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Comparative adhesive and migratory properties of mesenchymal stem cells from different tissues
Abstract
Background:
Mesenchymal stem cells (MSC) are used in therapy, often by injection into the blood.
Objective:
We aimed to compare the adhesive and migratory properties of MSC from umbilical cords (UCMSC), bone marrow (BMMSC) or trabecular bone (TBMSC), which might influence delivery to injured tissue.
Methods:
MSC were perfused through glass capillaries coated with matrix proteins, collagen or fibronectin, or albumin. Adherent cells were counted microscopically and their spreading analysed over time. MSC migration through 8 μm pore filters coated with the same proteins was analysed.
Results:
The number of MSC adhering to collagen was greater than fibronectin, decreased as wall shear rate increased from 17 to 70 s-1, and was in the order UCMSC>BMMSC>TBMSC. Conversely, spreading was more effective on fibronectin and was in the order BMMSC>TBMSC≥UCMSC. Migration was promoted by coating the lower surface of filters with either matrix protein, with UCMSC migrating more efficiently than BMMSC.
Conclusions:
MSC show origin-dependent variations in their efficiency of capture from flow and subsequent spreading or ability to migrate on matrix proteins. UCMSC showed most efficient capture from flow, which was followed by less spreading, but more rapid migration. These responses might be associated with more effective delivery from the circulation into damaged tissue.
... Dans notre travail axé sur l'ingénierie tissulaire osseuse et en nous basant sur ces résultats, nous avons choisi de privilégier des conditions de culture normoxiques pour nos CSM-PD.• Le support de culture : le collagène Différentes études se sont focalisées sur l'intérêt d'utiliser le collagène pour améliorer la culture primaire et le potentiel de différenciation des CSM(199,(304)(305)(306)(307)(308)(309)(310)(311)(312) et plus particulièrement des CSM-PD(269,280,313). Le collagène est en effet un composant majoritaire de la phase organique du tissu osseux ainsi que du tissu pulpaire.Réaliser un coating des plaques de culture avec du collagène permet en effet de recréer un microenvironnement favorable et proche de la MEC physiologique(268).Ainsi, il a été démontré que le collagène permet d'améliorer les capacités d'adhésion et de migration ou la prolifération des CSM de cordon ombilical(304), des CSM issues du muscle(306), des CSM dérivées de cellules induites pluripotentes (iPS) (311) et des CSM-MO(304,305,310,312). ...
... Dans notre travail axé sur l'ingénierie tissulaire osseuse et en nous basant sur ces résultats, nous avons choisi de privilégier des conditions de culture normoxiques pour nos CSM-PD.• Le support de culture : le collagène Différentes études se sont focalisées sur l'intérêt d'utiliser le collagène pour améliorer la culture primaire et le potentiel de différenciation des CSM(199,(304)(305)(306)(307)(308)(309)(310)(311)(312) et plus particulièrement des CSM-PD(269,280,313). Le collagène est en effet un composant majoritaire de la phase organique du tissu osseux ainsi que du tissu pulpaire.Réaliser un coating des plaques de culture avec du collagène permet en effet de recréer un microenvironnement favorable et proche de la MEC physiologique(268).Ainsi, il a été démontré que le collagène permet d'améliorer les capacités d'adhésion et de migration ou la prolifération des CSM de cordon ombilical(304), des CSM issues du muscle(306), des CSM dérivées de cellules induites pluripotentes (iPS) (311) et des CSM-MO(304,305,310,312). ...
... Dans notre travail axé sur l'ingénierie tissulaire osseuse et en nous basant sur ces résultats, nous avons choisi de privilégier des conditions de culture normoxiques pour nos CSM-PD.• Le support de culture : le collagène Différentes études se sont focalisées sur l'intérêt d'utiliser le collagène pour améliorer la culture primaire et le potentiel de différenciation des CSM(199,(304)(305)(306)(307)(308)(309)(310)(311)(312) et plus particulièrement des CSM-PD(269,280,313). Le collagène est en effet un composant majoritaire de la phase organique du tissu osseux ainsi que du tissu pulpaire.Réaliser un coating des plaques de culture avec du collagène permet en effet de recréer un microenvironnement favorable et proche de la MEC physiologique(268).Ainsi, il a été démontré que le collagène permet d'améliorer les capacités d'adhésion et de migration ou la prolifération des CSM de cordon ombilical(304), des CSM issues du muscle(306), des CSM dérivées de cellules induites pluripotentes (iPS) (311) et des CSM-MO(304,305,310,312). ...
Face à des pertes de substance osseuse de grande étendue dépassant les capacités naturelles de régénération de l’os natif, aucune solution thérapeutique, qu’il s’agisse de l’os autologue ou du l’utilisation de substituts osseux, n’est à ce jour pleinement satisfaisante, en particulier par l’absence de cellularisation du substitut et de sa vascularisation. La création d’un substitut osseux pré vascularisé en ingénierie tissulaire osseuse pourrait dépasser ces limites et autoriser une régénération tissulaire osseuse de qualité, favorisée par une vascularisation du greffon. Notre projet visait à développer ce substitut osseux pré vascularisé en combinant un biomatériau associant éponge de collagène et hydrogel d’alginate enrichi en hydroxyapatite (HA) et colonisé par des cellules souches mésenchymateuses de la pulpe dentaire (CSM-PD). La première partie de notre travail a permis de préciser les conditions environnementales pouvant être choisies pour l’utilisation des CSM-PD en ingénierie tissulaire osseuse, telles que la densité d’ensemencement, la teneur en glucose de l’environnement et le choix de nanovecteurs. Nous avons mis en lumière que les CSM-PD n’étaient pas impactées d’une part par des nanoliposomes de lécithine de colza (utilisables pour la vectorisation de molécules bioactives), et d’autre part qu’une faible teneur en glucose pouvait induire une différenciation ostéogénique de façon aussi performante qu’une forte teneur en glucose. La deuxième partie de notre travail a consisté en l’ensemencement des CSM-PD au sein de notre biomatériau innovant associant éponge de collagène et hydrogel d’alginate enrichi en HA. Nous avons observé une potentialisation des capacités de colonisation des biomatériaux et de la différenciation ostéogénique des CSM-PD et mis en évidence la présence de précurseurs de cellules endothéliales-like. Ces résultats encourageants mettent en lumière le potentiel d’utilisation des CSM-PD combinées à un biomatériau combinant collagène et hydrogel d’alginate enrichi en HA pour l’ingénierie osseuse pré-vascularisée.
... Collagen is one of the ECM proteins that play a part in cell activity. Collagen also plays essential roles in adhesion, migration, and ostoegenic differentiation [67][68]. Increased cellular collagen in growth and ostegenic culture of DP-MSCs with L. rhamnosus EPS is suggested to be a pomising regenerative inducer for preconditioning studies. ...
... Increased cellular collagen in growth and ostegenic culture of DP-MSCs with L. rhamnosus EPS is suggested to be a pomising regenerative inducer for preconditioning studies. It was reported that function of gingival and palatal MSCs was impaired in oral microbiological imbalance [68][69][70][71]. Up to now, it was discussed that delayed wound healing processed was attributed to the result of imbalanced flora [72][73][74][75]. ...
Abstract Among mesenchymal stem cell (MSCs) sources, the most interesting ones in tissue engineering and regenerative medicine are oromaxillofacial tissue. Of these, dental pulp derived (DP-MSCs) MSCs are part of interest. The substantial expansion of MSCs in vitro is a requirement site to achieve adequate cell numbers for cell-based therapy. However, the limited proliferation of MSCs diminishes with long term cell culture amplification. Therefore, natural agents are being investigated to increase proliferation and/or differentiation. Lactobacillus rhamnosus draws attention with the expolysaccharides (EPSs) it produces. EPSs have been attributed to have a significant role in probiotic activity; including immunomodulatory, antitumor, cholesterol lowering, biofilm reducing, antioxidant, antiallergic, and wound healing effects. However, there is incompetent knowledge discussing their effect on DP-MSCs. In this study we aimed to demonstrate the induced proliferation, differentiation and cellular collagen secretion of DP-MSCs in response to the L. rhamnosus E9 EPSs. 1000 μg/mL EPSs was determined as the effective concentration by using a real-time monitoring system. L. rhamnosus E9 EPS was able to accelarate ostegenic differentiation and secretion of cellular collagen. Indeed, EPS was able to decreased the calcium granules that occurs due to long term cultivation. By consideration of inducing proliferation, osteogenic differentiation and secretion of cellular collagen, L. rhamnosus EPS could be considered as good candidate for preconditioning agent and/or scaffold material.
