Cultured cell‐derived extracellular matrices to enhance the osteogenic differentiation and angiogenic properties of human mesenchymal stem/stromal cells

Cell‐derived extracellular matrix (ECM) consists of a complex assembly of fibrillary proteins, matrix macromolecules, and associated growth factors that mimic the composition and organization of native ECM micro‐environment. Therefore, cultured cell‐derived ECM has been used as a scaffold for tissue engineering settings to create a biomimetic micro‐environment, providing physical, chemical, and mechanical cues to cells, and support cell adhesion, proliferation, migration, and differentiation. Here, we present a new strategy to produce different combinations of decellularized cultured cell‐derived ECM (dECM) obtained from different cultured cell types, namely, mesenchymal stem/stromal cells (MSCs) and human umbilical vein endothelial cells (HUVECs), as well as the coculture of MSC:HUVEC and investigate the effects of its various compositions on cell metabolic activity, osteogenic differentiation, and angiogenic properties of human bone marrow (BM)‐derived MSCs, vital features for adult bone tissue regeneration and repair. Our findings demonstrate that dECM presented higher cell metabolic activity compared with tissue culture polystyrene. More importantly, we show that MSC:HUVEC ECM enhanced the osteogenic and angiogenic potential of BM MSCs, as assessed by in vitro assays. Interestingly, MSC:HUVEC (1:3) ECM demonstrated the best angiogenic response of MSCs in the conditions tested. To the best of our knowledge, this is the first study that demonstrates that dECM derived from a coculture of MSC:HUVEC impacts the osteogenic and angiogenic capabilities of BM MSCs, suggesting the potential use of MSC:HUVEC ECM as a therapeutic product to improve clinical outcomes in bone regeneration.     

Mesenchymal stem cells from rat olfactory bulbs can differentiate into cells with cardiomyocyte characteristics

Mesenchymal stromal/stem cells (MSCs) are widely distributed in different tissues such as bone marrow, adipose tissues, peripheral blood, umbilical cord and amnionic fluid. Recently, MSC‐like cells were also found to exist in rat olfactory bulb and are capable of inducing differentiation into mesenchymal lineages – osteocytes, chondrocytes and adipocytes. However, whether these cells can differentiate into myocardial cells is not known. In this study, we examined whether olfactory bulb‐derived MSCs could differentiate into myocardial cells in vitro. Fibroblast‐like cells isolated from the olfactory bulb of neonatal rats were grown under four conditions: no treatment; in the presence of growth factors (neuregulin‐1, bFGF and forskolin); co‐cultured with cardiomyocytes; and co‐cultured with cardiomyocytes plus neuregulin‐1, bFGF and forskolin. Cell differentiation into myocardial cells was monitored by RT–PCR, light microscopy immunofluorescence, western blot analysis and contractile response to pharmacological treatments. The isolated olfactory bulb‐derived fibroblast‐like cells expressed CD29, CD44, CD90, CD105, CD166 but not CD34 and CD45, consistent with the characteristics of MSCs. Long cylindical cells that spontaneously contracted were only observed following 7 days of co‐culture of MSCs with rat cardiomyocytes plus neuregulin‐1, bFGF and forskolin. RT–PCR and western blot analysis indicated that the cylindrical cells expressed myocardial markers, such as Nkx2.5, GATA4, sarcomeric α‐actinin, cardiac troponin I, cardiac myosin heavy chain, atrial natriuretic peptide and connexin 43. They also contained sarcomeres and gap junction and were sensitive to pharmacological treatments (adrenal and cholinergic agonists and antagonists). These findings indicate that rat olfactory bulb‐derived fibroblast‐like cells with MSC characteristics can differentiate into myocardial‐like cells. Copyright © 2013 John Wiley & Sons, Ltd.     

