Differentiation of mesenchymal stem cells into vascular cell lineages underinfluence of mechanical stimuli

M.G.J. BongersDecember 2004

BMTE05.03

MSc-thesisPart II

Committee:dr. C.V.C. Boutenprof. dr. ir. F.P.T. Baaijensprof. dr. M. J. Postprof. dr. ir. F.N. van de Vosse

Eindhoven University of TechnologyFaculty of Biomedical Engineering

Samenvatting

De laatste jaren worden multipotente stamcellen gebruikt als cellulaire component bij de tissueengineering van biologisch functionele vaatconstructen. Deze cellen kunnen differentieren in vel-erlei celtypen waaronder ook vasculaire cellen. In de loop der tijd zijn verschillende cytokinesen groeifactoren geıdentificeerd als stimulatoren van specifieke differentiatiemogelijkheden, zowelin vivo als in vitro. Recente studies tonen aan dat ook mechanische stimuli stamceldifferenti-atie kunnen induceren. Dit concept zou mogelijk toegepast kunnen worden in vasculaire tissueengineering. In deze studie is gekeken of zogenaamde mesenchymale stamcellen differentieren inendotheelcellen indien ze blootgesteld worden aan schuifspanning. Verder is onderzocht of dezecellen differentieren in vasculaire gladde spiercellen als gevolg van het opleggen van cyclische rek.Humane mesenchymale stamcellen (hMSCs), afkomstig van beenmerg, werden in monolaagkweekgebracht op respectievelijk collageen type I en fibronectine. Allereerst werd bepaald of deze cellenkunnen differentieren in endotheelcellen onder invloed van de aan het kweekmedium toegevoegdegroeifactor VEGF (vascular endothelial growth factor). Het endotheliale fenotype werd vast-gesteld door twee endotheliale eiwitten, VE-cadherin en CD31, fluorescent aan te kleuren en derespectievelijke intensiteiten door middel van confocaalmicroscopie te vergelijken met die vanendotheelcellen van humane coronaire vaten (hCAECs) evenals met hMSCs, gekweekt zonder to-evoeging van VEGF. In een ander experiment werden hMSCs gekweekt in monolaag gedurende 24uur blootgesteld aan een afschuifstroming (schuifspanning 0.5 Pa) die werd opgewekt door mid-del van een conus/vlakke plaat-systeem. Op de al eerder beschreven wijze werden VE-cadherinen CD31 expressie vergeleken met die van statisch gekweekte hMSCs en hCAECs. In het laat-ste experiment werden hMSCs gekweekt op flexibele membranen gecoat met collageen type I.Met behulp van een nauwkeurig aanstuurbare vacuumpomp werden deze membranen cyclischequi-biaxiaal gerekt gedurende 24 uur (zaagtandprofiel, 10% maximale rek). Nadien werden decytoskeletale eiwitten alfa smooth muscle actine (ASMA) en vimentine fluorescent aangekleurd.De expressiepatronen van deze eiwitten in het cytoplasma werden bestudeerd door middel vanconfocaalmicroscopie en vergeleken met die van statisch gekweekte cellen. Een FACS (fluores-cence activated cell sorter) werd gebruikt om de cellulaire fluorescentie-intensiteiten van ASMAen vimentine te kwantificeren, direct nadat de belasting was beeindigd, evenals na 48 uur ad-ditionele statische kweek. Eveneens werd celgranulariteit op dezelfde momenten kwantitatiefvergeleken met statisch gekweekte cellen. Het bleek dat hMSCs VE-cadherin en CD31 aan-maken onder invloed van VEGF op zowel collageen type I als fibronectine. Verder werden in deafwezigheid van serum vaatpatronen waargenomen op fibronectine. hMSCs gekweekt in de aan-wezigheid van schuifspanning brachten VE-cadherin en CD31 tot expressie op collageen type I.Verder paste de celmorfologie bij een endotheliaal fenotype en werden er celclusters waargenomen,mogelijk duidend op primitieve vaatpatronen. Endotheliale differentiatie van hMSCs gekweektop fibronectine kon niet vastgesteld worden. Cyclisch gerekte hMSCs werden gekenmerkt dooreen toename van de hoeveelheid ASMA en vimentine in de regio rond de celkern vergeleken metstatisch gekweekte cellen. Verder kende het cytoplasma van deze cellen een hogere granulariteit.Deze fenotypische eigenschappen zijn kenmerkend voor contractiele vasculaire gladde spiercellenen waren 48 uur na het beeindigen van de belasting nog steeds van kracht. Deze preliminaireresultaten ondersteunen het idee dat vasculaire differentiatie van mesenchymale stamcellen kanworden bewerkstelligd door het aanbrengen van mechanische stimuli. Schuifspanning en cyclis-che rek lijken geschikte stimuli voor het triggeren van differentiatie van hMSCs in respectievelijkendotheelcellen en vasculaire gladde spiercellen.

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Abstract

Background: In recent years, multipotent stem cells have been used as cell source for tissueengineering of biologically functional vessel substitutes. These cells can differentiate into manyspecialized cell lineages, including vascular cells. Over the years several cytokines and growthfactors have been identified as stimulators of particular differentiation pathways both in vivo andin vitro. Recent studies indicate that mechanical stimuli can trigger stem cell differentiation aswell, a concept which might eventually be exploited in vascular tissue engineering. The presentstudy was performed to investigate whether shear stress can induce endothelial differentiationof so-called human mesenchymal stem cells (hMSCs). Furthermore it was investigated if cyclictensile strain triggers differentiation into vascular smooth muscle cells.Methods: hMSCs from bone marrow were cultured in monolayer on both collagen type I andfibronectin. First it is was tested if these hMSCs were able of endothelial differentiation by sup-plementing culture medium with vascular endothelial growth factor (VEGF, 50 ng·ml−1). Theendothelial phenotype was confirmed by immunofluorescent labelling of the endothelial proteinsVE-cadherin and CD31 and comparison of the respective expression levels to those of humancoronary artery endothelial cells (hCAECs) and hMSCs cultured without VEGF by confocal mi-croscopy. In a second experiment hMSCs were exposed to constant fluid shear stress (0.5 Pa)induced by a cone-and-plate system for 24 hours followed by comparison of VE-cadherin andCD31 expression levels to those of hCAECs and statically cultured hMSCs.Finally, hMSCs were seeded on flexible silicone membranes coated with collagen-I. Using a dedi-cated straining system these membranes were cyclically stretched for 24 hours (triangular equib-iaxial straining regime, 10% peak strain). Afterwards cells were immunofluorescently stained forthe cytoskeletal proteins alpha smooth muscle actin (ASMA) and vimentin. Cytoplasmic ex-pression patterns of both proteins were studied on confocal microscope. In a similar experimentcellular fluorescence intensities of ASMA and vimentin were read by a fluorescence activated cellsorter (FACS) which was also used for comparing cell granularity to statically cultured cells.Additionally, ASMA and vimentin expression levels after 48 hours of additional static culturewere determined.Results: hMSCs exposed to VEGF expressed VE-cadherin and CD31 on both collagen-I andfibronectin. Vascular patterns were formed under no-serum conditions on fibronectin. hMSCsexposed to shear stress expressed both VE-cadherin and CD31 on collagen-I. Cell shape suitedan endothelial phenotype. Furthermore, cell clusters were observed which could be a sign ofearly vascular patterning. No signs of endothelial differentiation were found in the cultures onfibronectin. Cyclic straining of hMSCs resulted in an increased expression of ASMA and vimentinin the perinuclear cell region compared to statically cultured cells, accompanied by an increasedcytoplasmic granularity. These changed phenotypic characteristics fitted the contractile vascularsmooth muscle phenotype and were maintained up to 48 hours after ending the straining regime.Conclusion: These preliminary data provide evidence for the notion that vascular differentia-tion of mesenchymal stem cells can be directed by mechanical stimuli. Shear stress and cyclictensile strain seem to be appropriate stimuli for inducing respectively endothelial and vascularsmooth muscle differentiation of hMSCs.

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Contents

1 Introduction 7

2 Materials and Methods 9

2.1 Cells and culture media . . . . . . . . . . . . . . . . . . . . . . . . . . . . 9

2.2 Phenotype identification . . . . . . . . . . . . . . . . . . . . . . . . . . . . 9

2.2.1 Cellular proteins . . . . . . . . . . . . . . . . . . . . . . . . . . . . 9

2.2.2 Antibodies . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 10

2.3 Assessment of the in vitro angiogenic potency of hMSC . . . . . . . . . . 11

2.4 Shear stress experiments . . . . . . . . . . . . . . . . . . . . . . . . . . . . 11

2.4.1 Cone and plate system . . . . . . . . . . . . . . . . . . . . . . . . . 11

2.4.2 Cell seeding . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 14

2.4.3 Shear stress regime . . . . . . . . . . . . . . . . . . . . . . . . . . . 14

2.5 Cyclic equibiaxial strain experiments . . . . . . . . . . . . . . . . . . . . . 15

2.5.1 Flexercell system . . . . . . . . . . . . . . . . . . . . . . . . . . . . 15

2.5.2 Cell seeding and straining . . . . . . . . . . . . . . . . . . . . . . . 16

2.6 Data acquisition and analysis . . . . . . . . . . . . . . . . . . . . . . . . . 16

2.6.1 Fluorescence microscopy . . . . . . . . . . . . . . . . . . . . . . . . 16

2.6.2 Laser scanning confocal microscopy . . . . . . . . . . . . . . . . . . 17

2.6.3 Flow cytometric analysis . . . . . . . . . . . . . . . . . . . . . . . . 18

2.7 Statistical analysis . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 18

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3 Results 19

3.1 Assessment of the in vitro angiogenic potency of hMSC . . . . . . . . . . 19

3.1.1 Fibronectin substrate . . . . . . . . . . . . . . . . . . . . . . . . . 19

3.1.2 Collagen type I substrate . . . . . . . . . . . . . . . . . . . . . . . 21

3.2 Shear stress experiments . . . . . . . . . . . . . . . . . . . . . . . . . . . . 21

3.2.1 Collagen type I substrate . . . . . . . . . . . . . . . . . . . . . . . 21

3.2.2 Fibronectin substrate . . . . . . . . . . . . . . . . . . . . . . . . . 23

3.3 Cyclic equibiaxial strain experiments . . . . . . . . . . . . . . . . . . . . . 24

3.3.1 General observations and cell morphology . . . . . . . . . . . . . . 24

3.3.2 Expression of ASMA and vimentin . . . . . . . . . . . . . . . . . . 24

4 Discussion 29

5 Conclusion 33

A Freezing, thawing and culture protocols 39

A.1 Cryopreservation of hMSC . . . . . . . . . . . . . . . . . . . . . . . . . . . 39

A.2 Thawing hMSC and culture initiation . . . . . . . . . . . . . . . . . . . . 39

