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.     

Dynamic cell–cell interactions between cord blood haematopoietic progenitors and the cellular niche are essential for the expansion of CD34+, CD34+CD38− and early lymphoid CD7+ cells

Most clinical applications of haematopoietic stem/progenitor cells (HSCs) would benefit from their ex vivo expansion to obtain a therapeutically significant amount of cells from the available donor samples. We studied the impact of cellular interactions between umbilical cord blood (UCB) haematopoietic cells and bone marrow (BM)‐derived mesenchymal stem cells (MSCs) on the ex vivo expansion and differentiative potential of UCB CD34⁺‐enriched cells. UCB cells were cultured: (a) directly in contact with BM MSC‐derived stromal layers (contact); (b) separated by a microporous membrane (non‐contact); or (c) without stroma (no stroma). Highly dynamic culture events occurred in HSC‐MSC co‐cultures, involving cell–cell interactions, which preceded HSC expansion. Throughout the time in culture [18 days], total cell expansion was significantly higher in contact (fold increase of 280 ± 37 at day 18) compared to non‐contact (85 ± 25). No significant cell expansion was observed in stroma‐free cultures. CD34⁺ cell expansion was also clearly favoured by direct contact with BM MSCs (35 ± 5‐ and 7 ± 3‐fold increases at day 18 for contact and non‐contact, respectively). Moreover, a higher percentage of CD34⁺CD38^? cells was consistently maintained during the time in culture under contact (8.1 ± 1.9% at day 18) compared to non‐contact (5.7 ± 1.6%). Importantly, direct cell interaction with BM MSCs significantly enhanced the expansion of early lymphoid CD7⁺ cells, yielding considerably higher (×3–10) progenitor numbers compared to non‐contact conditions. These results highlight the importance of dynamic cell–cell interactions between UCB HSCs and BM MSCs, towards the maximization of HSC expansion ex vivo to obtain clinically relevant cell numbers for multiple settings, such as BM transplantation or somatic cell gene therapy. Copyright © 2009 John Wiley & Sons, Ltd.     

Ex vivo expansion of cord blood haematopoietic stem/progenitor cells under physiological oxygen tensions: clear‐cut effects on cell proliferation,differentiation and metabolism

Physiologically low O₂ tensions are believed to regulate haematopoietic stem cell (HSC) functions in the bone marrow (BM; 0–5%). In turn, placenta and umbilical cord are characterized by slightly higher physiological O₂ tensions (3–10%). We hypothesized that O₂ concentrations within this range may be exploited to augment the ex vivo expansion/maintenance of HSCs from umbilical cord (placental) blood (UCB). The expansion of UCB CD34⁺‐enriched cells was studied in co‐culture with BM mesenchymal stem/stromal cells (MSCs) under 2%, 5%, 10% and 21% O₂. 2% O₂ resulted in a significantly lower CD34⁺ cell expansion (25‐fold vs 60‐, 64‐ and 92‐fold at day 10 for 5%, 21%, 10% O₂, respectively). In turn, 10% O₂ promoted the highest CD34⁺CD90⁺ cell expansion, reaching 22 ± 5.4‐ vs 5.6 ± 2.4‐ and 5.7 ± 2.0‐fold for 2%, 5% and 21% O₂, respectively, after 14 days. Similar differentiation patterns were observed under different O₂ tensions, being primarily shifted towards the neutrophil lineage. Cell division kinetics revealed a higher proliferative status of cells cultured under 10% and 21% vs 2% O₂. Expectedly, higher specific glucose consumption and lactate production rates were determined at 2% O₂ when compared to higher O₂ concentrations (5–21%). Overall, these results suggest that physiological oxygen tensions, in particular 10% O₂, can maximize the ex vivo expansion of UCB stem/progenitor cells in co‐culture with BM MSCs. Importantly, these studies highlight the importance of exploiting knowledge of the intricate microenvironment of the haematopoietic niche towards the definition of efficient and controlled ex vivo culture systems capable of generating large HSCs numbers for clinical applications. Copyright © 2013 John Wiley & Sons, Ltd.     