Potential of VEGF‐encapsulated electrospun nanofibers for in vitro cardiomyogenic differentiation of human mesenchymal stem cells

Heart disease, especially myocardial infarction (MI), has become the leading cause of death all over the world, especially since the myocardium lacks the ability to regenerate after infarction. The capability of mesenchymal stem cells (MSCs) to differentiate into the cardiac lineage holds great potential in regenerative medicine for MI treatment. In this study, we investigated the potential of human MSCs (hMSCs) to differentiate into cardiomyogenic cell lineages, using 5‐azacytidine (5‐aza) on electrospun poly(ε‐caprolactone)–gelatin (PCL–gelatin) nanofibrous scaffolds. Immunofluorescence staining analysis showed that after 15 days of in vitro culture the hMSCs differentiated to cardiomyogenic cells on PCL–gelatin (PG) nanofibers and expressed a higher level of cardiac‐specific proteins, such as α‐actinin and troponin‐T, compared to the MSC‐differentiated CMs on tissue culture plates (control). To further induce the cardiac differentiation, vascular endothelial growth factor (VEGF) was incorporated into the nanofibers by blending or co‐axial electrospinning, and in vitro release study showed that the growth factor could cause sustained release of VEGF from the nanofibers for a period of up to 21 days. The incorporation of VEGF within the nanofibers improved the proliferation of MSCs and, more importantly, enhanced the expression of cardiac‐specific proteins on PG–VEGF nanofibers. Our study demonstrated that the electrospun PG nanofibers encapsulated with VEGF have the ability to promote cardiac differentiation of hMSCs, and might be promising scaffolds for myocardial regeneration. Copyright © 2015 John Wiley & Sons, Ltd.     

An in vitro expansion score for tissue‐engineering applications with human bone marrow‐derived mesenchymal stem cells

Human bone marrow‐derived mesenchymal stem cells (MSCs) have limited growth potential in vitro and cease to divide due to replicative senescence, which from a tissue‐engineering perspective has practical implications, such as defining the correct starting points for differentiation and transplantation. Time spent in culture before the loss of required differentiation potential is different and reflects patient variability, which is a problem for cell expansion. This study aimed to develop a score set which can be used to quantify the senescent state of MSCs and predict whether cells preserve their ability to differentiate to osteogenic, adipogenic and chondrogenic phenotypes, based on colony‐forming unit (CFU) assay, population doubling time (PDT), senescence‐associated β‐galactosidase (SA‐β‐Gal) activity, cell size, telomere length and gene expression of MSCs cultured in vitro over 11 passages. This set of morphological, physiological and genetic senescence markers was correlated to the ability of MSCs to differentiate. Differentiation efficiency was assessed by marker genes and protein expression. CFUs decreased with increasing passage number, whereas SA‐β‐Gal activity and PDT increased; however, the correlation with MSCs' differentiation potential was sometimes unexpected. The expression of genes related to senescence was higher in late‐passage cells than in early‐passage cells. Early‐passage cells underwent efficient osteogenic differentiation, with mid‐passage cells performing best in chondrogenic differentiation. Late‐passage cells preserve only adipogenic differentiation potential. Based on this marker set, we propose a senescence score in which combined markers give a reliable quality control of MSCs, not depending only on mechanistic passage number. Copyright © 2013 John Wiley & Sons, Ltd.     

Autonomous magnetic labelling of functional mesenchymal stem cells for improved traceability and spatial control in cell therapy applications

