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Keywords:

  • mesenchymal stem cells;
  • cell trafficking;
  • chemokines;
  • selectin;
  • integrin

Summary

  1. Top of page
  2. Summary
  3. Mesenchymal stem cells: markers and multipotency
  4. Do MSC occur naturally in the blood?
  5. Method of therapeutic administration of MSC
  6. Tracking of MSC
  7. Mechanism of therapeutic effect of MSC
  8. Mechanism of MSC homing to damaged tissue
  9. The role of selectins in MSC rolling
  10. Chemokine-mediated MSC activation and their role in MSC biology
  11. Integrins and their role in MSC firm-adhesion and transendothelial migration
  12. Concluding remarks
  13. Author's contributions
  14. Financial support
  15. References

The use of adult stem cells to regenerate damaged tissue circumvents the moral and technical issues associated with the use of those from an embryonic source. Mesenchymal stem cells (MSC) can be isolated from a variety of tissues, most commonly from the bone marrow, and, although they represent a very small percentage of these cells, are easily expandable. Recently, the use of MSC has provided clinical benefit to patients with osteogenesis imperfecta, graft-versus-host disease and myocardial infarction. The cellular cues that enabled the MSC to be directed to the sites of tissue damage and the mechanisms by which MSC then exert their therapeutic effect are becoming clearer. This review discusses the relative therapeutic importance of the ability of MSC to differentiate into multiple cell lineages or stimulate resident or attracted cells via a paracrine mode of action. It also reviews recent findings that MSC home to damaged tissues in a similar, but somewhat distinct, manner to that of leucocytes via the utilisation of adhesion molecules, such as selectins and integrins, and chemokines and their receptors in a manner reminiscent of leucocytes trafficking from the blood stream to inflammatory sites.

The regenerative potential of stem cells can only be realised through their efficient delivery to the required site. Recently, encouraging clinical studies have suggested that effective transfer of mesenchymal stem cells (MSC) can lead to therapeutic benefit in various, and ever expanding, pathologies. MSC are also known as marrow stromal cells but the latest nomenclature for these plastic-adherent stem cells, first identified by Friedenstein et al (1976), is multipotent mesenchymal stromal cells (Horwitz et al, 2005), and this denotation importantly retains the popularised acronym MSC (Caplan, 1991). MSC have a fibroblastic morphology and are most commonly derived as a heterogeneous population from bone marrow (BM) after removal of the non-adherent contaminating haematopoietic cells. Nevertheless, MSCs have also been isolated from several other sources, including adipose tissue (Zuk et al, 2002; Lee et al, 2004), scalp tissue (Shih et al, 2005), dermal tissue (Toma et al, 2001) and, albeit controversially, as will be discussed, peripheral blood (Fernandez et al, 1997). In addition to obtaining MSC from these adult tissues, MSC have also been found in various foetal tissues (In't Anker et al, 2003) not exclusive of the placenta (In't Anker et al, 2004), Wharton's jelly of the umbilical cord (Wang et al, 2004), umbilical veins and umbilical cord blood (Erices et al, 2000). The various methods for isolation and culture of MSC has recently been reviewed elsewhere (Beyer Nardi & da Silva Meirelles, 2006); briefly MSC represent a very small fraction of the cells from the afore mentioned sources with only 0·001–0·01% of the 1·077 g/ml density gradient-isolated cells from a BM aspirate belonging to this population (Pittenger et al, 1999). However, as their stem cell nature suggests, they have an outstanding expansive capacity with the possibility of generating a 500-fold increase in cell number over a 12-d period of in vitro culture (Sekiya et al, 2002). MSC isolation by plastic-adherence produces a somewhat heterogeneous population of cells; various methods have been used in an attempt to enrich a purer cell population, the most recent utilising the early embryonic glycolipid antigen SSEA-4 (Gang et al, 2007), which may be utilised further in the future.

Mesenchymal stem cells: markers and multipotency

  1. Top of page
  2. Summary
  3. Mesenchymal stem cells: markers and multipotency
  4. Do MSC occur naturally in the blood?
  5. Method of therapeutic administration of MSC
  6. Tracking of MSC
  7. Mechanism of therapeutic effect of MSC
  8. Mechanism of MSC homing to damaged tissue
  9. The role of selectins in MSC rolling
  10. Chemokine-mediated MSC activation and their role in MSC biology
  11. Integrins and their role in MSC firm-adhesion and transendothelial migration
  12. Concluding remarks
  13. Author's contributions
  14. Financial support
  15. References

Human MSC populations are distinguished from haematopoietic stem cells (HSC) and leucocytes by being negative for the cell surface markers CD14/CD34 and CD45, respectively and via their expression of a combination of other, albeit not specific to MSC, markers whose collective absence/presence defines the cell population (Pittenger et al, 1999). Surface markers that are commonly utilised to establish purified populations of MSCs are Stro-1, CD105, CD166 and, most recently, SSEA-4 (Table I). Stro-1 identifies non-haematopoietic stromal cell precursors in BM cell populations (Simmons & Torok-Storb, 1991). SH-2, SH-3 and SH-4 antibodies raised in mice immunised with MSC recognise, respectively, the antigens CD105 (endoglin), CD73 and SH-4 present on MSC (Haynesworth et al, 1992). Moreover, despite being predominantly associated with endothelial cells (Cheifetz et al, 1992), SH-2, an epitope present on the transforming growth factor-β receptor (Barry et al, 1999), is sold commercially to isolate MSC via positive selection. SB-10 is an antibody that recognises CD166 [activated leucocyte cell adhesion molecule (ALCAM)], this antigen was shown to be present on undifferentiated MSC and disappears following their differentiation to an osteogenic lineage (Bruder et al, 1997, 1998a). SSEA-4 expressing BM cells appeared to represent an MSC population devoid of haematopoietic cells (Gang et al, 2007). In addition, and as will be discussed in more detail later, MSC express a restricted spectrum of adhesion molecules and chemokine receptors that could also, potentially, be used to distinguish this population of cells (Majumdar et al, 2003; Honczarenko et al, 2006).

