in order to make sure that any remaining cells that could contribute to background staining were removed. Then, cells on the lower surface (migrated cells) were stained with 1 lg/ml Hoechst 33342 (Sigma-Aldrich) and the polyanionic dye Calcein AM from the LIVE/DEAD Viability/ Cytotoxicity Assay kit (Invitrogen), which produces an intense uniform green fluorescence in live cells. Migrated cells were quantified after photographing under a fluorescence microscope.

Cardiac, Endothelial, and Smooth Muscle Cell Differentiation of ISL1-hMSCs For cardiomyogenic differentiation, 2 3 104 cells were seeded onto each well of a 12-well plate. After overnight culture, hMSCs were treated with 10 lM 5-azacytidine (5-Aza; SigmaAldrich) for 24 hours and then maintained in culture for 4 weeks [21]. Samples were analyzed by assessing the expression of cardiomyocyte markers by immunocytochemistry. For endothelial cell differentiation, subconfluent hMSCs were cultivated in EGM-2 medium for 21 days [22]. The medium was changed every 2 days. After 3 weeks induction, cells were examined for the protein expression of endothelial cell markers by immunostaining and FACS analysis. For smooth muscle cell differentiation, hMSCs (2 3 103 cells per cm2) were seeded onto a six-well plate coated with 100 ng/ml fibronectin (Sigma-Aldrich) and induced to differentiation by incubating in serum-free medium containing 2.5 ng/ml transforming growth factor (TGF)-b1 (R&D Systems, Minneapolis, MN, http://www.rndsystems.com) for 6 days [23]. The medium was changed every 2 days. The phenotype of differentiated cells was assessed by immunofluorescence staining and qRT-PCR.

In Vitro Tube-Formation Assay In vitro formation of capillary-like structures by HUVECs was studied using growth factor-reduced Matrigel (BD Biosciences, diluted 1:1 on ice with cold DMEM) and Cytodex-3 Beads, a commercial product from Amersham Biosciences (Piscataway, NJ, http://www.amersham.com). Conditioned medium from ISL1-hMSCs was collected after 24 hours in culture; conditioned medium from nontransduced hMSCs served as a negative control. For Matrigel assay, after culturing HUVECs with conditioned media for 24 hours, cells (5 3 104 cells per well) C AlphaMed Press 2014 V

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were seeded onto 24-well plates coated with 200 ll diluted Matrigel in conditioned media. Cells were observed under a phase-contrast microscope after 4 hours of incubation at 37 C. Tube formation was evaluated and scored as described elsewhere [24]. For Cytodex-3 microcarriers assay, the detailed experiment procedure was as described [25]. Sprout formation and lumen formation of HUVECs in conditioned medium were visualized and analyzed under light microscope. The number of sprouts and lumens per bead was counted and analyzed [25].

In Vivo Angiogenesis Assay The in vivo hMSCs-induced angiogenesis model was prepared as previously described [6] with two kinds of materials, matrigel and hydrogel (Glycosan Biosystem, UT, http://www.glycosan. com/). In MATRIGEL assay, briefly, nontransduced hMSCs (2 3 106); ISL1-hMSCs (2 3 106); HUVECs (2 3 106); nontransduced hMSCs (1 3 106) and PKH26-labeled HUVECs (1 3 106); or ISL1hMSCs (1 3 106) and PKH26 labeled HUVECs (1 3 106) suspended in 50 ll PBS were mixed with 100 ll liquid Matrigel and subcutaneously injected into two groups of severe combined immunodeficient (SCID) mice (n 5 10) for 2 weeks. In hydrogel assay, nontransduced hMSCs (5 3 106) and hrGFP-labeled HUVECs (5 3 106); or ISL1-hMSCs (5 3 106) and hrGFP-labeled HUVECs (5 3 106) were suspended in 100 ll DG (degassed and deionized) water-reconstituted hydrogel (according to manufacturer’s instruction) and subcutaneously injected into two groups of SCID mice (n 5 10) for 2 months. After 14 days (matrigel assay) or 2 months (hydrogel assay), mice were sacrificed, Matrigel plugs were removed and fixed in 4% paraformaldehyde, and then embedded in optimum cutting temperature compound or paraffin for further sectioning and histological analysis. Immunohistochemical assessment of vascular density was performed by immunostaining for CD31 and counterstaining with hematoxylin. Quantitative analyses were performed under a light microscope (3200) in high-power fields (n 5 10 per mouse).