... 42Moreover, the adhesive and migratory properties of MSCs may influence their fate and efficacy when injected into a disease environment. Alanazi and colleagues recently found that UC-MSCs showed the most efficient capture from flow and less spreading but more rapid migration ability compared with BMSCs, which might be associated with more effective delivery from the circulation into damaged tissue.60 They further investigated whether cell size affected the level of adhesion by measuring the diameter of adherent cells from the images taken immediately at the end of a perfused bolus.The average diameters were less than those of the original samples, and the cell diameter of UC-MSCs was approximately 21.2 µm at the original sample and 17.8 µm when adherent to collagen, which was smaller than that of BMSCs.60 Cell behaviour differs between in vivo and in vitro cultures with a conventional two-dimensional culture system in terms of phenotypic marker expression, homing and migratory capacity.61,62 ...
... Alanazi and colleagues recently found that UC-MSCs showed the most efficient capture from flow and less spreading but more rapid migration ability compared with BMSCs, which might be associated with more effective delivery from the circulation into damaged tissue.60 They further investigated whether cell size affected the level of adhesion by measuring the diameter of adherent cells from the images taken immediately at the end of a perfused bolus.The average diameters were less than those of the original samples, and the cell diameter of UC-MSCs was approximately 21.2 µm at the original sample and 17.8 µm when adherent to collagen, which was smaller than that of BMSCs.60 Cell behaviour differs between in vivo and in vitro cultures with a conventional two-dimensional culture system in terms of phenotypic marker expression, homing and migratory capacity.61,62 ...
Transplantation of stem cells is a promising, emerging treatment for cardiovascular diseases in the modern era. Mesenchymal stem cells (MSCs) derived from the umbilical cord are one of the most promising cell sources because of their capacity for differentiation into cardiomyocytes, endothelial cells and vascular smooth muscle cells in vitro/in vivo. In addition, umbilical cord-derived MSCs (UC-MSCs) secrete many effective molecules regulating apoptosis, fibrosis and neovascularization. Another important and specific characteristic of UC-MSCs is their low immunogenicity and immunomodulatory properties. However, the application of UC-MSCs still faces some challenges, such as low survivability and tissue retention in a harmful disease environment. Gene engineering and pharmacological studies have been implemented to overcome these difficulties. In this review, we summarize the differentiation ability, secretion function, immunoregulatory properties and preclinical/clinical studies of UC-MSCs, highlighting the advantages of UC-MSCs for the treatment of cardiovascular diseases.
... Due to their rapid proliferation, strong adhesion, and migration capabilities, hUC-MSCs often form millimetre-sized aggregates before cell harvesting when microcarriers are poorly suspended. The formation of such aggregates introduces mass transfer limitations that impact cell viability and make cell digestion and isolation challenging (Ferrari et al. 2012;Alanazi et al. 2019;Zeddou et al. 2010; Luo et al. 2014). Increasing the impeller speed of spinner asks can enhance the degree of aggregate suspension and reduce the maximum size of aggregates (Jossen et al. 2016). ...
Human umbilical cord-derived mesenchymal stem cells (hUC-MSCs) are widely utilized in the field of regenerative medicine, particularly in cell therapy and tissue engineering. However, their application and development are hindered by limitations in cell expansion efficiency. When hUC-MSCs are cultured in suspension while attached to microcarriers, they tend to aggregate, and adjusting operational parameters fails to resolve the conflict between shear and aggregation. The microenvironment created by the original impeller is not suitable for cell growth. In this study, computational fluid dynamics (CFD) simulations were employed to investigate the flow field structure generated by the original impeller in the commercial spinner flasks. It was found that the flow field structure were unsuitable for the expansion of cells prone to aggregate. Consequently, a new impeller was designed to alter the flow field structure, aimed to promote aggregate suspension while maintaining a similar shear rate at the same rotation speed. Compared to the original impeller, the newly designed impeller demonstrated significant improvements. Notably, it reduced the size of aggregates, increased maximum cell density, and preserved cell stemness during cell expansion. Combining simulation results with experimental data, this study reveals that the degree of suspension of aggregates played a critical role in determining aggregate size. Additionally, the level of cell stemness was determined by modulating shear rate and the degree of aggregate suspension.
... The fate of cells injected into the circulation and the capacity of the cells to promote repair and immune regulation at the site of damage are affected by both properties. USMSC appears to adhere and migrate more rapidly from flow than BMMSC, but BMMSC surpasses USMSC in terms of spreading kinetics [9]. More vitally, we have Fig. 1 Classification of registered stem cell-related clinical trials in liver fibrosis or cirrhosis. ...
Cost-effective treatment strategies for liver fibrosis or cirrhosis are limited. Many clinical trials of stem cells for liver disease shown that stem cells might be a potential therapeutic approach. This review will summarize the published clinical trials of stem cells for the treatment of liver fibrosis/cirrhosis and provide the latest overview of various cell sources, cell doses, and delivery methods. We also describe the limitations and strengths of various stem cells in clinical applications. Furthermore, to clarify how stem cells play a therapeutic role in liver fibrosis, we discuss the molecular mechanisms of stem cells for treatment of liver fibrosis, including liver regeneration, immunoregulation, resistance to injury, myofibroblast repression, and extracellular matrix degradation. We provide a perspective for the prospects of future clinical implementation of stem cells.
... It has been further demonstrated that even the tissue origin of MSCs can influence their efficiency to spread, as well as their ability to migrate on matrix proteins. MSCs from different origins might display different adhesive properties [35]. In line with this observation, Sheriff et al. reported that umbilical cord MSCs (ucMSCs) caused platelet activation and had a greater ability to adhere to extracellular matrix proteins than bmMSCs [36]. ...
Purpose of Review
Mesenchymal stem or stromal cells (MSCs) are a heterogeneous group of non-hematopoietic, multipotent progenitor cells that share a multitude of similarities with fibroblasts and other cell types. The question of how to distinguish MSCs from other cell types lacks a definitive answer. Here, we discuss the differences and similarities between MSCs and other cell types with regard to their cellular characteristics, origins, and immune interactions. Furthermore, the distribution of MSCs and their regional effects within the body are outlined.
Recent Findings
We give insights into the current methods to track MSC distribution and homing within the body and review the mode of action of MSC therapy. The local and systemic effects of MSC therapy and possible alternatives such as the application of MSC-derived vesicles are also discussed.
Summary
Important mechanisms regarding MSC therapy are not yet completely understood. This review provides insights into the current methods used to better understand these issues, which is a prerequisite for the clinical application of MSC-based therapies.
... COL1 and FN are ECM proteins that contribute to cell activity and involve in regulating DPSCs. They play crucial roles in adhesion, migration, and osteogenic differentiation of stem cells (Alanazi et al., 2019;Kang et al., 2017;Mittag et al., 2012;Zheng et al., 2019). COL1 as collagen sponges is a natural material has the advantages of its biocompatibility, abundance, and its highly porous. ...