Table I.   Cell surface marker characterisation of MSC. The common cell surface markers used to select or exclude MSC from a heterogeneous cell population.
MSC selection markersMSC exclusion markers
  1. MSC, mesenchymal stem cells; IL-2R, interleukin-2 receptor; LPSR, lipopolysaccharide receptor; VCAM-1, vascular cell adhesion molecule-1; ALCAM, activated leucocyte cell adhesion molecule.

HLA-ABC (MHC Class I) 
CD90 (Thy-1)HLA-DR (MHC Class II)
CD105 (Endoglin/SH-2)CD4 (T-cell co-receptor)
CD106 (VCAM-1)CD14 (LPSR)
CD73 (SH-3)CD25 (IL-2R)
CD166 (ALCAM)CD45 (Leucocyte antigen)
SSEA-4CD34 (Haematopoietic marker)
Stro-1 
SH-4 

As previously alluded, MSC differentiate to osteogenic cells; their differentiation to bone, cartilage and fat has been extensively characterised and is induced in vitro using specific growth factors and chemical agents (Pittenger et al, 1999). Furthermore, identification of some of the signalling events that are important for lineage-specific adipogenic and osteogenic differentiation has commenced (Bruder et al, 1998a; Jaiswal et al, 2000). Even more interesting is the finding that these cells also appear to be capable of differentiating into cells outside of the ‘mesenchymal remit’. Neural (Kopen et al, 1999) and cardiac cells (Makino et al, 1999), skeletal muscle (Ferrari et al, 1998) and smooth muscle (Yang et al, 1999) have all been derived from MSC and the differentiation conditions required are summarised in a review by Minguell et al (2001). Indeed, in the case of neural cells derived from MSC, the progenitors have been observed to express surface markers of mature neural cells prior to any differentiation, correlating the surface marker expression with their extensive multipotentiality (Tondreau et al, 2004). The multipotentiality of MSC has understandably aroused great interest in the ability of MSC to differentiate into tissue cells in vivo for the therapeutic treatment of various pathologies. Indeed, clinical benefit has been observed following the intravenous (IV) administration of heterogeneous populations of MSC in patients with osteogenesis imperfecta (Horwitz et al, 1999, 2001) and severe acute graft-versus-host disease (Le Blanc et al, 2004), or in myocardial infarction (MI) following the intra-coronary introduction of MSC (Chen et al, 2004). In the first report, when c. 6 × 108 unmanipulated bone-marrow cells from a sibling donor were injected per kg IV into osteogenesis imperfecta patients (n = 3), their growth velocity, bone mineral content and frequency of fracture was markedly improved compared to age-matched control patients (n = 2) and similar to healthy children of the same age (Horwitz et al, 1999, 2001). Further, administration of 1 × 106 allogeneic MSC per kg of a patient's body weight led to a striking clinical recovery in their severe acute graft-versus host-disease of the gut and liver that was refractory to all previous therapy (Le Blanc et al, 2004). Similar to the study by Le Blanc et al MSC were again isolated by plastic adherence from bone-marrow isolates but in the third study mentioned Chen et al administered 8–10 × 109 cells, in the absence of anterior blood flow, approximately 18 d after acute transmural MI into the infarct-related coronary artery following angioplasty. This was consistently performed within 8 h of the initial infarct; the trial consisted of 34 patients receiving MSC therapy and 35 that received saline as controls. As in the previous trials, MSC infusion was well tolerated and led to improved cardiac function at 3 and 6 month follow-up when compared with the sham-treated control patients (Chen et al, 2004).

However, many questions have arisen following just these highlighted clinical studies: what is the best method of delivery of cells, how do the cells get to the sites of injury and via what mechanisms are they targeted and how do they exert their clinical improvement? Additionally, another aspect of utilising MSC in a clinical setting is establishing an effective dose and a relevant dosing regime. Patients with osteogenesis imperfecta who were administered MSC had a temporary increase in growth rate that was briefly faster than the median growth velocity of age- and sex-matched healthy controls (Horwitz et al, 2001). However, their growth, like that of untreated patients, did appear to plateau and it may be that repeated administration of MSC is required for long-term beneficial effect, which will only be identified in clinical trials with a substantial follow-up period.

Do MSC occur naturally in the blood?