ISL1 Promotes Vascularization in hMSCs

I (Fermentas, Glen Burnie, MD, http://www.thermoscientificbio. com/fermentas/). Reverse transcription was performed using a Quantitect Reverse Transcription kit (Qiagen, Valencia, CA, http://www.qiagen.com/), as described by the manufacturer, followed by qPCR using a DyNAmo ColorFlash SYBR Green qPCR kit (Thermo Fisher Scientific, Rutherford, NJ, https://www.thermofisher.com/) and the LightCycler 480 Detection System (Roche). The samples were transferred to the thermal cycler and DNA was amplified using the following thermocycling conditions: 40 cycles of denaturation at 95 C for 10 seconds, annealing at 60 C for 10 seconds, and extension at 72 C for 30 seconds. Glyceraldehyde-3-phosphate dehydrogenase served as an internal control. Primer details are provided in Supporting Information Table S2.

Immunofluorescence, Immunohistochemistry, and Western Blot

Neutralization assays were performed with hMSCsconditioned medium supplemented with anti-monocyte chemotactic protein 3 (MCP3) (10 lg/ml; R&D Systems).Tube formation, migration ability, and cell survival rate of HUVECs were evaluated and scored as indicated above.

Prior to immunofluorescence analyses, undifferentiated and differentiated ISL1-hMSCs or hMSCs were fixed in 4% (v/v) paraformaldehyde for 20 minutes and then permeabilized by incubating for 30 minutes at room temperature in PBS containing 0.1% (v/v) Triton X-100, goat serum, and 1% (w/v) bovine serum albumin (Sigma). Cells were next incubated overnight at 4 C with primary antibodies against a-myosin heavy chain (aMHC, 1:200; Chemicon), cardiac troponin T (cTnT), a-smooth muscle actin (aSMA, 1:200; Thermo Fisher Scientific, Fremont, CA), or CD31 (1:50; Abcam, Cambridge, U.K., http://www.abcam.com). Alexa Fluor-488- or Alexa Fluor594-conjugated secondary antibodies (1:1,000, anti-rabbit or anti-mouse; Invitrogen) were added and incubated at room temperature for 1 hour in the dark. Nuclei were counterstained with Hoechst 33342 (1:1,000; Sigma). For immunohistochemistry assessment, formalin-fixed and paraffin-embedded samples were deparaffinized and rehydrated. After rinsing with PBS, antigen retrieval was carried out by microwave treatment in 0.01 M sodium citrate buffer (pH 6.0) at 100 C for 15 minutes. Subsequent antibody incubation and nuclei counterstaining procedures were as described above. For Western blot assay, osteogenic or adipogenic cells were washed with cold PBS and directly lysed in 13 RIPA buffer and then centrifuged at 15,000g for 10 minutes at 4 C. Each supernatant was recovered as a total cell lysate. Equal amounts of protein were separated on SDS-PAGE and then electrotransferred to 0.45-lm pore-sized polyvinylidene difluoride membranes (Millipore, Bedford, MA, http://www.millipore.com). After the transfer, each membrane was blocked using a solution of 0.1% (v/v) Tween 20/TBS (TBS/T) containing 5% (w/v) nonfat milk powder for 1 hour at room temperature and then incubated with appropriate primary antibodies against RUNX2, OPN, PPARc, and FABP4 (all from Abcam) overnight at 4 C. Specifically bound primary antibodies were detected using peroxidase-coupled secondary antibodies and enhanced chemiluminescence signaling (Cell Signaling Technologies, Beverly, MA, http://www.cellsignal.com).