Collagen 1 (COL1) and fibronectin (FN) are extracellular matrix (ECM) proteins that contribute in cell activity and involve in regulating dental pulp cells (DPCs). The purpose of this study was to investigate the effect of COL1 and FN on the behavior of DPCs. Here, DPCs were grown under three different conditions: COL1 coating, FN coating and control group without coating. The proliferation and differentiation of DPCs were investigated. DPCs in osteogenic media were able to differentiate into osteoblastic phenotype. The morphological analysis revealed no obvious difference on the shape of cells. Cells had spread well on both coated and non‐coated culture plates with slightly more spreading in the coated plates after 24 h. The MTT analysis did not demonstrate a significant difference at 1 and 3 hours among the groups, but interestingly, the analysis disclosed more cells on the coated plates after longer cultures, which indicated a higher proliferative capacity in response to COL1 and FN. RT‐PCR, Western Blotting and mineralization assays did not reveal significant differences between the coated and non‐coated surfaces in relation to osteogenic differential potential. Our data suggested that the surface coating of COL1 and FN were able to promote cellular proliferation and the osteogenic differentiation tendency of DPCs was also observed in vitro. This article is protected by copyright. All rights reserved.
... Therefore, the number and survival time of transplanted stem cells are the critical factors to reverse PF. We suggest that the different effects may be due to differences in stem cell sources, resulting in diverse cell properties and various outcomes [51,52]. Next, we will examine and compare the efficacy of transplantation of bone marrow MSCs or adipose tissue MSCs in this animal model of BLM-induced one-sided and severe PF. ...
Pulmonary fibrosis (PF) is a progressive and irreversible condition with various causes, and no effective treatment has been found to rescue fibrotic lungs. Successful recovery from PF requires inhibiting inflammation, promoting collagen degradation and stimulating alveolar regeneration. Human umbilical mesenchymal stem cells (HUMSCs) not only regulate immune responses but also synthesize and release hyaluronan to improve lung regeneration. This study investigated the feasibility of HUMSC engraftment into rats with bleomycin (BLM)-induced PF to explore HUMSC therapeutic effects/outcomes.
Methods: A unique BLM-induced left-lung-dominated PF animal model was established. Rats were transplanted with low-dose (5×10⁶) or high-dose (2.5×10⁷) HUMSCs on Day 21 after BLM injection. Combinations in co-culture of pulmonary macrophages, fibroblasts, HUMSCs treated with BLM and the same conditions on alveolar epithelia versus HUMSCs were evaluated.
Results: Rats with high-dose HUMSC engraftment displayed significant recovery, including improved blood oxygen saturation levels and respiratory rates. High-dose HUMSC transplantation reversed alveolar injury, reduced cell infiltration and ameliorated collagen deposition. One month posttransplantation, HUMSCs in the rats' lungs remained viable and secreted cytokines without differentiating into alveolar or vascular epithelial cells. Moreover, HUMSCs decreased epithelial-mesenchymal transition in pulmonary inflammation, enhanced macrophage matrix-metallopeptidase-9 (MMP-9) expression for collagen degradation, and promoted toll-like receptor-4 (TLR-4) expression in the lung for alveolar regeneration. In coculture studies, HUMSCs elevated the MMP-9 level in pulmonary macrophages, released hyaluronan into the medium and stimulated the TLR-4 quantity in the alveolar epithelium.
Principal Conclusions: Transplanted HUMSCs exhibit long-term viability in rat lungs and can effectively reverse rat PF.
... However, these results were observed in animals and have not been confirmed in clinical studies, despite arguments confirming their predominance [108]. Some studies have confirmed the superiority of UC-MSCs over BM in terms of secretome [109] and migration speed [110]. An evaluation of the expression of 106 cytokines between UCB-derived and UC-derived MSCs showed differences in the excretion of the following: TSP-1, TSG-14, TIMP-1, IL-8, IL-6, CXCL1, GIF, and IGFBP3. ...
The aim of this narrative review is to report on the current knowledge regarding the clinical use of umbilical cord blood (CB) based on articles from PubMed and clinical trials registered on ClinicalTrials.gov. An increasing amount of evidence suggests that CB may be used for both early diagnostics and treatment of cerebral palsy. The acidity of CB and its biochemical parameters, including dozens of cytokines, growth factors, and other metabolites (such as amino acids, acylcarnitines, phosphatidylcholines, succinate, glycerol, 3-hydroxybutyrate, and O-phosphocholine) are predictors of future neurodevelopment. In addition, several clinical studies confirmed the safety and efficacy of CB administration in both autologous and allogeneic models, including a meta-analysis of five clinical trials involving a total of 328 participants. Currently, nine clinical trials assessing the use of autologous umbilical CB in children diagnosed with hypoxic-ischemic encephalopathy or cerebral palsy are in progress. The total population assessed in these trials exceeds 2500 patients.
Liver fibrosis is a reversible damage-repair response, the pathological features of which mainly include damage to hepatocytes, sinusoid capillarization, hepatic stellate cells activation, excessive accumulation of extracellular matrix and inflammatory response. Although some treatments (including drugs and stem cell therapy) for these pathological features have been shown to be effective, more clinical trials are needed to confirm their effectiveness. In recent years, nanomaterials-based therapies have emerged as an innovative and promising alternative to traditional drugs, being explored for the treatment of liver fibrosis diseases. Natural nanomaterials (including extracellular vesicles) and synthetic nanomaterials (including inorganic nanomaterials and organic nanomaterials) are developed to facilitate drug targeting delivery and combination therapy. In this review, the pathological features of liver fibrosis and the current anti-fibrosis drugs in clinical trials are briefly introduced, followed by a detailed introduction of the therapeutic nanoagents for the precise delivery of anti-fibrosis drugs. Finally, the future development trend in this field is discussed.
During the ex vivo expansion of umbilical cord-derived mesenchymal stem cells (hUCMSCs) in a stirred tank bioreactor, the formation of cell–microcarrier aggregates significantly affects cell proliferation and physiological activity, making it difficult to meet the quantity and quality requirements for in vitro research and clinical applications. In this study, computational fluid dynamic (CFD) simulations were used to investigate the effect of an impeller structure in a commercial spinner flask on flow field structure, aggregate formation, and cellular physiological activity. By designing a modified impeller, the aggregate size was reduced, which promoted cell proliferation and stemness maintenance. This study showed that increasing the stirring speed reduced the size of hUCMSC-microcarrier aggregates with the original impeller. However, it also inhibited cell proliferation, decreased activity, and led to spontaneous differentiation. Compared to low stirring speeds, high stirring speeds did not alter the radial flow characteristics and vortex distribution of the flow field, but did generate higher shear rates. The new impeller’s design changed the flow field from radial to axial. The use of the novel impeller with an increased axial pumping rate (Q z ) at a similar shear rate compared to the original impeller resulted in a 43.7% reduction in aggregate size, a 37.4% increase in cell density, and a better preservation of the expression of stemness markers (SOX2, OCT4 and NANOG). Increasing the Q z was a key factor in promoting aggregate suspension and size reduction. The results of this study have significant implications for the design of reactors, the optimisation of operating parameters, and the regulation of cellular physiological activity during MSC expansion.
Graphical Abstract
Mesenchymal stem cells (MSCs) are a population of primary and non-specialized cells, which can be isolated from various tissues. Currently, MSCs are key players in cellular therapy and regenerative medicine. However, the possibility of using MSCs in the treatment of many diseases needs to be preceded, though, by in-depth analysis of their properties, especially by determining the mechanism of tissue homing as well as the mechanism, due to which cells contribute to tissue regeneration. This review is intended to present information on recent findings regarding the mechanism of recruitment and tissue homing by MSCs and discuss current hypotheses for how MSCs can reach target tissues.
