Mesenchymal stem cells in vascular tissue engineeringA literature survey
M.G.J. BongersOctober 2004BMTE05.02
MSc-thesisPart I
Supervisor: prof. dr. M. J. Post
Eindhoven University of TechnologyFaculty of Biomedical Engineering
Contents
Introduction 3
1 Arterial blood vessels 4
1.1 Introduction . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 4
1.2 Structure and function of mature arteries . . . . . . . . . . . . . . . . . . 4
1.3 Vessel wall and mechanical forces . . . . . . . . . . . . . . . . . . . . . . . 6
1.4 Embryonic development of blood vessels . . . . . . . . . . . . . . . . . . . 7
1.4.1 Tube formation and stabilization . . . . . . . . . . . . . . . . . . . 7
1.4.2 Branching and maturation . . . . . . . . . . . . . . . . . . . . . . . 8
2 Tissue engineering of arterial vessel substitutes 10
2.1 Introduction . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 10
2.2 Tissue engineering of living grafts . . . . . . . . . . . . . . . . . . . . . . . 10
2.3 Stem cells and TEBVs . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 11
3 Mesenchymal stem cells 13
3.1 Introduction . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 13
3.2 Isolating and culturing MSC from human bone marrow . . . . . . . . . . 13
3.3 Characterising MSC . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 14
3.4 MSC, endothelial cells and vascular smooth muscle cells have a commonorigin . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 15
3.5 Cytokine-induced in vitro differentiation of MSC . . . . . . . . . . . . . . 17
3.6 Differentiation of MSC and mechanical stimuli . . . . . . . . . . . . . . . 17
Discussion 18
2
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, associated with high occurrence of thrombosis.It was therefore that scientists focussed onto the construction of biologically functionalvessel substitutes from vascular cells which allow remodelling in vivo.23,26,31,44 In recentyears, several investigators have started pursuing the use of stem cells as cell source forliving vessel grafts, because these undifferentiated cells allegedly can commit themselvesto a wide spectrum of cell lineages, including vascular cells.13,22 A particular type ofmultipotent stem cell, the mesenchymal stem cell, seems to have potential for applicationin clinical vessel engineering because it is constantly and readily available in all humans.The question remains though, how its lineage commitment to vascular cell types canbe controlled. In recent years several cytokines and growth factors have been identifiedwhich induce mesenchymal stem cells to differentiate into a specific (vascular) cell lin-eage.29,32,43 Interestingly enough, recent studies indicate that the differentiation of stemcells is related to cytoskeletal tension which is in turn determined by the mechanicalmicroenvironment.28,37 This relation being only recently established, science is still faraway from the revolutionary practice of controlling stem cell differentiation by means ofmechanical cues. This concept would have a major impact on vascular tissue engineeringas well. The construction of a vessel graft incorporating endothelium, smooth muscle andfibroblasts by applying different mechanical stimuli to mesenchymal stem cells within ascaffold is of course an appealing idea and creates and opens new avenues for scientificexploration.
This literature survey starts by describing the main structural and functional proper-ties of mature arterial vessels. Furthermore, it is elucidated how these structures formfrom stem cells within the embryo. Thereafter the interplay between the in vivo mechan-ical environment and the functioning of cells in the vascular wall is discussed. Chapter 3deals with the developments in vascular tissue engineering over the years and discussesthe application of stem cells within this discipline. A general background on mesenchy-mal stem cells is provided by chapter 4 which also discusses the alleged relation betweenmesenchymal stem cell differentiation and mechanical cues. By means of a general dis-cussion this leads to the formulation of a research question with associated hypotheses.This in order to contribute to the understanding of the relation between mechanicalstimuli and lineage commitment of mesenchymal stem cells in a vascular context.
3
Chapter 1
Arterial blood vessels
1.1 Introduction
The first organ to form within the embryo is the circulatory system, as oxygen andnutrient delivery and waste removal are essential to the development, functioning andsurvival of all other organs. The heart functions as the driving pump of blood flow,while the vessels distribute the blood among the different organ systems. The embryonicdevelopment of the vascular system is a complex yet not fully understood process, whichthis chapter tries to elucidate.First the function and structure of mature arteries is described both on a macroscopicand microscopic level. Thereafter, it is elucidated how these biological structures formwithin the embryo by the processes of vasculogenesis and angiogenesis. A basic notionof these events is fundamental to whomever would like to tissue-engineer blood vesselsusing stem cells.