A.3 Subculture of hMSC . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 40

B Markers 42

B.0.1 Endothelial cell markers . . . . . . . . . . . . . . . . . . . . . . . . 42

B.0.2 Vascular smooth muscle markers . . . . . . . . . . . . . . . . . . . 43

C Antibody titration 44

C.1 Introduction . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 44

C.2 CD31 / VE-cadherin antibody titration . . . . . . . . . . . . . . . . . . . 45

C.2.1 Results . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 45

C.3 ASMA / vimentin antibody titration . . . . . . . . . . . . . . . . . . . . . 46

D Coating procedures 48

D.1 Collagen type I coating . . . . . . . . . . . . . . . . . . . . . . . . . . . . 48

D.2 Fibronectin coating . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 49

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E Preparations for immunofluorescence microscopy 50

F Medium dynamic viscosity measurement 52

G Cone-plate system: pH and temperature measurement 54

G.1 Results . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 54

H Schematic Flexercell system 55

I Fluorescence activated cell sorting (FACS) 56

I.1 Concept . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 56

I.2 Cell preparations for FACS analysis . . . . . . . . . . . . . . . . . . . . . 57

I.3 Isotype FACS . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 58

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Chapter 1

Introduction

Tissue engineering of blood vessel substitutes has gained interest since the mid-eightiesof the past century. This was caused by the poor results which were achieved with theimplantation of synthetic ’static’ grafts. Small-caliber grafts (diameter < 5 mm) are typi-cally associated with high occurrence of thrombosis. Furthermore, these synthetic vesselslack the ability of growth and remodelling, therefore being unable to respond to the invivo hemodynamic environment. For these reasons scientists resorted to the constructionof biologically functional vessel substitutes from vascular cells which allow remodellingin vivo.23,27,33,41 In recent years, several investigators have started pursuing the useof stem cells as cell source for living vessel grafts, because these undifferentiated cellsallegedly can commit themselves to a wide spectrum of cell lineages, including vascularcells.15,21 A particular type of multipotent stem cell, the mesenchymal stem cell, seemsto have potential for application in clinical vessel engineering because it is constantlyand readily available in all humans. Furthermore it was shown that these cells depositan extracellular matrix with excellent mechanical properties for employment in vasculartissue engineering.20 Its multipotency has been proven by its ability to differentiate notonly into the ’nearest neighbours’ the connective-tissue cells, i.e. osteocytes, chondro-cytes and adipocytes, but for example also into cardiomyocytes, smooth muscle cells andendothelial cells.12 The question remains, how its lineage commitment to vascular celltypes can be controlled. In recent years several cytokines and growth factors have beenidentified which induce mesenchymal stem cells to differentiate into a specific (vascular)cell lineage.32,34,39 Interestingly, recent studies indicate that the differentiation of stemcells is related to cytoskeletal tension which is in turn determined by the mechanicalmicroenvironment.31,36 Considering the strong postnatal influence of the mechanicalenvironment on the functioning and remodelling of the vessel (see Part I) it might bequestioned if mechanical cues can influence the differentiation of adult mesenchymal stemcells within the vascular wall, for example for the purpose of remodelling or repair. Itis conceivable that pericytes in the adventitia can differentiate into smooth muscle cellsin response to typical local mechanical forces. For instance, differentiation into cartilagein response to compressive load has been shown.16 Furthermore it is known that mes-enchymal stem cells from bone-marrow circulate in the blood and nestle themselves invarious tissues.35 Most interestingly, these circulating mesenchymal stem cells migrateinto the subendothelial space and form endothelial cells, smooth muscle cells as well asfibroblasts.9,12 In this way these precursors contributed to the stabilization of myocar-

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dial infarction. The fluid flow patterns around such lesions and local forces in the vesselwall could accomplish this differential lineage commitment.The establishment of such in vivo mechanisms would have a major impact on vasculartissue engineering as well. The construction of a vessel graft incorporating endothelium,smooth muscle and fibroblasts by applying different mechanical stimuli to scaffold-seededmesenchymal stem cells seeded is of course an appealing idea. Such rather complicated(3D) tissue engineering approaches should however be preceded by in vitro studies whichinvestigate the feasibility of mechanically differentiating mesenchymal stem cells intovascular wall cells using relative simple and well-defined (2D) cell models.For these reasons and in order to contribute to the understanding of lineage commitmentof mesenchymal stem cells under influence of mechanical stimuli, the following researchquestion was formulated for the present study:

Can mechanical stimuli induce differentiation of mesenchymal stem cells into vascularwall cells?

Of particular interest are the typical mechanical forces experienced by the different arte-rial wall cells, as discussed in part I of this thesis. Therefore it is hypothesized that theapplication of fluid shear flow to mesenchymal stem cells results in their differentiationinto endothelial cells. Alternatively, it is hypothesized that the application of cyclic ten-sile strain causes mesenchymal stem cells to differentiate into smooth muscle cells. Thedifferentiation of mesenchymal stem cells into fibroblasts under influence of mechanicalstimuli is beyond the scope of this study.

In this study a well-defined commercial human mesenchymal stem cell line was used.It was first investigated if these cells were capable of endothelial differentiation and orga-nization into tube structures (vasculogenesis) in vitro under influence of an establishedpro-angiogenic cytokine vascular endothelial growth factor (VEGF) on both fibronectinand collagen type I culture substrates.In a second experiment it was investigated if endothelial differentiation of mesenchymalstem cells could be induced by applying a constant physiological level of shear stress (0.5Pa). Parameters of interest were the expression of the endothelial markers CD31 andVE-cadherin and cell morphology. A well-established in vitro system was used for theapplication of shear flow to cells cultured on either collagen type I and fibronectin.In the final experiment, the transformation of mesenchymal stem cells into vascularsmooth muscle cells under influence of physiological cyclic tensile strain was studied.Cells were cultured on collagen type I and strained using a well-defined commercialstraining system. Parameters of interest were the expression of the smooth muscle mark-ers α-smooth muscle actin (ASMA), vimentin and cell morphology.The experimental methods and techniques are described in chapter 2, whereas chapter3 presents the main findings. A general discussion (chapter 4) leads to the concludingchapter 5, in which also guidelines for future research are formulated.

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Chapter 2

Materials and Methods

This chapter describes the experimental methods and analysis techniques followed forthe angiogenesis experiment, the shear stress experiment and the equibiaxial strain ex-periment respectively.

2.1 Cells and culture media

In this study a commercial human mesenchymal stem cell line from Cambrex Bioscience(CB, De Petit-Rechain, Belgium) was used. These human mesenchymal stem cells (hM-SCs) were derived from a bone marrow biopsy of a 19 year old female human donorand frozen in passage 2. For passage 2 hMSCs the manufacturer guaranteed chondro-genic, osteogenic and adipogenic differentiation capacities. Furthermore the expression ofCD105 (endothelial marker), CD166 (monocyte marker), CD29 (beta-1-integrin), CD44(lymphocyte adhesion molecule) had been evidenced by flow cytometry. Hematopoieticmarkers CD14, CD34 and CD45 were not expressed.After population expansion and preliminary experiments, cells were refrozen in passage7 (see appendix A), such that passage 8 hMSC were used in the experiments describedhere.hMSCs were cultured in Mesenchymal Stem Cell Basal medium supplemented with 10%Mesenchymal Stem Cell Growth Supplement (serum) and aliquots of L-Glutamine andpenicillin-streptomycin solution (all included in BulletKit PT-3001, CB). Initial seedingdensity was 5 · 103 cells·cm−2 in all experiments.

2.2 Phenotype identification

2.2.1 Cellular proteins

Each cell type is characterized by an unique set of membrane and/or intracellular pro-teins, commonly referred to as cell markers. These cell markers can be ligated with fluo-rescently labelled antibodies which can be subsequently detected by immunofluorescence

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Table 2.1: Antibody properties

Primary antibody Secondary antibodyMarker Immunotype Dye Info Immunotype Dye InfoVE-cadherin IgG1, κ - BD, 555661 IgG1 FITC BD, 553443CD31 IgG1, κ PE BD, 555446 - - -ASMA IgG2a Cy3 SG, C6198 - - -Vimentin IgG1 - BD, 550513 IgG1 FITC BD, 553443isotype IgG1 FITC BD, 550616 - - -isotype IgG1 PE BD, 550617 - - -

microscopy or flow cytometry. For the identification of endothelial cells two junctionalproteins were chosen, being platelet endothelial cell adhesion molecule-1 (PECAM-1 orCD31) and vascular endothelial cadherin (VE-cadherin or CD144).Vascular smooth muscle cells were identified by expression of the cytoskeletal proteinvimentin and α-smooth muscle actin (ASMA), a protein belonging to cellular contractilemachinery. Cell morphology was used as supplementary characteristic of phenotype forboth endothelial and vascular smooth muscle cells. Appendix B provides backgroundinformation on all proteins of interest.

2.2.2 Antibodies

For immunofluorescent labelling both directly conjugated monoclonal mouse anti-humanantibodies were used as well as combinations of monoclonal primary mouse anti-humanantibodies probed with labelled secondary rat anti-mouse antibodies. Optimal antibodydilutions were determined in separate experiments, which are described in appendix C.A general background on antibodies and their application is provided here as well.CD31 was probed with R-Phycoerythrin (R-PE)-conjugated (IgG1, κ) antibody (1:200dilution, no. 555446, Becton Dickinson (BD), Alphen a/d Rijn, Netherlands). A primaryIgG1, κ antibody (1:200 dil., no. 555661, BD) was used for identifying VE-cadherin.For secondary labelling fluorescein isothiocyanate (FITC)-conjugated IgG1 monoclonal(1:100, no. 553443, BD) was chosen. ASMA was recognized by Cy3-conjugated IgG2a

antibody (1:250, C6198, Sigma-Aldrich (SG), Zwijndrecht, The Netherlands). PrimaryIgG1 antibody (1:250, no. 550513, BD) was used against vimentin and subsequentlyprobed with FITC-conjugated IgG1 antibody (1:100, no. 550513).Isotype control antibodies were a IgG1 PE-conjugated (no. 550617, BD) and IgG1 FITC-conjugated antibody (no. 550616, BD), which were used at the same dilution as therespective specific primary antibodies. An IgG2a Cy3 isotype control was not available.Table 2.1 summarizes the properties of all used antibodies.