El libro se encuentra distribuido en tres grandes capítulos, el primero de ellos relacionado con aspectos básicos como la clasificación de la Biotecnología, el estudio de las fermentaciones microbianas, las enzimas y sus métodos de purificación, el estudio de los ácidos nucleicos y de las células madre. En el segundo capítulo, nos hemos enfocado en las herramientas de la Biotecnología como los procesos de inmovilización de enzimas, la producción de proteínas recombinantes y el estudio de los péptidos bioactivos. Finalmente, en el capítulo tres, nos enfocamos en las aplicaciones de la Biotecnología Roja o Médica, como las herramientas biotecnológicas para la producción de vacunas, las aplicaciones clínicas de las células madre, la aplicación clínica de los biomateriales, aplicaciones de la metagenómica y la metabolómica, finalizando con la importancia del estudio de las plantas medicinales en general y la actividad biológica de plantas del género Tabebuia, que ha sido ampliamente estudiada ...
Increasing the migratory capacity of the implanted mesenchymal stem cells (MSCs) is a major challenge in developing successful cell transplantation therapies. Nevertheless, the regulatory factors involved in the migration of BMMSCs remain largely unknown. In this study, we studied the role of the peripherin (PRPH) gene in regulating the ability of Wuzhishan mini pig (WZSP) BMMSCs to migrate in vitro. Four different shRNA vectors directed against PRPH were designed and transfected into BMMSCs. The vector with the best interference effect was chosen to be used in the following experiments. The expression level of PRPH in BMMSCs was determined by quantitative real-time PCR and western blot analysis. The migration capacity of the BMMSCs was estimated using a scratch assay, a transwell in vitro migration model assay, and filamentous actin staining. The results showed that shRNA-mediated knockdown of the expression of the PRPH gene in BMMSCs reduced the ability of these cells to migrate. Overall, these results illustrate that the PRPH gene regulates the migration of BMMSCs in the WZSP.
Coronary heart disease is one of the leading causes of death in the United States. Recent advances in stem cell biology have led to the development and engineering of human pluripotent stem cell (hPSC)-derived cardiac cells and tissues for application in cellular therapy and cardiotoxicity studies. Initial studies in this area have largely focused on improving differentiation efficiency and maturation states of cardiomyocytes. However, other cell types in the heart, including endothelial and stromal cells, play crucial roles in cardiac development, injury response, and cardiomyocyte function. This review discusses recent advances in differentiation of hPSCs to cardiac stromal cells, identification and classification of cardiac stromal cell types, and application of hPSC-derived cardiac stromal cells and tissues containing these cells in regenerative and drug development applications.
We investigated the adhesive behaviour of mesenchymal stem cells (MSC) in blood, which might influence their fate when infused as therapy. Isolated human bone marrow (BM) or umbilical cord (UC) MSC adhered efficiently from flow to the matrix proteins, collagen or fibronectin, but did not adhere to endothelial selectins. However, when suspended in blood, BMMSC no longer adhered to collagen, while UCMSC adhered along with many aggregated platelets. Neither MSC adhered to fibronectin from flowing blood, although the fibronectin surface did become coated with a platelet monolayer. UCMSC induced platelet aggregation in platelet rich plasma, and caused a marked drop in platelet count when mixed with whole human or mouse blood in vitro, or when infused into mice. In contrast, BMMSC did not activate platelets or induce changes in platelet count. Interestingly, isolated UCMSC and BMMSC both adhered to pre-deposited platelets. The differences in behaviour in blood were attributable to expression of podoplanin (an activating ligand for the platelet receptor CLEC-2), which was detected on UCMSC, but not BMMSC. Thus, platelets were activated when bound to UCMSC, but not BMMSC. Platelet aggregation by UCMSC was inhibited by recombinant soluble CLEC-2, and UCMSC did not cause a reduction in platelet count when mixed with blood from mice deficient in CLEC-2. We predict that both MSC would carry platelets in the blood, but their interaction with vascular endothelium would depend on podoplanin-induced activation of the bound platelets. Such interactions with platelets might target MSC to damaged tissue, but could also be thrombotic. This article is protected by copyright. All rights reserved.
Mesenchymal stem cells (MSCs) are promising candidates for adult cell therapies in regenerative medicine. To fully exert their potential, efficient homing and migration toward lesion sites play an important role. Local transplantation deposits MSC in spatial proximity to the lesion, but often requires invasive procedures. Systemic administration routes are favored, but require the targeted extravasation of the circulating MSC at the site of injury. Transplanted MSC can indeed leave the blood flow and transmigrate through the endothelial barrier, and reach the lesion site. However, the underlying processes are not completely dissolved yet. Recent in vitro and in vivo research identified some key molecules scattered light on the extravasation mechanism. This review provides a detailed overview over the current knowledge of MSC transendothelial migration. We use the leukocyte extravasation process as a role model to build a comprehensive concept of MSC egress mechanisms from the blood stream and identified relevant similarities as well as important differences between the extravasation mechanisms. Stem Cells 2017;35:1446-1460.
In addition to being multi-potent, mesenchymal stem cells (MSCs) possess immunomodulatory functions that have been investigated as potential treatments in various immune disorders. MSCs can robustly interact with cells of the innate and adaptive immune systems, either through direct cell–cell contact or through their secretome. In this review, we discuss current findings regarding the interplay between MSCs and different immune cell subsets. We also draw attention to the mechanisms involved.
Mesenchymal stromal cells (MSC) are tissue-resident stromal cells capable of modulating immune responses, including leukocyte recruitment by endothelial cells (EC). However, the comparative potency of MSC from different sources in suppressing recruitment, and the necessity for close contact with endothelium remain uncertain, although these factors have implications for use of MSC in therapy. We thus compared the effects of MSC isolated from bone marrow, Wharton’s jelly, and trabecular bone on neutrophil recruitment to cytokine-stimulated EC, using co-culture models with different degrees of proximity between MSC and EC. All types of MSC suppressed neutrophil adhesion to inflamed endothelium but not neutrophil transmigration, whether directly incorporated into endothelial monolayers or separated from them by thin micropore filters. Further increase in the separation of the two cell types tended to reduce efficacy, although this diminution was least for the bone marrow MSC. Immuno-protective effects of MSC were also diminished with repeated passage; with BMMSC, but not WJMSC, completing losing their suppressive effect by passage 7. Conditioned media from all co-cultures suppressed neutrophil recruitment, and IL-6 was identified as a common bioactive mediator. These results suggest endogenous MSC have a homeostatic role in limiting inflammatory leukocyte infiltration in a range of tissues. Since released soluble mediators might have effects locally or remotely, infusion of MSC into blood or direct injection into target organs might be efficacious, but in either case, cross-talk between EC and MSC appears necessary.
Mesenchymal stem cells (MSCs) have anti-inflammatory and immunosuppressive properties and may be useful in the therapy of diseases such as arteriosclerosis. MSCs have some ability to traffic into inflamed tissues, however to exploit this therapeutically their migratory mechanisms need to be elucidated. This study examines the interaction of murine MSCs (mMSCs) with, and their migration across, murine aortic endothelial cells (MAECs), and the effects of chemokines and shear stress. The interaction of mMSCs with MAECs was examined under physiological flow conditions. mMSCs showed lack of interaction with MAECs under continuous flow. However, when the flow was stopped (for 10 min) and then started, mMSCs adhered and crawled on the endothelial surface, extending fine microvillous processes (filopodia). They then spread extending pseudopodia in multiple directions. CXCL9 significantly enhanced the percentage of mMSCs adhering, crawling and spreading and shear forces markedly stimulated crawling and spreading. CXCL9, CXCL16, CCL20 and CCL25 significantly enhanced transendothelial migration across MAECs. The transmigrated mMSCs had down-regulated receptors CXCR3, CXCR6, CCR6 and CCR9. This study furthers the knowledge of MSC transendothelial migration and the effects of chemokines and shear stress which is of relevance to inflammatory diseases such as arteriosclerosis.