  1. Top of page
  2. Summary
  3. Mesenchymal stem cells: markers and multipotency
  4. Do MSC occur naturally in the blood?
  5. Method of therapeutic administration of MSC
  6. Tracking of MSC
  7. Mechanism of therapeutic effect of MSC
  8. Mechanism of MSC homing to damaged tissue
  9. The role of selectins in MSC rolling
  10. Chemokine-mediated MSC activation and their role in MSC biology
  11. Integrins and their role in MSC firm-adhesion and transendothelial migration
  12. Concluding remarks
  13. Author's contributions
  14. Financial support
  15. References

The concept of endogenously produced MSC that circulate in the peripheral blood is contentious; initial studies that reported MSC in the blood have been difficult to replicate. Fernandez et al identified cells in peripheral blood from breast cancer patients mobilised after treatment with granulocyte-colony stimulating factor (G-CSF) or granulocyte-macrophage colony stimulating factor that were negative for CD14, CD34 and CD45 but were positive for the MSC marker CD106, as recognised by the SH-2 and SH-3 monoclonal antibodies (Fernandez et al, 1997). These cells represented 0·63% of the total cells and were found to form adherent, fibroblastic-like populations after a 20 d culture. However, in a similar study, Lazarus et al (1997) collected blood from G-CSF treated cancer patients but there were very few adherent cells, none of which had MSC morphology nor formed bone in vivo. Wexler et al (2003) were also unable to identify MSC in peripheral blood. Whilst some suggest the different results may be attributed to different mobilisation and culture conditions (Roufosse et al, 2004), others suggest that the cells in the primary report by Fernandez et al (1997) may, in fact, have been monocytes (Purton et al, 1998).

Nevertheless, not all attempts to isolate MSC from peripheral blood have been unsuccessful. Circulating fibroblastic-like cells with osteogenic and adipogenic potential that resemble, but are distinguishable from MSC due to the absence of Stro-1 and endoglin, have been found in the blood of four animal species, including human (Kuznetsov et al, 2001). Similarly, Zvaifler et al (2000) isolated fibroblast-like cells expressing CD105 from the peripheral blood of healthy volunteers that were capable of showing features of osteoblasts or adipocytes, after appropriate culture conditions. Moreover, both Mansilla et al (2006) and Wang et al (2006) have reported interesting results regarding the natural presence of MSC in samples of human peripheral blood. Mansilla et al (2006) found significantly higher numbers of MSC in blood samples taken from acute burn patients than in healthy controls and observed that the percentage of MSCs in the blood correlated with the severity of the burn, which the authors propose could be a mechanism in the regeneration process. Wang et al (2006), on the other hand, reported a reduction in the circulating pool of MSC in patients 1-week after MI and suggested that the MSC may have been recruited to the damaged myocardium. There are also several reports of the derivation of MSC from circulating fetal blood or umbilical cord (Ye et al, 1994; Campagnoli et al, 2000, 2001; Erices et al, 2000), and of the presence of MSC in the peripheral blood of other species (Huss et al, 2000; Wu et al, 2003). The two studies by Mansilla et al (2006) and Wang et al (2006) suggest that injury/trauma might evoke the release of MSC; thus systemic administration of MSC might enhance the presence of the circulating MSC, which may ultimately lead to an enhanced therapeutic response if the cells can be efficiently targeted to the damaged tissues.

Method of therapeutic administration of MSC

  1. Top of page
  2. Summary
  3. Mesenchymal stem cells: markers and multipotency
  4. Do MSC occur naturally in the blood?
  5. Method of therapeutic administration of MSC
  6. Tracking of MSC
  7. Mechanism of therapeutic effect of MSC
  8. Mechanism of MSC homing to damaged tissue
  9. The role of selectins in MSC rolling
  10. Chemokine-mediated MSC activation and their role in MSC biology
  11. Integrins and their role in MSC firm-adhesion and transendothelial migration
  12. Concluding remarks
  13. Author's contributions
  14. Financial support
  15. References

Various routes of administration have been used to deliver MSC; systemic delivery circumvents the problems associated with site-specific delivery, such as tissue damage and the unsuitability of delivering multiple doses. Site-specific delivery has the advantage of delivering large numbers of cells directly to the required site; it could be envisaged that this would not be the case for MSC delivered systemically, whereas, in reality, systemically delivered MSC have only been found to home to damaged- and not healthy-tissue. One point to note is that the microenvironment of a solid tumour closely resembles that of an injured tissue; whilst MSC have been shown to inhibit tumour cell proliferation in vitro they had an opposite effect when injected into mice alongside tumour cells (Ramasamy et al, 2006) and this must be an important topic of consideration before MSC are utilised systemically. Due to the inherent differences, understanding which of the two routes of delivery is most suitable is complex and has rarely been performed in parallel studies in the same model. Barbash et al (2003) found systemic delivery of MSC resulted in low levels of cells reaching the heart following MI, compared with efficient translocation of MSC to the infarcted tissue after their intra-ventricular infusion. Nevertheless, more recently, McFarlin et al (2006) demonstrated that, although the number and frequency of MSC delivery was different, fascial wound healing in rats was accelerated with a similar level of magnitude after systemic or local delivery of MSC, indicating that either mechanism of delivery is capable of conveying the beneficial effects of MSC (McFarlin et al, 2006). Successful systemic delivery of MSC is dependent upon efficient homing of the cells to the required site. In this respect, the migration of MSC from the circulation into damaged or pathological tissues leading to therapeutic effects has been documented (Chen et al, 2001; Horwitz et al, 2002; Ortiz et al, 2003; Wu et al, 2003). More importantly, these studies documented the absence of non-specific trafficking of MSC to undamaged tissue, i.e. MSC were selectively recruited only to sites where therapeutic intervention was required. The methods used to track the cells and attempts to optimise this process are outlined later in this review.