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2.3 Assessment of the in vitro angiogenic potency of hMSC

It was investigated if the hMSC provided by Cambrex were capable of endothelial dif-ferentiation and organization into tube structures in vitro. It is known that during ves-sel formation (vasculogenesis) in the embryo, mesenchymal stem cells differentiate intoendothelial cells and form endothelial tubes from which later on mature vessels arise.Vascular endothelial growth factor (VEGF) is an important stimulator of this process(see literature review, chapter 2). The angiogenic potency of hMSCs cultured on bothcollagen type I and fibronectin was evaluated.Passage 8 hMSC were seeded on both fibronectin-coated and collagen-I-coated (both 5µg·cm−2) culture dishes and cultured in standard medium supplemented with 50 ng·ml−1

VEGF. The coating procedure is described in appendix D. As negative control serveda culture without VEGF addition. To find out if the action of VEGF was affected bythe presence of serum, a culture in VEGF-supplemented serum-free medium was set up.Furthermore, it was determined if the absence of serum affected cell viability by includ-ing a culture in serum-free medium without VEGF addition.All media were changed on day 4, while fresh VEGF was added on day 0, 3, 4, 5 and6. On day 7, cultures were morphologically studied by microscopy, whereupon cells werewashed, fixed and stained for VE-cadherin and CD31 according to the protocol in ap-pendix E. Finally, fluorescence data was collected using confocal microscope (see section1.6.2). To enable evaluation of this data, endothelial expression levels of VE-cadherinand CD31 were determined in a separate experiment (see appendix C).

2.4 Shear stress experiments

2.4.1 Cone and plate system

For the application of shear stress to hMSCs cultured in monolayer, a cone and platesystem was used. This device is commonly used for studying the response of cells tofluid shear flow.4–7,10,13,14,28,29 The principle of this loading apparatus originates fromthe rheometer, a device which is used to determine fluid viscosity. In typical viscositymeasurements, a sample of fluid is loaded between to closely separated circular horizontalplates. During rotation of the bottom plate viscous forces in the sample exert torque onthe upper plate which is translated into the dynamic viscosity of the sample.Such a device can however also be used for shearing a sample and the underlying surface.In this case the bottom plate remains static, while the upper plate rotates and exertsshear load on the sample. In this situation (uniform gap height) the imposed shear stressdepends linearly on the radial position from the center of the plate, while circumferentialplate velocity does as well. By using a cone as top geometry the linear increase in gapheight compensates for the increase in plate velocity from center to edge. For this reasonshear rate is uniform over the gap as is consequently the case for shear stress.

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Figure 2.1: Right: Cross-section of cone-plate system fitted on 6-wells culture multidish.Shown are a cone geometry positioned above well bottom and Transwell insertrespectively. Left: Close-up of gap. ε = gap error, α = cone angle, r = radialposition from center.

Figure 2.2: Cone and plate system built by Instrument Development Engineering & Evaluation,Maastricht University.

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This is however only valid under the following assumptions:

• The sample is a Newtonian incompressible fluid.

• R = r2ωα2/12ν << 1, such that fluid inertia is negligible. The parameter Ris the cone-and-plate analogy of the Reynolds number and measures the ratio ofcentrifugal and viscous forces acting on the fluid.7 ω denotes angular velocity, νdenotes kinematic viscosity (=dynamic viscosity/density (η/ρ)).

• Cone angle (α in figure 2.1) < 0.1 rad.

• No slip conditions on the cone and bottom surface.

In the present study a truncated cone geometry was used, which means that the tipof the cone is flattened (see figure 2.1). This is commonly done for practical reasons.First, it is impossible to make a ’perfect’ tip out of polycarbonate on a lathe for reasonsof precision. Secondly, the absence of direct contact between cone and bottom plateprevents the wear of both and destruction of the cell monolayer. It can be derived thatin this case for the shear stress τ in the gap holds:

τ = ηΩr

ε + αr(2.1)

Where:η = 7.7 · 10−4 Pa.s (dynamic viscosity) (for shear rate 464 - 1000 s−1, determined bymeasurement, see appendix F)ω = angular velocity of cone surface [rad·s−1]r = radial position [m] (see figure 2.1)ε = gap error, i.e. height of virtual tip above plate [m]α = 0.029 rad

If the gap error ε is set to zero, i.e. the virtual cone tip is positioned exactly at thebottom plate, then equation (2.1) reduces to:

τ = ηΩα

(2.2)

In this case shear stress is uniform throughout the gap.For the present study a new cone and plate system was designed and constructed byInstrument Development Engineering & Evaluation (ID, Maastricht University). Thissystem could simultaneously load all wells of a 6-wells plate (Nunctm (see figure 2.2).Polycarbonate cone geometries (d=20 mm) were fixed into teflon-lubricated bearingsand secured on the upper side after accurate height adjustment. A Maxon DC motor(Maxon Motor Benelux B.V., Enschede, The Netherlands) was used to drive the conesvia a transmission belt. Rotational velocities of motor and cones was identical. A controlsystem maintained a steady rotational speed.In preliminary experiments it was determined that culture medium pH and temperaturewere not affected by cone rotation (see appendix G).

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Figure 2.3: Culturing of hMSCs on Transwell membranes.

2.4.2 Cell seeding

Passage 8 hMSCs were seeded either in either collagen-I-coated 6-wells plates or onfibronectin-coated Transwell membranes (Costar, Badhoevedorp, The Netherlands) asshown in figure 2.3. Coating was performed according to the protocol mentioned earlier(see appendix D). The use of Transwells was based on the experience, that sometimes,by piercing the well bottom, cones damaged themselves as well as cell culture. Thesoft flexible Transwell membranes, intended to be used for cocultures, prevented at leastdamage to the cone geometry.Prior to seeding cells Transwells were glued to the well edges using Loctite primaire770 and Loctite adhesif instantane 406 (Loctite, France). Evaporation of toxic gluecomponents was allowed for several days after fixation. Per well 1.5 ml of medium wasadded, either on the well bottom or on top of the membrane. In addition, the spaceunder the Transwell membrane was filled with 2.6 ml of medium.The experiment on collagen type I included 3 dynamic cultures, 3 static cultures, 1positive control and 1 isotype control culture. The fibronectin experiment included 2dynamic cultures, 2 static cultures, 1 positive control culture and 1 isotype control.Both positive control cultures were supplemented with VEGF (50 ng·ml−1) on days 0and 3-6. A medium change was performed on day 4 of culture.

2.4.3 Shear stress regime

The shear stress regime was started on day 7 of culture. Within a LAF cabinet the conegeometries were carefully fitted on the wells, with maximal separation from the cells.Thereafter, the gap between cone surface and cell layer was reduced to 50 µm on confo-cal microscope. The cone geometry was winded over its thread, while the cone/mediumand medium/bottom interfaces were monitored. This meant that gap error ε equalledzero and that the virtual cone tip was positioned exactly on top of the cells.The complete setup was placed water-level within an incubator (37°C, 5% CO2) togetherwith the static control cultures. Cone speed was gradually increased to 184 RPM, equiv-alent to a shear stress of 0.5 Pa. After 24 hours the motor was carefully stopped and thesystem was removed from the well plates within a LAF cabinet.Cells were then washed, fixed and subsequently stained for CD31 and VE-cadherin ac-

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Figure 2.4: Concept of the Flexercell system. Left: Culture plates are placed on a base platefitted with loading posts, which connects to the vacuum system. The system issealed against leaking by gaskets around the culture plates. Right: (Cross-section)Silicone membranes are stretched over a lubricated (silicone gel) circular loadingpost by the application of vacuum. Attached cells situated above the loading postat peak strain are subjected to an equibiaxial strain field.

cording to the protocol in appendix E. Fluorescent images of the collagen-I cultures werecollected using a fluorescence microscope (see section 2.6.1). Fluorescence data from thefibronectin experiment on the other hand, were collected by confocal microscopy (seesection 2.6.2).

2.5 Cyclic equibiaxial strain experiments

The experiment described in this section was performed twice using respectively im-munofluorescence microscopy and flow cytometry for analysis.

2.5.1 Flexercell system

For the purpose of applying well-defined cyclic equibiaxial strain to hMSC the commercialFX-4000Ttm system (Flexcell International Corp., Hillsborough, NC, USA) was used(see appendix H for a schematic). This system uses a vacuum pump to create sub-atmospheric pressure beneath flexible silicone culture membranes. As a consequencethe membranes stretch over a lubricated loading post (see figure 2.4). This createsan equibiaxial membrane strain in the area situated above the loading post at peakstrain, which consequently is felt by attached cells as well. The characteristic of anequibiaxial strain is that radial strains equals circumferential strain. This has beenexperimentally and numerically validated for the membranes used with this sytem.19

The time-dependent character of the pressure wave imposed by the vacuum system canbe defined via a dedicated software package. This software incorporates calibrationdata of pressure against strain magnitude for several types of loading posts and culturemembranes.

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Figure 2.5: Triangular straining regime, 1 Hz, peak strain: 10%.

2.5.2 Cell seeding and straining

Bioflex 6-wells dishes with collagen type I coated culture membranes were obtained fromDunn Labortechnik (Asbach, Germany). hMSC were seeded in passage 8 or 11 (at adensity of 5·103 cells.cm−2 in 3 ml culture medium per well. Cultures were incubatedat 37°C / 5% CO2. Medium was changed on day 4. A positive control was created bysupplementing cells with 10 ng·ml−1 PDGF-B on days 0, 3-6 to stimulate ASMA andvimentin expression (n=1). On day 7 the well bottoms of the static cultures (n=5) andthe positive control cultures were sealed with a plug such that the membrane did notexperience the pressure waves imposed by the vacuum system. Dynamic cultures (n=6)were exposed to a 1 Hz triangular-shaped pressure wave with a peak strain of 10% (seefigure 2.5) for 24 hours. Cells were then prepared for fluorescence analysis and collectionof fluorescence intensity profiles on confocal microscope according to the protocol inappendix E. Alternatively, in the experiment involving flow cytometric analysis, a staticand dynamic population were made by pooling 4 static and 4 dynamic cultures, whichwere then prepared for FACS analysis (see section 2.6.3 and appendix I). The remaining2 static and 2 dynamic cultures were additionally cultured statically for 48 hours priorto treatment and analysis.

2.6 Data acquisition and analysis

2.6.1 Fluorescence microscopy

Fluorescent images were collected on a Zeiss Axiovert 200M fluorescence microscope. Theavailable filter sets did not allow separate detection of FITC and PE. Both fluorophoreswere excited in the range 450-490 (bandpass filter) which includes their excitation peak of488 nm. Fluorescence was collected above 515 nm using a long pass filter. The emission

16

Table 2.2: Filter settings for confocal immunofluorescence microscopy

Fluorophore Excitation wavelength Emission filterFITC 488 nm 505-530 BPPE 488 nm 585 LPCy3 543 nm 560-615 BP

Figure 2.6: Collecting cell fluorescence intensity profiles of cells. The black arrow representsa typical curve along which fluorescence intensities were collected and averaged.Typical CD31 and VE-cadherin profiles of an endothelial cell are shown at the leftof the figure.

signal consequently is a combination of both the FITC emission spectrum (typically480-600 nm) and the PE emission spectrum (typically 540-650 nm). This means thatexpression of CD31 and VE-cadherin could not be independently monitored.