SUMMARY: Mesenchymal stem cells (MSCs) are primarily fibroblast-like cells. Yet, once studied under conditions of shear stress when flowing along endothelial cells in vitro or in blood vessels, as well as in classic migration assays such as chemotaxis assays, MSCs have recently been found to function similarly to leukocytes in many ways. Firstly, MSCs express several homing receptors which are typically activated during extravasation of leukocytes. Secondly, some of these receptors are definitely functional, and required for their tissue localization in certain physiological or pathological contexts. Clinical protocols have in the last few years provided the first data on whether and how human MSCs may work in patients once delivered locally e.g. by injection, or systemically via the intra-arterial or intravenous route. Still, analysis of the ability of MSCs to activate specific homing receptors has up to now received relatively little attention. Moreover, maintenance or alterations of homing receptor expression or functions during good manufacturing practice (GMP) preparation steps, and documentation of presence and function of individual pathways on MSC preparations for clinical use are often missed. Hence, we review here mechanisms predicted to be relevant for adhesion, migration, and homing competence of MSCs. We also discuss some early data on homing of MSCs, deduced from preclinical experiments and from the few clinical studies with MSCs. Finally, we introduce some assays which could be applied to monitor preservation of the homing capacity of MSCs during GMP preparation.
Cell migration is a highly integrated multistep process that orchestrates embryonic morphogenesis; contributes to tissue repair
and regeneration; and drives disease progression in cancer, mental retardation, atherosclerosis, and arthritis. The migrating
cell is highly polarized with complex regulatory pathways that spatially and temporally integrate its component processes.
This review describes the mechanisms underlying the major steps of migration and the signaling pathways that regulate them,
and outlines recent advances investigating the nature of polarity in migrating cells and the pathways that establish it.
Mesenchymal stem cells (MSCs) are multipotent cells found in several adult tissues. Transplanted allogeneic MSCs can be detected in recipients at extended time points, indicating a lack of immune recognition and clearance. As well, a role for bone marrow-derived MSCs in reducing the incidence and severity of graft-versus-host disease (GVHD) during allogeneic transplantation has recently been reported; however, the mechanisms remain to be investigated. We examined the immunomodulatory functions of human MSCs (hMSCs) by coculturing them with purified subpopulations of immune cells and report here that hMSCs altered the cytokine secretion profile of dendritic cells (DCs), naive and effector T cells (T helper 1 [T(H)1] and T(H)2), and natural killer (NK) cells to induce a more anti-inflammatory or tolerant phenotype. Specifically, the hMSCs caused mature DCs type 1 (DC1) to decrease tumor necrosis factor alpha (TNF-alpha) secretion and mature DC2 to increase interleukin-10 (IL-10) secretion; hMSCs caused T(H)1 cells to decrease interferon gamma (IFN-gamma) and caused the T(H)2 cells to increase secretion of IL-4; hMSCs caused an increase in the proportion of regulatory T cells (T(Regs)) present; and hMSCs decreased secretion of IFN-gamma from the NK cells. Mechanistically, the hMSCs produced elevated prostaglandin E2 (PGE(2)) in co-cultures, and inhibitors of PGE(2) production mitigated hMSC-mediated immune modulation. These data offer insight into the interactions between allogeneic MSCs and immune cells and provide mechanisms likely involved with the in vivo MSC-mediated induction of tolerance that could be therapeutic for reduction of GVHD, rejection, and modulation of inflammation.
The ability of human mesenchymal stem cells (hMSC) to differentiate into osteoblasts was examined through the use of osteogenic induction medium (MSCOIM) cultures. hMSC first attached to the dish surface and exhibited fibroblast-like spindle shapes, and after proliferation, formed cuboidal shapes. Calcium assays and the use of von Kossa and alizarin red S staining showed that hMSC were capable of mineralization when cultured in MSCOIM. Gene expressions of Cbfa-1 and BMP-4, which are markers for osteogenic differentiation, were also increased during the hMSC differentiation into osteoblasts. When compared to albumin (Alb)-coated dishes, microscopic observation documented enhanced cell attachment and spreading when hMSC were cultured on fibronectin (FN)-coated dishes. Adherent cell numbers also exhibited a greater increase on the FN-coated dishes during earlier culture stages than that seen for the Alb-coated dishes. These findings suggest that hMSC have the capability to differentiate into osteoblasts and that FN can stimulate the attachment and spreading of the hMSC.
Circulating stem cells home within the myocardium, probably as the first step of a tissue regeneration process. This step requires adhesion to cardiac microvascular endothelium (CMVE). In this study, we studied mechanisms of adhesion between CMVE and mesenchymal stem cells (MSCs). Adhesion was studied in vitro and in vivo. Isolated 1,1'-dioctadecyl-3,3,3',3'-tetramethylindocarbocyanine perchlorate-labeled rat MSCs were allowed to adhere to cultured CMVE in static and dynamic conditions. Either CMVE or MSCs were pretreated with cytokines [IL-1beta, IL-3, IL-6, stem cell factor, stromal cell-derived factor-1, or TNF-alpha, 10 ng/ml]. Control or TNF-alpha-treated MSCs were injected intracavitarily in rat hearts in vivo. In baseline in vitro conditions, the number of MSCs that adhered to CMVE was highly dependent on the flow rate of the superfusing medium but remained significant at venous and capillary shear stress amplitudes. Activation of both CMVE and MSCs with TNF-alpha or IL-1beta before adhesion concentration dependently increased adhesion of MSCs at each studied level of shear stress. Consistently, in vivo, activation of MSCs with TNF-alpha before injection significantly enhanced cardiac homing of MSCs. TNF-alpha-induced adhesion could be completely blocked by pretreating either CMVE or MSCs with anti-VCAM-1 monoclonal antibodies but not by anti-ICAM-1 antibodies. Adhesion of circulating MSCs in the heart appears to be an endothelium-dependent process and is sensitive to modulation by activators of both MSCs and endothelium. Inflammation and the expression of VCAM-1 but not ICAM-1 on both cell types have a regulatory effect on MSC homing in the heart.
Human mesenchymal stem cells (MSCs) are increasingly being considered in cell-based therapeutic strategies for regeneration of various organs/tissues. However, the signals required for their homing and recruitment to injured sites are not yet fully understood. Because stromal-derived factor (SDF)-1 and hepatocyte growth factor (HGF) become up-regulated during tissue/organ damage, in this study we examined whether these factors chemoattract ex vivo-expanded MSCs derived from bone marrow (BM) and umbilical cord blood (CB). Specifically, we investigated the expression by MSCs of CXCR4 and c-met, the cognate receptors of SDF-1 and HGF, and their functionality after early and late passages of MSCs. We also determined whether MSCs express matrix metalloproteinases (MMPs), including membrane type 1 (MT1)-MMP, matrix-degrading enzymes that facilitate the trafficking of hematopoietic stem cells. We maintained expanded BM- or CB-derived MSCs for up to 15-18 passages with monitoring of the expression of 1) various tissue markers (cardiac and skeletal muscle, neural, liver, and endothelial cells), 2) functional CXCR4 and c-met, and 3) MMPs. We found that for up to 15-18 passages, both BM- and CB-derived MSCs 1) express mRNA for cardiac, muscle, neural, and liver markers, as well as the vascular endothelial (VE) marker VE-cadherin; 2) express CXCR4 and c-met receptors and are strongly attracted by SDF-1 and HGF gradients; 3) express MMP-2 and MT1-MMP transcripts and proteins; and 4) are chemo-invasive across the reconstituted basement membrane Matrigel. These in vitro results suggest that the SDF-1-CXCR4 and HGF-c-met axes, along with MMPs, may be involved in recruitment of expanded MSCs to damaged tissues.