qRT-PCR Analyses

Statistical Analysis

Total RNA was extracted from multipotent and differentiated hMSCs using a commercial kit (Roche, Indianapolis, IN, http:// www.roche-applied-science.com). Any contaminating genomic DNA was eliminated by digesting RNA preparations with DNase

All results presented represent data collected from at least three independent experiments. Statistical analyses of data used ANOVA. A p value .05, Fig. 2A). There was no statistically significant difference in PDT between control cells and ISL1-hMSCs at passage 5 (33.8 6 1.36 hours vs. 33.1 6 2.01 hours) and passage 10 (36.8 6 2.11 hours vs. 37.7 6 1.68 hours) (p > .05, Supporting Information Fig. S3). To demonstrate the multipotency of ISL1-hMSCs, we cultured cells under conditions that promote differentiation into

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osteogenic or adipogenic lineages. Culturing in osteogenic medium for 2 weeks induced the differentiation of ISL1-hMSCs into osteoblasts, as confirmed by strong Alizarin Red S staining (Fig. 2B). No statistically significant difference was found in the results of Alizarin Red S staining and the expression of osteogenesis related proteins including RUNX2, COL1A1, OPN, and Osteocalcin (p > .05, Supporting Information Fig S4A–S4D). These results demonstrated that the ISL1-hMSCs have shown no change in osteogenic differentiation capacity as compared with control hMSCs. Similarly, oil red O staining revealed the presence of lipid droplets in the cytoplasm of differentiated ISL1-hMSCs on day 21 of adipogenic differentiation (Fig. 2C). There are no difference in the result of oil red O staining and the expression level of adipogenesis marker PPARc, LPL, and FABP4 between ISL1 group and control group (p > .05, Supporting Information Fig. S5A–S5C). These results demonstrated that the ISL1-hMSCs have shown no change in adipogenic differentiation capacity as compared with control hMSCs (p > .05). These differentiation assays confirmed that ISL1-hMSCs maintained the phenotype and multipotency of parental hMSCs. The results of immunostaining assay after neural differentiation for 2 weeks showed that ISL1-hMSCs retained ISL1 expression, while control cells were ISL1-negative. Moreover, both cell types contained similar population of TUBB3 and neurofilament light chain protein positive cells, however, no SOX2 or HB9 cells were present in the culture in both groups (p > .05) (Supporting Information Fig. S6A). qRT-PCR detection revealed that differentiated cells in both groups expressed similar levels of PAX6, TUBB3, and MAP2 (p > .05). However, other motor neuron related genes including ISL2, LHX3, LHX4, HB9, and CHAT were not detected in control cells and ISL1-hMSCs (Supporting Information Fig. S6B). We additionally tested whether ISL1 overexpression altered cell survival or migration capacity. As shown in Figure 2D, culturing under serum-deprived conditions for 48 hours did not C AlphaMed Press 2014 V

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Figure 2. Characterization of ISL1-hMSCs. (A): Cell-proliferation ability was analyzed by CCK8 assay. (B): Alizarin Red S staining of osteogenic-differentiated SIL1-hMSCs. (C): Oil red O staining of adipogenic-differentiated, transduced human mesenchymal stem cells (hMSCs). (D): ISL1-overexpressing hMSCs and control hMSCs were cultured in serum-free medium for 48 hours. Cell survival was tested using a LIVE/DEAD viability/cytotoxicity kit. (E): Migration assays were performed in transwell dishes with 8-lm pore filters. Migrated cells were identified by Hoechst and Calcein AM staining. No significant differences in proliferation, survival, or migration capacity were found between control hMSCs and ISL1-hMSCs (p > .05). Scale bar 5 50 lm. Abbreviations: CCK8, cell counting kit-8; DAPI, 40 ,6-diamidino-2-phenylindole; ISL1, islet-1; PI, propidium iodide.

significantly increase cell death rates in either ISL1-hMSCs (7.51% 6 0.94%) or control hMSCs (7.18% 6 0.73%; p > .05). Similarly, transwell migration assays showed no significant difference between the percentage of transmigrated ISL1-hMSCs (10.54% 6 0.89%) and control hMSCs (11.01% 6 1.08%) after incubation for 6 hours (p > .05; Fig. 2E). Collectively, these results indicated that ISL expression did not influence the survival or migration potential of hMSCs.