Although systemic delivery of MSC is well described, the majority of studies involving MSC transplantation have delivered the cells directly to the tissue of interest, and observations by Rombouts and Ploemacher (2003) and Barbash et al (2003) support this approach. It was noted that, at least in the case of cells derived from the mouse, the ability of MSC to home to BM after transplantation into irradiated mice can be affected by in vitro culture. The authors suggest this could be due to decreased expression of relevant adhesion molecules, which may include, as discussed later, the reduction of chemokine receptor expression in cells that occurs with their extended in vitro culture (Rombouts & Ploemacher, 2003). Furthermore, Barbash et al (2003) observed that MSC intravenously injected in rats was limited by entrapment of the cells in the lung. MSC entrapment in the lung has not been reported in humans and has been successfully circumvented in rats by the use of a vasodilator (Gao et al, 2001). Nevertheless, direct transplantation of MSC circumvents these potential caveats and numerous MSC transplantation studies for the treatment of animal models of disorders as diverse as glaucoma (Yu et al, 2006) and regeneration of periodontal tissue (Yamada et al, 2006), using this method of delivery, exist in the literature. However, the majority of studies have concentrated upon regeneration of the myocardium or neural tissue after infarction/injury. In recent reports, Miyahara et al (2006) transplanted a monolayer of MSC upon post-ischaemic scarred myocardium and observed reversal of the scar tissue and improved cardiac function (Miyahara et al, 2006). Moreover, Hou et al (2007) injected MSC into the myocardial infarct zone and witnessed improved left ventricular function of the rat heart. The interesting aspect of these, and other studies, regards the mechanism of the observed pathological improval following MSC transplant. With the knowledge that MSC are capable of differentiating, in a suitable environment, into, in this case, myocytes in vitro following culture in media containing 5-azacytidine (a demethylating agent) or following culture with myocardial tissue supernatant, it was quite reasonably speculated that differentiated resident MSC replace the damaged tissue. Cell tracking has indeed confirmed that the MSC remain in the damaged tissue, albeit they are capable of a degree of migration (Kopen et al, 1999) and their survival is variable (Pereira et al, 1995; Elzaouk et al, 2006).

Tracking of MSC

  1. Top of page
  2. Summary
  3. Mesenchymal stem cells: markers and multipotency
  4. Do MSC occur naturally in the blood?
  5. Method of therapeutic administration of MSC
  6. Tracking of MSC
  7. Mechanism of therapeutic effect of MSC
  8. Mechanism of MSC homing to damaged tissue
  9. The role of selectins in MSC rolling
  10. Chemokine-mediated MSC activation and their role in MSC biology
  11. Integrins and their role in MSC firm-adhesion and transendothelial migration
  12. Concluding remarks
  13. Author's contributions
  14. Financial support
  15. References

The detection of MSC migration into tissues has been achieved after tissue harvest and subsequent analysis of the samples by various methods. These include the detection of MSC by fluorescent or genetic techniques following transfection of the cells prior to implantation with vectors designed to express fluorescent or other specific markers not present in native host tissue or by the utilisation of sex-mismatched MSC and subsequent chromosomal investigations. It is more favourable, and less invasive, to monitor MSC homing in vivo, but this has been difficult as nuclear scintigraphic tracking, for example, is limited by its poor spatial resolution and radionuclide decay. Nevertheless, the development of more powerful magnetic resonance imaging (MRI) and methods to deliver suitable magnetic particles into cells has, more recently, allowed live cell imaging in vivo with an accurate, albeit not single cell, level of resolution that has validated the approach of introducing MSC via the circulation. Iron-oxide can generate MRI contrast by disturbing the local magnetic field and amplifying the decreased signal intensity, known as T2*. Thus, iron-oxide labelled cells appear as hypointense areas under MRI (Bulte et al, 2001). Iron-oxides have been transfected into MSC using cationic transfection reagents, principally protamine sulphate (Arbab et al, 2004a) or poly-l-lysine (Kraitchman et al, 2003). The presence of iron-oxides within MSC does not affect their viability, their proliferation or, albeit controversially regarding chondrogenic differentiation, their ability to differentiate (Arbab et al, 2004a; Arbab et al, 2004b, 2005; Bulte et al, 2004). As such, they have been used efficiently to track MSC within various tissues (Bos et al, 2004). Arbab et al (2004a) then hypothesised that using the magnetic properties of the iron-oxides might allow the use of a strong external magnet to direct labelled cells to a designated place, such as damaged tissue. Following iron-oxide labelled MSC introduction via the tail vein into rats, MSC migration to, and retention within, the liver was increased when a circular magnet was placed directly over the liver (Arbab et al, 2004a). This gives rise to the possibility of being able to deliver stem cells to a specific site, which may augment the natural homing of MSC to damaged tissue that has previously been documented (Hauger et al, 2006).