To explore the initial steps by which transplanted mesenchymal stem cells (MSCs) interact with the vessel wall in the course of extravasation, we studied binding of human MSCs to endothelial cells (ECs). In a parallel plate flow chamber, MSCs bound to human umbilical vein ECs (HUVECs) similar to peripheral-blood mononuclear cells (PBMCs) or CD34(+) hematopoietic progenitors at shear stresses of up to 2 dynes/cm(2). This involved rapid extension of podia, rolling, and subsequent firm adhesion that was increased when ECs were prestimulated with TNF-alpha. MSC binding was suppressed when ECs were pretreated with function-blocking anti-P-selectin antibody, and rolling of MSCs was induced on immobilized P-selectin, indicating that P-selectin was involved in this process. Preincubation of HUVECs with anti-VCAM-1 or of MSCs with anti-VLA-4 antibodies suppressed binding of MSCs to HUVECs but did not enhance inhibition by anti-P-selectin, indicating that both P-selectin and VCAM-1 are equally required for this process. Intravital microscopy demonstrated the capacity of MSCs to roll and adhere to postcapillary venules in vivo in a mouse model in a P-selectin-dependent manner. Thus, MSCs interact in a coordinated fashion with ECs under shear flow, engaging P-selectin and VCAM-1/VLA-4.
Human umbilical cord (UC) has been a tissue of increasing interest in recent years. Many groups have shown the stem cell potency of stromal cells isolated from the human UC mesenchymal tissue, namely, Wharton's jelly. Since UC is a postnatal organ discarded after birth, the collection of cells does not require an invasive procedure with ethical concerns. Stromal cells, as the dominant cells of this fetus-derived tissue, possess multipotent properties between embryonic stem cells and adult stem cells. They bear a relatively higher proliferation rate and self-renewal capacity. Although they share common surface markers with bone marrow-derived MSCs, they also express certain embryonic stem cell markers, albeit in low levels. Without any spontaneous differentiation, they can be successfully differentiated into mature adipocytes, osteoblasts, chondrocytes, skeletal myocytes, cardiomyocytes, neurons, and endothelial cells. While causing no immunorejection reaction, they effectively function in vivo as dopaminergic neurons, myocytes, and endothelial cells. Given these characteristics, particularly the plasticity and developmental flexibility, UC stromal cells are now considered an alternative source of stem cells and deserve to be examined in long-term clinical trials. This review first aims to document the published findings so far regarding the nature of human UC stroma with special emphasis on the spatial distribution and functional structure of stromal cells and matrix, which serves as a niche for residing cells, and, secondly, to assess the in vitro and in vivo experiments in which differential stem cell potencies were evaluated.
Disclosure of potential conflicts of interest is found at the end of this article.
The immune system safeguards organ integrity by employing a balancing act of inflammatory and immunosuppressive mechanisms designed to neutralize foreign invaders and resolve injury. Maintaining or restoring a state of immune homeostasis is particularly challenging at barrier sites where constant exposure to immunogenic environmental agents may induce destructive inflammation. Recent studies underscore the role of epithelial and mesenchymal barrier cells in regulating immune cell function and local homeostatic and inflammatory responses. Here, we highlight immunoregulatory circuits engaging epithelial and mesenchymal cells in the intestine, airways, and skin and discuss how immune communications with hematopoietic cells and the microbiota orchestrate local immune homeostasis and inflammation.
Mesenchymal stem cells (MSC) possess a range of immunomodulatory properties which they exert via soluble mediators and through direct cell-cell contact. Due to these immune regulatory properties, the safety and clinical efficacy of MSC treatment has been tested in a number of autoimmune disorders. In this review we analyse the current data from early phase trials into Crohn's disease, systemic lupus erythematosus, and rheumatoid arthritis. In general, no adverse side-effects were observed in patients treated with MSC; however, their clinical efficacy is difficult to interpret. Systemic or site-specific administration of MSC has been reported to exert potent immunomodulatory effects in 7 of the 11 trials discussed. Nonetheless the mechanism(s) by which MSC exert their regulatory effects in vivo remain largely unknown. We discuss potential limitations or safety concerns associated with MSC therapy, including the heterogeneity of MSC and their potential contribution to disease pathogenesis, which need be considered when designing future clinical trials, along with the need to standardise trial design. Although we are bridging the translational gap between scientific observations on MSC function and clinical applications for therapy, our understanding of basic MSC biology is still limited. Despite these issues, large, double-blinded, multicentre clinical trials are already underway. Further research into the endogenous function of MSC is required to elucidate the mechanism by which therapeutic MSC are acting.
Stromal cells regulate the recruitment of circulating leukocytes during inflammation through cross-talk with neighboring endothelial cells. Here we describe two in vitro “vascular” models for studying the recruitment of circulating neutrophils from flow by inflamed endothelial cells. A major advantage of these models is the ability to analyze each step in the leukocyte adhesion cascade in order, as would occur in vivo. We also describe how both models can be adapted to study the role of stromal cells, in this case mesenchymal stem cells (MSC), in regulating leukocyte recruitment.
Cell migration directed by spatial cues, or taxis, is a primary mechanism for orchestrating concerted and collective cell movements during development, wound repair, and immune responses. Compared with the classic example of amoeboid chemotaxis, in which fast-moving cells such as neutrophils are directed by gradients of soluble factors, directed migration of slow-moving mesenchymal cells such as fibroblasts is poorly understood. Mesenchymal cells possess a distinctive organization of the actin cytoskeleton and associated adhesion complexes as its primary mechanical system, generating the asymmetric forces required for locomotion without strong polarization. The emerging hypothesis is that the molecular underpinnings of mesenchymal taxis involve distinct signaling pathways and diverse requirements for regulation.
Using bipolar co-ordinates, an exact solution of Stokes equations is obtained for the translational and rotational velocities of a neutrally buoyant sphere moving in proximity to a single plane wall under the influence of a simple shearing flow. The solution, valid for small shear Reynolds numbers, applies for all ratios of sphere radius to distance of its center from the wall. This formal solution is supplemented by two asymptotic solutions: (i) a lubrication-theory-like approximation applicable to the case where the sphere is very near to the wall; (ii) a “method of reflections” approximation, valid for the opposite case. Agreement with limited experimental data currently available in the literature is shown to be good, though the question of the true, limiting behavior of a sphere “touching” a wall remains unresolved.
Mesenchymal stem cells (MSC) have immuno-modulatory properties, but their effects on endothelial cells (EC) and recruitment of leukocytes are unknown. We cocultured human bone marrow-derived MSC with EC, and found that MSC could down-regulate adhesion of flowing neutrophils or lymphocytes, and their subsequent transendothelial migration. This applied for EC treated with tumour necrosis factor-α (TNF), interleukin-1β (IL-1) or TNF and interferon-γ combined. Supernatant from cocultures also inhibited endothelial responses. This supernatant had much higher levels of IL-6 than supernatant from cultures of the individual cells, which also lacked inhibitory functions. Addition of neutralising antibody against IL-6 removed the bioactivity of the supernatant and also the immunomodulatory effects of coculture. Studies using siRNA showed that IL-6 came mainly from the MSC in coculture, and reduction in production in MSC alone was sufficient to impair the protective effects of coculture. Interestingly, siRNA knock-down of IL-6-receptor expression in MSC as well as EC inhibited anti-inflammatory effects. This was explained when we detected soluble IL-6R-receptor in supernatants and showed that receptor removal reduced the potency of supernatant. Neutralisation of transforming growth factor-β indicated that activation of this factor in coculture contributed to Il-6 production. Thus, crosstalk between MSC and EC caused up regulation of production of IL-6 by MSC which in turn down regulated the response of EC to inflammatory cytokines, an effect potentiated by MSC release of soluble IL-6R. These studies establish a novel mechanism by which MSC might have protective effects against inflammatory pathology and cardiovascular disease. Stem Cells 2013.
Integrin receptors provide a dynamic, tightly-regulated link between the extracellular matrix (or cellular counter-receptors) and intracellular cytoskeletal and signalling networks, enabling cells to sense and respond to their chemical and physical environment. Talins and kindlins, two families of FERM-domain proteins, bind the cytoplasmic tail of integrins, recruit cytoskeletal and signalling proteins involved in mechanotransduction and synergize to activate integrin binding to extracellular ligands. New data reveal the domain structure of full-length talin, provide insights into talin-mediated integrin activation and show that RIAM recruits talin to the plasma membrane, whereas vinculin stabilizes talin in cell-matrix junctions. How kindlins act is less well-defined, but disease-causing mutations show that kindlins are also essential for integrin activation, adhesion, cell spreading and signalling.