ISL1 Enhanced the Smooth Muscle Differentiation Capacity of hMSCs ISL1 has been shown to play an essential role in heart embryogenesis, and ISL1-positive cells have been demonstrated to be cardiovascular progenitors [10]. C AlphaMed Press 2014 V

Accordingly, we sought to determine whether ISL1 overexpression in hMSCs enhanced the cardiovascular-differentiation potential of these cells. ISL1-hMSCs were induced to differentiate into cardiomyocytes, endothelial cells, and smooth muscle cells as described in Materials and Methods section. After treating with10 lM 5-Aza for 24 hours and then culturing for an additional 4 weeks, cells were analyzed by immunofluorescence using antibodies against the cardiovascular lineage markers aMHC and cTnT. These immunofluorescence analyses showed that a very small proportion of ISL1-hMSCs gave rise to aMHCpositive (0.052% 6 0.004%) or cTnT-positive (0.083% 6 0.016%) cardiomyocyte-like cells in vitro; comparable results were obtained for control hMSCs (aMHC, 0.047% 6 0.006%; cTnT, 0.075% 6 0.012%; p > .05; Fig. 3A–3C). For endothelium STEM CELLS

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Figure 3. Cardiovascular differentiation of ISL1-hMSCs. (A): Cardiac differentiation was assessed by immunofluorescence using antiaMHC and anti-cTnT antibodies. (B): After differentiation, aMHC-positive cells constituted about 0.047% 6 0.006% and 0.052% 6 0.004% of control human mesenchymal stem cells (hMSCs) and ISL1-hMSCs, respectively (p > .05). (C): The percentage of cTnT positive cells was 0.075% 6 0.012% in the control hMSCs group and 0.083% 6 0.016% in the ISL1-overexpressing group (p > .05). (D): Endothelial cell differentiation was assessed by CD31 immunostaining. (E): Approximately, 1% of cells in both control hMSCs and ISL1-hMSCs groups expressed CD31 and there was no significant difference between the two groups (p > .05). (F): Fluorescence activated cell sorting analyses indicated that about 0.1% of cells in both groups were CD34-positive (0.121% 6 0.009% and 0.114% 6 0.014% for control hMSCs and ISL1-hMSCs groups, respectively). (G): Smooth muscle differentiation was evaluated by immunofluorescence detection of aSMA expression. (H): A quantitative reverse transcriptase polymerase chain reaction analysis of differentiated cells revealed aSMA expression in differentiated cells from the ISL1-overexpressing hMSCs group compared with the parental hMSCs group (*, p < .05). Scale bar 5 50 lm. Abbreviations: aMHC, a-myosin heavy chain; aSMA, a-smooth muscle actin; cTnT, cardiac troponin T; DAPI, 40 ,6-diamidino-2-phenylindole; ISL1, islet-1.

differentiation, ISL1-hMSCs and control hMSCs were cultured in EGM-2 medium for 21 days and then analyzed for the expression of CD31 and CD34. Immunostaining for CD31 expression and FACS analysis for CD34 expression revealed no significant differentiation of either control hMSCs or ISL1-hMSCs into endothelial cells; the percentages of CD31-positive cells were 1.342% 6 0.098% and 1.527% 6 0.167% for control hMSCs and ISL1-hMSCs, respectively, and the corresponding values for CD34 were 0.121% 6 0.009% and 0.114% 6 0.014% (p > .05; Fig. 3D–3F). TGF-b1 has been reported to efficiently induce the differentiation of hMSCs into smooth muscle cells [20]. After culturing in serum-free medium supplemented with 2.5 ng/ml TGF-b1 for 6 days, differentiated cells lost their fibroblast appearance and exhibited large and flatten morphology. Immunofluorescence analyses revealed that significantly more ISL1hMSCs gave rise to cells positive for the smooth muscle cell marker aSMA (38.23% 6 3.93%) compared with control hMSCs (23.46% 6 2.78%; p < .05; Fig. 3G, 3H). This result was further validated by qRT-PCR detection of aSMA expression (Fig. 3I).

