TISSUE-SPECIFIC STEM CELLS

Proangiogenic and Prosurvival Functions of Glucose in Human

Mesenchymal Stem Cells Upon Transplantation

MICKAEL DESCHEPPER, MATHIEU MANASSERO, KARIM OUDINA, JOSEPH PAQUET,

LAURENT-EMMANUEL MONFOULET, MORAD BENSIDHOUM, DELPHINE LOGEART-AVRAMOGLOU, HERVE PETITE

Laboratoire de Bioing�enierie et Biom�ecanique Ost�eo-articulaire UMR CNRS 7052 (B2OA),

Universit�e Denis-Diderot, Facult�e de M�edecine Lariboisiere-Saint-Louis, Paris, France

Key Words. Human mesenchymal stem cells • Marrow stromal cells • Anoxia • Glucose • Ischemia • Angiogenesis

ABSTRACT

A major limitation in the development of cellular therapiesusing human mesenchymal stem cells (hMSCs) is cell sur-vival post-transplantation. In this study, we challenged thecurrent paradigm of hMSC survival, which assigned a piv-otal role to oxygen, by testing the hypothesis that exogenousglucose may be key to hMSC survival. We demonstratedthat hMSCs could endure sustained near-anoxia conditionsonly in the presence of glucose. In this in vitro cell model,the protein expressions of Hif-1a and angiogenic factors

were upregulated by the presence of glucose. Ectopicallyimplanted tissue constructs supplemented with glucoseexhibited four- to fivefold higher viability and were morevascularized compared to those without glucose at day 14.These findings provided the first direct in vitro and in vivodemonstration of the proangiogenic and prosurvival func-tions of glucose in hMSC upon transplantation and identifiedglucose as an essential component of the ideal scaffold fortransplanting stem cells. STEM CELLS 2013;31:526–535

Disclosure of potential conflicts of interest is found at the end of this article.

INTRODUCTION

Mesenchymal stem cells (MSCs) hold considerable promisein bioengineering because of their ability to differentiate intovarious phenotypes. To date, MSCs have not met this prom-ise, in part due to their high death rate upon transplantation[1–5]. In fact, cell transplantation strategies to replace dam-aged lost tissues are limited by the inability of MSCs to resistcell death in engineered construct after implantation.

A likely explanation for this limited cell survival is that,upon implantation, MSCs encounter an ischemic environmentcomposed of both low oxygen tension, nutrient deprivation[6]. Although either one of these insults has the potential toaffect cell survival significantly, most studies have focused onthe role of oxygen, because it modulates several critical cellu-lar processes (e.g., cell adhesion [7, 8], metabolism, prolifera-tion, and differentiation [9, 10]). In addition, oxygen is apoorly diffusive molecule whose passive diffusion from capil-laries is limited to 100–200 lm [11, 12]. In fact, this limiteddiffusibility of oxygen poses a crucial challenge to tissueengineering, and it is held responsible for the difficulty inobtaining functional tissue construct of pertinent volume forclinical application [13–15].

In this study, we challenged the current paradigm thatassigns a pivotal role to oxygen alone. We hypothesized that

exogenous glucose (the main ‘‘metabolic fuel’’ source forMSCs) plays a key role in the survival of human MSCs(hMSCs) under transplantation conditions. We exposedhMSCs in vitro to a sustained, near-anoxic environment andassessed the influence of exogenous glucose on (a) cell viabil-ity, (b) expression of the hypoxia inducible factor Hif-1a, (c)secretion of angiogenesis proteins, and (d) functions pertinentto new tissue formation. We further investigated the criticalcontribution of glucose to in vivo survival of implantedhMSCs and to peri-implant vascularization in an ectopicmouse model.

MATERIALS AND METHODS

Chemicals and Molecular Assay

Details regarding chemicals and respective suppliers were as fol-lows: Alpha minimum essential medium without glucose (aMEM0glc), Dominique Dutscher (Brumath, France, http://www.dut-scher.com). Glucose, Mannitol, deferoxamine, and hyaluronicacid, Sigma-Aldrich (St. Louis, MO, http://www.sigmaaldrich.com); antibiotics, trypsin, and fetal bovine serum (FBS), PAA(Pasching, Austria, http://www.paa.at); Guava reagents for flowcytometry and angiogenesis protein assay, Millipore (Bedford,MA, http://www.millipore.com); CellTiter-Glo Luminescent cell

Author contributions: M.D.: conception and design, collection and/or assembly of data, data analysis and interpretation, and manuscriptwriting; M.M. and K.O.: collection and/or assembly of data; J.P. and L.E.M.: conception and design and data analysis andinterpretation; M.B.: provision of study material or patients; D.L-A.: data analysis and interpretation and financial support; H.P.:conception and design, data analysis and interpretation, financial support, manuscript writing, and final approval of manuscript.