We investigated rheological adaptation of leukocytes and platelets for their adhesive functions in inflammation and haemostasis respectively. Adhesion and margination of leukocytes or platelets where quantified for blood perfused through capillaries coated with P-selectin or collagen, when flow rate or suspending phase viscosity, or red cell aggregation or rigidity were modified. Independent variation of shear rate and shear stress indicated that the ability of platelets to attach at higher values than leukocytes was largely attributable to their smaller size reducing their velocity before attachment and especially, drag experienced afterwards. Increasing red cell aggregation increased the number of marginated and adhering leukocytes, but inhibited platelet adhesion without effect on the number marginated. Increasing red cell rigidity tended to inhibit leukocyte adhesion but promote platelet adhesion. The effects on platelets may be explained by changes in the depth of the near-wall, red cell-depleted layer; broadening (or narrowing) this layer to greater (or less) than the platelet diameter would decrease (or increase) the normal force applied by red cells and make attachment less (or more) efficient. Thus, different adhesive capabilities of leukocytes and platelets may arise from their differences in size, both directly because of influence on cell velocity and force experienced at the wall, and indirectly through effects of size on margination in the bloodstream and interaction with the cell-free layer. In addition, red cell aggregation (of hitherto uncertain physiological significance) may be useful in promoting leukocyte adhesion in inflamed venules but inhibiting unwanted platelet deposition in veins.
Systemically administered adult mesenchymal stem cells (MSCs), which are being explored in clinical trials to treat inflammatory disease, exhibit the critical ability to extravasate at sites of inflammation. We aimed to characterize the basic cellular processes mediating this extravasation and compare them to those involved in leukocyte transmigration. Using high-resolution confocal and dynamic microscopy, we show that, like leukocytes, human bone marrow-derived MSC preferentially adhere to and migrate across tumor necrosis factor-α-activated endothelium in a vascular cell adhesion molecule-1 (VCAM-1) and G-protein-coupled receptor signaling-dependent manner. As several studies have suggested, we observed that a fraction of MSC was integrated into endothelium. In addition, we observed two modes of transmigration not previously observed for MSC: Paracellular (between endothelial cells) and transcellular (directly through individual endothelial cells) diapedesis through discrete gaps and pores in the endothelial monolayer, in association with VCAM-1-enriched "transmigratory cups". Contrasting leukocytes, MSC transmigration was not preceded by significant lateral migration and occurred on the time scale of hours rather than minutes. Interestingly, rather than lamellipodia and invadosomes, MSC exhibited nonapoptotic membrane blebbing activity that was similar to activities previously described for metastatic tumor and embryonic germ cells. Our studies suggest that low avidity binding between endothelium and MSC may grant a permissive environment for MSC blebbing. MSC blebbing was associated with early stages of transmigration, in which blebs could exert forces on underlying endothelial cells indicating potential functioning in breaching the endothelium. Collectively, our data suggest that MSC transmigrate actively into inflamed tissues via both leukocyte-like and novel mechanisms. STEM CELLS2012;30:2472-2486.
Integrin-based adhesion has served as a model for studying the central role of adhesion in migration. In this article, we outline modes of migration, both integrin-dependent and -independent in vitro and in vivo. We next discuss the roles of adhesion contacts as signaling centers and linkages between the ECM and actin that allows adhesions to serve as traction sites. This includes signaling complexes that regulate migration and the interplay among adhesion, signaling, and pliability of the substratum. Finally, we address mechanisms of adhesion assembly and disassembly and the role of adhesion in cellular polarity.
Cell migration during vascular remodelling is regulated by crosstalk between growth factor receptors and integrin receptors, which together coordinate cytoskeletal and motogenic changes. Here, we report extracellular matrix (ECM)-directed crosstalk between platelet-derived growth factor receptor (PDGFR)-β and α5β1-integrin, which controls the migration of mesenchymal stem (stromal) cells (MSCs). Cell adhesion to fibronectin induced α5β1-integrin-dependent phosphorylation of PDGFR-β in the absence of growth factor stimulation. Phosphorylated PDGFR-β co-immunoprecipitated with α5-integrin and colocalised with α5β1-integrin in the transient tidemarks of focal adhesions. Adhesion to fibronectin also strongly potentiated PDGF-BB-induced PDGFR-β phosphorylation and focal adhesion kinase (FAK) activity, in an α5β1-integrin-dependent manner. PDGFR-β-induced phosphoinositide 3-kinase (PI3K) and Akt activity, actin reorganisation and cell migration were all regulated by fibronectin and α5β1-integrin. This synergistic relationship between α5β1-integrin and PDGFR-β is a fundamental determinant of cell migration. Thus, fibronectin-rich matrices can prime PDGFR-β to recruit mesenchymal cells at sites of vascular remodelling.
A bioabsorbable nanofibrous scaffold was developed for early adhesion of mesenchymal stem cells (MSCs). Collagen nanofibers with diameters of 430 +/- 170 nm were fabricated by electrospinning. Over 45% of the MSC population adhered to this collagen nanofiber after 30 min at room temperature. Remarkably, collagen-coated P(LLA-CL) electrospun nanofibers were almost as efficient as collagen nanofibers whereas collagen cast film did not enhance early capture when it was applied on cover slips. The adhesive efficiency could be further increased to over 20% at 20 min and over 55% at 30 min when collagen nanofibers were grafted with monoclonal antibodies recognizing CD29 or CD49a. These data demonstrate that the early adhesive behavior is highly dependent on both the surface texture and the surface chemistry of the substrate. These findings have potential applications for early capture of MSCs in an ex vivo setting under time constraints such as in a surgical setting.
The study of MSC trafficking is clinically relevant for minimally invasive cell therapy to promote regeneration of damaged tissue, to treat inflammation, and to promote angiogenesis. However, these studies are complicated by the diverse methods used to culture, characterize, and deliver MSCs and by the variety of methods used to assess homing events. This review provides a critical analysis of the methods used to track homing of exogenously infused MSCs and discusses strategies for enhancing their trafficking to particular tissues.
The authors are grateful for financial support from the National Institutes of Health (grants GM23244 and GM53905), and to very helpful comments on the manuscript from Elliot Elson, Vlodya Gelfand, Paul Matsudaira, Julie Theriot, and Sally Zigmond. D. A. L. and A. F. H. would also like to thank Alan Wells, and Anna Huttenlocher and Rebecca Sandborg, respectively, for stimulating conversations on this subject, and Sean Palecek for Figure 2Figure 2. Finally, we extend our apologies to all our colleagues in the field whose work we were unable to cite formally because of imposed reference limitations.
Integrin-mediated adhesion is a critical regulator of cell migration. Here we demonstrate that integrin-mediated adhesion to high fibronectin concentrations induces a stop signal for cell migration by inhibiting cell polarization and protrusion. On fibronectin, the stop signal is generated through alpha 5 beta 1 integrin-mediated signaling to the Rho family of GTPases. Specifically, Cdc42 and Rac1 activation exhibits a biphasic dependence on fibronectin concentration that parallels optimum cell polarization and protrusion. In contrast, RhoA activity increases with increasing substratum concentration. We find that cross talk between Cdc42 and Rac1 is required for substratum-stimulated protrusion, whereas RhoA activity is inhibitory. We also show that Cdc42 activity is inhibited by Rac1 activation, suggesting that Rac1 activity may down-regulate Cdc42 activity and promote the formation of stabilized rather than transient protrusion. Furthermore, expression of RhoA down-regulates Cdc42 and Rac1 activity, providing a mechanism whereby RhoA may inhibit cell polarization and protrusion. These findings implicate adhesion-dependent signaling as a mechanism to stop cell migration by regulating cell polarity and protrusion via the Rho family of GTPases.