Immunomodulatory effect of human bone marrow‐derived mesenchymal stromal/stem cells on peripheral blood T cells from rheumatoid arthritis patients

Rheumatoid arthritis (RA) is a Th1/Th17‐mediated autoimmune disease whose current treatment, consisting in the blockage of inflammatory cytokines by disease‐modifying antirheumatic drugs, is not effective for all patients. The therapeutic potential of mesenchymal stromal/stem cells' (MSCs) immunomodulatory properties is being explored in RA. Here, we investigate the effect of human bone marrow (BM)‐MSCs on the expression of cytokines involved in RA physiopathology by the distinct functional compartments of CD4⁺ and CD8⁺ T cells from RA patients. Peripheral blood mononuclear cells from healthy individuals (n = 6) and RA patients (n = 12) were stimulated with phorbol myristate acetate plus ionomycin and cultured in the presence/absence of BM‐MSCs. The expression of (interleukin) IL‐2, tumor necrosis factor alpha (TNF‐α), and interferon‐gamma (IFN‐γ) was evaluated in naive, central memory, effector memory, and effector CD4⁺ and CD8⁺ T cells, whereas IL‐6, IL‐9, and IL‐17 expression was measured in total CD4⁺ and CD8⁺ T cells. mRNA expression of IL‐4, IL‐10, transforming growth factor beta (TGF‐β), cytotoxic T‐lymphocyte‐associated antigen 4, and/or forkhead box P3 was quantified in fluorescence‐activated cell sorting‐purified CD4⁺ T cells, CD8⁺ T cells, and CD4⁺ Treg. BM‐MSCs inhibited the production of TNF‐α, IL‐17, IL‐6, IL‐2, IFN‐γ, and IL‐9 by T cells from RA patients, mainly by reducing the percentage of cells producing cytokines. This inhibitory effect was transversal to all T cell subsets analyzed. At mRNA level, BM‐MSCs increased expression of IL‐10 and TGF‐β by CD4⁺ and CD8⁺ T cells. BM‐MSCs displayed a striking inhibitory action over T cells from RA patients, reducing the expression of cytokines involved in RA physiopathology. Remarkably, BM‐MSC‐derived immunomodulation affected either naive, effector, and memory T cells.     

A rapid method for obtaining mesenchymal stem cells and platelets from bone marrow aspirate

Mesenchymal stem cells (MSCs) and platelet‐rich plasma (PRP) are currently used alone or in combination for therapeutic applications especially for bone repair. We tested whether MSCs can be isolated from bone marrow (BM) aspirate using a commercially available kit commonly used to obtain PRP from peripheral blood (PB). Results revealed that mononuclear cells and platelets from both PB and BM could be efficiently isolated by obtaining a mononuclear and platelet rich fraction (PB‐MPRF and BM‐MPRF, respectively). Starting with comparable volumes, the number of platelets increased 1.5‐fold in BM‐MPRF compared to PB‐MPRF. The number of clonogenic cells in BM‐MPRF samples was significantly higher than whole BM samples as revealed by CFU‐F assay (54.92 ± 8.55 CFU‐F/1.5 x 10⁵ nucleated cells and 32.50 ± 12.43 CFU‐F/1.5 x 10⁵ nucleated cells, respectively). Cells isolated from BM‐MPRF after in vitro expansion fulfilled the definition of MSCs by phenotypic criteria, and differentiated along osteogenic, adipogenic and chondrogenic lineages following induction. Results showed that the kit isolated MSCs and platelets from BM aspirate. Isolated MSCs were further expanded in a laboratory and BM‐MPRF was used clinically following BM withdrawal for rapid intra‐operative cell therapy for the treatment of bone defects. Copyright © 2012 John Wiley & Sons, Ltd.     