Correspondence: Herve Petite, Ph.D., Laboratoire de Bioing�enierie et Biom�ecanique Ost�eo-articulaire UMR CNRS 7052 (B2OA),Universit�e Denis-Diderot, Facult�e de M�edecine Lariboisiere-Saint-Louis, Paris, France. Telephone: 0157278533; Fax: 0157278533;e-mail: herve.petite@univ-paris-diderot.fr Received July 23, 2012; Revised November 8, 2012; accepted for publication November 14,2012; first published online in STEM CELLS EXPRESS December 7, 2012. VC AlphaMed Press 1066-5099/2012/$30.00/0 doi: 10.1002/stem.1299

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assay, Promega (Mannheim, Germany, http://www.promega.com);enzyme-linked immunosorbent assay (ELISA) assay, Invitrogen(Saint Aubin, France, http://www.invitrogen.com), and Hif-1aActivation Kit, Thermo Scientific (Quebec, Canada).

Near-Anoxic Conditions

Near-anoxia was established, and maintained, in a sterile, 37�C,humidified environment using a hypoxic incubator (BINDER CO2

incubators CB-210; Binder Scientific, France, http://www.binder-world.com).

Pericellular pO2 was measured using an oxygen electrodeconnected to an Oxylab pO2 meter (Oxford Optronix; Oxford,U.K., http://www.oxford-optronix.com). To ensure sustained near-anoxia as well as cell-driven nutrient depletion, the culture platescontaining the cells were not disturbed and the supernatant me-dium was not changed for the duration of the study.

Cells and Cell Culture

hMSCs were isolated from bone marrow obtained as discardedtissue during routine bone surgery from five donors by the Lari-boisiere Hospital (Paris, France). hMSCs were isolated using aprocedure adapted from literature reports [16]. Cell passages 2–3were used for the experiments that are described in the sectionsthat follow. For the in vitro part of the study, MSCs obtainedfrom five donors were used. Each test was conducted in triplicateusing cells from each one of the five donors. For the in vivo partof the study, each test was conducted in sextuplicate using a poolof MSCs from the five donors who were also used in the in vitropart of this study.

hMSC Viability Under Sustained Near-Anoxiain Either the Presence or Absence of Glucose

hMSCs (1.25 � 104 cells per centimeter square) from each donorwere seeded into individual wells of a 24-well plate, culturedovernight, washed twice with phosphate buffer solution (PBS),and maintained in near-anoxia under aMEM supernatant mediumin either the absence or presence (1 or 5 g/l) of glucose. Cell via-bility and glucose and lactate levels were determined at days 3,7, 14, and 21 of culture unless otherwise stated.

Determination of Glucose, lactate, and ATP

Glucose and lactate levels were monitored using a biomedicalARCHITECT C8000 (Abbott Diagnostic) robot. Intracellular ATPcontent was quantified using the CellTiter-Glo Luminescent cellassay (Promega), according to manufacturer’s instructions. Datawere expressed as ‘‘fold increase’’ compared to data obtained onday 0.

Flow Cytometry

Viable and apoptotic cells were identified using the Viacountassay and the annexinV-phycoerythrin (PE)/7 amino-actinomycin(7-AAD) assay, respectively, according to the manufacturer’sinstructions. Briefly, the Viacount assay is based on incorporationof fluorescent propidium iodide (PI) after loss of cell membraneintegrity, and the annexinV-PE/7-AAD assay is based on the factthat, in early apoptosis, annexin V binds to phosphatidylserineresidues on the outer leaflet of the cell membrane, and that 7-AAD is excluded by viable cells. The cell cycle phase of hMSCswas identified using the Guava Cell Cycle Assay according to themanufacturer’s instructions. Briefly, the Cell Cycle Assay usesPI, a nuclear DNA stain, to identify cell-cycle phase. Restingcells (G0/G1) contain two copies of each chromosome. Cyclingcells synthesize chromosomal DNA (S-phase), which results inincreased fluorescence intensity. When all chromosomal DNA hasdoubled (G2/M phase), the cells fluoresce with twice the intensityof the initial population. Data were collected using a GuavaEasy-CyteTM PCA-96 System and were analyzed assessed usingViacount and Nexin and Cell cycle software, respectively, toquantify cell viability, apoptosis, and cell cycle.

Proteins Expression

Adenosine monophosphate-activated protein kinase (AMPK) ac-tivity, extracellular angiopoietin-2, and vascular endothelialgrowth factor (VEGF)-C concentration were determined byELISA analysis. hMSCs were exposed to 0% O2 in the absenceor in the presence of glucose (0.1, 1, and 5 g/L) for 3 and 7 days.At the prescribed time, hMSCs were lysed using cell lysis buffercontaining phenylmethanesulfonylfluoride and a protease inhibi-tor. Each cell extract was centrifuged and treated according tomanufacturer’s instructions.

hMSCs Viability upon Reperfusion

After 21 days of exposure to sustained near-anoxia, hMSCs weretransferred to normoxic (21% O2) conditions with aMEM con-taining 1 g/l of glucose and 10% FBS. Controls were hMSCs cul-tured in normoxic conditions at all times.