Mesenchymal stem cells (MSCs) represent a valuable resource for regenerative medicine treatments for orthopaedic repair and beyond. Following developments in isolation, expansion and differentiation protocols, efforts to promote clinical translation of emerging cellular strategies now seek to improve cell delivery and targeting. This study shows efficient live MSC labelling using silica‐coated magnetic particles (MPs), which enables 3D tracking and guidance of stem cells. A procedure developed for the efficient and unassisted particle uptake was shown to support MSC viability and integrity, while surface marker expression and MSC differentiation capability were also maintained. In vitro, MSCs showed a progressive decrease in labelling over increasing culture time, which appeared to be linked to the dilution effect of cell division, rather than to particle release, and did not lead to detectable secondary particle uptake. Labelled MSC populations demonstrated magnetic responsiveness in vitro through directed migration in culture and, when seeded onto a scaffold, supporting MP‐based approaches to cell targeting. The potential of these silica‐coated MPs for MRI cell tracking of MSC populations was validated in 2D and in a cartilage repair model following cell delivery. These results highlight silica‐coated magnetic particles as a simple, safe and effective resource to enhance MSC targeting for therapeutic applications and improve patient outcomes. © 2016 The Authors Journal of Tissue Engineering and Regenerative Medicine Published by John Wiley & Sons Ltd.     

SIRT1‐dependent anti‐senescence effects of cell‐deposited matrix on human umbilical cord mesenchymal stem cells

Human umbilical cord‐derived mesenchymal stem cells (UC‐MSCs) are considered an attractive cell source for tissue regeneration. However, environmental oxidative stress can trigger premature senescence in MSCs and thus compromises their regenerative potential. Extracellular matrix (ECM) derived from MSCs has been shown to facilitate cell proliferation and multi‐lineage differentiation. This investigation evaluated the effect of cell‐deposited decellularized ECM (DECM) on oxidative stress‐induced premature senescence in UC‐MSCs. Sublethal dosages of H₂O₂, ranging from 50 μm to 200 μm , were used to induce senescence in MSCs. We found that DECM protected UC‐MSCs from oxidative stress‐induced premature senescence. When treated with H₂O₂ at the same concentration, cell proliferation of DECM‐cultured UC‐MSCs was twofold higher than those on standard tissue culture polystyrene (TCPS). After exposure to 100 μm H₂O₂, fewer senescence‐associated β‐galactosidase‐positive cells were observed on DECM than those on TCPS (17.6  ±  4.0% vs. 60.4  ±  6.2%). UC‐MSCs cultured on DECM also showed significantly lower levels of senescence‐related regulators, such as p16^INK4α and p21. Most importantly, DECM preserved the osteogenic differentiation potential of UC‐MSCs with premature senescence. The underlying molecular mechanisms involved the silent information regulator type 1 (SIRT1)‐dependent signalling pathway, confirmed by the fact that the SIRT1 inhibitor nicotinamide counteracted the DECM‐mediated anti‐senescent effect. Collagen type I, rather than fibronectin, partially contributed to the protective effect of decellularized matrix. These findings provide a new strategy of using stem cell‐deposited matrix to overcome the challenge of cellular senescence and to facilitate the clinical application of MSCs in regenerative medicine. Copyright © 2017 John Wiley & Sons, Ltd.     

Persistence of fluorescent nanoparticle‐labelled bone marrow mesenchymal stem cells in vitro and after intra‐articular injection

Mesenchymal stem cells (MSCs) improve the osteoarthritis condition, but the fate of MSCs after intra‐articular injection is unclear. We used fluorescent nanoparticles (quantum dots [QDs]) to track equine MSCs (QD‐labelled MSCs [QD‐MSCs]) in vivo after intra‐articular injection into normal and osteoarthritic joints. One week after injection of QD‐MSCs, unlabelled MSCs, or vehicle, we determined the presence of QD‐MSCs in synovium and articular cartilage histologically. In vitro, we evaluated the persistence of QDs in MSCs and whether QDs affected proliferation, immunophenotype, or differentiation. In joints injected with QD‐MSCs, labelled cells were identified on the synovial membrane and significantly less often on articular cartilage, without differences between normal and osteoarthritic joints. Joints injected with QD‐MSCs and MSCs had increased synovial total nucleated cell count and protein compared with vehicle‐injected joints. In vitro, QDs persisted in nonproliferating cells for up to 8 weeks (length of the study), but QD fluorescence was essentially absent from proliferating cells within two passages (approximately 3 to 5 days). QD labelling did not affect MSC differentiation into chondrocytes, adipocytes, and osteocytes. QD‐MSCs had slightly different immunophenotype from control cells, but whether this was due to an effect of the QDs or to drift during culture is unknown. QD‐MSCs can be visualized in histological sections 1 week after intra‐articular injection and are more frequently found in the synovial membrane versus cartilage in both normal and osteoarthritic joints. QDs do not alter MSC viability and differentiation potential in vitro. However, QDs are not optimal markers for long‐term tracking of MSCs, especially under proliferative conditions.     

