Dose-Dependent Effect of Mesenchymal Stromal Cell Recruiting Chemokine CCL25 on Porcine Tissue-Engineered Healthy and Osteoarthritic Cartilage.

Thymus-expressed chemokine (CCL25) is a potent cell attractant for mesenchymal stromal cells, and therefore it is a candidate for in situ cartilage repair approaches focusing on the recruitment of endogenous repair cells. However, the influence of CCL25 on cartilage is unknown. Accordingly, in this study, we investigated the effect of CCL25 on tissue-engineered healthy and osteoarthritic cartilage. Porcine chondrocytes were cultured in a three-dimensional (3D) micromass model that has been proven to mimic key-aspects of human cartilage and osteoarthritic alterations upon stimulation with tumor necrosis factor-α (TNF-α). Micromass cultures were stimulated with CCL25 (0, 0.05, 0.5, 5, 50, 500 nmol/L) alone or in combination with 0.6 nmol/L TNF-α for seven days. Effects were evaluated by life/dead staining, safranin O staining, histomorphometrical analysis of glycosaminoglycans (GAGs), collagen type II (COL2A1) real-time RT-PCR and Porcine Genome Array analysis. 500 nmol/L CCL25 led to a significant reduction of GAGs and COL2A1 expression and induced the expression of matrix metallopeptidases (MMP) 1, MMP3, early growth response protein 1 (EGR1), and superoxide dismutase 2 (SOD2). In concentrations lower than 500 nmol/L, CCL25 seems to be a candidate for in situ cartilage repair therapy approaches.

Hyaluronic Acid Influence on Normal and Osteoarthritic Tissue-Engineered Cartilage.

The aim of this study is to identify gene expression profiles associated with hyaluronic acid (HA) treatment of normal and osteoarthritis (OA)-like tissue-engineered cartilage. 3D cartilage micromasses were treated with tumour necrosis factor-α (TNF-α) (OA-inducer) and/or HA for 7 days. Viability was examined by PI/FDA staining. To document extracellular matrix (ECM) formation, glycosaminoglycans (GAG) were stained with Safranin-O and cartilage-specific type II collagen was detected immunohistochemically. Genome-wide gene expression was determined using microarray analysis. Normal and OA-like micromasses remained vital and showed a spherical morphology and homogenous cell distribution regardless of the treatment. There was no distinct difference in immunolabeling for type II collagen. Safranin-O staining demonstrated a typical depletion of GAG in TNF-α-treated micromasses (-73%), although the extent was limited in the presence of HA (-39%). The microarray data showed that HA can influence the cartilage metabolism via upregulation of TIMP3 in OA-like condition. The upregulation of VEGFA and ANKRD37 genes implies a supportive role of HA in cartilage maturation and survival. The results of this study validate the feasibility of the in vitro OA model for the investigation of HA. On the cellular level, no inhibiting or activating effect of HA was shown. Microarray data demonstrated a minor impact of HA on gene expression level.

Quality Assessment of Surgical Disc Samples Discriminates Human Annulus Fibrosus and Nucleus Pulposus on Tissue and Molecular Level.

A discrimination of the highly specialised annulus fibrosus (AF) and nucleus pulposus (NP) cells in the mature human intervertebral disc (IVD) is thus far still not possible in a reliable way. The aim of this study was to identify molecular markers that distinguish AF and NP cells in human disc tissue using microarray analysis as a screening tool. AF and NP samples were obtained from 28 cervical discs. First, all samples underwent quality sorting using two novel scoring systems for small-sized disc tissue samples including macroscopic, haptic and histological evaluation. Subsequently, samples with clear disc characteristics of either AF or NP that were free from impurities of foreign tissue (IVD score) and with low signs of disc degeneration on cellular level (DD score) were selected for GeneChip analysis (HGU1332P). The 11 AF and 9 NP samples showed distinctly different genome-wide transcriptomes. The majority of differentially expressed genes (DEGs) could be specifically assigned to the AF, whereas no DEG was exclusively expressed in the NP. Nevertheless, we identified 11 novel marker genes that clearly distinguished AF and NP, as confirmed by quantitative gene expression analysis. The novel established scoring systems and molecular markers showed the identity of AF and NP in disc starting material and are thus of great importance in the quality assurance of cell-based therapeutics in regenerative treatment of disc degeneration.

Chemokine CCL25 Induces Migration and Extracellular Matrix Production of Anulus Fibrosus-Derived Cells.