2.6.2 Laser scanning confocal microscopy

Sample slides were visualized on a Zeiss Axiovert 100 M laser scanning confocal micro-scope using a 10X objective (NA = 0.3). Table 2.2 summarizes the used excitation andemission settings for all fluorophores used in this study. Note that on confocal micro-scope FITC was always used in combination with either PE or Cy3, such that overlapof emission spectra was minimal. Fluorescence intensity profiles of individual cells werecollected as follows. Manually, a line was drawn which spanned the cell area in thelongest possible way. This procedure is elucidated in figure 2.6. Fluorescence intensitiesfor each dye were read at each pixel on this line and averaged.

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2.6.3 Flow cytometric analysis

Cells were analyzed using a fluorescent activated cells sorter (FACS). A general intro-duction to this technique is provided in appendix I. Briefly, cells were washed in PBSand fixed in 4% paraformaldehyde for 20 minutes at room temperature. Thereafter,cells were permeabilized in 0.1% Triton X-100 for 5 minutes at room temperature. Cellswere incubated with antibodies for vimentin (prim. 1:250, sec. 1:100), ASMA (1:150)or isotype control for 30 minutes on ice per step. An isotype control for Cy3 was notavailable.Samples were centrifuged and resuspended in FACS buffer (0.5% BSA, 0.05% Sodiumazide in PBS). Subsequently, analysis was performed on a FACS Vantage system (BD)using CellQuest Pro analysis software (BD). Excitation was set at 488 nm (Argon laser),while fluorescence was collected using a 515-545 nm bandpass filter for the FITC signaland a 543-627 nm bandpass filter for the Cy3 signal. Forward scatter (indicative of cellsize) and side scatter (indicative of cell granularity) signals were collected as well. Com-pensation, i.e. correction for the overlap of emission spectra resulting in spill fluorescencein both channels, was set based on PE-labelled and FITC-labelled uniform beads. A pos-itive control for Cy3 was not available. The Cy3 and PE fluorescence emission spectraare however similar such that FITC-PE spill approximately equals FITC-Cy3 spill andvice versa. Compensation set on the basis of PE and FITC beads should consequentlybe correct. Compensation settings were as follows: FL1(FITC)-%FL2(Cy3) = 1.6% andFL2-%FL1 = 25.6% at 639 V for FL1 and 601 V for FL2.

2.7 Statistical analysis

All numerical results are presented as mean ± S.D.. Statistical analysis was not per-formed, while statistical tests lack power because of small sample sizes. For this reasonthere was no added value in using them.

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Chapter 3

Results

This chapter is divided into three sections, which present the respective results of theangiogenic experiment, the shear stress experiment and cyclic strain experiments.

3.1 Assessment of the in vitro angiogenic potency of hMSC

3.1.1 Fibronectin substrate

VEGF/serum cultures appeared morphologically different from no-VEGF/serum cul-tures. In addition to the typical fibroblastic shaped cells, also spindle-shaped, oval andcircular cells were present (figure 3.1 (a)). From the fluorescence data it follows thatthe mean VEGF/serum intensities for VE-cadherin and CD31 exceed those from no-VEGF/serum cultures. Together these observations suggest that hMSC are transformingtowards an endothelial phenotype.VEGF/no-serum cultures consisted primarily of elliptical and circular cells. Most inter-estingly, cells had organized in clusters and arrays (figure 3.1 (b)). The accompanyingfluorescence data (figure 3.2 (a)) learned that mean VE-cadherin and CD31 intensitiesexceed the defined threshold for endothelial differentiation (see appendix C). These ob-servations indicate that the majority of cells had an endothelial phenotype and that thesecells participated in vasculogenesis, i.e. the formation of vascular patterns by (precursorsof) endothelial cells.Finally, it appeared that no-VEGF/no-serum cultures resembled no-VEGF/serum cul-tures with respect to morphology and marker expression levels (not shown). There wereno signs of (excessive) cell necrosis. Cell viability therefore seemed not to be affected bythe absence of serum.

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(a) VEGF/serum, fibronectin (b) VEGF/no-serum, fibronectin

Figure 3.1: Appearance of VEGF/serum and VEGF/no-serum cultures on day 7.

(a) n=5 for all groups (b) from left to right: n=4, n=2, n=4

Figure 3.2: Mean fluorescence data for angiogenic experiment on both fibronectin and collagentype I. n represents number of cells included.

20

3.1.2 Collagen type I substrate

It appeared that after preparation few cells were left in all cultures. For this reason typ-ical morphological cell characteristics could not be determined. Marker expression wasnevertheless quantified for the cells present as can be seen in figure 3.2 (b). CD31 ex-pression by cells from VEGF-serum and VEGF-noserum cultures was relatively strongerthan for cells from no-VEGF-serum cultures. This was also valid for VE-cadherin ex-pression, although the differences were subtle. None of the experimental groups reachedthe threshold for endothelial differentiation. This indicates that the angiogenic potentialof hMSCs cultured on collagen type I is limited compared to fibronectin.

3.2 Shear stress experiments

3.2.1 Collagen type I substrate

All cultures had low till moderate confluence (no numerical data available) at the timeof shearing (day 7). On day 8 statically cultured cells generally had a fibroblastic mor-phology typical for cultured hMSCs (figure 3.3 (a)). Fluorescence microscopy revealedthe absence of endothelial markers as follows from figure 3.3 (b).VEGF-supplemented cells morphologically resembled the VEGF/serum cultures on fi-bronectin and collagen type I (see previous section), with spindle-shaped, oval and cir-cular cells present (figure 3.3 (c)). The expression of endothelial markers was evidencedby fluorescence microscopy (figure 3.3 (d)).Cells from dynamic culture (n=2) were typically elongated, with a cigar-like appearance(figure 3.3 (e)). Cell clusters were present as well. Marker fluorescence intensities weresimilar to the positive control and higher than the static culture (figure 3.3 (f)). Inter-estingly, brightest fluorescence was found in cell clusters.These observations suggest that the application of shear stress at a magnitude of 0.5 Pa tohMSCs cultured on collagen type I resulted in endothelial differentiation. Furthermore,the observed cell clusters might be a sign of vascular patterning.

21

(a) static, phase-contrast (b) static, CD31

(c) positive, phase-contrast (d) positive, CD31

(e) dynamic, phase-contrast (f) dynamic, CD31

Figure 3.3: Results shear stress experiment, collagen type I. Field of view for phase-contrastand fluorescent images is the same.

22

3.2.2 Fibronectin substrate

Cultures on fibronectin were near confluence at the time of shearing. On day 8, dynam-ically cultured cells had a fibroblastic morphology typical for statically cultured cells(compare figures 3.4 (a)/(b)). An inspection of figure 3.5 learns that there were no re-markable differences between static cultured and dynamically cultured cells with respectto the cellular expression levels of VE-cadherin and CD31.These results thus suggest that the application of 0.5 Pa shear stress did not induceendothelial differentiation and vascular patterning of hMSCs cultured on fibronectin.

(a) static, fibronectin (b) dynamic, fibronectin

Figure 3.4

Figure 3.5: Mean fluorescence intensities for VE-cadherin and CD31 of 40 cells of static anddynamic cultures, fibronectin.)

23

3.3 Cyclic equibiaxial strain experiments

3.3.1 General observations and cell morphology

At the end of the strain regime both static and dynamic cultures had similar conflu-encies (assessed qualitatively). Furthermore they both incorporated cells with a typicalfibroblastic shape as well as spindle-shaped cells. Dynamically cultured cells generallyhad a granular appearance whereas statically cultured cells were more or less transpar-ent (compare figure 3.6 (a)/(c)). This observation corresponds with the outcome of flowcytometric analysis. A comparison of figure 3.6 (b) and figure 3.6 (d) learns that the ma-jority (90%) of statically cultured cells had a low side-scatter intensity, i.e. light is onlyweakly scattered while passing through the cells. On the other hand, 53% of the dynam-ically cultured cells had a high side scatter compared to statically cultured cells, whichindicates that the cytoplasmic structure is more complex. Interestingly, these differentialside-scatter intensities were also typical for cultures which were statically cultured for 48hours after straining was stopped.

3.3.2 Expression of ASMA and vimentin

It appeared that after 8 days of static culture cells weakly expressed both ASMA andvimentin (figure 3.7 (a)) compared to the isotype control culture (not shown). This alsofollows from the fluorescence data presented in figure 3.8. Sparsely, relative large cellswith randomly orientated actin stress fibers were found (indicated by white arrows).These cells had diffuse weak vimentin expression as well.For FACS analysis cell debris was excluded by gating based on isotype control fluores-cence dot plots (see appendix I). Based on the fluorescence dot plot of the dynamicpopulation discussed later (see figure 3.7 (d)) a double positive region (cluster to theright of figure) and double negative region (lower left quadrant) were defined. Eventsoccurring in these regions were included in numerical analysis. It appeared that 90%of statically cultured cells did not express ASMA or vimentin whereas 10% exhibited arelative strong expression of ASMA of vimentin (scattered data points on the right sideof figure 3.7 (b).Immunofluorescence images and fluorescent dot plots from dynamically cultures areshown in figures 3.7 (a) and 3.7 (c) respectively. It appeared that dynamically culturedcells generally strongly expressed vimentin in the perinuclear region and weaker towardsto cell edges. Furthermore, ASMA expression in the perinuclear (central) region wasstrong compared to statically cultured cells. With respect to the cellular distributionsof these proteins cells resemble contractile vascular smooth muscle cells in culture.42

It could not be observed if proteins were organized into filaments or stress fibers. Large,stress fiber incorporating cells were found with a similar frequency as in static cul-tures. Fluorescence data from confocal microscopy also shows stronger expression ofboth ASMA and vimentin by dynamically cultured cells compared to statically culturedcells. The positive control resembled static culture morphologically (not shown) as wellas with respect to ASMA and vimentin expression (see figure 3.8).FACS analysis revealed the presence of a double-positive cell population in dynamic cul-ture (cluster to the right of figure 3.7 (d) ASMA and vimentin fluorescence intensities of

24

similar magnitude. This population constituted 53% of the total cell number, whereas46% of the total population did express neither ASMA nor vimentin.It appeared that cultures which were cultured statically for 48 hours after being strainedyielded similar results. This suggests together with the maintained granularity that theapplication of cyclic strain induced a definitive change of cellular phenotype.

25

(a) (b)

(c) (d)

Figure 3.6: Left: Phase contrast images of statically (a) and dynamically cultured (c) passage8 hMSC. Right: side-scatter (SSC) versus FITC fluorescence (vimentin) data fromFACS analysis for a similar experiment using passage 11 hMSC: (b) static culture,mainly cells with low cytoplasmic granularity (d) dynamic culture, two clusterswith a low and high cytoplasmic granularity respectively.

26

Figure 3.7: Mean fluorescence intensities of 10 cells for ASMA and vimentin for isotype control,static culture, dynamic culture and positive (PDGF-B) control.