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ISL1 Improved the Angiogenic Properties of hMSCs In Vitro Next, we examined whether conditioned medium from ISL1overexpressing hMSCs affected the survival, migration, and/or tube-formation ability of HUVECs. For cell survival assays, HUVEC viability was determined after culturing in serum-free conditioned medium from ISL1-hMSCs or control hMSCs for 24 hours. We found that more than 90% of HUVECs (91.27% 6 6.91%) survived in the ISL1-hMSCs group, whereas only about 50% (50.14% 6 4.76%) of cells cultured in conditioned medium from control hMSCs survived (p < .01; Fig. 4A). These results indicate that ISL1 influences the paracrine activity of hMSCs and augments the survival of HUVECs under serum-starved condition. To determine whether paracrine factors in conditioned medium from ISL1-hMSCs affect the migration of HUVECs, we performed transwell migration assays. These assays revealed that the migratory capacity of HUVECs towards conditioned C AlphaMed Press 2014 V

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Figure 4. In vitro angiogenesis assay of ISL1-hMSCs and control human mesenchymal stem cells (hMSCs). (A): Human umbilical vein endothelial cells (HUVECs) were cultured in CM from ISL1-hMSCs or control hMSCs. The cell survival rate was 91.27% 6 6.91% in the ISL1-hMSCs group, whereas only about 50% (50.14% 6 4.76%) HUVECs in the control hMSCs group survived (**, p < .01). (B): Transwell assays showed that the migratory capacity of HUVECs toward CM from ISL1-hMSC cultures was 2.58 6 0.45-fold higher than that toward control group CM (*, p < .05). (C): The tube-formation ability of HUVECs increased 4.18 6 0.51-fold in CM from ISL1-hMSCs cultures compared to that for HUVECs grown in CM from control hMSCs (*, p < .05). Scale bar 5 50 lm. Abbreviations: CM, conditioned medium; DAPI, 40 ,6-diamidino-2-phenylindole; ISL1, islet-1; PI, propidium iodide.

medium from ISL1-hMSC cultures was significantly greater (2.58 6 0.45-fold; p < .05) than that towards condition medium from parental hMSCs (Fig. 4B). Tube-formation capacity is an important functional property of endothelial cells that is activated during angiogenesis. To determine whether the tube formation-ability of HUVECs was affected by paracrine factors released from ISL1-hMSCs, we cultured HUVECs on Matrigel in the presence of conditioned media from ISL1-hMSC cultures. These experiments showed that media conditioned by ISL1-overexpressing hMSCs enhanced the tube-formation ability of HUVECs (4.18 6 0.51fold; p < .05) as early as 4 hours after seeding on Matrigel (Fig. C AlphaMed Press 2014 V

4C). The Cytodex-3 Beads-based tube formation assay also showed that there was more sprout formation (Supporting Information Fig. S8A, S8B) and lumen formation (Supporting Information Fig S8A, S8C) of HUVECs in conditioned medium from ISL1 group than control after 48 hours culture (p < .01).