Phenotype Determination

The phenotype of the isolated hMSCs was determined using flowcytometry. For this purpose, hMSCs were pretreated with theappropriate monoclonal antibody in the dark, at room tempera-ture, for 30 minutes. Details regarding antibodies used and suppli-ers were as follows: PE-conjugated antibodies against CD31,CD73 (BD Pharmingen, San Diego, http://www.bdbiosciences.com/index_us.shtml), CD45, CD105 (Beckman Coulter, Fullerton,CA, http://www.beckmancoulter.com); fluorescein isothiocyanate(FITC)-conjugated antibodies against CD90 (Beckman Coulter)and CD34 (Beckman Dickinson). Nonspecific fluorescencewas detected using goat anti-mouse immunoglobulin (BeckmanCoulter).

Effects of Glucose on Hif-1a Expression andBioactivity

Expression and bioactivity of Hif-1a were determined by threedifferent methods, all with the following procedures in common:hMSCs were exposed to either 21% or 0% O2 in the absence orin the presence of glucose (0.1, 1, and 5 g/l) for 3 days. The posi-tive control for Hif-1a expression was obtained by adding 500lM of deferoxamine (Sigma) to the supernatant medium ofhMSCS cultured under normoxic (i.e., 21% oxygen) condition.Method-specific details follow.

Immunochemistry of Hif-1a

Hif-1a expression was assessed using the Hif-1a Activation Kit(Thermo Scientific), following the manufacturer’s instructions.hMSCs were examined using confocal microscopy (LSMZEISS 510).

Western Blot and Hif-1a Expression Analysis

Hif-1a expression was determined using a previously publishedtechnique [17]. Antibodies against Hif-1a (1:1,000; Novus Biologi-cal) were used for immunoblotting. Specific bioluminescence sig-nals was detected by bioluminescence (BLI) using an IVIS LuminaBioluminescent imaging system (Xenogen, Caliper Life Science,Tremblay-en-France, France, http://www.caliperls.com) and werequantified by the IVIS Lumina imaging software (Living ImageSoftware, version 3.1).

Hif-1a Bioactivity

hMSCs were transfected with a pGL3/5HRE-CMV-Luc plasmidkindly donated by Dr. Masahiro Hiraoka containing five hypoxiaresponsive elements (HRE) sequences. Transfection was accom-plished using the Amaxa Nucleofector II Device nucleoporationsystem (Lonza Walkersville, Walkersville, MD, http://www.lon-za.com) following the manufacturer’s instructions. Bioactivity ofHif-1a was assessed by BLI.

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Assessment of MSCs Survival in 3D Constructs

Animal Model. The in vivo effect of glucose supplementationon cell viability was assessed using a mouse transplantationmodel (8-week-old female nih/nu/ xid/bg mice; Harlan-SpragueDawley, Indianapolis, IN, http://www.harlan.com). All animalprocedures were performed in compliance with institutional pub-lished guidelines (Directive du Conseil 24.11.1986. 86/609/CEE).

Tissue Construct Preparation. Cylindrical polyacrylonitrile-sodium methallyl sulfonate (PASM) scaffolds (diameter: 7 mm;height: 5 mm) were donated by Dr. Jiri Honiger (Saint-AntoineHospital, Paris, France). The scaffolds were prepared by phaseinversion of a polymer solution containing 6% acrylonitrile andsodium methallylsulfonate copolymer, 91% dimethylsulfoxide,and 3% physiological saline (0.9% NaCl) solution (w/w/w) [1].The scaffolds had pores with a mean diameter of 500–1,000 mmand a porosity of 77%. They were sterilized via immersion in10% Dialox in physiological saline (v/v). After a thorough rinseof the PASM scaffolds using sterile physiological saline, hMSCs(3 � 105 cells) genetically modified by rMLV-LTR-eGFP-luc ret-roviral vector were seeded by injection and were cultured inaMEM (containing 10% FBS) at 37�C overnight.

In Vivo Experiments. At the time of implantation, 100 ll of ei-ther hyaluronic acid (2%) or fibrin gel (9 mg/ml) containing either0 or 10 g/l of glucose was gently injected inside each cell-contain-ing construct. Two cell constructs (loaded vs. not loaded with glu-cose) were implanted subcutaneously per animal on six mice andwere imaged by BLI at days 1, 4, 7, and 14 postimplantation.

Assessment of HRE Expression in Vivo

The effect of glucose on HRE expression was assessed in vivo.Briefly, 3 � 105 hMSCs transfected with pGL3/5HRE-CMV-Lucwere seeded by injection into cylindrical PASM scaffolds andwere cultured in aMEM (containing 10% FBS) at 37�C over-night. At the time of implantation, these scaffolds were embeddedinto fibrin gel (9 mg/ml) containing either 0 or 10 g/l glucose.Two cell constructs per animal (loaded vs. not loaded with glu-cose) were implanted subcutaneously on six mice and wereimaged by BLI at days 1, 4, 7, 10, and 14 postimplantation.