VEGF overexpression improves mesenchymal stem cell sheet transplantation therapy for acute myocardial infarction

Cell sheet‐based tissue engineering shows great potential in the treatment of ischaemic heart disease. However, treatment efficacy is compromised by low blood and nutrient supply. The aim of this study was to investigate the effect of pro‐angiogenic vascular endothelial growth factor (VEGF)‐modified mesenchymal stem cell (MSC) sheet transplantation therapy in ischaemic heart failure. Rat MSCs were manipulated to overexpress the VEGF gene. In vitro, the antiapoptotic and paracrine effects were assessed under hypoxic conditions. In vivo, we evaluated the therapeutic effect of VEGF‐modified MSC sheet therapy in a rat model of acute myocardial infarction (AMI). Forty‐five Wistar rats were divided into three groups; one group underwent AMI (control), another underwent AMI and WT sheet transplantation (WT‐MSC) and a third group underwent AMI and VEGF sheet transplantation (VEGF‐MSC). Echocardiography was performed after 3, 10 and 28 days. Samples for histological analysis were collected at the end of the study. The VEGF gene protected MSCs against apoptosis. In vitro, VEGF overexpression significantly reduced MSC apoptosis compared with wild‐type and enhanced VEGF secretion under hypoxic conditions. Capillary density in the infarct border zone was higher in VEGF‐MSC‐transplanted animals than in WT‐MSC‐treated animals. Furthermore, VEGF‐MSC‐transplanted animals had a smaller infarct size than WT‐MSC‐treated animals and exhibited remarkable functional recovery. These findings support the premise that transplantation of proangiogenic gene‐modified MSCs may be valuable for mediating substantial functional recovery after AMI. Copyright © 2012 John Wiley & Sons, Ltd.     

Comparison of different culture conditions for human mesenchymal stromal cells for clinical stem cell therapy

Objective. Mesenchymal stromal cells (MSCs) from adult bone marrow (BM) are considered potential candidates for therapeutic neovascularization in cardiovascular disease. When implementing results from animal trials in clinical treatment, it is essential to isolate and expand the MSCs under conditions following good manufacturing practice (GMP). The aims of the study were first to establish culture conditions following GMP quality demands for human MSC expansion and differentiation for use in clinical trials, and second to compare these MSCs with MSCs derived from culture in four media commonly used for MSC cultivation in animal studies simulating clinical stem cell therapy. Material and methods. Human mononuclear cells (MNCs) were isolated from BM aspirates by density gradient centrifugation and cultivated in a GMP‐accepted medium (EMEA medium) or in one of four other media. Results. FACS analysis showed that the plastic‐adherent MSCs cultured in EMEA medium or in the other four media were identically negative for the haematopoietic surface markers CD45 and CD34 and positive for CD105, CD73, CD90, CD166 and CD13, which in combined expression is characteristic of MSCs. MSC stimulation with vascular endothelial growth factor (VEGF) increased expression of the characteristic endothelial genes KDR and von Willebrand factor; the von Willebrand factor and CD31 at protein level as well as the capacity to develop capillary‐like structures. Conclusions. We established culture conditions with a GMP compliant medium for MSC cultivation, expansion and differentiation. The expanded and differentiated MSCs can be used in autologous mesenchymal stromal cell therapy in patients with ischaemic heart disease.     