Mesenchymal stem cells protect from acute liver injury by attenuating hepatotoxicity of liver natural killer T cells in an inducible nitric oxide synthase‐ and indoleamine 2,3‐dioxygenase‐dependent manner

The effects of mesenchymal stem cells (MSCs) on the phenotype and function of natural killer T (NKT) cells is not understood. We used concanavalin A (Con A) and α‐galactosylceramide (α‐GalCer)‐induced liver injury to evaluate the effects of MSCs on NKT‐dependent hepatotoxicity. Mouse MSCs (mMSCs) significantly reduced Con A‐ and α‐GalCer‐mediated hepatitis in C57Bl/6 mice, as demonstrated by histopathological and biochemical analysis, attenuated the influx of inflammatory [T‐bet⁺, tumour necrosis factor‐α (TNF‐α), interferon‐γ (IFN‐γ)‐producing and GATA3⁺, interleukin‐4 (IL‐4)‐producing] liver NKT cells and downregulated TNF‐α, IFN‐γ and IL‐4 levels in the sera. The liver NKT cells cultured in vitro with mMSCs produced lower amounts of inflammatory cytokines (TNF‐α, IFN‐γ, IL‐4) and higher amounts of immunosuppressive IL‐10 upon α‐GalCer stimulation. mMSC treatment attenuated expression of apoptosis‐inducing ligands on liver NKT cells and suppressed the expression of pro‐apoptotic genes in the livers of α‐GalCer‐treated mice. mMSCs reduced the cytotoxicity of liver NKT cells against hepatocytes in vitro. The presence of 1‐methyl‐dl ‐tryptophan, a specific inhibitor of indoleamine 2,3‐dioxygenase (IDO), or l ‐N^G‐monomethyl arginine citrate, a specific inhibitor of inducible nitric oxide synthase (iNOS), in mMSC‐conditioned medium injected into α‐GalCer‐treated mice, counteracted the hepatoprotective effect of mMSCs in vivo and restored pro‐inflammatory cytokine production and cytotoxicity of NKT cells in vitro. Human MSCs attenuated the production of inflammatory cytokines in α‐GalCer‐stimulated human peripheral blood mononuclear cells in an iNOS‐ and IDO‐dependent manner and reduced their cytotoxicity against HepG2 cells. In conclusion, MSCs protect from acute liver injury by attenuating the cytotoxicity and capacity of liver NKT cells to produce inflammatory cytokines in an iNOS‐ and IDO‐dependent manner.     

Engineered extracellular matrix components do not alter the immunomodulatory properties of mesenchymal stromal cells in vitro

Mesenchymal stromal cells (MSCs) have emerged as promising candidates for regenerative therapies, including tissue engineering. Recently it has been reported that engineered extracellular matrix (ECM) components support the differentiation of MSCs into osteocytes and chondrocytes, indicating that ECM components may represent attractive carriers for MSC transplants to repair damaged tissues. However, little is known about the impact of engineered ECM components on the immunosuppressive properties of MSCs, which may essentially contribute to the prevention of allogeneic MSC transplant rejection. In the present study, we explored the potential of fibronectin, fibrillar collagen I, tropocollagen and collagen I/heparin to influence the immunosuppressive capacities of MSCs. We found that these ECM components do not modulate the capability of MSCs to inhibit the proliferation of anti‐CD3/anti‐CD28 antibody‐stimulated CD4⁺ and CD8⁺ T cells and of lymphocytes in a mixed lymphocyte reaction. In addition, the potential of MSCs to impair the production of immunostimulatory IL‐12 and to improve the release of immunosuppressive IL‐10 by 6‐sulpho LacNAc⁺ (slan) dendritic cells (DCs), representing a pro‐inflammatory subset of human blood DCs, was not altered by the ECM components. Furthermore, ECM components do not influence the ability of MSCs to inhibit the slanDC‐induced proliferation of CD4⁺ T cells. In conclusion, the used engineered ECMs maintain important immunosuppressive properties of MSCs, which support their suitablility as carriers for MSC transplants in tissue engineering. Copyright © 2012 John Wiley & Sons, Ltd.     