Assessment of Implanted Constructs Vascularization21 Days After Implantation

The in vivo effect of glucose on vascularization was histologi-cally assessed. Briefly, 21 days postimplantation, constructs wereexplanted, fixed in 0.4% Paraformaldehyde (PFA) for 12 hours,and embedded in paraffin; thin (8 lm) sections of each constructwere stained with hematoxylin, eosin, and safran. Peripheral vas-cularization was quantified in the area within 1 mm around eachconstruct. Vascularization was also immunohistochemicallyassessed using FITC-conjugated Griffonia Simplicifolia IsolectineB4 (Sigma) according to the manufacturer’s instructions. Briefly,embedded-paraffin-sections were stained with Isolectine B4 (dilu-tion of 1/250), in the dark, at room temperature for 1 hour. Sec-tions were examined using a fluorescent microscope (NikonEclipse TE2000-U; Nikon, Champigny sur Marne, France) fittedwith a digital camera (DXM1200F). The Nikon NIS element F2.20 software was used for imaging and analysis.

Statistical Analysis

Numerical data are expressed as the mean 6 SD. Statistical analy-ses were performed with ANOVA for in vitro data and Mann–Whit-ney U tests for in vivo data (GraphPad Prism Software). Asterisksindicate significant differences, as follows: *, p < .05; **, p < .001.Significant differences among the data of the same group are indi-cated by the symbol ‘‘#’’ for p < .001 in comparison with day 3.

RESULTS

Establishment of Near-Anoxia and Ischemia

To validate our in vitro model, hMSCs in a-MEM withoutserum in the absence or presence (either 1 or 5 g/l) of glucosewere cultured in a humidified, 37�C, 5% CO2, 95% N2

incubator set at 0% oxygen for 21 days. The time course ofoxygen tension, lactate (a hallmark of anaerobic metabolism),and glucose were determined by testing samples of superna-tant cell medium (Fig. 1A). To prevent reoxygenation, toensure cell-driven nutrient depletion, the culture plates con-taining the cells were not disturbed, and the medium was notchanged over the course of the experiments.

In this experimental setting, when the cells were transferredto the environment of 0% oxygen, the oxygen tension droppedfrom 21% to 2.8% within 2 hours (Fig. 1B). After 72 hours,the oxygen level reached 0.12% and remained constant at thislevel for the duration of the experiments. This result demon-strated that hMSCs were exposed to sustained, near-anoxia.

Based on the time course of glucose concentrationchanges in the medium, hMSCs cultured in the absence ofglucose faced abrupt ischemia from day 0 (Fig. 1C). hMSCscultured in the presence 1 g/l of glucose faced completedepletion of glucose by day 14, and hMSCs cultured in thepresence 5 g/l of glucose did not face glucose exhaustionduring the entire 21 days of the experiment. The kinetics oflactate revealed that hMSCs had shifted to anaerobic metabo-lism, and the lactate accumulated in the supernatant mediumonly in the presence of glucose (Fig. 1D). In these conditions,hMSCs culture with 0, 1, and 5 g/L of glucose, respectively,faced abrupt ischemic like, cell driven ischemic, and near-anoxic like conditions for the duration of the experiments.

Abrupt and Cell-Driven Ischemia, but not bySustained Near-Anoxia Affected hMSC Viability

Cell death during ischemia might result from either a shortageof glucose or exposure to near-anoxia. To assess the respec-tive roles of near-anoxia and glucose concentrations under theconditions tested, we compared the survival rates of hMSCscultured with or without glucose.

Under abrupt ischemic conditions (0 g/l glucose), thenumber of hMSCs decreased over 21 days (Fig. 2A). In addi-tion, the ATP content per cell drastically decreased from day3 to day 21 (Fig. 2B). Under cell-driven ischemic conditions(1 g/l glucose), hMSCs survived for 7 days; then, the numberof viable cells significantly decreased from day 7 to day 21.The ATP content per cell remained stable for the first 7 daysbut rapidly decreased from days 7 to 21 of culture. Undernear-anoxic conditions (5 g/l glucose), the number of viablehMSCs and the ATP content per cell remained stable for theduration of the experiment. Interestingly, at day 3, althoughATP content per cell was similar in all conditions tested, adownregulation of phosphorylated AMPK (a key sensor offuel and energy status in cell) was already observed (Fig. 2C).

Quantification of Annexin V expression (a marker of apo-ptosis) in hMSCs revealed that, after 3 days of either near-an-oxia or ischemia, only 15% of hMSCs were Annexin V-posi-tive (Fig. 2D). These results were corroborated by theabsence of induction of phosphatidylinositide 3-kinase(PI3K)/mammalian target of rapamycin (mTOR) pathway [18](as measured by phosphorylated p70S6K data not shown). Incontrast, after 21 days in near-anoxia, the number of AnnexinV-positive cells was significantly lower than that observed inhMSCs exposed to abrupt and cell driven ischemia (19% vs.45%, p < .001; 19% vs. 38%, p < .001, respectively).