27

(a) (b)

(c) (d)

Figure 3.8: Left: Expression of ASMA and vimentin by static (a) and dynamic (c) cultures.green = ASMA, red = vimentin, orange = colocalisation of vimentin and ASMA.Right: Cy3 (ASMA, FL2 channel) fluorescence versus FITC (vimentin, FL1 chan-nel) fluorescence data from flow cytometry analysis for a similar experiment usingpassage 11 hMSC. (b) Statically cultured cells are primarily (90%) in the lower leftquadrant (double negative). (d) A substantial percentage (53%) of the dynamicallycultured cells is in the upper right quadrant (double positive).

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Chapter 4

Discussion

The results from the angiogenesis experiment demonstrated that human mesenchymalstem cells can differentiate into cells with an endothelial phenotype and form vascularpatterns in vitro. The in vitro transformation of hMSCs into endothelial cells underinfluence of VEGF was recently reported by Oswald et al. as well.34 They also foundthat the differentiated cells were able of forming vascular patterns. Together these find-ings justify a study focussed on endothelial differentiation of hMSCs under influence ofshear stress. Namely, the occurrence of angiogenesis can only be attributed to the ex-pression of VEGF-receptors Flt-1 and Flk-1 by hMSCs (see Part I). When VEGF binds,the receptor is tyrosine phosphorylated which triggers the associated signalling pathwaysthat accomplish endothelial differentiation. It has been reported that shear stress caninduce tyrosine phosphorylation of the Flk-1 receptor.22,43 Consequently, investigationof the applicability of shear stress for induction of endothelial differentiation of hMSCsis reasonable.Nonetheless, some remarks regarding the results from the angiogenesis experiment canbe made. One might argue that the observed cell cluster formation was due to cellnecrosis caused by the absence of serum. In that case non-specific antibody bindingand thus fluorescence can substantially increase. Reduced viability of fibroblastic cellsis furthermore accompanied by diminished cell spread, resulting in elliptically shapedcells.40 These observations may together lead to false identification of endothelial cells.This explanation however is extremely unlikely as cell viability was maintained underno-VEGF/no-serum conditions. Hence, antibody binding was primarily specific, whichmeans that angiogenesis was taking place.It appeared as well that the angiogenic potency of hMSCs cultured on collagen type I waslimited. The VEGF-supplemented control culture from the shear stress experiment how-ever showed that VE-cadherin and CD31 expression were substantial even in the presenceof serum. It is conceivable that the low cell density in this particular experiment pre-cluded an angiogenic response. The limited angiogenic response of hMSCs cultured oncollagen-I is unexpected in the light of the findings of Qian et al.24 They demonstratedthat hMSCs cultured on both fibronectin and collagen-I and polystyrene have identicalphenotypes and expansion efficiencies. Additionally it was found in the present studythat human coronary artery endothelial cells (hCAECs) grow well on collagen-I. Forthese reasons, collagen-I and fibronectin are supposed to have similar (pro-)angiogeniccapacities.

29

Finally, the limited effect of VEGF in the presence of serum can be explained by the pres-ence of VEGF-antagonists or general angiogenesis inhibitors in the serum. The eventualeffect of VEGF will be limited in this case, certainly since VEGF has a typical half-lifeof 30 minutes in culture medium.

The shear stress experiments on collagen-I clearly indicated that hMSCs can differen-tiate into endothelium in response to appropriate mechanical cues. The expression ofthe specific endothelial markers CD31 and VE-cadherin, as well as the morphologicalcharateristics support this phenotypic qualification.This is an interesting finding, which to the best of my knowledge has never been reportedbefore. Morphological characteristics generally suited an endothelial phenotype but cannot be put forward as strong evidence for endothelial differentiation. Interestingly, inthe cultures subjected to shear stress cell clustering was observed. This could be a signof vascular patterning.The shear stress experiment with fibronectin did not reveal any changes in marker expres-sion levels and cell morphology. Surprisingly, it appeared that VE-cadherin expressionof statically cultured cells was at endothelial level. Based on the earlier observation thatCD31 and VE-cadherin expression were coupled in the angiogenesis experiment and thefact that cell shape was fibroblastic, this apparent basal expression of a typical endothe-lial marker can not have biological relevance. It should be attributed to fluorescence ofthe Transwell culture membrane in the range of phycoerythrin (PE). This explanationis supported by the fact that this basal expression was not observed in the angiogenicexperiment involving cells of equal passage seeded on the bottom of 6-wells plates.Shear stress data are preliminary with few observations and should therefore not be over-interpreted. However, the possibility that endothelial differentiation of MSC depends onvery specific combinations of mechanical stimuli and coatings warrants detailed study,as cytoskeletal tension seems to be an important regulator.31,36 It is interesting to relatethe findings of the present study to those from a recent study by Yamamoto et al.43

They investigated the effect of fluid shear flow on the expression of endothelial markersand the formation of vascular patterns by endothelial progenitor cells (EPCs), i.e. stemcells which have gained a partial endothelial phenotype and are supposed to originatefrom the hematopoietic stem cell compartment. It appeared that shear stress upregu-lated VE-cadherin expression and enhanced tube formation by EPCs. If further researchvalidates the findings from the present study, it might eventually be established that(precursors of) endothelial cells derive from mesenchymal stem cells as well.

The application of cyclic equibiaxial strain to hMSCs resulted in increased vimentin andASMA expression, which was most pronounced in the perinuclear region. These cellsmodulate between a synthetic (matrix-deposition/proliferation) state and a contractilestate both in vivo and in vitro that are both associated with typical protein expressionprofiles and organizations.38 The organization of ASMA and vimentin observed in thepresent study corresponds well with the state of these proteins in contractile vascularsmooth muscle cells. Vimentin is typically found in a dense network in the perinuclearregion. ASMA is present in stress fibers which are thickest in de perinuclear region.42 Forthe present study it is a question whether these proteins were present in monomeric formor incorporated in a network or stress fibers respectively. A vimentin mesh could not beobserved at the magnification used for fluorescent imaging. It is known that the majorpart of cellular vimentin of mesenchymal cells is in polymerized state, incorporated into

30

intermediate filaments.1 This notion is supported by the side-scatter data from FACSanalysis. The relative high side scatter of a subpopulation of dynamically cultured cellscorresponds with a spatial complex structure of the cytoplasm, thus likely representedby a vimentin network.Such an explanation does not hold for ASMA. At the used magnification stress fibersconsisting of polymerized actin were discernable in some cells of both static and dynamiccultures. The increased ASMA expression of dynamically cultured cells was however notaccompanied by a visible increase of the number of actin stress fibers. Therefore, it ismust be concluded that the application of cyclic strain resulted in an increase of thecellular amount of unpolymerized ASMA. Interestingly, actin stress fibers are typicallygenerated in alignment with the direction of principal strain to provide cell stiffness. Apreferential direction of strain is by definition not present in an equibiaxial strain field.For this reason it would be interesting to investigate if ASMA organizes into stress fibersin response to an uniaxial strain field.It appeared from the positive control that the PDGF-B did not affect the expressionof ASMA and vimentin. PDGF-B was chosen based on reports that it triggers smoothmuscle differentiation of mesenchymal precursors.8,32 The effect of mechanical strain onASMA and vimentin expression was however evident such that retrospectively a positivecontrol was not needed.The results from FACS analysis require some discussion as two experimental artifactsmight be falsely attributed biological relevance. First, it appeared that dynamic cultureincorporates a small population of vimentin-positive/ASMA-negative cells. It should benoted that Cy3 (ASMA) excitation at 488 nm is not as efficient as it is at 543 nm. Withoptimal excitation the double positive cell cluster would move up, such that the apparentvimentin-positive/ASMA-negative cells fall nicely into the upper right quadrant.Secondly, the double negative population found in dynamic culture did not representunresponsive cells but cells that were not subjected to the loading regime. Rememberthat the part of the membrane positioned above the loading post at maximal strain ex-perienced the uniform equibiaxial strain field. The remainder of the cells (approximately50% under the current conditions) experienced static conditions and were therefore ex-pected to be double negative. This corresponds with the observation that about 50% ofthe cells fell in the double-negative quadrant.

It is interesting to relate the results of the present study to data from literature. Galmicheet al. showed that marrow stromal cells in long-term culture (up to 7 weeks) expressedASMA and that this expression increased with the number of adherent interdigitatedcells.30 Additionally, confluent cultures also expressed desmin, calponin, caldesmon andmyosin light chain kinase, which indicates that adherent marrow stromal cells in long-term culture adopt a phenotype similar to that of vascular smooth muscle cells. Anexplanation for this phenomenon has however never been given. An interesting theorycan however be established based on the findings of the present study. It has been re-ported that contractility of both mesenchymal stem cells and fibroblasts relates directlyto the expression of ASMA.2,3 It is known that fibroblasts in culture produce strong con-tractile forces which enable deformation of silicon culture substrates.2 Newly initiatedmarrow stromal cultures typically contain up to 1% of ASMA-positive fibroblastic cells.30

Contractile forces generated by these fibroblastic cells might be expected to strain inter-digitated mesenchymal stem cells. Extrapolating the findings of the present study MSCthen respond by increasing ASMA expression which consequently results in increased

31

contractility. As the number of interdigitated cells increases because of mitosis the num-ber of contractile ASMA-expressing cells increases as well if the proposed mechanismprevails. The theory thus states that tensile strain is responsible for the transformationof mesenchymal stem cells into vascular smooth muscle cells in long-term culture. Itshould however be validated that desmin, calponin, caldesmon and myosin light chainkinase respond to tensile strain as well.

32

Chapter 5

Conclusion

In conclusion, the preliminary data presented in this study support the hypothesis thatdifferentiation of mesenchymal stem cells can be directed by mechanical stimuli. The ex-pression of endothelial markers observed in the shear stress experiment involving collagentype I support the hypothesis that fluid shear flow induces endothelial differentiation ofhuman mesenchymal stem cells. Alternatively, the hypothesis that cyclic tensile straininduces differentiation of human mesenchymal stem cells into vascular smooth musclecells is supported by the upregulation of ASMA and vimentin and their cellular distri-butions as observed in the cyclic strain experiment.However, more important than answering this question is defining the added value ofthe present study for vascular tissue engineering. With respect to this the most im-portant conclusion that can be extracted from the findings of this study is that bothshear stress and tensile strain modulate the expression of cell markers which are essen-tial for the fulfillment of the in vivo function of respectively endothelial and smoothmuscle cells. Namely, VE-cadherin and CD31 are essential proteins for the regulationof vascular permeability, whereas α-smooth muscle actin is a major component of thecontractile apparatus of smooth muscle cells. This is an important finding while, in vivofunctionality is important for clinical application of tissue-engineered vascular constructs.Nevertheless, further research using a monolayer cell model is needed before the findingsof the present study can be translated into differentiation protocols usable in the 3Denvironment of a cell-seeded scaffold.