Phenotypic and functional comparison of optimum culture conditions for upscaling of bone marrow‐derived mesenchymal stem cells

Human adult bone marrow‐derived mesenchymal stem cells (MSCs) are a promising tool in the newly emerging avenue of regenerative medicine. MSCs have already been translated from basic research to clinical transplantation research. However, there is still a lack of consensus on the ideal method of culturing MSCs. Here we have compared different culture conditions of human MSCs with an attempt to preserve their characteristics and multi‐lineage differentiation potential. We compare the different basal culture media DMEM‐F12, DMEM‐high glucose (DMEM‐HG), DMEM‐low glucose (DMEM‐LG), knock‐out DMEM (DMEM‐KO) and Mesencult^® on the proliferation rate, surface markers and differentiation potentials of MSCs. At every fifth passage until the 25th passage, the differentiation potential and the presence of a panel of surface markers was observed, using flow cytometry. We also compared the characteristics of human MSCs when cultured in reduced concentrations of fetal bovine serum (FBS), knockout serum replacement (KO‐SR) and human plasma. Data indicate that the presence of serum is essential to sustain and propagate MSCs cultures. The choice of basal medium is equally important so as to preserve their characteristics and multipotent properties even after prolonged culture in vitro. With MSCs emerging as a popular tool for regenerative therapies in incurable diseases, it is essential to be able to obtain a large number of MSCs that continue to preserve their characteristics following passaging. The data reveal the optimum basal medium for prolonged culture of MSCs while retaining their ability to differentiate and hence this may be used for up‐scaling to provide sufficient numbers for transplantation. Copyright © 2009 John Wiley & Sons, Ltd.     

Engineering cartilaginous grafts using chondrocyte‐laden hydrogels supported by a superficial layer of stem cells

During postnatal joint development, progenitor cells that reside in the superficial region of articular cartilage first drive the rapid growth of the tissue and later help direct the formation of mature hyaline cartilage. These developmental processes may provide directions for the optimal structuring of co‐cultured chondrocytes (CCs) and multipotent stromal/stem cells (MSCs) required for engineering cartilaginous tissues. The objective of this study was to engineer cartilage grafts by recapitulating aspects of joint development where a population of superficial progenitor cells drives the development of the tissue. To this end, MSCs were either self‐assembled on top of CC‐laden agarose gels (structured co‐culture) or were mixed with CCs before being embedded in an agarose hydrogel (mixed co‐culture). Porcine infrapatellar fat pad‐derived stem cells (FPSCs) and bone marrow‐derived MSCs (BMSCs) were used as sources of progenitor cells. The DNA, sGAG and collagen content of a mixed co‐culture of FPSCs and CCs was found to be lower than the combined content of two control hydrogels seeded with CCs and FPSCs only. In contrast, a mixed co‐culture of BMSCs and CCs led to increased proliferation and sGAG and collagen accumulation. Of note was the finding that a structured co‐culture, at the appropriate cell density, led to greater sGAG accumulation than a mixed co‐culture for both MSC sources. In conclusion, assembling MSCs onto CC‐laden hydrogels dramatically enhances the development of the engineered tissue, with the superficial layer of progenitor cells driving CC proliferation and cartilage ECM production, mimicking certain aspects of developing cartilage. Copyright © 2015 John Wiley & Sons, Ltd.     