528 Proangiogenic and Prosurvival Functions of Glucose

To exclude that the effect of glucose on MSC viabilitywas driven by the glucose-mediated hypertonicity of the cellenvironment, hMSCs were cultured under near anoxic condi-tions in the presence of 5 g/l mannitol (Fig. 2A). Under theseconditions, hMSCs viability was not statistically differentfrom the one observed in the absence of glucose suggestingthat hypertonicity is not cytoprotective. Moreover, to verifythat only glucose, not glutamine (an alternative source forATP production), is responsible of cell survival in ischemia,the role of glutamine was evaluated in the in vitro cell modeltested in this study. We found that adding glutamine in thesupernatant medium did not prevent cell death and did notmaintain the ATP content (Supporting Information Fig. 1).Taken together, these results demonstrated that, in the near-absence of oxygen, glucose depletion (abrupt or cell driven)affected hMSC viability. In contrast, hMSCs remained viablein sustained near-anoxia conditions in the presence ofglucose.

hMSCs Exposed to Near-Anoxia Remained Viableand Retained Proliferative Ability After Reperfusion

To assess the functional status of hMSCs after a 21-dayexposure to either sustained near-anoxia, abrupt ischemia, orcell-driven ischemia, we simulated the conditions of bloodreperfusion, and then assessed select hMSC stem cell functionusing observation of morphology, CD marker characterization,and cell cycle analysis.

We found that hMSCs cultured under either abrupt orcell-driven ischemia did not proliferate upon reperfusion (Fig.3A). In contrast, hMSCs exposed to near-anoxia for 21 daysexhibited normal fibroblast-like morphology after reperfusion(Fig. 3A). Cell cycle analyses showed that these hMSCs pro-liferated at a normal rate, that is, the percentage of mitoticcells was similar to that observed under standard cell cultureconditions (Fig. 3B). Flow cytometry analysis revealed that,after 21 days under near-anoxic conditions and 3 days after

Figure 1. Establishment of near-anoxia and ischemia. (A): Outline of the experimental protocol used to induce near anoxic and/or ischemicenvironments. (B): Time course of oxygen tension in the presence (solid line) and absence (dashed line) of hMSCs. (C): Time course of residualglucose and of (D) lactate production when hMSCs were cultured with 0, 1, or 5 g/l glucose (red, green, and blue bars, respectively). Data showthat hMSCs were exposed to a pO2 less than to 0.13%, shifted to anaerobic metabolism (lactate production), and were exposed either to abruptischemia (0 g/l glucose), cell-driven ischemia (1 g/l glucose), or near-anoxia (5 g/l glucose). Data are represented as mean 6 SD. *, p < .05; **,p < .001. Abbreviation: hMSCs, human mesenchymal stem cells.

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Figure 2. Abrupt or cell-driven ischemia, but not sustained near-anoxia, affected hMSC viability. (A): Quantification of cell viability, (B) per-cent of ATP per cell, (C) phospholylated AMPK activity when hMSCs were either maintained in abrupt ischemia (red bar), cell-driven ischemia(green bar), near-anoxia (blue), or near-anoxia in the presence of manitol (shaded bar). (D): FACS analysis of apoptotic hMSCs (stained byAnnexin-V) when hMSCs were either maintained in ischemia or near-anoxia. hMSCs survived and maintained their ATP content for 21 daysunder sustained near-anoxia only when cultured in the presence of 5 g/l of glucose. In contrast, hMSCs cultured without or with 1 g/l of glucosedid not survive. Data are represented as mean 6 SD. *, p < .05; **, p < .001 and significant differences among the data of the same group areindicated by the symbol ‘‘&’’ for p < .001 in comparison with day 3. Abbreviation: MSCs, mesenchymal stem cells.

Figure 3. In vitro assessment of MSC viability after reperfusion. (A): Cell proliferation and morphology before reperfusion and 7 days afterreperfusion compared to freshly cultured hMSCs under standard culture conditions (control), light microscopy magnification: �10. (B): Percent-age of hMSCs in mitosis and (C) flow cytometry analysis of hMSCs phenotype markers for expression in hMSCs cultured under standard cultureconditions (i.e., 21% oxygen, medium supplemented with 10% FBS and 1 g/l of glucose), hMSCs exposed to 21 days of near-anoxia in the pres-ence of glucose (5 g/l), and hMSCs cultured 3 days after reperfusion. Data show that hMSCs when cultured 21 days under near-anoxia in thepresence of glucose, hMSCs kept their phenotypic markers and their ability to proliferate. Data are represented as mean 6 SD. *, p < .05; **, p< .001. Abbreviation: hMSC, human mesenchymal stem cells.

530 Proangiogenic and Prosurvival Functions of Glucose

reperfusion, the cells were positives for CD73, CD90, andCD105, and negatives for CD31, CD34, and CD45 (Fig. 3C).Taken together, these data confirmed that in sustainednear-anoxic conditions the presence of a glucose supply wasnecessary to ensure cell viability and function.