1.2 Structure and function of mature arteries
The arterial system basically serves as a transport and distribution system for bloodtowards organs. The proximal part of this system is formed by elastic arteries and func-tions as a compliance chamber which accomodates the large pressure and flow gradientsimposed by the pumping heart. The distal part is a variable peripheral resistance whichmaintains a relative high diastolic blood pressure for essential organs such as brains andkidneys. Both functions are reflected within the structure of these vessels. An the macro-scopic level all arteries and large arterioles share a typical three-layered wall structureconsisting of an intima, media and adventitia.
The innermost layer or intima, directly bordering the vessel lumen (see figure 1.1), isformed by a single layer of endothelial cells, called the endothelium and a thin layer con-sisting of collagen and elasting fibers, the basement membrane.5 The endothelium’s mainfunctions are regulation of vascular permeability and prevention of thrombus formation.Various protein junctions between neighboring endothelial cells control the intercellular
4
Figure 1.1: The composition of a medium-size mature muscular artery in side view and crosssection view. Pictures taken from ’Netter’s Atlas of Human Physiology’ by J.T.Hansen and ’Molecular regulation of vessel maturation’ by R.K. Jain (NatureMedicine, 2003)
space and in this way the ability of blood proteins and cells to enter the subendothelialspace.25 The antithrombogenic function of the endothelium is provided by the smoothendothelial surface itself, which averts activation of the clotting system. Secondly, theglycocalyx, a structure consisting of various glycosaminoglycans bound to the endothe-lial surface, repulses platelets and clotting factors. Finally, the protein thrombomod-ulin bound to the endothelium slows the clotting process by binding the clotting factorthrombin which circulates in the blood. The thrombomodulin-thrombin complex alsoinactivates other clotting factors.5 Endothelial cells divide slowly under physiologicalconditions, about fifty times in a lifetime, but have a high potential for proliferationand migration when the endothelial surface has been damaged.1 Within the media, themiddle wall layer, elastic laminae alternate with layers of smooth muscle cells. Vascularsmooth muscle lacks the typical striations of skeletal muscle that are caused by a longitu-dinal arrangement of its contractile filaments. Within smooth muscle, actin and myosinfilaments are spirally arranged. For this reason, force generation by smooth muscle is notas powerful as by skeletal muscle. However, this typical configuration of the contractilesystem enables an increased shortening range compared to skeletal muscle.1
From the promixal towards the distal side of the arterial system the amount of smoothmuscle cells increases while the number of elastic laminae reduces. Within the promixalelastic arteries, smooth muscle cells regulate vessel stiffness by increasing and decreasingthe separation of the elastic laminae. In this way blood flow is controlled.20 The moredistally located muscular arteries control the distribution of blood to the organ systems.They incorporate up to 40 layers of circumferentially orientated smooth muscle whichregulate the tonus of the vessel wall tonus. Arterioles, the smallest vessels of the arterialsystem, mainly consist of smooth muscle cells. Smooth muscle contraction here resultsin lumen narrowing, which increases the peripheral resistance.
5
The outer layer, the adventitia, formes a connective tissue incorporating longitudinallyorientated collagen and elastin, which support the vessel. This layer is present in allarterial vessels except the small arterioles.20 The adventitia homes fibroblasts, whichsynthesizes collagen and elastin, the building materials for the extracellular matrix.30
The other constituents of the adventitia are pericytes, mesenchymal precursor cells ofvascular smooth muscle, vasa vasorum, which provide the vessel’s own blood and nutri-ents supply and vasomotor neurons which branch through the adventitia and innervatesmooth muscle within the media.
1.3 Vessel wall and mechanical forces
The walls of the vessel system are continuously exposed to mechanical forces imposed bycirculating blood. These mechanical loads have a cyclic character directly related to therhythm of the pumping heart and can be divided into both time-dependent shear stressand transmural pressure, as depicted in figure 1.2. The magnitude of the wall shearstress has a periodic character, with maxima ranging from near 0 Pa proximally up to3 Pa distally.7,25 Shear stress is elicited by blood flowing along the endothelium. Thisstretches the cell membrane of the endothelial cells. Changes within local flow patternsare sensed via receptors within the cell membrane and directly affect internal processeslike reorganization of the cytoskeleton.24,25 Endothelial cells for example always orien-tate their longitudinal axis parallel to the direction of flow, i.e. stress vector, τw(t).