Agonism of Wnt–β‐catenin signalling promotes mesenchymal stem cell (MSC) expansion

Promoting mesenchymal stem cell (MSC) proliferation has numerous applications in stem cell therapies, particularly in the area of regenerative medicine. In order for cell‐based regenerative approaches to be realized, MSC proliferation must be achieved in a controlled manner without compromising stem cell differentiation capacities. Here we demonstrate that 6‐bromoindirubin‐3′‐oxime (BIO) increases MSC β‐catenin activity 106‐fold and stem cell‐associated gene expression ~33‐fold, respectively, over untreated controls. Subsequently, BIO treatment increases MSC populations 1.8‐fold in typical 2D culture conditions, as well as 1.3‐fold when encapsulated within hydrogels compared to untreated cells. Furthermore, we demonstrate that BIO treatment does not reduce MSC multipotency where MSCs maintain their ability to differentiate into osteoblasts, chondrocytes and adipocytes using standard conditions. Taken together, our results demonstrate BIO's potential utility as a proliferative agent for cell transplantation and tissue regeneration. Copyright © 2013 John Wiley & Sons, Ltd.     

Modulation of chondrogenic differentiation of human mesenchymal stem cells in jellyfish collagen scaffolds by cell density and culture medium

Studies on tissue‐engineering approaches for the regeneration of traumatized cartilage focus increasingly on multipotent human mesenchymal stem cells (hMSCs) as an alternative to autologous chondrocytes. The present study applied porous scaffolds made of collagen from the jellyfish Rhopilema esculentum for the in vitro chondrogenic differentiation of hMSCs. Culture conditions in those scaffolds differ from conditions in high‐density pellet cultures, making a re‐examination of these data necessary. We systematically investigated the influence of seeding density, basic culture media [Dulbecco's modified Eagle's medium (DMEM), α‐minimum essential medium (α‐MEM)] with varying glucose content and supplementation with fetal calf serum (FCS) or bovine serum albumin (BSA) on the chondrogenic differentiation of hMSCs. Gene expression analyses of selected markers for chondrogenic differentiation and hypertrophic development were conducted. Furthermore, the production of cartilage extracellular matrix (ECM) was analysed by quantification of sulphated glycosaminoglycan and collagen type II contents. The strongest upregulation of chondrogenic markers, along with the highest ECM deposition was observed in scaffolds seeded with 2.4 × 10⁶ cells/cm³ after cultivation in high‐glucose DMEM and 0.125% BSA. Lower seeding densities compared to high‐density pellet cultures were sufficient to induce in vitro chondrogenic differentiation of hMSCs in collagen scaffolds, which reduces the amount of cells required for the seeding of scaffolds and thus the monolayer expansion period. Furthermore, examination of the impact of FCS and α‐MEM on chondrogenic MSC differentiation is an important prerequisite for the development of an osteochondral medium for simultaneous osteogenic and chondrogenic differentiation in biphasic scaffolds for osteochondral tissue regeneration. Copyright © 2015 John Wiley & Sons, Ltd.     

Decellularized extracellular matrix gelloids support mesenchymal stem cell growth and function in vitro

Volumetric muscle loss (VML) injuries are irrecoverable due to a significant loss of regenerative elements, persistent inflammation, extensive fibrosis, and functional impairment. When used in isolation, previous stem cell and biomaterial‐based therapies have failed to regenerate skeletal muscle at clinically relevant levels. The extracellular matrix (ECM) microenvironment is crucial for the viability, stemness, and differentiation of stem cells. Decellularized‐ECM (D‐ECM) scaffolds are at the forefront of ongoing research to develop a viable therapy for VML. Due to the retention of key ECM components, D‐ECM scaffolds provide an excellent substrate for the adhesion and migration of several cell types. Mesenchymal stem cells (MSCs) possess regenerative and immunomodulatory properties and are currently under investigation in clinical trials for a wide range of medical conditions. However, a major limitation to the use of MSCs in clinical applications is their poor viability at the site of transplantation. In this study, we have fabricated spherical scaffolds composed of gelatin and skeletal muscle D‐ECM for the adhesion and delivery of MSCs to the site of VML injury. These spherical scaffolds termed “gelloids” supported MSC survival, expansion, trophic factor secretion, immunomodulation, and myogenic protein expression in vitro. Future studies would determine the therapeutic efficacy of this approach in a murine model of VML injury.     