Glucose Regulated Hif-1a, Angiopoietin-2,and VEGF-C Expression

The hypoxia-inducible factor Hif-1a is a major regulator ofthe cellular response to hypoxia because it activates the tran-

scription of many genes, including those involved in cellsurvival, angiogenesis, oxygen delivery, and metabolic adap-tation to hypoxia. To evaluate the effect of glucose on Hif-1a expression, immunohistochemistry was performed onhMSCs cultured for 3 days under various conditions includ-ing the following: standard cell culture conditions in the ab-sence (negative control) or presence (positive control) of de-feroxamine and near-anoxia in the absence or presence ofglucose (1 and 5 g/l) (Fig. 4A). Under normoxia, no expres-sion of Hif-1a was detected; however, in the presence of

Figure 4. Glucose regulated Hif-1a, angiopoietin, and VEGF-C expression. (A): Confocal imaging, magnification: �40, (B) representativeresults of Western blot for Hif-1a, (C) quantification and bioactivity of Hif-1a in hMSCs cultured under normoxia without (gray bar) and with(black bar) DFO (positive control) and near-anoxia with either 0, 0.1, 1, or 5 g/l of glucose (red, purple, green, and blue bars, respectively), (D)angiopoietin-2 and (E) VEGF-C in the supernatant medium of mesenchymal stem cells (MSCs) cultured under near-anoxia with either 0, 0.1, 1,or 5 g/l of glucose (red, purple, green, and blue bars, respectively) at day 3 (empty bars) and day 7 (gridded bars). For the first time, these dataprovided evidence that glucose concentration affects Hif-1a expression and bioactivity when hMSCs under near-anoxia. Data are represented asmean 6 SD. *, p < .05; **, p < .001. Abbreviations: BLI, bioluminescence; DFO, deferoxamine; HIF, hypoxia inducible factor; VEGF, vascularendothelial growth factor.

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deferoxamine (positive control for Hif-1a expression), Hif-1a was localized in the cell nucleus. Under near-anoxia, Hif-1a was always localized in the nucleus, regardless of glucoseconcentration. Most importantly, analysis of images acquiredwith the same exposure settings showed that Hif-1a expres-sion was positively correlated with increases in glucose con-centration in the supernatant medium (Fig. 4A). This obser-vation was also confirmed by Western blot analysis (Fig.4B).

The effect of glucose concentration on Hif-1a bioactivitywas also evaluated by transfecting hMSCs with the reporterplasmid, pGL3/5HRE.CMVmp-Luc, which contained fivecopies of the hypoxia responsive element (HRE), aDNA binding sequence for Hif-1a. The intensity of theHif-1a-induced signal was directly correlated to the glucoseconcentration present in the supernatant media of hMSC cul-tures exposed to near-anoxia for 3 days. These results showedthat the increases in Hif-1a expression resulted in increasedbioactivity (Fig. 4C).

To further investigate the role of glucose concentrationon angiogenesis, the expression of angiopoietin-2 andVEGF-C was quantified by ELISA assays. Results showedthat glucose significantly enhanced the amount of angiopoie-tin in hMSC cultures exposed to near-anoxia at 3 and 7 days(a fourfold increase when compared to results obtained with-out glucose) (Fig. 4D). Moreover, in the presence of glucose,VEGF-C level increase in hMSC exposed to near-anoxia at

3 and 7 days. Taken together, these results provided evi-dence that the glucose concentration enhance Hif-1a expres-sion and secretion of angiogenic factors (such as angiopoie-tin and VEGF-C).

Glucose Enhanced In Vivo hMSCs Survivalin 3D Constructs

To extend the results of the in vitro studies to the in vivo set-ting, we assessed hMSC survival in an ectopic transplantationmouse model. Briefly, eGFP-Luc hMSCs expressing constitu-tively the green fluorescent protein and the gene of luciferase(eGFP-Luc hMSCs) were seeded into PASM scaffold in theabsence of glucose. The hMSC PSAM scaffolds wereimplanted ectopically in the back of mice, and the BLI ofeach implant (a noninvasive measurement of transplantedhMSC viability) was quantified on days 1, 4, 7, and 14 post-implantation. The number of viable cells decreased from days1 to 14 (Fig. 5A). On day 14, only 15% viable cells weredetected.

In order to limit glucose (which is a small, highly diffu-sive molecule) leakage outside the cell-containing construct,glucose was loaded into either fibrin or hyaluronic acidhydrogels with which the PASM scaffold were filled beforeimplantation. The number of viable cells in PASM scaffoldsloaded with fibrin alone significantly decreased from days 1to 14, and, on day 14, only 15% viable cells were detected

Figure 5. Glucose enhances in vivo cell survival in a 3D construct. (A): Quantification of cell viability of hMSCs seeded in PASM scaffoldsalone (i.e., not filled with a hydrogel). (B): Imaging and viability quantification of hMSCs seeded in PASM scaffolds filled with fibrin gel (loaded[black bars] or not [white bars] with glucose). (C): Imaging and viability quantification of hMSC seeded in PASM scaffolds filled with hyal-uronic acid (2%) loaded (black bars) or not (white bars) with glucose. A striking increase of cell viability was observed in cell constructs loadedwith glucose independent of the type of hydrogel (i.e., hyaluronic acid or fibrin gel) used. At day 14, a four- to fivefold increase in cell numbersobtained for cell-containing constructs loaded with glucose when compared to the respective control (i.e., cell-containing constructs without glu-cose). Data are represented as mean 6 SD. *, p < .05; **, p < .001.