Mesenchymal stem cells from CD34− human umbilical cord blood

Umbilical cord blood (UCB) is well known to be a rich source of stem cells especially for haematopoietic stem cells (HSCs). Recently, mesenchymal stem cells (MSCs) have also been shown to exist in cord blood. Although MSCs have been described by a subset of surface antigens after expansion, little is known about the cell surface phenotype of undifferentiated MSCs. The aim of this study therefore was to clarify whether undifferentiated MSCs are resident among CD34^? UCB cells. CD34⁺ cells were separated from UCB mononuclear cells (MNCs) by magnetic sorting and the CD34^? cell fractions were cultured in Dulbecco's modified Eagle's medium (DMEM) with 10% foetal calf serum (FCS) and basic‐fibroblast growth factor. Isolated CD34⁺ cells were also cultured in the same medium. Adherent fibroblast‐like cells at passage 3–4 were analyzed by fluorescence‐activated cell sorting (FACS) for MSC marker expression , and standard adipogenic, osteogenic and chondrogenic assays were used to investigate their differentiation potentials. After 4–5 weeks in culture, the cells from the CD34^? fraction became confluent with flat and fibroblast‐like morphology. These cells were positively stained for the mesenchymal cell markers CD29, CD73 and CD105. In adipogenic differentiation, the cells showed oil red O positive and expressed FABP4, adipsin and proliferation‐activated receptor γ‐2 (PPARγ2 genes) associated with adipogenesis. In osteogenic differentiation, calcium accumulation and osteocalcin were detected. The cells grown in chondrogenic conditions were positively stained for human aggrecan and expressed collagen type II and Sox‐9 genes. In contrast, cells from the CD34⁺ fraction failed to generate any cells with MSC morphology under the same culture conditions. Our results showed that UCB contained MSCs which are only resident in the CD34^? fraction. The MSCs could be induced to differentiate into at least three lineage cell types, adipocytes, osteoblasts and chondrocytes.     

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.     

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.     

Synthesis and secretome release by human bone marrow mesenchymal stem cell spheroids within three‐dimensional collagen hydrogels: Integrating experiments and modelling

Myocardial infarction results in loss of cardiac cell types, inflammation, extracellular matrix (ECM) degradation, and fibrotic scar. Transplantation of bone marrow‐derived mesenchymal stem cells (BM‐MSCs) is being explored as they could differentiate into cardiomyocyte‐like cells, integrate into host tissue, and enhance resident cell activity. The ability of these cells to restore lost ECM, remodel the inflammatory scar tissue, and repair the injured myocardium remains unexplored. We here elucidated the synthesis and deposition of ECM (e.g., elastin, sulfated glycosaminoglycans, hyaluronan, collagen type III, laminin, fibrillin, lysyl oxidase, and nitric oxide synthases), matrix metalloproteinases (MMPs) and their inhibitors (TIMPs), and other secretome (cytokines, chemokines, and growth factors) in adult human BM‐MSC spheroid cultures within three‐dimensional collagen gels. The roles of species‐specific type I collagen and 5‐azacytadine were assessed over a 28‐day period. Results revealed that human collagen (but not rat‐derived) suppressed MSC proliferation and survival, and MSCs synthesized and released a variety of ECM proteins and secretome over the 28 days. Matrix deposition is at least an order of magnitude lower than their release levels at every time point, most possibly due to elevated MMP levels and interleukins with a concomitant decrease in TIMPs. Matrix synthesis over the 28‐day period was fitted to a competitive inhibition form of Michaelis–Menten kinetics, and the production and decay rates of ECM, MMPs, and TIMPs, along with the kinetic model parameters quantified. Such an integrated experimental and modelling approach would help elucidate the critical roles of various parameters (e.g., cell encapsulation and delivery vehicles) in stem cell‐based transplantation therapies.     