ISL1 Enhanced the In Vivo Angiogenesis Properties of hMSCs Previous reports have demonstrated that hMSCs function as perivascular cells and facilitate angiogenesis in an ischemia model in vivo [26]. Here, we first tested whether ISL1 expression reinforced the angiogenic effect of hMSCs in vivo using a STEM CELLS

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Figure 5. In vivo angiogenesis assay. (A): H&E staining of Matrigel plugs from mice implanted with human mesenchymal stem cells (hMSCs), ISL1-hMSCs, or HUVECs alone for 2 weeks showed that functional vessels were extremely rare in these samples. (B): Cotransplantation of hMSCs and HUVECs increased the number of functional blood vessels formed, as revealed by fluorescence detection of PKH26-labeled HUVECs. The number of viable HUVECs and the size of Matrigel plugs were further increased in the ISL1-hMSCs group compared with the control hMSCs group. (C): Immunostaining for CD31 indicated that blood vessel density in the ISL1-overexpressing hMSC group was 2.08 6 0.33-fold higher than that in the control hMSC group (*, p < .05). (D): Immunostaining assays showed that more CD31- and aSMA-positive cells were present in Matrigel plugs from mice implanted with ISL1-expressing hMSCs than in plugs from mice implanted with control hMSCs. Scale bar 5 50 lm. Abbreviations: DAPI, 40 ,6-diamidino-2-phenylindole; HUVECs, human umbilical vein endothelial cells; H&E, hematoxylin and eosin; ISL1, islet-1; aSMA, a smooth muscle actin.

Matrigel angiogenesis assay. We found that hMSCs, ISL1hMSCs, or HUVECs implanted alone formed virtually no functional vessels after 2 weeks (Fig. 5A). However, HUVECs (labeled with the fluorescence indicator PKH26) coimplanted with hMSCs assembled into a greater number of functional blood vessels (Fig. 5B). Notably, more viable PKH26-labeled HUVECs were found and Matrigel plugs were larger in the ISL1-hMSCs group than in the control hMSCs group (Fig. 5B). The histological findings were confirmed by a quantitative analysis, which showed that blood vessel density in the HUVECs/ISL1-hMSCs coimplanted group was 2.08 6 0.33-fold higher than that in the HUVECs/hMSCs coimplanted group (p < .01; Fig. 5C). Immunostaining also showed a higher percentage of CD31- and aSMA-positive cells in plugs composed of ISL1-overexpressing hMSCs than in plugs from the control hMSCs group (Fig. 5D). In hydrogel assay, the results of HE staining and CD31 immunostaining revealed higher blood vessel density in ISL11HUVECs group (143 6 17 microvessels per mm2) than in control group (68 6 9 microvessels per mm2) (p < .01). In addition, when observed under the fluorescent microscope directly, more viable hrGFP positive HUVECs were found in ISL1-hMSCs group than in control group (Supporting Information Fig. S7).

ISL1 Expression Resulted in Elevated Expression of MCP3 in hMSCs To establish the involvement of cytokines secreted from ISL1hMSCs in enhancing the angiogenic capacity of HUVECs, we

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examined 43 different angiogenic cytokines in conditioned medium from ISL1-hMSCs and control hMSCs cultures using a human angiogenesis antibody array. Based on the threshold definition, the expression of MCP3 was significantly increased (2.75 6 0.34-fold) in conditioned medium from ISL1-hMSCs cultures compared with that in medium from control cultures (p < .05; Fig. 6A). A qRT-PCR analysis of ISL1-hMSCs and control hMSCs also verified this result (Fig. 6B) and further showed that expression of intercellular adhesion molecule-1 (ICAM-1) was upregulated in ISL1-hMSCs (2.31 6 0.42-fold compared with control hMSCs; p < .05; Supporting Information Fig. S2). Finally, we tested the impact of secreted MCP3 on the biological properties of HUVECs using a neutralizing antibody against MCP3. These neutralization assays showed that the cell survival rate, migration, and tube-formation ability of HUVECs cultured in conditioned medium from ISL1-hMSCs cultures was significantly reduced by the addition of neutralizing antibody to the conditioned medium (Figs. 6C, 7A, 7B). In Cytodex-3 Beads-based tube formation assay, when anti-MCP3 antibody was added in the conditioned medium from ISL1 transduced cells, the sprout formation and lumen formation decreased obviously (p < .01) and was comparable to that of control group (p > .05) (Supporting Information Fig. S8).