Mechanism of therapeutic effect of MSC

  1. Top of page
  2. Summary
  3. Mesenchymal stem cells: markers and multipotency
  4. Do MSC occur naturally in the blood?
  5. Method of therapeutic administration of MSC
  6. Tracking of MSC
  7. Mechanism of therapeutic effect of MSC
  8. Mechanism of MSC homing to damaged tissue
  9. The role of selectins in MSC rolling
  10. Chemokine-mediated MSC activation and their role in MSC biology
  11. Integrins and their role in MSC firm-adhesion and transendothelial migration
  12. Concluding remarks
  13. Author's contributions
  14. Financial support
  15. References

Mesenchymal stem cells have been shown to differentiate into lineage-specific cells under the microenvironment into which they are transplanted; this has been demonstrated by the detection of various markers of the differentiated cells utilising reverse transcription polymerase chain reaction and immunostaining. However, it remains controversial as to whether the upregulation of certain markers constitutes complete cellular differentiation and the assumption of all the pertinent cellular characteristics. Whilst some would consider the expression of, for example, α-smooth muscle actin to be a marker showing that MSC have differentiated into cardiac myocytes, others do not (Dai et al, 2005), and, to our knowledge, the detection of differentiated MSC capable of action potential propagation, surely the hallmark of a mature cardiomyocyte, has only been demonstrated in two of dozens of studies (Li et al, 2006; Pijnappels et al, 2006). Similarly, Choi et al (2006) observed positive immunostaining of hMSC for various neuronal markers after their incubation in neuronal induction media for 24 h but proteomics, DNA microarray analysis and the absence of a voltage-dependent inward current suggested that the cells had little resemblance of true neurones. Other reports suggest only a relatively small number of MSC differentiate at all, and/or they do not fully integrate with host tissue (Noiseux et al, 2006). Further, the observations of Krampera et al (2006), that extended exposure of MSC to neural differentiation media resulted in massive cellular apoptosis and that the differentiation process was reversible upon returning the MSC to basal media provides further strong evidence to question the occurrence of full MSC differentiation. Yu et al (2006) did not observe the differentiation of MSC in their experimental glaucoma model despite the observed reduction in retinal ganglion cell (RGC) death, which they attributed to the detection of increased levels of an RGC survival factor and other cytokines. Similarly, Togel et al (2005) suggests that the improvement in kidney function observed in their rat model of renal failure was due to the presence of MSC altering the resident cell's production of pro-inflammatory to anti-inflammatory cytokines, such that they observed the downregulation of interleukin (IL)-1β, tumour necrosis factor (TNF)-α, interferon-γ and inducible nitric oxide synthase and the upregulation of IL-10, basic fibroblast growth factor, transforming growth factor-β and the anti-apoptotic gene Bcl-2. The extensive immunomodulatory properties of MSC are likely to be important in these settings (Rasmusson, 2006), but a review of this topic is beyond the scope of this review and have been recently considered elsewhere (Stagg, 2007).

Nevertheless, the most compelling evidence is apparent from studies in which MSC-conditioned media has been injected into infarct sites following MI. In these studies, MSC-conditioned media, derived from 12 h of in vitro culture of MSC, limited the number of apoptotic cells and the infarct area in addition to improving left ventricular function. This indicated that secreted factors from MSC were responsible for the observed cardiac improvements after MI (Gnecchi et al, 2005, 2006). Thus, it has been interesting to observe the increased propensity of manuscripts concluding that the beneficial effects of MSC can be attributed to two possible mechanisms of action: both the paracrine mechanism, which has become increasingly plausible, and the in situ differentiation of MSC to replace damaged tissue. A paracrine mode of action is, of course, unsurprising as the beneficial effects of MSC on haematopoiesis have long since been characterised and attributed to soluble factors released from MSC (Weimar et al, 1998).

The dissection of the transcription factors responsible for the differentiation of MSC, as has begun for other stem cells (Ivanova et al, 2006), might enable the production of genetically modified MSC incapable of differentiation into a particular lineage, as has been elegantly performed, using RNA interference, by Xu et al (2006). For instance, cells incapable of differentiating into a cardiac lineage could be transplanted into animal models of myocardial ischaemia and their effect could be compared with the beneficial one imparted by wild-type MSC. Beneficial effects would then be attributable only to a paracrine mechanism of action so long as the relevant effector cytokines, such as vascular endothelial growth factor, are produced in the un-differentiated cells, which could be confirmed in vitro after maintenance of the cells in an environment inducive to cell differentiation, such as co-culture of MSC with cardiomyoctes (Yoon et al, 2005). This would appear to be the major unexplained factor in the field and the delineation would surely substantially extend the understanding of the clinical benefits of MSC.

Mechanism of MSC homing to damaged tissue

  1. Top of page
  2. Summary
  3. Mesenchymal stem cells: markers and multipotency
  4. Do MSC occur naturally in the blood?
  5. Method of therapeutic administration of MSC
  6. Tracking of MSC
  7. Mechanism of therapeutic effect of MSC
  8. Mechanism of MSC homing to damaged tissue
  9. The role of selectins in MSC rolling
  10. Chemokine-mediated MSC activation and their role in MSC biology
  11. Integrins and their role in MSC firm-adhesion and transendothelial migration
  12. Concluding remarks
  13. Author's contributions
  14. Financial support
  15. References

The process of leucocyte homing to specific inflammatory sites in response to inflammatory stimuli is a well characterised sequential process that has been shown to involve selectins, chemokines, integrins and other adhesion molecules (Butcher, 1991; Springer, 1994). HSC are also known to be recruited from the blood vessels into the surrounding tissues in a process largely reliant on similar processes to that of leucocytes (Chute, 2006). As MSC are known to be selectively recruited to damaged tissue it is a fair assumption to suppose that they utilise comparable mechanisms of recruitment. Some obvious differences between the recruitment of MSC and leucocytes/HSC might be that leucocytes employ L- and E-selectin in the initial rolling stage (as described later in this review). L-selectin expression is low or absent on the surface of MSC (although its message is abundant in the cytoplasm) and the role of E-selectin, as yet, has not been fully determined (Ruster et al, 2006). Similarly, with respect to other adhesion molecules on MSC, one obvious absentee is platelet/endothelial cell adhesion molecule 1 (PECAM-1)/CD34, which is reported to play an important role in leucocyte transmigration across the endothelium (Muller et al, 1993). As such, MSC recruitment may occur via a similar mechanism to leucocytes, albeit utilising a distinct set of adhesion molecules.