Bone marrow‐on‐a‐chip: Long‐term culture of human haematopoietic stem cells in a three‐dimensional microfluidic environment

Multipotent haematopoietic stem and progenitor cells (HSPCs) are the source for all blood cell types. The bone marrow stem cell niche in which the HSPCs are maintained is known to be vital for their maintenance. Unfortunately, to date, no in vitro model exists that accurately mimics the aspects of the bone marrow niche and simultaneously allows the long‐term culture of HSPCs. In this study, a novel three‐dimensional coculture model is presented, based on a hydroxyapatite coated zirconium oxide scaffold, comprising of human mesenchymal stromal cells (MSCs) and cord blood derived HSPCs, enabling successful HSPC culture for a time span of 28 days within the microfluidic multiorgan chip. The HSPCs were found to stay in their primitive state (CD34⁺CD38^?) and capable of granulocyte, erythrocyte, macrophage, megakaryocyte colony formation. Furthermore, a microenvironment was formed bearing molecular and structural similarity to the in vivo bone marrow niche containing extracellular matrix and signalling molecules known to play an important role in HSPC homeostasis. Here, a novel human in vitro bone marrow model is presented for the first time, capable of long‐term culture of primitive HSPCs in a microfluidic environment.     

Integrin α4 impacts on differential adhesion of preadipocytes and stem cells on synthetic polymers

Stem cells represent an ideal cell source for tissue engineering and regenerative medicine, because they can be readily isolated, expanded, differentiated and transplanted. For stem cell‐based therapies, biomaterials are required to allow for a spatial distribution of the stem cells within a defined area in the body. In our recent studies, we analysed the interaction of a large panel of stem cell types with an array of biomaterials and demonstrated that a rational prediction of stem cell behaviour on a specific biomaterial is so far not possible. Interestingly, even ontogenetically related stem cell types, such as mesenchymal stem cells (MSCs), preadipocytes and dental pulp stem cells (DPSCs), exhibit distinct adhesion properties on the very same biomaterial surface. Therefore, we investigated integrin and extracellular matrix (ECM) protein expression of stem cells to relate gene expression to adhesion behaviour. MSCs, preadipocytes and DPSCs were cultured on selected synthetic polymers, such as Texin, a thermoplastic polyurethane, poly(dimethyl siloxane) (PDMS), poly‐d,l ‐lactic acid (PDLLA) and l ‐lactic acid‐trimehylene carbonate (Resomer® LT706). Integrins and ECM proteins were analysed by RT–PCR, real‐time PCR and immunohistochemistry. Analysis of several adhesion molecules yielded that only one molecule, integrin α4, might play a significant role in differential adhesion on polymers for preadipocytes compared to DPSCs and MSCs. Thus, our studies on the molecular interactions of stem cells and polymers are expected to lead to a more profound understanding of the stem cell–biomaterial interactions to eventually allow for a rational biomaterial design. Copyright © 2012 John Wiley & Sons, Ltd.     

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.     

In vitro differentiation of bone marrow derived porcine mesenchymal stem cells to endothelial cells

Mesenchymal stem cells (MSCs) hold potential for the regeneration of damaged tissues in cardiovascular diseases. In this study, we investigated the potential of porcine MSCs to differentiate into endothelial cells (ECs) in vitro. The cultured bone marrow‐derived cells were CD11b^–CD34^–CD44⁺CD45^–CD90⁺ and showed mesodermal lineage differentiation, which is characteristic of MSCs. The MSCs were induced to differentiate into ECs using endothelial growth medium (EGM), with and without high concentrations of VEGF (EGM + VEGF; 50 ng/ml). Endothelial basal medium (EBM) without growth factors served as the control. The EC differentiation was assessed by the presence of vWF, ability to take up acetylated LDL, in vitro angiogenesis assay, flow cytometry and qPCR of EC markers vWF, VE‐cadherin, PECAM‐1, VEGF‐R1 and VEGF‐R2 after 10 days of stimulation. Cells cultured in EGM + VEGF medium demonstrated higher amounts of DiI‐AcLDL‐positive cells and enhanced the presence of vWF (90%), VE‐Cadherin‐ (60%) and PECAM‐1 (48%)‐positive cells, than in EBM. These cells showed profuse sprouting of capillary tubes and closed polygon formation in the angiogenesis assay. There was 1.5–2‐fold increase in the mRNA expression of endothelial markers in the cells stimulated with EGM + VEGF medium when compared to control. The results demonstrate the ability of porcine MSCs to differentiate into ECs under in vitro inducing conditions. The differentiated cells would provide new options for re‐endothelialization following interventional procedures and tissue engineering. Copyright © 2012 John Wiley & Sons, Ltd.     