Future studies

I recommend to repeat the shear stress experiments and elucidate the role of cultureconfluence at the time of shear stress initiation on the eventual angiogenic response.Additionally, I advise to investigate the effect of a physiologically more realistic time-dependent cyclic shearing regime.In order to confirm the vascular smooth muscle phenotype, it is recommended to includeadditional cell markers. Suggested are calponin, caldesmon and myosin light chain ki-nase. Furthermore, the relation between protein organization and the nature of the strainfield deserves study. With respect to this, the response to an uniaxial cyclic straining

33

regime could be studied, as this is a better representation of the in vivo mechanical loadon the medial layer. With the excellent matrix generating properties of mesenchymalstem cells in mind, it is likely that tissue engineers eventually will be able to construct bi-ologically functional blood vessel substitutes by mechanically conditioning mesenchymalstem cells.

34

Bibliography

[1] Alberts, B., Bray, D., Lewis, J., Raff, M., Roberts, K., Watson, J., Molecular biologyof the cell, Garland Publishing Inc., 1994

[2] B. Hinz, G. Celetta, J. T. G. G. C. C., Alpha-Smooth Muscle Actin Expression Up-regulates Fibroblast Contractile Activity, Molecular Biology of the Cell, 12(9):2730–2741, 2001

[3] B. Kinner, J.M. Zaleskas, M. S., Regulation of Smooth Muscle Actin Expression andContraction in Adult Human Mesenchymal Stem Cells, Experimental Cell Research,278(1):72–83, 2002

[4] Blackman, B., Barbee, K., Thibault, L., In Vitro Cells Shearing Device to Investigatethe Dynamic Response of Cells in a Controlled Hydrodynamic Environment, Annalsof Biomedical Engineering, 28(4):363–372, 2000

[5] Bronneberg, D., MMP-2 and MMP-9 Regulation of a Vascular Coculture Systemunder Shear Stress, Master’s thesis, Eindhoven University of Technology, 2003

[6] Brown, T., Techniques for mechanical stimulation of cells in vitro: a review, Journalof Biomechanics, 33(1):3–14, 2000

[7] Bussolari, S., Dewey Jr, C., Gimbrone Jr., M., Apparatus for subjecting living cellsto fluid shear stress, Review of Scientific Instruments, 53(12):1851–4, 1982

[8] Carmeliet, P., One cell, two fates, Nature, 408(6808):43–45, 2000

[9] Davani, S., Marandin, A., Mersin, N., Royer, B., Kantelip, B., Herve, P., Etievent,J., Kantelip, J., Mesenchymal Progenitor Cells Differentiate into an EndothelialPhenotype, Enhance Vascular Density, and Improve Heart Function in a Rat Cel-lular Cardiomyoplasty Model, Circulation, 108(suppl.II):253–258, 2003

[10] Davies, P., Mundel, T., Barbee, K., A Mechanism for Heterogeneous EndothelialResponses to Flow In Vivo and In Vitro, Journal of Biomechanics, 28(12):1553–1560, 1995

[11] Dejana, E., Bazzoni, G., Lampugnani, M., Vascular Endothelial (VE)-Cadherin:Only an Intercellular Glue? - Review, Experimental Cell Research, 252(1):13–19,1999

[12] Forrester, J., Price, M., Makkar, R., Stem Cell Repair of Infarcted Myocardium,Circulation, 108:1139–1145, 2003

35

[13] G.A. Skarja, R.L. Kinlough-Rathbone, D. P. F. R. J. B., A cone-and-plate devicefor the investigation of platelet biomaterial interactions, Journal of Biomedical Ma-terials Research, 34:427–438, 1997

[14] H. Shankaran, S. N., Effect of secondary flow on biological experiments in the cone-plate viscometer: Methods for estimating collision frequency, wall shear stress andinter-particle interactions in non-linear flow, Biorheology, 38:275–304, 2001

[15] Hoerstrup, S., Kadner, A., Breymann, C., Maurus, C., Guenter, C., Sodian, R.,Visjager, J., Zund, G., Turina, M., Living, Autologous Pulmonary Artery ConduitsTissue Engineered From Human Umbilical Cord Cells, The Annals of ThoracicSurgery, 74(1):46–52, 2002

[16] Huang, C., Hagar, K., Frost, L., Sun, Y., Cheung, H., Effects of cyclic compressiveloading on chondrogenesis of rabbit bone-marrow derived mesenchymal stem cells,Stem Cells, 22(3):313–23, 2004

[17] Huntley, G., Gil, O., Bozdagi, O., The Cadherin Family of Cell Adhesion Molecules:Multiple Roles in Synaptic Plasticity, The Neuroscientist, 8(3):221–33, 2002

[18] Jackson, D., The unfolding tale of PECAM-1 - Minireview, FEBS Letters, 540(3):7–14, 2003

[19] J.P. Vande Geest, E.S. Di Martino, D. V., An analysis of the complete strainfield within Flexercell membranes JB 2003-67 Rev. 2, Journal of Biomechanics,37(12):1923–8, 2004

[20] Kadner, A., Hoerstrup, S., Zund, G., Eid, K., Maurus, C., Melnitchouk, S.,Grunenfelder, J., Turina, M., A new source for cardiovascular tissue engineering:human bone marrow stromal cells, European Journal of Cardio-thoracic Surgery,21(6):1055–1060, 2002

[21] Kaushal, S., Amiel, G., Guleserian, K., Shapira, O., Perry, T., Sutherland, F.,Rabkin, E., Moran, A., Schoen, F., Atala, A., Soker, S., Bischoff, J., Mayer Jr.,J., Functional small-diameter neovessels created using endothelial progenitor cellsexpanded ex vivo, Nature Medicine, 7(9):1035–40, 2001

[22] K.D. Chen, Y.S. Li, M. K. S. L. S. Y. S. C. J. S., Mechanotransduction in responseto shear stress. Roles of receptor tyrosine kinases, integrins, and Shc., The Journalof Biological Chemistry, 274(26):18393–18400, 1999

[23] Koike, N., Fukumura, D., Gralla, O., Au, P., Schechner, J., Jain, R., Tissue engi-neering: creation of long-lasting blood vessels, Nature, 428(6979):138–139, 2004

[24] L. Qian, W. S., Improving the expansion and neuronal differentiation of mesenchy-mal stem cells through culture surface modification, Biomaterials, 25(7-8):1331–7,2004

[25] Lelkes, P., Mechanical forces and the endothelium, Harwood academic publishers,1999

36

[26] Leroux, C., Cordier, G., Mercier, I., Chastang, J., Lyon, M., Querat, G., Greenland,T., Vigne, R., Mornex, J., Ovine aortic smooth muscle cells allow the replication ofvisna-maedi virus in vitro, Archives of Virology, 140(1):1–11, 1995

[27] L’Heureux, N., Paquet, S., Labbe, R., Germain, L., Auger, F., A completely bi-ological tissue-engineered human blood vessel, The FASEB Journal, 12(1):47–56,1998

[28] M. Flores-Guzman, P. Gutierrez-Rodrıguez, H. M., Apparatus for subjecting livingcells to fluid shear stress, Archives of Medical Research, 33(2):107–114, 2002

[29] M. Papadaki, L. M., Quantitative Measurement of Shear-Stress Effects on Endothe-lial Cells, Methods in Molecular Medicine, 18:577–591, 1998

[30] M.C. Galmiche, V.E. Koteliansky, J. B. P. H. P. C., Stromal Cells From HumanLong-Term Marrow Cultures Are Mesenchymal Cells that Differentiate Following aVascular Smooth Muscle Differentiation Pathway, Blood, 82(1):66–76, 1993

[31] McBeath, R., Pirone, D., Nelson, C., Bhadriraju, K., Chen, C., Cell shape, cy-toskeletal tension, and RhoA regulate stem cell commitment, Developmental Cell,6(4):483–95, 2004

[32] Minguell, J., Erices, A., Conget, P., Mesenchymal Stem Cells, Experimental Biologyand Medicine, 226(6):507–520, 2001

[33] Niklason, L., Gao, J., Abbott, W., Hirschi, K., Houser, S., Marini, R., Langer, R.,Functional Arteries Grown in Vitro, Science, 284(5413):489–493, 1999

[34] Oswald, J., Boxberger, S., Jorgensen, B., Feldmann, S., Ehninger, G., Bornhauser,M., Werner, C., Mesenchymal stem cells can be differentiated into endothelial cellsin vitro, Stem Cells, 22(3):377–84, 2004

[35] Roufosse, C., Direkze, N., Otto, W., Wright, N., Circulating mesenchymal stem cells,The International Journal of Biochemistry & Cell Biology, 36(4):585–597, 2004

[36] Settleman, J., Tension precedes commitment-even for a stem cell, Molecular Cell,14(2):148–50, 2004

[37] Sigma-Aldrich, Product Information belonging to product C6198

[38] Sobue, K., Hayashi, K., Nishida, W., Expressional regulation of smooth muscle cell-specific genes in assocation with phenotypic modulation, Molecular and CellularBiochemistry, 190(1-2):105–118, 1999

[39] Wakitani, S., Saito, T., Caplan, A., Myogenic cells derived from rat bone marrowmesenchymal stem cells exposed to 5-azacytidine, Muscle Nerve, 18(12):1417–26,1995

[40] W.C. Lan, W.H. Lan, C. C. C. H. M. C. J. J., The effects of extracellular citric acidacidosis on the viability, cellular adhesion capacity and protein synthesis of culturedhuman gingival fibroblasts, Australian Dental Journal, 44(2):123–30, 1999

[41] Weinberg, C., Bell, E., A blood vessel model constructed from collagen and culturedvascular cells, Science, 231(4736):397–400, 1986

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[42] Worth, N., Rolfe, B., Song, J., Campbell, G., Vascular smooth muscle cell pheno-typic modulation is associated with reorganisation of contractile and cytoskeletalproteins, Cell Motility and the Cytoskeleton, 49(3):130–145, 2001

[43] Yamamoto, K., Takahashi, T., Asahara, T., Ohura, N., Sokabe, T., Kamiya, A.,Ando, J., Proliferation, Differentiation, and Tube Formation by Endothelial Pro-genitor Cells in Response to Fluid Shear Stress, Journal of Applied Phsyiology,95(5):2081–8, 2003

38

Appendix A

Freezing, thawing and cultureprotocols

A.1 Cryopreservation of hMSC

Reagents:

• Mesenchymal stem cell growth medium (CB)

• Dimethyl sulfoxide (DMSO)

• Human serum albumin (HSA)

1. Prepare freezing medium by mixing 85% (v/v) MSCGM, 10% (v/v) DMSO and5% HSA.

2. Suspend cells at 5 · 103 to 2 · 104 cells·ml−1

3. Add cell suspension in aliquots of 1 ml to cryovials.

4. Store the vials in a freezing canister at -70°C.

5. Within 24 hours, place the vials in liquid nitrogen (-200°C) for long-term preser-vation.

A.2 Thawing hMSC and culture initiation

Media/solutions:

• Bulletkit PT-3001 (CB)

39

Preparations:

• Thaw Serum, L-glutamine and Pen/Strep Singlequots and add them to the basalmesenchymal stem cell medium.