The role of selectins in MSC rolling

  1. Top of page
  2. Summary
  3. Mesenchymal stem cells: markers and multipotency
  4. Do MSC occur naturally in the blood?
  5. Method of therapeutic administration of MSC
  6. Tracking of MSC
  7. Mechanism of therapeutic effect of MSC
  8. Mechanism of MSC homing to damaged tissue
  9. The role of selectins in MSC rolling
  10. Chemokine-mediated MSC activation and their role in MSC biology
  11. Integrins and their role in MSC firm-adhesion and transendothelial migration
  12. Concluding remarks
  13. Author's contributions
  14. Financial support
  15. References

Ruster et al (2006) recently proposed that selectins play an important role in the trafficking of MSCs. Using intravital microscopy they observed intravenously administered MSC rolling along the walls of vessels in the ear veins of mice; this observation was significantly diminished under the same conditions in P-selectin deficient mice (Ruster et al, 2006). Furthermore, in other experiments in this study a neutralising P-selectin antibody reduced MSC rolling upon human umbilical vein endothelial cells (HUVECs) in an in vitro assay under shear flow conditions. A novel lectin ligand expressed by MSCs was proposed to be the counter-ligand for endothelially expressed P-selectin, as neither P-selectin glycoprotein ligand-1 (PSGL-1) nor the alternative ligand CD24 were present on MSC. These data suggest MSC, like leucocytes, roll upon endothelial cells as the first stage in their recruitment. As regards the other selectins, E- and L-selectins have been reported to be absent or present in low amounts on the surface of MSC and, together with their counterligands, may not be as important as P-selectin in the trafficking of MSC (Bruder et al, 1998b; Pittenger et al, 1999; Ruster et al, 2006). During rolling it is likely that MSC encounter chemokines, which induce various cellular responses via their 7-transmembrane spanning G protein-coupled receptors including integrin activation/upregulation and co-ordinated cell movement.

Chemokine-mediated MSC activation and their role in MSC biology

  1. Top of page
  2. Summary
  3. Mesenchymal stem cells: markers and multipotency
  4. Do MSC occur naturally in the blood?
  5. Method of therapeutic administration of MSC
  6. Tracking of MSC
  7. Mechanism of therapeutic effect of MSC
  8. Mechanism of MSC homing to damaged tissue
  9. The role of selectins in MSC rolling
  10. Chemokine-mediated MSC activation and their role in MSC biology
  11. Integrins and their role in MSC firm-adhesion and transendothelial migration
  12. Concluding remarks
  13. Author's contributions
  14. Financial support
  15. References

The role that chemokines and their receptors play in the targeting of leucocytes to areas of inflammation, infection or injury is well characterised (Miyasaka & Tanaka, 2004). As chemokine receptors are expressed on the cell surface of MSC, and their stimulation has been shown to induce cell migration, it seems likely that they play a similar role in directing MSC. MSC have been shown to express a variety of chemokine receptors (see Table II), and to date CCR1, CCR4, CCR6, CCR7, CCR9, CCR10, CXCR4, CXCR5, CXCR6, CX3CR1 have been detected on human MSC (Wynn et al, 2004; von Luttichau et al, 2005; Sordi et al, 2005; Honczarenko et al, 2006; Ruster et al, 2006) with CCR2, CCR5, CXCR4, CX3CR1 being present on their rat counterparts (Ji et al, 2004). It is not clear why the reported chemokine receptor repertoire of MSC has been inconsistent as the isolation and culture conditions are largely similar (Table II). It may be that the heterogenic nature of a typical MSC population obscures the detection of a distinct receptor repertoire. Alternatively, because the level of expression can be relatively low, the antibodies used may not have been sensitive enough to detect receptor expression. Nevertheless, the functionality of the various chemokine receptors has been demonstrated using conventional in vitro assays of chemokine-mediated MSC migration and chemokine-mediated increases in the intracellular concentration of calcium using an appropriate ligand (Kortesidis et al, 2005). However, chemokine receptor expression has been shown to diminish with in vitro culture such that at passage 16, and above, decreased receptor expression led to a corresponding decrease in chemotactic responsiveness of the cells (Honczarenko et al, 2006). As such, it will be important to define the molecular events governing chemokine receptor expression.

Table II.   Chemokine receptors detected on the cell-surface of hMSC. The chemokine receptors found to be present or absent upon the cell-surface of MSC, as assessed by flow cytometry, are illustrated alongside the MSC source, method of isolation, growth conditions and passage number.
ReferenceChemokine receptor repertoire*Receptors absentMSC sourceIsolation procedureGrowth mediaPassage
  1. MSC, mesenchymal stem cells; DMEM, Dulbecco's modified Eagle's media; α-MEM, α-minimum essential medium; FCS, fetal calf serum; NA, not available.

  2. *Data in parentheses represents the percentage of the MSC cell population that were found to express the given chemokine receptor.