Quantification of MSCs involved in wound healing: use of SIS to transfer MSCs to wound site and quantification of MSCs involved in skin wound healing

Mesenchymal stem cells (MSCs) are known to be effective in wound healing, but not much has been reported on quantitative correlations between MSCs injected into the wound site and MSCs that actually participate in wound healing. This study traced MSCs participating in wound healing by using small intestinal submucosa (SIS) as a cell carrier, identified their moving path and calculated the number of MSCs involved in wound healing. First, MSCs were isolated from the nude mouse and 1 × 10⁶ cells were seeded onto the centre of the SIS. MSC‐seeded SIS complexes were injected onto full‐thickness skin wounds made on the dorsum of nude mice. Tracing of MSC‐seeded SIS complex transplanted to the wound site revealed that 27.6% of the MSCs were migrated to the wound site at the first attempt. Second, repeated injection of additional MSCs did not increase the number of MSCs participating in wound healing beyond a certain constant maximum amount. The number of MSCs present in the wound site remains constant in the range 2–3 × 10⁵ from day 1 to day 10. The expression of skin regeneration‐related growth factors was confirmed by real‐time polymerase chain reaction (PCR) and enzyme‐linked immunosorbent assay (ELISA). MSCs participating in wound healing were found not only to suppress inflammation of the wound but also to increase the skin regeneration‐related growth factors that enable the recovery of the skin. An optimal number of about 3 × 10⁵ MSCs injected into the site was found to adapt themselves to the skin wound‐healing process effectively. Copyright © 2012 John Wiley & Sons, Ltd.     

Transplantation of porcine umbilical cord matrix mesenchymal stem cells in a mouse model of Parkinson's disease

The present study compared mesenchymal stem cells derived from umbilical cord matrix (UCM‐MSCs) with bone marrow (BM‐MSCs) of miniature pigs on their phenotypic profiles and ability to differentiate in vitro into osteocytes, adipocytes and neuron‐like cells. This study further evaluated the therapeutic potential of UCM‐MSCs in a mouse Parkinson's disease (PD) model. Differences in expression of some cell surface and cytoplasm specific markers were evident between UCM‐MSCs and BM‐MSCs. However, the expression profile indicated the primitive nature of UCM‐MSCs, along with their less or non‐immunogenic features, compared with BM‐MSCs. In vitro differentiation results showed that BM‐MSCs had a higher tendency to form osteocytes and adipocytes, whereas UCM‐MSCs possessed an increased potential to transform into immature or mature neuron‐like cells. Based on these findings, UCM‐MSCs were transplanted into the right substantia nigra (SN) of a mouse PD model. Transplantation of UCM‐MSCs partially recovered the mouse PD model by showing an improvement in basic motor behaviour, as assessed by rotarod and bridge tests. These observations were further supported by the expression of markers, including nestin, tyrosine hydroxylase (TH), neuronal growth factor (NGF), vascular endothelial growth factor (VEGF) and interleukin‐6 (IL‐6), at the site of cell transplantation. Our findings of xenotransplantation have collectively suggested the potential utility of UCM‐MSCs in developing viable therapeutic strategies for PD. Copyright © 2011 John Wiley & Sons, Ltd.     

The neural plasticity of early‐passage human bone marrow‐derived mesenchymal stem cells and their modulation with chromatin‐modifying agents