Transmural pressure includes hydrostatic pressure relative to the atmospheric pressurewithin the surrounding tissues, potential energy pressure induced by the pumping heartand dynamic pressure arising from moving blood. Taken together this results in a radialload on the vessel wall, with typical diastolic aortic value around 12 kPa and systolicpeaks up to 18 kPa.42 The transmural pressure circumferentially strains the vessel wall,up to 10% within the human aorta.10 The resulting stress is buttressed by the medialwall layer, which is reflected in the circumferential orientation of smooth muscle cells.Longitudinally straining of the vessel is largely impeded by surrounding connective tis-sues. Minor longitudinal stress is buttressed by the longitudinally orientated collagenstress fibers within the adventitia.The mechanical forces experienced by the living constituents of the vessel wall changeperiodically on a time scale of seconds due to the rhythm of the pumping heart as wellas chronically on time scale of years due to pathological conditions. Vascular cells willalways try to counteract the changing mechanical conditions they experience. In a hyper-tensive situation for example, increased circumferential stress within the media inducessmooth muscle cells to synthesize matrix. In this way the media enlarges, which reduceshoop stress.The response of both endothelial and smooth muscle cells to stress and strain has beenand is being extensively studied both in vivo and in vitro.17,27,39,40 However, in vitromodels do not necessarily resemble the in vivo situation. Straining of in vitro culturedsmooth muscle cells for example results in protein synthesis and increased proliferation,whereas the stretching of intact aortic vessels results in smooth muscle cells producingproteins without proliferating.24 Despite this discrepancy it is evident that the mechanic(micro)environment and vascular wall cells affect and control each other inextricably.
6
Figure 1.2: Illustration of the mechanical forces acting on a straight part of the vessel wall ina local coordinate system. p represents transmural pressure, τw wall shear stress,εφφ circumferential strain, εzz longitudinal strain.
1.4 Embryonic development of blood vessels
1.4.1 Tube formation and stabilization
Oxygen delivery to growing and developing embryonic tissues is initially sufficiently pro-vided by diffusion solely. However, when embryonic tissues enlarge, tissue hypoxia devel-ops while diffusion flux decreases with increasing diffusion distance. Driven by subnormaloxygen tension (< 21% O2) cells increase the production of HIF-1α, which on its turnstimulates the production and secretion of vascular endothelial growth factor (VEGF).VEGF stimulates stem cells from the mesodermal germ layer to differentiate into en-dothelial cells by binding to their Flk1-receptors.2 In addition VEGF associates withFlt1-receptors, which stimulates proliferation of the newly formed endothelial cells andtheir assembly into tubular structures (figure 1.3 A). This process of de novo generationof primitive blood vessels is called vasculogenesis.Endothelial tubes cannot exist without the presence of pericytes.6,8 It has been shownthat nascent vessels lacking pericytes regress without VEGF being present due to apop-tosis of endothelial cells (figure 1.3 D). Vessels associated with pericytes in contrast areno longer dependent on VEGF6 (as indicated in figure 1, bottom). The growth fac-tors PDGF-β (platelet-derived growth factor) and TGF-β (transforming growth factor)are pivots in this process of vessel stabilization. PDGF-β secreted by endothelial cellsattracts mural cells and supports their proliferation, whereas TGF-β promotes genera-
7
Figure 1.3: Formation of immature vasculature. (A) Endothelial tubes form by vasculogenesisor angiogenesis. Long-term exposure to VEGF results in vessel regression (D). Thedecreasing dependence of VEGF with vessel maturation is indicated. Stabilizationis accomplished by generation of basement membrane / ECM and association withmural cells (B). Stabilized vessels can degrade their ECM and loosen pericytesunder influence of VEGF and Angiopoietin-2 for the purpose of vessel branching andremodelling. Picture modified from ’Blood vessel maturation: vascular developmentcomes of age’ by Darland and D’Amore (Nature, 1999).
tion of extracellular matrix (ECM) and differentiation of mesenchymal stem cells intopericytes (figure 1.3 B). Binding of Angiopoietin-1 to Tie-2 receptors promotes the in-teraction between endothelial cells and mural cells. In this way the primitive vessels sealthemselves against leaking (figure 1.3 C).