Honczarenko et al (2006)CCR1 (60·0 ± 18·0) CCR7 (67·0 ± 4·0) CCR9 (57·0 ± 4·0) CXCR4 (43·0 ± 13·0) CXCR5 (70·0 ± 14·0) CXCR6 (43·0 ± 7·0)CCR2 CCR3 CCR4 CCR5 CCR6 CCR8 CXCR1 CXCR2 CXCR3 CX3CR1BM from healthy volunteersAdherence of mononuclear cellsDMEM + 10% FCS2
von Luttichau et al (2005)CCR1 (NA) CCR4 (NA) CCR7 (NA) CCR10 (NA) CXCR5 (NA)CXCR3 CXCR4BM from healthy volunteersAdherence of mononuclear cellsDMEM + 10% FCSNA
Sordi et al (2005)CCR1 (1·8 ± 2·7) CCR7 (2·0 ± 2·4) CXCR4 (26·0 ± 2·0) CXCR6 (22·0 ± 5·4) CX3CR1 (20·0 ± 9·0)CCR2 CCR6 CCR8 CCR9 CXCR1 CXCR3 CXCR5 XCR1Commercial source (Cambrex)Unknownα-MEM + 10% FCS≥2
Ruster et al (2006)CCR6 (NA) CXCR4 (1·8 to 26·0)No others examinedBM from hip replacement patientsAdherence of mononuclear cellsDMEM + 20% FCS5–9
Wynn et al (2004)CXCR4 (1·0 to 3·9)No others examinedBM from healthy volunteersAdherence of mononuclear cellsDMEM + 10% FCS1–7

Chemokine-mediated MSC migration has also been demonstrated in vivo. After MI, levels of CXCL12 protein have been observed to rise significantly in the left ventricle of mice. Its expression was restricted to cardiomyocytes and blood vessels in the infarct zone but not in remote areas of the myocardium (Abbott et al, 2004). Abbott et al (2004) administered BM-derived MSC intravenously into mice 48 h after inducing a MI by suture of the left anterior descending coronary artery or, as a control, in sham operated animals. They observed MSC migration within 72 h to the left ventricle only in animals that developed infarcted tissue. The importance of CXCL12 and its receptor, CXCR4, in this migration was confirmed via the administration of a specific CXCR4 receptor antagonist, AMD3100, which significantly inhibited MSC migration to the infarct site. Furthermore, when the myocardium was transduced with an adenoviral vector containing CXCL12, which led to a 2·5-fold increase in CXCL12 expression, MSC detection in the heart was significantly increased. These data suggest that CXCL12 interacting with CXCR4 was critical in the migration of MSC to the infracted heart but was not sufficient to induce homing in the absence of injury (Abbott et al, 2004). In support of these findings, CXCL12 levels in humans have also been observed to rise in patients after MI. In these patients, circulating levels of MSC fluctuated and it is interesting to speculate that the fluctuations were caused by CXCL12-induced recruitment of MSC to the damaged myocardium as part of the body's response to injury (Wang et al, 2006).

An important role for chemokine involvement in mediating MSC migration to the brain is also evident. After ischaemic brain injury, the level of CCL2 was observed to increase significantly in ischaemic brain tissue extract (Wang et al, 2002a). The brain tissue extract was chemotactic for MSC in vitro and this migration was significantly diminished in the presence of a neutralising CCL2 antibody and is thus likely to be mediated by its receptor CCR2, which was found to be expressed on MSC in this study (Wang et al, 2002a). CCL3 and CXCL8 may also be important agents that mediate MSC migration to damaged cerebral tissue (Wang et al, 2002b).

In addition to their role in mediating cell migration, chemokines may also play important autocrine and paracrine roles. CXCL12 promotes the growth, survival and development of MSC (Kortesidis et al, 2005). MSC are known to be able to synthesise this chemokine, which may thus act in an autocrine manner via CXCR4 (Kortesidis et al, 2005). Similarly, the anti-proliferative effects of MSC on T lymphocytes may be via chemokines, such as CCL1, acting in a paracrine manner either on the T lymphocytes or via the recruitment of regulatory T-lymphocytes that subsequently induce T-cell anergy (Batten et al, 2006). Chemokines are also recognised as primary inducers of integrin upregulation following their interaction with their cell surface receptors and various downstream signalling events. Integrins are known to mediate the firm adhesion of leucocytes to endothelial cells and play an important role in their transendothelial migration. It is likely they play a similar role for MSC.

Integrins and their role in MSC firm-adhesion and transendothelial migration

  1. Top of page
  2. Summary
  3. Mesenchymal stem cells: markers and multipotency
  4. Do MSC occur naturally in the blood?
  5. Method of therapeutic administration of MSC
  6. Tracking of MSC
  7. Mechanism of therapeutic effect of MSC
  8. Mechanism of MSC homing to damaged tissue
  9. The role of selectins in MSC rolling
  10. Chemokine-mediated MSC activation and their role in MSC biology
  11. Integrins and their role in MSC firm-adhesion and transendothelial migration
  12. Concluding remarks
  13. Author's contributions
  14. Financial support
  15. References