532 Proangiogenic and Prosurvival Functions of Glucose

(Fig. 5B). In contrast, the number of viable cells in PASMscaffolds loaded with fibrin and glucose remained constantfrom days 1 to 7 but decreased thereafter. Importantly, thenumber of viable cells on day 14 in PASM scaffolds loadedwith fibrin was five times higher in the presence of glucosethan that observed in the absence of glucose. To confirm thepositive influence of glucose on hMSC viability, we con-ducted a similar study with hyaluronic acid as the deliverysystem for glucose. Of interest, in these conditions, the num-ber of viable cells increased from days 1 to 7 and remainedconstant between days 7 and 14 (Fig. 5C). Importantly, thepercentage of viable cells on day 14 was 4.5-fold higher inthe presence of glucose than that observed in the absence ofglucose. Taken together, these results established, for the first

time, that the presence of glucose in engineered scaffoldcould significantly limit and even prevent massive cell deathupon implantation of cell containing constructs.

Glucose Enhanced HIF-1 Bioactivity andVascularization of 3D Construct

To evaluate the effect of glucose on HIF-1 bioactivity, HRE-Luc hMSCs were seeded into PASM scaffolds, embedded ina fibrin gel, and implanted ectopically in the back of mice ineither the presence or in the absence of glucose. The BLI ofeach implant was quantified on days 1, 4, 7, 10, and 14 (Fig.6A). In the absence of glucose, the BLI signal remained con-stant from day 1 to 14. In contrast, in the presence of glucose,

Figure 6. Glucose enhanced hypoxia inducible factor 1a (Hif-1a) bioactivity and vascularization of 3D constructs: (A): Quantification andimaging (at day 14) of Hif-1a expression by human mesenchymal stem cell (hMSC) seeded in poly-acrylonitrile-sodium methallyl sulfonate scaf-folds filled with fibrin gel loaded (black bars) or not (white bars) with glucose. (B): Representative histology results of peripheral vascularization(black arrows) of MSCs containing constructs loaded with glucose after 2 weeks of subcutaneous implantation in mice. Stain: Hematoxylin-Eo-sin-Safran. Magnification: �2 including magnification (�10) of region of interest. (C): Representative photograph of the 3D construct whenloaded without (left photograph) or with (right photograph) glucose at day 14 postimplantation. (D): Immunostaining of blood vessels using iso-lectin B4, magnification: �10. (E): Peripheral blood vessels quantification of the cell containing constructs filled with either hyaluronic acid orfibrin gel and loaded without or with glucose. A striking increase of peripheral blood vessels was observed in cell-containing constructs loadedwith glucose independently of the type of the hydrogel used (i.e., hyaluronic acid or fibrin gel). Data are represented as mean 6 SD. *, p < .05;**, p < .001. Abbreviation: BLI, bioluminescence.

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the BLI signal increased from day 1 to 10 but remained stableat day 14. At day 14, the BLI signal was seven times higherin the presence of glucose (2.12 vs. 15; p < .001). Taken to-gether, these data suggest that the presence of glucoseenhance Hif-1a bioactivity.

To evaluate the effect of glucose on vascularization ofimplanted 3D construct they were imaged (Fig. 6C),explanted, embedded, included, and stained by Hematoxylin-Eosin-Safran (Fig. 6B). The number of peripheral blood ves-sels was determined from histological sections (1 mm thin)from tissue surrounding each implant. In the absence of glu-cose, few peripheral blood vessels were observed. In contrast,when the constructs were supplemented with either hyaluronicacid and glucose or with fibrin gel and glucose, the numberof peripheral blood vessels increased significantly (2 vs. 4; p< .01 and 2 vs. 9; p < .001, respectively) (Fig. 6E). The pres-ence of blood vessels was confirmed immunochemically bystaining endothelial cells with isolectine B4 (Fig. 6D).

DISCUSSION

In order to realize the full therapeutic potential of hMSCs,their survival rate upon implantation must be improved. Toachieve this important goal, we require a better understandingof the underlying mechanisms of cell death upon implanta-tion, and we must develop a niche that reduces hMSC sensi-tivity to ischemia.