Intervertebral disc degeneration is a major source of back pain. For intervertebral disc regeneration after herniation a fast closure of anulus fibrosus (AF) defects is crucial. Here, the use of the C-C motif chemokine ligand 25 (CCL)25 in comparison to differentiation factors such as transforming growth factor (TGF)ß3, bone morphogenetic protein (BMP)2, BMP7, BMP12, and BMP14 (all in concentrations of 10, 50 and 100 ng/mL) was tested in an in vitro micro mass pellet model with isolated and cultivated human AF-cells (n = 3) to induce and enhance AF-matrix formation. The pellets were differentiated (serum-free) with supplementation of the factors. After 28 days all used factors induced proteoglycan production (safranin O staining) and collagen type I production (immunohistochemical staining) in at least one of the tested concentrations. Histomorphometric scoring revealed that TGFß3 delivered the strongest induction of proteoglycan production in all three concentrations. Furthermore, it was the only factor able to facilitate collagen type II production, even higher than in native tissue samples. CCL25 was also able to induce proteoglycan and collagen type I production comparable to several BMPs. CCL25 could additionally induce migration of AF-cells in a chemotaxis assay and therefore possibly aid in regeneration processes after disc herniation by recruiting AF-cells.

Cardiac migration of endogenous mesenchymal stromal cells in patients with inflammatory cardiomyopathy.

Introduction. Mesenchymal stromal cells (MSC) have immunomodulatory features. The aim of this study was to investigate the migration and homing potential of endogenous circulating MSC in virus negative inflammatory cardiomyopathy (CMi). Methods. In 29 patients with (n = 23) or without (n = 6) CMi undergoing endomyocardial biopsies (EMB), transcardiac gradients (TCGs) of circulating MSC were measured by flow cytometry from blood simultaneously sampled from aorta and coronary sinus. The presence of MSC in EMB, cardiac inflammation, and SDF-1α mRNA expression were detected via immunohistochemistry and real-time PCR. Results. MSC defined as CD45(-)CD34(-)CD11b(-)CD73(+)CD90(+) cells accounted for 0.010 [0.0025-0.048]%/peripheral mononuclear cell (PMNC) and as CD45(-)CD34(-)CD11b(-)CD73(+)CD105(+) cells for 0.019 [0.0026-0.067]%/PMNC, both with similar counts in patients with or without cardiac inflammation. There was a 29.9% (P < 0.01) transcardiac reduction of circulating MSC in patients with CMi, correlating with the extent of cardiac inflammation (P < 0.05, multivariate analysis). A strong correlation was found between the TCG of circulating MSC and numbers of MSC (CD45(-)CD34(-)CD90(+)CD105(+)) in EMB (r = -0.73, P < 0.005). SDF-1α was the strongest predictor for increased MSC in EMB (P < 0.005, multivariate analysis). Conclusions. Endogenous MSC continuously migrate to the heart in patients with CMi triggered by cardiac inflammation.

Suitability of porcine chondrocyte micromass culture to model osteoarthritis in vitro.

In vitro tissue models are useful tools for the development of novel therapy strategies in cartilage repair and care. The limited availability of human primary tissue and high costs of animal models hamper preclinical tests of innovative substances and techniques. In this study we tested the potential of porcine chondrocyte micromass cultures to mimic human articular cartilage and essential aspects of osteoarthritis (OA) in vitro. Primary chondrocytes were enzymatically isolated from porcine femoral condyles and were maintained in 96-multiwell format to establish micromass cultures in a high-throughput scale. Recombinant porcine tumor necrosis factor alpha (TNF-α) was used to induce OA-like changes documented on histological (Safranin O, collagen type II staining), biochemical (hydroxyproline assay, dimethylmethylene blue method), and gene expression level (Affymetrix porcine microarray, real time PCR) and were compared with published data from human articular cartilage and human micromass cultures. After 14 days in micromass culture, porcine primary chondrocytes produced ECM rich in proteoglycans and collagens. On gene expression level, significant correlations of detected genes with porcine cartilage (r = 0.90), human cartilage (r = 0.71), and human micromass culture (r = 0.75) were observed including 34 cartilage markers such as COL2A1, COMP, and aggrecan. TNF-α stimulation led to significant proteoglycan (-75%) and collagen depletion (-50%). Comparative expression pattern analysis revealed the involvement of catabolic enzymes (MMP1, -2, -13, ADAM10), chemokines (IL8, CCL2, CXCL2, CXCL12, CCXL14), and genes associated with cell death (TNFSF10, PMAIPI, AHR) and skeletal development (GPNMB, FRZB) including transcription factors (WIF1, DLX5, TWIST1) and growth factors (IGFBP1, -3, TGFB1) consistent with published data from human OA cartilage. Expression of genes related to cartilage ECM formation (COL2A1, COL9A1, COMP, aggrecan) as well as hypertrophic bone formation (COL1A1, COL10A1) was predominantly found decreased. These findings indicating significant parallels between human articular cartilage and the presented porcine micromass model and vice versa confirm the applicability of known cartilage marker and their characteristics in the porcine micromass model. TNF-α treatment enabled the initiation of typical OA reaction patterns in terms of extensive ECM loss, cell death, formation of an inflammatory environment through the induction of genes coding for chemokines and enzymes, and the modulation of genes involved in skeletal development such as growth factors, transcription factors, and cartilage ECM-forming genes. In conclusion, the porcine micromass model represents an alternative tissue platform for the evaluation of innovative substances and techniques for the treatment of OA.