Matrix‐directed differentiation of human adipose‐derived mesenchymal stem cells to dermal‐like fibroblasts that produce extracellular matrix

Commercially available skin substitutes lack essential non‐immune cells for adequate tissue regeneration of non‐healing wounds. A tissue‐engineered, patient‐specific, dermal substitute could be an attractive option for regenerating chronic wounds, for which adipose‐derived mesenchymal stem cells (ADMSCs) could become an autologous source. However, ADMSCs are multipotent in nature and may differentiate into adipocytes, osteocytes and chondrocytes in vitro, and may develop into undesirable tissues upon transplantation. Therefore, ADMSCs committed to the fibroblast lineage could be a better option for in vitro or in vivo skin tissue engineering. The objective of this study was to standardize in vitro culture conditions for ADMSCs differentiation into dermal‐like fibroblasts which can synthesize extracellular matrix (ECM) proteins. Biomimetic matrix composite, deposited on tissue culture polystyrene (TCPS), and differentiation medium (DM), supplemented with fibroblast‐conditioned medium and growth factors, were used as a fibroblast‐specific niche (FSN) for cell culture. For controls, ADMSCs were cultured on bare TCPS with either DM or basal medium (BM). Culture of ADMSCs on FSN upregulated the expression of differentiation markers such as fibroblast‐specific protein‐1 (FSP‐1) and a panel of ECM molecules specific to the dermis, such as fibrillin‐1, collagen I, collagen IV and elastin. Immunostaining showed the deposition of dermal‐specific ECM, which was significantly higher in FSN compared to control. Fibroblasts derived from ADMSCs can synthesize elastin, which is an added advantage for successful skin tissue engineering as compared to fibroblasts from skin biopsy. To obtain rapid differentiation of ADMSCs to dermal‐like fibroblasts for regenerative medicine, a matrix‐directed differentiation strategy may be employed. Copyright © 2014 John Wiley & Sons, Ltd.     

Improved expansion of human bone marrow‐derived mesenchymal stem cells in microcarrier‐based suspension culture

Human bone marrow‐derived mesenchymal stem cells (hBM‐MSCs) have potential clinical utility in the treatment of a multitude of ailments and diseases, due to their relative ease of isolation from patients and their capacity to form many cell types. However, hBM‐MSCs are sparse, and can only be isolated in very small quantities, thereby hindering the development of clinical therapies. The use of microcarrier‐based stirred suspension bioreactors to expand stem cell populations offers an approach to overcome this problem. Starting with standard culture protocols commonly reported in the literature, we have successfully developed new protocols that allow for improved expansion of hBM‐MSCs in stirred suspension bioreactors using CultiSpher‐S microcarriers. Cell attachment was facilitated by using intermittent bioreactor agitation, removing fetal bovine serum, modifying the stirring speed and manipulating the medium pH. By manipulating these parameters, we enhanced the cell attachment efficiency in the first 8 h post‐inoculation from 18% (standard protocol) to 72% (improved protocol). Following microcarrier attachment, agitation rate was found to impact cell growth kinetics, whereas feeding had no significant effect. By serially subculturing hBM‐MSCs using the new suspension bioreactor protocols, we managed to obtain cell fold increases of 10³ within 30 days, which was superior to the 200‐fold increase obtained using the standard protocol. The cells were found to retain their defining characteristics after several passages in suspension. This new bioprocess represents a more efficient approach for generating large numbers of hBM‐MSCs in culture, which in turn should facilitate the development of new stem cell‐based therapies. Copyright © 2012 John Wiley & Sons, Ltd.     