• Equilibrate medium at room temperature.

Procedure:

• Remove cryovials from liquid nitrogen. Aseptically twist the cap a quarter turn torelieve internal pressure and then retighten.

• Dip the bottom of the vial in a 37°C water bath and swirl gently until the lastpiece of ice vanishes.

• Desinfect the vial with 70% ethanol and add the thawed cell suspension asepticallyto 5 ml of medium.

• Centrifuge for 5 minutes at 500 x g.

• Resuspend the cells in a minimum volume of medium and count the cells (dead/live).

• Plate the cells at 5 · 103 to 6 · 103 cells·ml−2. Add 0.5 ml medium per cm2.

• Incubate cultures at 37°C, 5% C02 and 90% humidity.

A.3 Subculture of hMSC

Media/solutions:

• PBS

• Mesenchymal stem cell medium

• Trypsin (for hMSC only, CC-3232, Cambrex)

Preparations:

• Equilibrate all solutions and media at room temperature.

Procedure:

• Aseptically remove and discard the culture medium.

• Wash the monolayer in PBS. Add PBS to the sides and rock gently. Remove anddiscard the PBS.

40

• Add Trypsin solution (0.05 ml·cm−2) and rock flask such that the whole cell layeris covered. Incubate for maximal 15 minutes at room temperature. By gentlytapping the flask cells will detach faster. Once >90% of the cells is rounded anddetached, flip the flasks vertically to allow the cells to drain. Add a volume ofculture medium to dilute the trypsin.

• Centrifuge for 5 minutes at 600 x g.

• Resuspend cells in a minimal volume of medium and count cells.

• Plate the cells at 5 · 103 to 6 · 103 cells.cm2. Add 0.5 ml medium per cm2.

• Incubate cultures at 37°C, 5% C02 and 90% humidity.

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Appendix B

Markers

B.0.1 Endothelial cell markers

It appeared from literature that commonly used identification markers for endothelialcells are junctional proteins.15,21,33 Both platelet endothelial cell adhesion molecule-1 (PECAM-1 or CD31) and vascular endothelial cadherin (VE-cadherin, cadherin-5 orCD144) are such junctional proteins, which provide intercellular contact.

CD31

Platelet endothelial cell adhesion molecule-1 (PECAM-1) or CD31 is a member of theimmunoglobulin (Ig) superfamily and is involved in mediation of vascular permeability,endothelial cell migration, angiogenesis and migration of cells (neutrophils and mono-cytes) into the subendothelial space. It is responsive to mechanical disturbation of thecell membrane. CD31 is strongly expressed by endothelial cells (106 units per cells) andto a smaller extent by immune cells and hematopoietic cells. From in-vitro endothelialcell cultures it appears that within isolated cells CD31 is diffusely dispersed over the cellmembrane, while at intercellular contact it concentrates on the contact border, which isalso the case in vivo.18,25

VE-cadherin

Vascular endothelial cadherin, VE-cadherin, cadherin-5 or CD144, is a member of thecadherin superfamily of transmembrane glycoproteins. It is exclusively expressed byendothelial cells and is found within adherens junctions, where it is connects to thecytoskeleton and transfers information to the nucleus.11 VE-cadherin mainly facilitatesmutual interactions between endothelial cells (homophilic interactions,17 enabling theregulation of vascular permeability as was evidenced by several in vivo blocking studies.Furthermore, animal knock-out studies showed that VE-cadherin is essential for normalembryonic vascular development, embryonic survival and also postnatal angiogenesis.11

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B.0.2 Vascular smooth muscle markers

For identification of vascular smooth muscle cells among others α-smooth muscle actin(ASMA), part of the contractile apparatus, and vimentin, a largely cytoskeletal protein,are used.21,26,33,42

α-smooth muscle actin (ASMA)

Alpha smooth muscle actin, a 42 kDa protein, belongs to the family of actins, whichtogether contribute up to 5% of the total cellular protein content. Actins are incorporatedwithin the microfilaments of the cytoskeleton and therefore enable functioning of thecontractile apparatus. Alpha smooth muscle actin is one of the six different isoforms ofactin that are found within the cells of higher eucaryotic species and it is highly smooth-muscle specific. It is however also found in pericytes, myofibroblasts and myoepithelialcells.1,37

Vimentin

This 54 kDa protein is found within the intermediate filaments of cells. These cytoskeletalfilaments provide cells with tensile strength. Vimentin is mainly found within cells whichstem from the embryonic mesoderm, smooth muscle cells included.1

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Appendix C

Antibody titration

C.1 Introduction

Antibodies are generated by the immune system of animals to eliminate microorganismsand foreign molecules, which are together referred to as antigens (antibody-generating).Each antibody recognizes and binds to a specific antigen, a property which is used inimmunofluorescence techniques. By labelling an antibody with a fluorescent dye andthereafter exciting it, the specific structures to which the antibody is directed can beidentified in for example thin tissue slides or cell cultures.All antibodies can be divided into three distinct regions. First there is the part whichis specifically matched with the antigen it is meant to bind. This is called the Fab part.Secondly, their is the part which bears the excitable fluorescent dye. The third ’leg’ isdenoted by Fc and binds to Fc-receptors on cells. This type of binding is called unspecificbecause it not involves the antigen of interest. Consequently, it is important to quantifythe contribution of fluorescence caused by non-specific binding. Therefore a so-calledisotype control is included in each immunofluorescence experiment. This means that cellsare separately incubated with an unspecific antibody of the same immunoglobulin-familythat is labelled with the same dye as the specific antibody. Non-specific fluorescencedefines the lower limit of the ’fluorescence scale’ used for interpretation of the actual data.On the same time, positive controls, mostly cells for which the antigen is characteristic,are used to determine the upper limit of the fluorescence scale by labelling them withthe specific antibody.The labelling of cellular antigens with fluorescent antibodies can be performed in twodifferent ways. It is possible to use an antibody which is directly conjugated with afluorescent dye, or alternatively first incubate with a primary unlabelled specific antibody.This is thereafter probed with a matched fluorescent labelled antibody. The first methodis faster, but the fluorescence signal is generally weaker. The second method takeslonger, but yields a stronger fluorescence intensity and allows the use of different dyes incombination with the primary antibody.

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C.2 CD31 / VE-cadherin antibody titration

It suits to perform an antibody titration for each antibody used in a particular experi-ment. In such an experiment the optimal dilution of an antibody is determined by usingcells which express the antigen to which the antibody is directed. Fluorescence intensitytypically increase with titre (µg antibody per µl), however there exists a saturating titrewhere fluorescence intensity reaches a plateau. This is the optimal antibody dilution,which is the best trade-off between resolution and cost.

The optimal dilutions of both directly PE-conjugated anti-CD31 antibody and the pri-mary antibody against VE-cadherin were determined using human coronary artery en-dothelial cells (hCAECs). Additionally, the data of this experiment was used to deter-mine threshold intensities for both markers. These represent the minimal fluorescenceintensities of endothelial cells. Judgement of endothelial differentiation of hMSC underinfluence of both VEGF and shear stress is then made possible (see main thesis text).hCAECs were used in passage 8. Origin of the donor was unknown. hCAECs were, onceremoved from liquid nitrogen, carefully thawed to room temperature and subsequentlycultured in Endothelial Cell Basal Medium-2 (CC-3156, CB), supplemented with aliquotsof hEGF, Hydrocortisone, GA-1000, VEGF, hFGF-B, R3-IGF-1, ascorbic acid and 5%fetal bovine serum (FBS). These were all included in SingleQuot kit(R) CC-4147 (CB).For the experiments cells were seeded at a density of 5 · 103 cells·ml−1 onto the wells of a96 wells plate coated with collagen type I (C9791, Sigma Aldrich, for coating proceduresee appendix D). A general medium change was performed on day 4 of culture.On day 7, cells were washed, fixed and stained according to the protocol in appendixE. The tested antibody dilutions for the PE-conjugated antibody against CD31 and theprimary antibody against VE-cadherin were 1:20, 1:50, 1:100, 1:200, 1:500 and 1:1000.The same dilutions were tested for the isotype controls for PE and FITC. Per dilutionthree replicates were used (n=3). The secondary FITC-conjugated antibody used forVE-cadherin labelling was used in a 1:100 dilution in all cases.Per replicate 5 cell fluorescence profiles were collected, yielding a total of 15 intensityprofiles per antibody dilution.

C.2.1 Results

Figure C.1 a/b show the mean FITC and PE fluorescence intensities of HCAECs for theantibody titres 1:20, 1:50, 1:100, 1:200, 1:500 and 1:1000, here expressed as µg antibodyper ml PBS, on a logarithmic scale. It appears from the isotype control data seriesthat non-specific fluorescence is similar for all antibody titres. Positive fluorescenceintensities first increase with antibody titre up to 0.5 µl per 100 ml, whereas at highertitre values fluorescence quenches. It was expected that fluorescence intensity wouldsaturate. Nonetheless, for all titres a clear distinction between positive and non-specificfluorescence is present. Based on these results, a moderate antibody titre of 1:200 waschosen for both primary antibodies (against CD31 and VE-cadherin). This seems a goodtrade-off between cost and resolution. The 1:100 dilution of the secondary FITC-labelledsuited and was used for all other experiments.Based on the fluorescence intensities of the positive controls, a threshold intensity of 125

45

dimensionless units was chosen as the minimum for endothelial cells.

C.3 ASMA / vimentin antibody titration

The antibody titration for the Cy3-conjugated antibody against ASMA and primaryantibody against vimentin was qualitative. Briefly, hMSCs were cultured for 7 dayson Flexercell silicone membranes. A stimulator of smooth muscle differentiation was notadded as it was found in preliminary experiments that some, relative large hMSC expressvimentin and have clear actin (ASMA) stress fibers. After fixation and permeabilization,cells were stained. Dilutions for both primary antibodies were 1:50, 1:100, 1:150, 1:200and 1:250. The secondary FITC-conjugated antibody was used at the following dilutions:1:100, 1:250, 1:400, 1:500. To efficiently use the rather expensive Flexercell membranes,multiple dilutions were tested on each membrane. Each antibody solution was added indroplets to a separate piece of paraffine which was then placed on the cells. The differentparts of the membrane were cut out and transferred to coverslips.Thereafter, fluorescence was read on confocal microscope. Results were inconsistent, i.e.high dilutions giving high fluorescence and vice versa. It is likely that antibody solutionsmixed on the membrane. A qualitative comparison of fluorescence intensities resulted inthe selection of a 1:250 dilution for the primary vimentin antibody, a 1:100 dilution forthe secondary FITC-labelled antibody and a 1:150 dilution for the Cy3-antibody againstASMA.