1.4.2 Branching and maturation
The newly formed primitive vessels start branching in order to form a vascular networkwithin their homing organ. This process involves proliferation, migration and apop-tosis of pericytes and endothelial cells via and favoured by constituents of the ECM.Angiopoietin-2 competes with its antagonist Angiopoietin-1 for Tie-2 binding sites whichcounteracts the stabilizing Angiopoietin-1 / Tie -2 signal. At the same time the ECMis degraded in a controlled manner by combined action of proteases and protease in-hibitors. Together these destabilizing events loosen pericytes and mural cells from theunderlying endothelial layer. Proangiogenic factors like VEGF, which are released frommatrix during its degradation, stimulate the uncovered endothelial cells to proliferateand migrate via the matrix and to form new vessel sprouts. The formation of vesselsfrom existing ones is called angiogenesis. Vessel branching is further regulated by vesselpruning, which involves controlled cell apoptosis, and by mutual repellence of arteriousand venous cells.18 With time large-diameter vessels form by additional generation of
8
ECM, elastic laminae in case of an artery and the acquisition of extra layers of muralcells. Eventually, the complex process as described here leads to the establishment of awell-organized interconnected system of arteries, arterioles, capillaries, venules and veins.The full postnatal maturation of the arterial wall takes up to twenty years.20 Angiogen-esis however continues throughout a human’s lifetime in physiological circumstances likewound healing as well as pathological conditions, for example tumor growth.
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Chapter 2
Tissue engineering of arterialvessel substitutes
2.1 Introduction
The need for arterial substitutes in the clinic is ongoing and unfortunately increasing,caused by the increasing occurence of atherosclerosis in recent years. Current thera-pies include replacement of occluded arteries by autologous veins or synthetic grafts.Unfortunately, both therapies have important drawbacks. Veins functioning under arte-rial environmental conditions will damage over time resulting in disfunction. (Coated)synthetic grafts on the other side are associated with problems such as inflammatoryresponses, infections and thrombosis. It is therefore that scientists resorted to the devel-opment of ’living’ grafts, which have a lower risk of thrombosis and allow growth, repairand remodelling, which marked the birth of vascular tissue engineering. In this chapterit is discussed where the developments within this discipline have led to thus far.
2.2 Tissue engineering of living grafts
In 1986, Weinberg and Bell were the first to present a completely tissue engineered bloodvessel (TEBV).44 As is the common approach in tissue engineering, the constructionof this TEBV involved the seeding cells within a stabilising matrix or scaffold whichdegrades over time. Its supporting function is gradually taken over by the extracellularmatrix (ECM) that is deposited by the cells. Although this vessel possessed a typicalthree-layered arterial wall structure, its burst strength was not a physiological level,which would certainly impede clinical application. However, this study stimulated othersto engineer similar TEBVs. A major goal hereby was the optimization of the mechanicalintegrity in order to enable long term in vivo functioning. Eventually it was shownthat by optimizing the deposition of extracellular matrix by vascular cells a physiologicalburst strength could be achieved. In 1998, Heureux presented a TEBV able to withstandan internal pressure of 2000 mmHg, achieved by supplementing the culture media withascorbic acid, which enhanced matrix deposition by SMCs and fibroblasts (see figure
10
Figure 2.1: Example of a tissue engineered blood vessel (TEBV) fabricated by Heureux et al.(The FASEB Journal, 1998)
2.1).26 A year later, Niklason et al. showed that an EC/SMC seeded scaffold transformedinto a TEBV with a rupture strength of 2150 mmHg within 8 weeks, by actively perfusingthe graft with a medium that enhanced collagen synthesis by SMC. The engineered vesselstructurally and functionally (i.e. contractive responsive) resembled native arteries.31 Astep forward towards the clinical relevance of TEBVs had certainly been made. However,at this point, with clinical application in mind, attention had to be paid to the bio-compatibility of TEBVs. The usage of allogeneic cells is inevitably associated withinflammatory responses and graft rejection by the body. Autologous vascular cells canbe harvested in situ from living vessels, which subsequently can be expanded in vitroand seeded on a scaffold. Yet, this means that intact vessels need to be disrupted,which of course is not preferential. Fortunately, the human body possesses a ratherinexhaustible source of so-called stem cells, which can be harvested from bone-marrowor peripheral blood with relative ease and without inflicting severe damage to the body.The application of these cells in vascular tissue engineering till the present day forms thetopic of the next section.