To date, the precise interactions between bone marrow stromal cells and the extracellular matrix that govern stromal cell development remain unclear. The integrin super-family of cell-surface adhesion molecules represents a major pathway used by virtually all cell types to interact with different extracellular matrix components. In this study, purified populations of stromal precursor cells were isolated from the STRO-1-positive fraction of normal human marrow, by fluoresence-activated cell sorting, and then assayed for their ability to initiate clonogenic growth in the presence of various integrin ligands. Bone marrow-derived stromal progenitors displayed differential growth to fibronectin, vitronectin, and laminin, over collagen types I and III, but showed a similar affinity for collagen type IV. The integrin heterodimers alpha1beta1, alpha2beta1, alpha5beta1, alpha6beta1, alpha(v)beta3, and alpha(v)beta5 were found to coexpress with the STRO-1 antigen on the cell surface of CFU-F, using dual-color analysis. Furthermore, only a proportion of stromal precursors expressed the integrin alpha4beta1, while no measurable levels of the integrin alpha3beta1 could be detected. Subsequent adhesion studies using functional blocking antibodies to different integrin alpha/beta heterodimers showed that stromal cell growth on collagen, laminin, and fibronectin was mediated by multiple beta1 integrins. In contrast, cloning efficiency in the presence of vitronectin was mediated in part by alpha(v)beta3. When human marrow stromal cells were cultured under osteoinductive conditions, their ability to form a mineralized matrix in vitro was significantly diminished in the presence of a functional blocking monoclonal antibody to the beta1 integrin subunit. The results of this study indicate that beta1 integrins appear to be the predominant adhesion receptor subfamily utilized by stromal precursor cells to adhere and proliferate utilizing matrix glycoproteins commonly found in the bone marrow microenvironment and bone surfaces. Furthermore, these data suggest a possible role for the beta1 integrin subfamily during the development of stromal precursor cells into functional osteoblast-like cells.
In their roles as major adhesion receptors, integrins signal across the plasma membrane in both directions. Recent structural and cell biological data suggest models for how integrins transmit signals between their extracellular ligand binding adhesion sites and their cytoplasmic domains, which link to the cytoskeleton and to signal transduction pathways. Long-range conformational changes couple these functions via allosteric equilibria.
Discovery of new genes and proteins directly supporting leukocyte adhesion is waning, whereas there is heightened interest in the cell mechanics and receptor dynamics that lead from transient tethering via selectins to affinity shifts and adhesion strengthening through integrins. New optical tools enable real-time imaging of leukocyte rolling and arrest in parallel plate flow channels (PPFCs), and detection of single-molecule force spectroscopy provides an inner view of the intercellular adhesive contact region. Leukocyte recruitment during acute inflammation is triggered by ligation of G protein-coupled chemotactic receptors (GPCRs) and clustering of selectins. This, in turn, activates beta(2)-integrin (CD18), which facilitates cell capture and arrest in shear flow. This review provides a conceptual model for the molecular events supporting leukocyte recruitment.
The considerable therapeutic potential of human multipotent mesenchymal stromal cells (MSC) has generated markedly increasing interest in a wide variety of biomedical disciplines. However, investigators report studies of MSC using different methods of isolation and expansion, and different approaches to characterizing the cells. Thus it is increasingly difficult to compare and contrast study outcomes, which hinders progress in the field. To begin to address this issue, the Mesenchymal and Tissue Stem Cell Committee of the International Society for Cellular Therapy proposes minimal criteria to define human MSC. First, MSC must be plastic-adherent when maintained in standard culture conditions. Second, MSC must express CD105, CD73 and CD90, and lack expression of CD45, CD34, CD14 or CD11b, CD79alpha or CD19 and HLA-DR surface molecules. Third, MSC must differentiate to osteoblasts, adipocytes and chondroblasts in vitro. While these criteria will probably require modification as new knowledge unfolds, we believe this minimal set of standard criteria will foster a more uniform characterization of MSC and facilitate the exchange of data among investigators.
Mesenchymal stem cells (MSCs) show differentiation capacity along mesenchymal lineages and have the potential to aid tissue regeneration. MSC transplantation strategies are therefore currently being assessed following injury to various organs. However, potential MSC migration to these organs after intravenous (IV) MSC injection continues to be impeded by cell trapping within the lung.
Mouse MSCs were isolated, purified, transfected with firefly luciferase, and labeled with CSFE. Their size was assessed in vitro. To estimate the diameter of mouse pulmonary capillaries, fluorescence-labeled microspheres of different sizes were injected with or without sodium nitroprusside (SN) pretreatment. The lungs were harvested after 30 seconds and mean numbers of trapped microspheres per high-power field (HPF) were calculated. After IV injection of MSC suspensions (with or without SN), their dynamic distribution was monitored by in vivo luciferine imaging as well as by histopathology.
The diameter of suspended MSCs in vitro was 15 to 19 microm. Whereas nearly no 4-microm microspheres could be detected in lung sections, the numbers of trapped 10- and 15-microm microspheres could be significantly decreased by prior SN injection from 19.3 +/- 11.1 to 6.0 +/- 1.6 cells/HPF (P = .004) and from 34.9 +/- 11.9 to 25.6 +/- 8.1 cells/HPF (P = .028), respectively. Within seconds after MSC IV injection, the vast majority of cells was found in the lungs. However, cell trapping in the pulmonary microvasculature was significantly reduced by pre-treatment with SN.
We demonstrate that cell trapping in lungs can be reduced with IV SN pretreatment, increasing MSC passage through the lung capillaries, and potentially facilitating cell access to injured organs.
Mesenchymal stem cells were initially characterized as plastic adherent, fibroblastoid cells. In recent years, there has been an increasing focus on mesenchymal stem cells since they have great plasticity and are potential for therapeutic applications. Mesenchymal stem cells or mesenchymal stem cell-like cells have been shown to reside within the connective tissues of most organs. These cells can differentiate into osteogenic, adipogenic and chondrogenic lineages under appropriate conditions. A number of reports have also indicated that these cells possess the capacity to trans-differentiate into epithelial cells and lineages derived from the neuro-ectoderm, and in addition, mesenchymal stem cells can migrate to the sites of injury, inflammation, and to tumors. These properties of mesenchymal stem cells make them promising candidates for use in regenerative medicine and may also serve as efficient delivery vehicles in site-specific therapy.
For successful systemic stem cell therapy, mesenchymal stem cells (MSCs) must transmigrate across the endothelium and invade their target tissue. To date, most of the underlying mechanisms of transmigration and invasion remain to be elucidated. Improving our knowledge on these core processes might elevate the efficiency of stem cell therapy. Our aim was therefore to characterize key mechanisms involved in transmigration and invasion of MSCs. Co-cultivation experiments infer that MSCs integrate into the endothelial monolayer. However, the time course of adhesion, integration and transmigration depends on the endothelial phenotype and is most effective in venous vessels of the myocardium. Thus, a variable capacity for transmigration exists within the vasculature. Additionally, three-dimensional systems reveal that MSCs penetrate the endothelium and invade the surrounding tissue via plasmic podia. Furthermore, transmigration not only requires the interaction of vascular cell adhesion molecule-1 (VCAM-1) and very late antigen-4 (VLA-4) as verified by blocking experiments, but also triggers the clustering of beta1 integrins. In addition, in situ zymographies infer the activation of gelatinases at sites of MSC invasion into myocardial tissue. As evidenced by ELISA, MSCs secrete matrix metalloproteinase (MMP)-2 but not MMP-9. Finally, media containing additional cytokines accelerate the transmigration. Concluding, key players involved in transmigration and invasion of MSCs are the endothelial phenotype, VCAM-1/VLA-4, beta1 integrins, MMP-2 secretion and cytokines.
Treatment of chronic wounds with bone marrow-derived cells
- Badiavas
Concise review: MSC adhesion cascade-insights into homing and transendothelial migration
- Nitzsche