46

(a)

(b)

Figure C.1: (a) Antibody titration curves for both FITC/VE-cadherin and PE/CD31. Notethe logarithmic scale of the x-axis. The arrow indicates the chosen dilution 1:200.

47

Appendix D

Coating procedures

D.1 Collagen type I coating

Reagents:

• 10 mg Collagen type I (C9791, Sigma Aldrich)

• 0.1 M acetic acid solution

Procedure:

1. Add collagen to 10 ml 0.1 M acetic acid to obtain 0.1 % (w/v) collagen solution.Allow this solution to stir at room temperature for 1-3 hours until collagen iscompletely dissolved.

2. Prepare a working solution by diluting 1:10 in PBS.

3. Add 0.5 ml of the working solution to each well of a 6 well multidish and allowthe collagen to bind for several hours at 37°C or overnight at 2-8°C. The densityof the coating will be 5 µg·cm−2

4. Remove excess liquid from the coated surface and allow it to dry overnight.

5. Sterilise the coated surface by exposing it to UV light in a sterile tissue culturehood for several hours.

6. Before introducing cells and medium rinse the surface with sterile EBSS or PBS.

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D.2 Fibronectin coating

Solutions:

• Sterile fibronectin solution (1 mg·ml−1, F0895, Sigma Aldrich), stored at 4°C.

• Sterile EBBS or PBS.

Procedure:

1. Calculate the volume of fibronectin solution needed to achieve the desired coatingdensity [µg·cm−2] on the type of culture dish used.

2. Dilute the appropriate volume of fibronectin solution in a minimum volume ofEBBS or PBS and add it to the culture dish.

3. Allow to surfaces to dry for at least 45 minutes at room temperature.

4. Remove excess fibronectin solution.

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Appendix E

Preparations forimmunofluorescence microscopy

Reagents:

• PBS

• Bovine serum albumin (BSA)

• 4% paraformaldehyde

• Triton X-100

• Antibodies against markers of interest / corresponding isotype control antibodies

• Anti-fading solution: polyvinyl alcohol mounting medium with DABCO (10981,Fluka Biochemica, Switzerland)

Preparations:

• Prepare PFA according to the following protocol:

1. Within a fume cabinet add 4 g PFA powder to 100 ml PBS.

2. Heat up to about 70°C while gently stirring .

3. Cool solution to room temperature and filter it into a sterile bottle.

4. Store PFA solution in the fridge at 4°C for one month or in freezer at -20°C forprolonged periods.

• Prepare blocking solutions: 0.5% (v/v) BSA in PBS

• Prepare permeabilization buffer: 0.1% Triton X-100 in PBS

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• Dilute antibodies at proper concentrations in BSA / PBS solution.

Protocol:

Steps to follow for all markers used:

• General remark: keep antibodies ’wet’ !

• Wash cells in PBS.

• Fix cells in PFA solution for 20 minutes at room temperature.

Additional step for intracellular proteins (ASMA, vimentin):

• Incubate cells with permeabilization buffer for 5 minutes at room temperature.

Steps to follow for all markers used:

• Wash three times using blocking solution.

• Incubate cells with primary or isotype control antibodies at proper dilutions onice in the dark (wrapped in aluminium foil) for 20 minutes.

• Wash three times using blocking solution

• Incubate cells with secondary antibodies at proper dilutions on ice in the dark for20 minutes.

• Wash cells in PBS (5 mins)

Final step for cells cultured on Transwell membranes:

• Carefully cut the membrane out of the Transwell insert and transfer them to acover glass.

• Add a few drops of anti-bleaching solution, fit a coverslip and seal using nailpolish.

Final step for cells cultured on dishes:

• Add a few drops of anti-bleaching solution, fit a coverslip and seal using nailpolish.

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Appendix F

Medium dynamic viscositymeasurement

The dynamic viscosity η (37°C) of supplemented Cambrex mesenchymal stem cell culturemedium was determined on an Ares parallel disk rheometer (Ares, Scientific Rheometer).A sample of culture medium was loaded onto the heated baseplate whereupon the parallelplate geometry was carefully lowered until sample contact was made. The upper platewas then put into spinning mode such that the sample adopted a homogeneous circularshape. The upper plate was incrementally lowered until the sample precisely filled thegap, with a spherical fluid meniscus on the edge. A dehydration cap was fitted aroundthe plate geometry and sample.The actual measurement involved a shear rate sweep test, in which shear rate ranged from10 s−1 up to 1000 s−1 and vice versa, with six measurements per decade. The systemdetermined the torque imposed on the upper plate, which was translated to dynamicviscosity. The dynamic behaviour of medium viscosity is depicted in figure F.1. In theregion depicted on the right of figure F.1 (464-100 s−1) dynamic viscosity was nearlyindependent of shear rate and equaled 7.7 · 103 Pa.s. From equation 2. in section 2.4.1of the main text it can be calculated that in order to achieve a shear stress of 0.5 Pa,angular velocity ω should equal 184 RPM.

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Figure F.1: Dynamic viscosity versus shear rate (Cambrex supplemented mesenchymal stemcell medium, 37 °C).

53

Appendix G

Cone-plate system: pH andtemperature measurement

In these preliminary experiments it was determined if culture medium pH and temper-ature was affected during by operation of the rotating system. A medium-filled 6-wellsplate (2 ml per well) was fitted with the cone and plate system and transferred to anincubator at 37°C / 5% CO2. Cone rotational speed was maintained at 184 RPM for24 hours. At t=0 hours and t=24 hours medium pH was determined using pH indicatorpaper. In a separate experiment, medium temperature was monitored for 3 hours usinga thermocouple (Hasco Z251/1) while the rotating system was active at 184 RPM (37°C/ 5% CO2).

G.1 Results

Six independent pH measurements showed that at t=0 pH equaled 7.2 ± 0.2 units and 7.4± 0.6 units at t=24 hours. This difference is neither physically relevant nor statisticallysignificant (P>0.05, paired t-test). It followed from the temperature measurement thatafter a short equilibration period, temperature maintained at 37°C during 3 hours ofmonitoring.These experiments learn that both pH and temperature can be regarded as experimentalconstants.

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Appendix H

Schematic Flexercell system

55

Appendix I

Fluorescence activated cell sorting(FACS)

I.1 Concept

This section discusses the general idea of fluorescence activated cell sorting, which isillustrated by figure I.1(a). A fluorescence activated cell sorter or FACS is a device whichallows the isolation of specific cells out of a mixed cell population. Cells are identifiedby their light scattering properties and/or fluorescence emitted by excited dye-labelledantibodies attached to their surface. Briefly, a cell suspension is injected into the fluidicssystem, the ’wet’ part of the FACS. In a small funnel called ’nozzle’ it encounters thesheath fluid, a salt solution (see figure I.1(b)). With a proper adjustment of both sheathfluid pressure and sample pressure, cells are hydrodynamically focussed in a thin verticalcolumn, the core stream, which is enclosed by a shell of sheath fluid. The diameterof the nozzle tip opening is typically 50-400 µm such that cells fit through. One byone the cells then pass through the focus point of a laser and scatter the laser light.The light which is scattered in the direction of the laser beam is called Forward Scatter(FSC) and is a measure of cell size, whereas the light scattered to the sides is called SideScatter (SSC) which is related to cell granularity. If cells are conjugated with fluorescentlabelled antibodies fluorescence is emitted as well. The different signals are collectedand digitized by separate detectors on a per cell basis, which data is subsequently storedin a data file in the form of distinct ’events’. A typical FACS allows cell populationsto be with processing speeds of hundreds or even thousands of cells per second. Yetit possesses another interesting feature, which is the possibility to sort cells based onthe signals they generate. After passing the laser focus point cells can be isolated intodroplets which break off from the liquid stream. Droplets containing cells which meet thedefined sorting criteria receive either a positive or negative charge before detaching fromthe liquid stream. Droplets without cells or with cells which do not meet the definedcriteria remain uncharged. An electric field deviates the charged droplets to either of twocollection tubes standing besides the stream, while uncharged droplets just obey gravityand fall into a waste container. Sorted cells can be sorted again using other criteria orbe subcultured. In the present study cell populations are only characterized, not sorted.

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(a) (b)

Figure I.1: (a) The general concept of fluorescence activated cell sorting. See text for explana-tion. (b) Close-up of nozzle.

I.2 Cell preparations for FACS analysis

Reagents:

• Trypsine

• PBS

• Bovine serum albumin (BSA)

• 4% paraformaldehyde

• Triton X-100

• Sodium azide

• Antibodies against markers of interest / corresponding isotype control antibodies

Preparations:

• Prepare PFA fixation buffer according to the protocol in appendix E.

• Prepare FACS buffer: 0.5% BSA, 0.05% sodium azide in PBS.

• Prepare permeabilization buffer: 0.1% Triton X-100 in PBS.

• Dilute antibodies at proper concentrations in FACS buffer solution.

57

Protocol:

Note: Next to your samples of interest, make sure to reserve cells for an isotype controlsample.

Steps to follow for all markers used:

• Trypsinize cells and collect 1− 3 · 103 cells per sample in a 15 ml Falcon tube.Wash cells in PBS.

• Fix cells by adding 100 µl of fixation buffer to cells, vortex and incubate at roomtemperature for 20 minutes.

Additional step for intracellular proteins (ASMA, vimentin):

• Add 1 ml of permeabilization buffer to each tube, centrifuge and aspiratesupernatant.

• Resuspend cells in 100 µl of permeabilization buffer and incubate at roomtemperature for 5 minutes.

Steps to follow for all markers used:

• Wash cells with 2 ml FACS buffer. Centrifuge and aspirate supernatant.

• Add primary antibody / isotype control antibody, vortex and incubate on ice for30 minutes.

• Wash cells with 2 ml FACS buffer. Centrifuge and aspirate supernatant.

• Add secondary antibody, vortex and incubate on ice for 30 minutes.

• Wash cells with 2 ml FACS buffer. Centrifuge and aspirate supernatant.

• Resuspend cells in 1 ml FACS buffer in appropriate FACS Falcon tubes andanalyze within 1 hour at room temperature. Stored at 4°C samples can beanalyzed up to one week.

I.3 Isotype FACS

From the isotype (unspecific binding) control FL2-FL1 dot plot (figure I.2) it appearsthat cells are mainly located in the lower left quadrant or upper right quadrant. Theserepresent a double negative subpopulation and double positive subpopulation respec-tively. The existence of a double positive population in an isotype control is unwantedbut not unusual. A possible cause may be undercompensation, which means that sub-stantial spill of PE-fluorescence is present in the FITC-channel and vice versa. Thedetector voltages then need to be lowered such that this double-positive cluster fits into

58

(a)

Figure I.2: Dot plot of ASMA-fluorescence versus vimentin-fluorescence of isotype control.

the lower left quadrant. The system was however properly compensated in advance us-ing labelled commercial beads (see main thesis text). The double-positive populationtherefore most likely represent dead cells, which are associated with unspecific antibodybinding and fluorescence.

59