2.3 Stem cells and TEBVs
Stem cells constitute a mixed population of cells and are generally distinguished fromother cell types by two characteristics. First, stem cells possess a relatively unlimitedcapacity for self-renewal by means of cell division compared to the lifespan of animals.Secondly, they are able to differentiate, under appropriate conditions, from cells with anunspecialized phenotype into committed cells capable of performing specific functions.1
If these properties can be exploited and controlled, these cells can constitute a perfectautologous cell source for tissue engineers of all disciplines. In recent years, several sub-populations of the stem cell pool have been used in the fabrication of TEBV’s. Kaushal
11
et al. for example seeded decellularized grafts with endothelial progenitor cells (EPCs),a subset of the human stem cell population which can easily be isolated from periph-eral blood. These grafts were preconditioned into a perfusion system and subsequentlyimplanted in carotid position in a sheep model. The seeded TEBVs showed excellent anti-thrombogenic properties compared to unseeded grafts and the luminal surface proved tobe functioning like endothelium up to 130 days after implantation. In addition the vesselhad gained a medial layer incorporating functional smooth muscle cells.22 While EPCare continuously circulating in the human blood, they form a readily available cell sourcefor vascular tissue engineers. The usefulness of another type of autologous stem cells,so-called umbilical cord cells (UCC), in vessel engineering was investigated by Hoerstrupet al. They produced large caliber arterial conduits by seeding human umbilical cordcells on a scaffold and applying gradually increasing pulsatile nutrient medium flow.13
TEBVs grown and conditioned in this way resembled the native pulmonary artery struc-ture and tensile strength after 14 days of culture, which showed that umbilical cord stemcells are also promising candidates for vascular tissue engineering. However, the usageof UCCs in clinic will likely be associated with practical and financial problems, whilethe umbilical cord of individuals will have to be preserved at birth, should it serve as anautologous cell source later in life.Yet there is another subset of the human stem cell population which should receive at-tention by vascular tissue engineers, which are the mesenchymal stem or precursor cells,also referred to with bone-marrow stromal cells. Kadner et al. recently investigatedthe potential of bone-marrow stromal cells, as cell source for vascular tissue engineeringand showed that constructs seeded with these cells cells developed a matrix compositionsimilar to that of constructs seeded with vascular cells,21 which is a promising result.From here on we will use the term ’mesenchymal stem cells’ or MSC to avoid confusion.The next chapter focusses on the intriguing properties of this type of stem cell.
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Chapter 3
Mesenchymal stem cells
3.1 Introduction
Several types of stem cells and precursor cells are present in many if not all adult tissuesfor the purpose of tissue renewal and repair. Tissue-specific precursor cells possess theability to differentiate into the committed cells of the homing tissue, while multipotentstem cells, which are mainly located in the bone-marrow can commit themselves to awider spectrum of cell types. The multipotent stem cell pool in the adult bone marrowcompartment consists of two sub-populations, the hematopoietic stem cells, which arebeyond the scope of this discussion, and the mesenchymal stem cells (MSC), which haveour attention.Adult MSC from bone-marrow prove their true stem cell nature by the fact that a sin-gle MSC is self-renewing and that it can differentiate into a wide variety of lineages.34
Namely, over the years it become evident by several in vitro and in vivo studies thatadult MSC are able to form cells of various connective tissues, including adipocytes,osteocytes, skeletal myocytes, cardiomyocytes, fibroblasts, chondrocytes, tenocytes andbone-marrow stromal cells.29,33,34,41 Clinically, MSC have already been used for thetreatment of children’s osteogenesis imperfecta3 and the regeneration of damaged artic-ular cartilage35 and tendon.4
Worth noting is the phenomenon that mesenchymal stem cell are able to commit them-selves to cell lineages which are not from mesenchymal origin. This socalled plasticityis a controversial and dubious phenomenon which has also been reported for other stemcell populations.14,16,19,29 Considering their (in vivo) multipotency, MSC truly haveunexploited potential for tissue engineering in general, especially if we regard their easyisolation and expansion procedures, which are the topic of the next section.