Mesenchymal stem cells (MSCs) in their immature state express a variety of genes of the three germ layers at relatively low or moderate levels that might explain their phenomenal plasticity. Numerous recent studies have demonstrated that under the appropriate conditions in vitro and in vivo the expression of different sets of these genes can be upregulated, turning MSCs into variety of cell lineages of mesodermal, ectodermal and endodermal origin. While transdifferentiation of MSCs is still controversial, these unique properties make MSCs an ideal autologous source of easily reprogrammable cells. Recently, using the approach of cell reprogramming by biological active compounds that interfere with chromatin structure and function, as well as with specific signalling pathways that promote neural fate commitment, we have been able to generate neural‐like cells from human bone marrow (BM)‐derived MSCs (hMSCs). However, the efficiency of neural transformation of hMSCs induced by this approach gradually declined with passaging. To elucidate the mechanisms that underlie the higher plasticity of early‐passage hMSCs, comparative analysis of the expression levels of several pluripotent and neural genes was conducted for early‐ and late‐passage hMSCs. The results demonstrated that early‐passage hMSCs expressed the majority of these genes at low and moderate levels that gradually declined at late passages. Neural induction further increased the expression of some of these genes in hMSCs, accompanied by morphological changes into neural‐like cells. We concluded that low and moderate expression of several pluripotent and neural genes in early‐passage hMSCs could explain their higher plasticity and pliability for neural induction. Copyright © 2012 John Wiley & Sons, Ltd.     

Reverse remodeling is associated with changes in extracellular matrix proteases and tissue inhibitors after mesenchymal stem cell (MSC) treatment of pressure overload hypertrophy

Changes in ventricular extracellular matrix (ECM) composition of pressure overload hypertrophy determine clinical outcomes. The effects of mesenchymal stem cell (MSC) transplantation upon determinants of ECM composition in pressure overload hypertrophy have not been studied. Sprague–Dawley rats underwent aortic banding and were followed by echocardiography. After an absolute decrease in fractional shortening of 25% from baseline, 1 × 10⁶ MSC (n = 28) or PBS (n = 20) was randomly injected intracoronarily. LV protein analysis, including matrix metalloproteinases (MMP‐2, MMP‐3, MMP‐6, MMP‐9) and tissue inhibitors of metalloproteinases (TIMP‐1, TIMP‐2, TIMP‐3), was performed after sacrifice on postoperative day 7, 14, 21 or 28. Left ventricular levels of MMP‐3, MMP‐6, MMP‐9, TIMP‐1 and TIMP‐3 were demonstrated to be decreased in the MSC group compared with controls after 28 days. Expression of MMP‐2 and TIMP‐2 remained relatively stable in both groups. Successful MSCs delivery was confirmed by histological analysis and visualization of labelled MSCs. In this model of pressure overload hypertrophy, intracoronary delivery of MSCs during heart failure was associated with specific changes in determinants of ECM composition. LV reverse remodeling was associated with decreased ventricular levels of MMP‐3, MMP‐6, MMP‐9, TIMP‐1 and TIMP‐3, which were upregulated in the control group as heart failure progressed. These effects were most significant at 28 days following injection. Copyright © 2008 John Wiley & Sons, Ltd.     

A local application of mesenchymal stem cells and cyclosporine A attenuates immune response by a switch in macrophage phenotype

The immunosuppressive effects of systemically administered mesenchymal stem cells (MSCs) and immunosuppressive drugs have been well documented. We analysed the mechanisms underlying the therapeutic effect of MSCs applied locally in combination with non‐specific immunosuppression in a mouse model of allogeneic skin transplantation. The MSC‐seeded and cyclosporine A (CsA)‐loaded nanofibre scaffolds were applied topically to skin allografts in a mouse model and the local immune response was assessed and characterized. MSCs migrated from the scaffold into the side of injury and were detected in the graft region and draining lymph nodes (DLNs). The numbers of graft‐infiltrating macrophages and the production of nitric oxide (NO) were significantly decreased in recipients treated with MSCs and CsA, and this reduction correlated with impaired production of IFNγ in the graft and DLNs. In contrast, the proportion of alternatively activated macrophages (F4/80⁺CD206⁺ cells) and the production of IL‐10 by intragraft macrophages were significantly upregulated. The ability of MSCs to alter the phenotype of macrophages from the M1 type into an M2 population was confirmed in a co‐culture system in vitro. We suggest that the topical application of MSCs in combination with CsA induces a switch in macrophages to a population with an alternatively activated 'healing' phenotype and producing elevated levels of IL‐10. These alterations in macrophage phenotype and function could represent one of the mechanisms of immunosuppressive action of MSCs applied in combination with CsA. Copyright © 2015 John Wiley & Sons, Ltd.