Mesenchymal stem cells are known to express various integrin molecules and their roles have begun to be elucidated. Pittenger et al embarked on the first study of integrin expression upon MSC and noted the presence of α1, α2, α3, aα, αv, β1, β3 and β4 alongside other adhesion molecules ICAM-1, ICAM-3, VCAM-1, ALCAM and endoglin/CD105 (Pittenger et al, 1999). The presence of β1 integrins is in agreement with Ruster et al (2006) who observed expression of the β1 integrin VLA-4/CD49d on approximately 50% of their MSC population. Neutralising antibodies to this integrin were incubated with MSC to demonstrate that they firmly adhered to endothelial cells, in the presence of shear flow, in a VLA-4 dependent manner. Further, treatment of endothelial cells with a blocking antibody to its counterpart adhesion molecule, namely VCAM-1, similarly decreased MSC adherence. This suggests a dependence upon the VLA-4/VCAM-1 axis for efficient MSC adherence to endothelial cells. Therefore, Ruster et al (2006) demonstrated that MSC are capable of rolling and adhering to the cells that line blood vessels in vitro and in vivo in a P-selectin and VLA-4/VCAM-1 dependent manner (see Fig 1).

image

Figure 1.  Transendothelial migration of mesenchymal stem cells (MSC). The process of MSC transendothelial migration appears to be similar to that of leucocytes and HSC, albeit under the control of specific adhesion molecules elucidated largely by Ruster et al (2006); Segers et al (2006) and Schmidt et al (2006). 1 Has been shown to be inhibited by: a neutralising P-selectin antibody, fucosidase or O-sialoglucoprotein-endopeptidase treatment of MSC, the absence of Ca2+/Mg2+, and significantly reduced in P-selectin −/− mice; 2 is predicted to occur via the interaction of endothelially expressed chemokines interacting with their cognate receptors on MSC, e.g. CXCL12/CXCR4 (see Table II); 3 has been shown to be inhibited by neutralising antibodies to VLA-4 and VCAM-1; 4 is indicated by electron microscopy and MSC trafficking to tissue after systemic administration.

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Mesenchymal stem cells have the capability to cross the blood–brain barrier (BBB) in animal models. When these cells were injected intravenously into the tail veins of rats they were later observed in the brain after artificial cerebral ischaemia (Chen et al, 2001). Similarly, MSC have been shown to migrate to the injured myocardium (Devine et al, 2001; Jiang et al, 2005) so it was not surprising that, more recently, the first microscopic evidence of the ability of MSC to transmigrate across the endothelial barrier was reported (Schmidt et al, 2006). They assembled a co-culture of endothelial cells (derived from differentiated embryonic stem cells) and MSC from human BM aspirates. Without stimulating the cells, MSC showed morphological changes after 30 min that resulted in contact with the endothelium and, after 2 h, subsequent flattening and integration within the endothelial monolayer. These results established that, albeit under static conditions in vitro, MSC could make the efficient cell–cell contact required to commence the transmigration of endothelial cells. These data were supported by further experiments that involved cannulating the aorta of mice and perfusing MSC with constant recirculation for up to 120 min. After this time, the hearts were analysed and it was revealed that the endothelial tight junctions had been abolished and MSC had become associated with the endothelial cells. Electron microscopy revealed that, even after 30 min of perfusion, approximately 30% of the perfused MSC had transmigrated across the endothelium – this percentage rose to 50% after 60 min but remained constant thereafter (Schmidt et al, 2006). Elsewhere, transmigration across the endothelium into cardiac tissue of MSC injected into the ventricular cavity has been observed in rats pre-treated with TNF-α or in rats 24 h after ischaemia (30 min)-reperfusion injury (Segers et al, 2006). Whilst the adhesion molecules and mechanisms involved in MSC diapedesis are, as yet, unclear, the molecules identified to date that regulate the various other stages of MSC recruitment from the blood stream to the surrounding tissues, summarised in Fig 1, have furthered our understanding of this process and extended our appreciation of the ability to administer MSC systemically as a potential therapeutic strategy.

Concluding remarks

  1. Top of page
  2. Summary
  3. Mesenchymal stem cells: markers and multipotency
  4. Do MSC occur naturally in the blood?
  5. Method of therapeutic administration of MSC
  6. Tracking of MSC
  7. Mechanism of therapeutic effect of MSC
  8. Mechanism of MSC homing to damaged tissue
  9. The role of selectins in MSC rolling
  10. Chemokine-mediated MSC activation and their role in MSC biology
  11. Integrins and their role in MSC firm-adhesion and transendothelial migration
  12. Concluding remarks
  13. Author's contributions
  14. Financial support
  15. References

The current interest and enthusiasm in MSC appears to be unbounded; this is a result of the historic observation that it was possible to isolate a population of stem cells with the ability to be transplanted in an autologous or allogeneic manner capable of mediating the improvement of damaged tissue without inducing an immune response. With publications on this subject appearing daily, it is a fast-moving field in which many of the unanswered questions raised above, such as the mechanism of MSC recruitment and the means by which MSC exert their beneficial effects, are likely to be answered in the near future. This information will no doubt stimulate the progression of small, un-blinded clinical trials into larger, multi-centric, randomised, double-blinded, placebo-matched trials with the hope that the results will indicate that the administration of MSC will be a revolutionary advance in the therapeutic intervention of various diseases or tissue damage.

References

  1. Top of page
  2. Summary
  3. Mesenchymal stem cells: markers and multipotency
  4. Do MSC occur naturally in the blood?
  5. Method of therapeutic administration of MSC
  6. Tracking of MSC
  7. Mechanism of therapeutic effect of MSC
  8. Mechanism of MSC homing to damaged tissue
  9. The role of selectins in MSC rolling
  10. Chemokine-mediated MSC activation and their role in MSC biology
  11. Integrins and their role in MSC firm-adhesion and transendothelial migration
  12. Concluding remarks
  13. Author's contributions
  14. Financial support
  15. References
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