DISCUSSION In this study, we reported the successful transfer of ISL1 into hMSCs by lentiviral transduction and confirmed ISL1 overexpression by immunostaining and qRT-PCR. We found that the C AlphaMed Press 2014 V

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Figure 6. Antibody array and effects of anti-MCP3 neutralizing antibody in cell-survival assays. (A): A human angiogenesis antibody array identified a significant increase in the expression of MCP3 in CM from ISL1-hMSCs cultures compared with medium conditioned by control hMSCs. (B): Quantitative reverse transcriptase polymerase chain reaction analyses confirmed that MCP3 expression was higher in ISL1-hMSCs than control hMSCs (*, p < .05). (C): HUVECs survival in CM from ISL1-hMSCs cultures, with and without the addition of anti-MCP3 neutralizing antibody, was assessed. The cell survival rate was significantly decreased in cultures supplemented with anti-MCP3 antibody (**, p < .01). Scale bar 5100 lm. Abbreviations: CM, conditioned medium; DAPI, 40 ,6-diamidino-2-phenylindole; ISL1, islet-1; MCP3, monocyte chemoattractant protein-3; PI, propidium iodide.

resulting transduced cells retained the characteristics of hMSCs, exhibiting both self-renewal and multipotency. We found that ISL1 enhanced the smooth muscle-differentiation ability of hMSCs but did not alter the capacity of hMSCs to differentiate into cardiomyocytes or endothelial cells. More importantly, ISL1-hMSCs expressed higher levels of the proangiogenic protein, MCP3, which greatly enhanced the survival, migration, and tube-formation ability of HUVECs in vitro. In vivo angiogenesis assays further demonstrated that overexpression of ISL1 enhanced the angiogenic properties of hMSCs. ISL1 also plays an important role in the development of motor neurons and endocrine islet cells during embryogenesis. Pfaff et al. [10] found that ISL1 is required for the generation of motor neurons—a prerequisite for the subsequent differentiation of engrailed-1-positive interneurons in the neural tube. Ahlgren et al. [27] analyzed the role of ISL1 in acinar and islet cell differentiation in the pancreas in mice with a targeted disruption of ISL1. They found that the dorsal pancreatic mesenchyme did not form and there was a complete loss of differentiated islet cells in ISL1-null embryos. They inferred that ISL1 is essential for the generation of all endocrine islet cells. Guo et al. [28] found that ISL1 transduction C AlphaMed Press 2014 V

promoted adult pancreatic islet cell proliferation, probably by activating c-Myc and cyclinD1 transcription through direct binding to their promoters. In contrast, the current study showed that ISL1 expression did not promote the proliferation of hMSCs. To date, most studies have focused on ISL1 involvement in embryonic pathways of cardiovascular development. Whether ISL1 influences postnatal angiogenesis of hMSCs had not been determined. Barzelay et al. [29] first reported that ISL1 expression in rat MSCs (rMSCs) upregulated CD31 expression and enhanced the tube-formation ability of rMSCs, but this latter study did not address the in vivo angiogenic properties of ISL1rMSCs. Using immunofluorescence and FACS analyses, we found no evidence for upregulation of CD31 or CD34 expression in ISL1-hMSCs cultured in the undifferentiated state or under endothelial cell differentiation-inducing conditions, which was consistent with previous reports [6]. The tube-formation ability of ISL1-hMSCs was also similar to that of control hMSCs (p > .05, data not shown). In contrast, our results from in vitro and in vivo assays indicated that ISL1-hMSCs differentiated to perivascular cells (smooth muscle cells) more efficiently, a property that could help to stabilize engineered blood vessels. STEM CELLS

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Figure 7. Effects of anti-MCP3 neutralizing antibody in tube-formation and migration assays. (A): In Matrigel plug assays, the tubeformation ability of human umbilical vein endothelial cells (HUVECs) grown in CM from ISL-human mesenchymal stem cells (hMSCs) cultures was significantly impaired by addition of an anti-MCP3 antibody (*, p < .05). (B): In transwell assays, the percentage of migrated HUVECs in CM from ISL-hMSCs cultures was significantly impaired by addition of an anti-MCP3 antibody (*, p < .05). Scale bar 5100 lm. Abbreviations: CM, conditioned medium; DAPI, 40 ,6-diamidino-2-phenylindole; ISL1, islet-1; MCP3, monocyte chemoattractant protein-3.