3.2 Isolating and culturing MSC from human bone marrow
Bone-marrow is aspirated from a donor’s posterior iliac crest using a syringe filled withheparin in order to prevent blood clotting. This aspirate is washed in phosphate buffered
13
Figure 3.1: Monolayer culture of human MSC passage 3, day 1
saline (PBS) and subjected to density gradient centrifugation, a procedure which sep-arates erythrocytes from nuclear cells. If the nuclear cells are carefully aspirated andcultured, mesenchymal stem cells will attach to the culture dish, spread like fibroblastsand form colonies within 4-6 days. Non-adherent cells are removed with medium changes.Before colonies reach other, cells are trypsinized and subcultured in a 1:3 ratio. Fromthen on cells are passaged at 80-90% confluence. The serum which supplements theculture medium is carefully tested in advance in order to maintain an undifferentiatedphenotype. It has been observed that after cell passage 7 the multi-potency of MSCdevalues and that phenotype changes. However, a normal karyotype and telomeraseactivity are maintained up to passage 12.29,33
3.3 Characterising MSC
MSC in in vitro culture are adherent, contact-inhibited cells with generally a spindle-shaped morphology (see figure 3.1). The MSC phenotype can be confirmed by checkingthe chondrogenic, adipogenic and osteogenic potency of a batch of cells while for thispurpose well-established protocols are available (see section 3.5).It is however difficult to come up with a generally accepted set of cellular antigens thatuniquely identify MSC. The probing of MSC with antibodies against 70 different cellularantigens has indicated that these cells express several surface proteins associated withother committed cell types but in different combinations,34 i.e. MSC express markerswhich are characteristic for endothelial, epithelial and muscle cells.3,41
It is common practice to distinguish MSC from contaminating hematopoietic cells by
14
selecting non-adherent cells which do not express specific hematopoietic markers. MSCfreshly isolated by bone-marrow puncture, do not express the hematopoietic markerCD50.41 Later on, after culture, the hematopoietic markers CD34, CD14 and CD45disappear.3,34 The identification of MSC by bone-marrow punctures based on positiveexpression of markers is still problematic. There is however increasing support for thenotion that expression of STRO-1, SH2, SH3 and SH4 identifies MSC in in vitro cul-ture.3
It is probably due to the ill-defined nature of MSC that investigators nowadays oftenuse commercially available mesenchymal stem cell lines, which have a guaranteed pu-rity. Others however isolate cells from animals or humans and characterize them usingflow cytometry according to their own defined sets of markers. This makes it rathertroublesome to compare the results of different studies.
3.4 MSC, endothelial cells and vascular smooth musclecells have a common origin
All cells and tissues of the adult body derive from the so-called embryonic germ lay-ers. These three cell layers are present in the gastrula stage of embryonic developmentand represent three distinct cell lineages, denoted by endoderm (inner layer), mesoderm(middle layer) and ectoderm (outer layer) (see figure 3.2). Each layer gives rise to anunique set of adult tissues as illustrated by table 3.1. It appears from this table thatadult bone marrow cells (including mesenchymal stem cells), endothelial cells, vascularsmooth muscle cells and fibroblasts all derive from the mesoderm. This close embryonicrelationship justifies studies focussed on guiding differentiation of adult mesenchymalstem cells into vascular cells.