Modulation of mesenchymal stem cell actin organization on conventional microcarriers for proliferation and differentiation in stirred bioreactors

Tissue engineering applications require an appropriate combination of a cell population, biochemical factors and scaffold materials. In this field, mesenchymal stem cells (MSCs) emerge as an attractive cell population, due to their ready availability and their potential to be differentiated into various mesodermal cell types. Commercially available microcarriers have been recently recognized as an efficient tool for the propagation of such cells compared to traditional monolayer culture, enabling efficient scale‐up and serving as a cell delivery system. The organization of actin as well as the induction of its effectors was previously shown to affect dramatically both proliferation and differentiation of MSCs in monolayer culture. To achieve mass scale production of differentiated cells derived from MSCs in scalable stirred bioreactors, this work aims at rationally screening microcarriers based on the characterization of actin organization. First, among the various supports tested, gelatin‐based microcarriers were found to be most suitable for MSC expansion, due to their best‐adapted actin organization compared to monolayer cultures. Secondly, the proper actin organization on Cultispher‐S was closely linked to its ability to bind serum adhesion molecules enabling Rho GTPase activation. Finally, by modulating actin behaviour, it was feasible to efficiently guide MSC differentiation on microcarriers. Taken together, these results show that controlling actin behaviour is a good strategy toward mass scale sequential expansion followed by differentiation of MSCs in a microcarrier based bioreactor. Copyright © 2012 John Wiley & Sons, Ltd.     

Liver extracellular matrix promotes BM‐MSCs hepatic differentiation and reversal of liver fibrosis through activation of integrin pathway

In cell‐based therapies for liver injuries, the clinical outcomes are closely related to the surrounding microenvironment of the transplanted bone marrow mesenchymal stem cells (BM‐MSCs). However, whether liver‐specific ECM (L‐ECM), as one of major microenvironment signals, could regulate the therapeutic effect of BM‐MSCs through changing their biological characteristics is unclear. This study aimed to investigate the hepatogenicity and underlying mechanism of L‐ECM as well as its potential regulative role in the MSC‐based liver recovery. L‐ECM was prepared by homogenization of decellularized whole porcine liver. After three‐dimensional culture with or without the presence of L‐ECM, BM‐MSCs expressed hepatocyte‐specific genes and proteins in an L‐ECM concentration‐dependent manner. Further analysis showed that L‐ECM could activate specific types of integrins (ITGs) as well as their downstream signalling pathways. When the cell/ECM interaction was enhanced by incorporating BM‐MSCs with Mn²⁺, ITGs were activated and the hepatogenic capacity of L‐ECM was improved. The regeneration of rat livers from either acute or chronic fibrosis could also be accelerated after transplantation of Mn²⁺‐treated BM‐MSCs. L‐ECM therefore promotes hepatic differentiation of BM‐MSCs via the ITG pathway and plays a therapeutically beneficial role for stem cell‐based liver regeneration. Copyright © 2016 John Wiley & Sons, Ltd.     

An in vitro model of mesenchymal stem cell targeting using magnetic particle labelling

The specific targeting of cells to sites of tissue damage in vivo is a major challenge precluding the success of stem cell‐based therapies. Magnetic particle‐based targeting may provide a solution. Our aim was to provide a model system to study the trapping and potential targeting of human mesenchymal stem cells (MSCs) during in vitro fluid flow, which ultimately will inform cell targeting in vivo. In this system magnet arrays were used to trap superparamagnetic iron oxide particle‐doped MSCs. The in vitro experiments demonstrated successful cell trapping, where the volume of cells trapped increased with magnetic particle concentration and decreased with increasing flow rate. Analysis of gene expression revealed significant increases in COL1A2 and SOX9. Using principles established in vitro, a proof‐of‐concept in vivo experiment demonstrated that magnetic particle‐doped, luciferase‐expressing MSCs were trapped by an implanted magnet in a subcutaneous wound model in nude mice. Our results demonstrate the effectiveness of using an in vitro model for testing superparamagnetic iron oxide particles to develop successful MSC targeting strategies during fluid flow, which ultimately can be translated to in vivo targeted delivery of cells via the circulation in a variety of tissue‐repair models. Copyright © 2012 John Wiley & Sons, Ltd.