Moreover, the paracrine effects of ISL1-hMSCs greatly promoted the survival, migration, and tube-formation ability of HUVECs in vitro, and facilitated blood vessel formation in vivo. Angiogenesis is a normal and vital process during growth and development, as well as in wound healing, tissue ischemia, and cancer. It is a complicated process involving endothelial cell proliferation, migration, and sprouting that ultimately leads to the formation of functional new vessels [30]. Here, we demonstrated that ISL1 expression in hMSCs promoted the angiogenic properties of these cells in a direct and paracrine manner. We found that expression of the adhesion molecule ICAM-1 was upregulated. ICAM-1 is a member of the Ig superfamily of Ca21-dependent transmembrane proteins whose increased expression has been shown to facilitate leukocyte-mediated angiogenesis [31, 32]. A previous study also suggested that ICAM-1 is essential during angiogenesis by regulating endothelial cell motility through a nitric oxidedependent pathway [33]. Importantly, antibody array data also showed that ISL1 transduction increased the expression of the proangiogenic cytokine MCP3, supporting the proangiogenic effects of ISL1 overexpression. MCP3 has been reported to act as a homing factor for circulating angiogenic cells, stimulating the migration of these cells in vitro, and inducing the

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formation of blood vessels in Matrigel plugs implanted in mice [34]. Huang et al. [35] found that MCP3 is critical for the high apoptosis resistance of chemokine [C-C motif] receptor 1-overexpressing MSCs. In our study, neutralization tests showed that the pro0angiogenic effects of MCP3 were abolished by the addition of an anti-MCP3 antibody, as demonstrated by in vitro survival, migration and tube-formation assays. These results, which were consistent with previous reports, further support the enhanced angiogenic function of ISL1-hMSCs, although additional studies will be required to elucidate the underlying mechanism. Likewise, trophic and/or antiapoptotic factors secreted by ISL1-hMSCs that help HUVECs survive under serum-starved conditions in vitro as well as in vivo remain to be identified.

CONCLUSION In conclusion, the findings of the present study provide new insights into the molecular mechanisms by which ISL1 mediates angiogenesis in vitro and in vivo. In addition, because of their multipotency, low immunogenicity, amenability to ex vivo expansion and genetic modification, hMSCs have great potential as a cellular therapy for use in regenerative medicine and are C AlphaMed Press 2014 V

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currently in clinical development [7]. Our results may provide new tools for the studies of ischemic diseases treatment.

ACKNOWLEDGMENTS This work was supported by the National Basic Research Program of China (2012CBA01302, 2012CB619105), the National Natural Science Foundation of China (81170272, 81370389, 31171398, 81270646, 81271265), Key Clinical Program of the Ministry of Health (254004), the Natural Science Foundation of Guangdong Province (S2013030013305), the Key Scientific and Technological Projects of Guangdong Province (2007A0 32100003, 2011A030300010), Key Scientific and Technological Program of Guangzhou City (2010U1-E00551, 201300000089).

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AUTHOR CONTRIBUTIONS J.L. and W.L.: collection and/or assembly of data, data analysis and interpretation, and manuscript writing; Y.W., W.F., P. L., and W.L.: collection and/or assembly of data; D.Y., R.F., M.F., C.H., and Z.D.: provision of study material; G.W. and A.P.X.: conception and design and final approval of manuscript. J.L. and W.L. contributed equally to this work.

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The authors indicate no potential conflicts of interest.

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