Figure 3.2: Cross-section of a frog gastrula
15
Table 3.1: Overview of adult tissues originating from the three germ layers
Endoderm
inner lining of digestive tractliver, pancreasinner lining of respiratory tractlarynx, trachea, lungurinary bladder, urethra, vaginamost glands
Mesoderm
vascular systembone (marrow), cartilagemuscle (all types)several connective tissuesgonads
Ectoderm
skin, hair, nailsbrain, nervous systemeyesears
16
3.5 Cytokine-induced in vitro differentiation of MSC
For the differentiation of MSC into members of the connective tissue family (adipocytes,chondrocytes and osteoblasts) well-established protocols exists. Adipogenic differen-tiation requires postconfluent cultures and the presence of isobutylmethylxantine andinsulin in serum-containing medium. MSC differentiate into chondrocytes when pel-leted and provided with serum-free medium containing a member of the transforminggrowth factor-β (TGF-β) superfamily, i.e. TGF-β1, TGF-β2 or TGF-β3. Osteogenesisis stimulated by supplying monolayer-cultured MSC with medium containing serum, β-glycerol-phosphate, ascorbic acid-2-phosphate and dexamethason.Next to these generally accepted differentiating treatments there are scattered reportson differentiation of MSC into for examples tenocytes, bone marrow stromal cells, skele-tal muscle cells, smooth muscle cells and cardiac muscle cells by culturing in specificsupplemented media.29
3.6 Differentiation of MSC and mechanical stimuli
Well-established protocols for in-vitro differentiation of bone-marrow derived mesenchy-mal stem cells into pure populations of osteocytes, chondrocytes and adipocytes based onmodification of the culture medium currently exist.33 In recent years evidence has beenprovided that mechanical stimuli are also able to influence differentiation of stem cellsin general.11,37 Simmons et al. recently showed that this is also valid for mesenchymalstem cells. They reported that equibiaxial cyclic strain applied to human mesenchymalstem cells cultured in osteogenic medium decreased proliferation and increased depositionof mineralized matrix, a late differentiation marker of osteogenesis.38 Nonetheless, thecontrol of mesenchymal stem cell differentiation by means of mechanical cues alone hasreceived little attention. A literature search yielded only one study which investigatedthis topic. Recently, Huang et al. studied the isolated effect of cyclic compressive loadingon the chondrogenic differentiation of rabbit bone-marrow derived MSC and comparedthis with typical TGF-β-induced chondrogenic differentiation of the same MSC. It wasfound that both treatments yielded a similar chondrocytic phenotype, which shows thatcompressive loading alone can induce chondrocytic differentiation of rabbit MSC.15 Thisjustifies further investigation of the relation between mechanical stimuli and differentia-tion patterns of MSC.
17
Discussion
It appeared that several types of stem cells have potential for application in vasculartissue engineering. For the present study however we disregard endothelial precursorcells and umbilical cord cells, and concentrate on the usefulness of mesenchymal stemcells for vascular tissue engineering.Chapter 2 mentioned the study by Kadner et al.21 which indicated that scaffold-seededmesenchymal stem cells deposit an extracellular matrix with excellent mechanical prop-erties for vascular application. Furthermore it appeared that they share a commonembryonic origin with vascular wall cells. Therefore, this study should be continued byinvestigation focussed on the question how to control the transformation of these mes-enchymal stem cells into functional vascular wall cells.Considering the strong postnatal influence of the mechanical environment on the func-tioning and remodelling of the vessel as discussed in chapter 2 it might be questioned ifmechanical cues can influence the differentiation of adult mesenchymal stem cells withinthe vascular wall, for example for the purpose of remodelling or repair. It is conceivablethat pericytes in the adventitia can differentiate into smooth muscle cells in response totypical local mechanical forces. For instance, differentiation into cartilage in response tocompressive load has been shown.15 Furthermore it is known that mesenchymal stemcells from bone-marrow circulate in the blood and nestle themselves in various tissues.36
Most interestingly, these circulating mesenchymal stem cells migrate into the suben-dothelial space of the vessel wall and form endothelial cells, smooth muscle cells as wellas fibroblasts.9,12 In this way these precursors contributed to the stabilization of my-ocardial infarction. The fluid flow patterns around such lesions and local forces in thevessel wall could accomplish this differential lineage commitment. For these reasons andin order to contribute to the understanding of lineage commitment of mesenchymal stemcells under influence of mechanical stimuli, I formulated the following research question:
Can mechanical stimuli induce differentiation of mesenchymal stem cells into vascularwall cells?
Explicitly, I hypothesize that the application of fluid shear flow to mesenchymal stemcells results in their differentiation into endothelial cells. Furthermore I hypothesize thatthe application of cyclic strain causes mesenchymal stem cells to differentiate into smoothmuscle cells.
The reader is referred to part II of this thesis, which discusses the experimental ap-proach used to validate these hypotheses and answer the proposed research question.
18
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