In this study, we developed an in vitro model to investi-gate the deleterious effects of ischemia on hMSC survival andfunction under conditions of near-anoxia, that is, low pO2,Hif-1a expression, and anaerobic metabolism. In the absenceof glucose, most hMSCs cultured under near-anoxia diedwithin 14 days; this result established glucose as a key playerin hMSC survival. An alternative explanation might be that asudden metabolic shift induced by abrupt depletion of glucosehad caused massive cell death. To exclude this alternativepossibility, hMSCs were cultured in near-anoxia in the pres-ence of 1 g/l glucose. Under these conditions, hMSCs faced aprogressive cell-driven exhaustion of glucose. Thus, by day14, near-anoxic (low pO2) conditions switched to ischemic(low pO2 and glucose depletion) conditions (Fig. 2). Ischemiacaused an early AMPK upregulation, cell shrinking, reducedcell viability, and resulted in low ATP content. We alsodefinitively ruled out a possible effect of glucose-mediatedhypertonicity on cell viability (Fig. 2A) and the contributionof glutamine as an alternative nutrient source in this model(Supporting Information data 1). When exposed to glutaminealone under near-anoxic conditions, hMSCs died at day 7.These results provided evidence that both abrupt andcell-driven glucose depletion led to massive cell death andvalidated the present model.

To confirm that glucose, not near-anoxia, was the key fac-tor in the cell death process, hMSCs were exposed to severe,continuous (21 days) near-anoxic condition in the presence ofglucose (5 g/l). Under these conditions, MSCs remained via-ble, expressed characteristic phenotypic markers and prolifer-ated in vitro (after simulation of reperfusion) (Fig. 3). Thesefindings challenged the traditional view that severe near-an-oxia per se is responsible for the massive MSC deathobserved postimplantation [15, 19]. The results of this studyprovided evidence that hMSCs can withstand exposure tosevere, continuous near-anoxia, provided that glucose is avail-able during all the duration of the experiment.

When hMSCs were exposed to near-anoxia in the pres-ence of various glucose concentrations, a dose effect on Hif-

1a (a key regulator of the cellular response to hypoxia [20])expression and bioactivity was observed. This result providedthe first evidence that glucose, in addition to supplying fuelfor ATP production, is required for the hMSC response tonear-anoxia through Hif-1a activation (Fig. 4) and confirmedprevious studies with carcinoma cells [21].

A key observation of this study is that glucose supple-mentation of cell-containing constructs in vivo resulted in afour- to fivefold increase of the bioluminescent signaldirectly correlated with the percentage of present viable cells[18, 22]. These results are the first direct demonstration thatglucose per se significantly reinforced the ability of hMSCsto survive in vivo transplantation. They are important in thecontext of tissue engineering, because they identified glucoseas an essential component of the ‘‘ideal niche’’ to reduceMSC sensitivity to ischemia. More interestingly, the pres-ence of glucose strongly enhanced peripheral vascularizationof the implanted tissue constructs (Fig. 6). This desirableresult might be due to in vivo overexpression of Hif-1a,which activates a large panel of genes involved in angiogen-esis [23] including angiopoietin and VEGF-C which wereboth overexpressed in vitro in the presence of glucose undernear anoxic condition (Fig. 4D, 4E). In addition, exposure tohigh glucose concentrations may induce locally protein gly-cation, which affects the local immune surveillance.Although, this outcome is unlikely in this study due to thelimited duration of the experiment, the occurrence of such aglycation process cannot be excluded during long-termexperiments (for review [24]).

In the presence of glucose, the number of hMSCs in theconstructs on day 14 was, at best, similar to that of MSCsin constructs at day 0. Although we cannot exclude the pos-sibility that only a transient exposure to glucose is neededto reinforce hMSC survival in vivo, we speculate that, thefull potential of glucose in fostering MSC survival and con-struct vascularization after implantation will be realizedwith advanced drug delivery systems that entrap glucoseand release it at a rate that matches MSC demand for glu-cose over a long period of time. An alternative, but notmutually exclusive, explanation for this observation mightbe that cell death occurred after several insults, includingthe absence of glucose, metabolic waste accumulation withconcomitant pH changes, and induction of inflammatoryreaction [6, 25].

This study was the first to provide in vitro and in vivodemonstrations that glucose (but not glutamine) significantlyenhanced the ability of hMSCs to survive in a near-anoxicenvironment. At last but not the least, in vitro and in vivoglucose supply significantly enhances Hif-1a expression andangiogenesis by the secretion of angiogenic factors such asangiopoietin and VEGF-C. This finding provides valuableinsights to current understanding of the mechanisms underly-ing MSC death upon implantation. Our in vivo results pro-vided evidence that glucose improved the viability of a tis-sue-engineered construct after implantation. Theseexperiments demonstrated the feasibility of our strategy andhave important implications for applications of major clini-cal impact.

ACKNOWLEDGMENTS

We thank Dr. J. Honniger for donating the PASM scaffolds;Professor R. Bizios for valuable comments on the manuscript; Y.Calando for immunochemistry advice regarding Hif-1a and the

534 Proangiogenic and Prosurvival Functions of Glucose

IFR65 platform for the quantification of angiopoietin andVEGF-C. We also acknowledge the financial support from theFonds d’amorcage Bioth�erapie BTH06003, the ANR 07-RIB-011-01 MYOCELLOS and ANR 08-TECS-004 GLASSBONE,and the Contrat d’Interface AP-HP/INSERM.

DISCLOSURE OF POTENTIAL

CONFLICTS OF INTEREST

The authors indicate no potential conflicts of interest.

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