A P19 and P19CL6 cell-based complementary approach to determine paracrine effects in cardiac tissue engineering.

The negligible self-repair potential of the myocardium has led to cell-based tissue engineering approaches to restore heart function. There is more and more consensus that, in addition to cell development, paracrine effects in particular play a pivotal role in the repair of heart tissue. Here, we present two complementary murine P19 and P19CL6 embryonic carcinoma cell-based in vitro test approaches to study the potential of repair cells and the factors secreted by these cells to induce cardiomyogenesis. P19 cells were 3-dimensionally cultured in hanging drops and P19CL6 cells in a monolayer. Both systems, capable of inducible differentiation towards the cardiomyogenic lineage shown by the appearance of beating cells, the expression of connexin 43 and cardiac troponins T and I, were used to test the cardiomyogenesis-inducing potential of human cardiac-derived adherent proliferating (CardAP) cells, which are candidates for heart repair. CardAP cells in coculture as well as CardAP cell-conditioned medium initiated beating in P19 cells, depending on the cell composition and concentration of the medium. CardAP cell-dependent beating was not observed in P19CL6 cultures, but connexin 43 and cardiac troponin formation as well as expression of GATA-binding protein 4 indicated the dose-dependent stimulatory cardiomyogenic effect of human CardAP cells. In summary, in different ways, P19 and P19CL6 cells have shown their capability to detect paracrine effects of human CardAP cells. In a complementary approach, they could be beneficial for determining the stimulatory cardiomyogenic potential of candidate cardiac-repair cells in vitro.

Chitosan-based injectable hydrogel as a promising in situ forming scaffold for cartilage tissue engineering.

Chitosan-beta glycerophosphate-hydroxyethyl cellulose (CH-GP-HEC) is a biocompatible and biodegradable scaffold exhibiting a sol-gel transition at 37°C. Chondrogenic factors or mesenchymal stem cells (MSCs) can be included in the CH-GP-HEC, and injected into the site of injury to fill the cartilage tissue defects with minimal invasion and pain. The possible impact of the injectable CH-GP-HEC on the viability of the encapsulated MSCs was assessed by propidium iodide-fluorescein diacetate staining. Proliferation of the human and rat MSCs was also determined by MTS assay on days 0, 7, 14 and 28 after encapsulation. To investigate the potential application of CH-GP-HEC as a drug delivery device, the in vitro release profile of insulin was quantified by QuantiPro-BCA™ protein assay. Chondrogenic differentiation capacity of the encapsulated human MSCs (hMSCs) was also determined after induction of differentiation with transforming growth factor ß3. MSCs have very good survival and proliferative rates within CH-GP-HEC hydrogel during the 28-day investigation. A sustained release of insulin occurred over 8 days. The CH-GP-HEC hydrogel also provided suitable conditions for chondrogenic differentiation of the encapsulated hMSCs. In conclusion, the high potential of CH-GP-HEC as an injectable hydrogel for cartilage tissue engineering is emphasised.

Transdifferentiation of mesenchymal stem cells-derived adipogenic-differentiated cells into osteogenic- or chondrogenic-differentiated cells proceeds via dedifferentiation and have a correlation with cell cycle arresting and driving genes.

It is generally accepted that after differentiation bone marrow mesenchymal stem cells (MSC) become lineage restricted and unipotent in an irreversible manner. However, current results imply that even terminally differentiated cells transdifferentiate across lineage boundaries and therefore act as a progenitor cells for other lineages. This leads to the questions that whether transdifferentiation occurs via direct cell-to-cell conversion or dedifferentiation to a progenitor cells and subsequent differentiation, and whether MSC potency decreases or increases during differentiation. To address these questions, MSC were differentiated into adipogenic lineage cells, followed by dedifferentiation. The process of dedifferentiation was also confirmed by single cell clonal analysis. Finally the dedifferentiated cells were used for adipogenesis, osteogenesis and chondrogenesis. Histology, FACS, qPCR and GeneChip analyses of undifferentiated MSC, adipogenic-differentiated and dedifferentiated cells were performed. Interestingly, gene profiling and bioinformatics demonstrated that upregulation (DHCR24, G0S2, MAP2K6, SESN3) and downregulation (DST, KAT2, MLL5, RB1, SMAD3, ZAK) of distinct genes have an association with cell cycle arrest in adipogenic-differentiated cells and perhaps narrow down the lineage potency. However, the upregulation (CCND1, CHEK, HGF, HMGA2, SMAD3) and downregulation (CCPG1, RASSF4, RGS2) of these genes have an association with cell cycle progression and maybe motivate dedifferentiation of adipogenic-differentiated cells. We found that dedifferentiated cells have a multilineage potency comparable to MSC, and also observed the associative role of proliferation genes with cell cycle arrest and progression. Concluded, our results indicate that transdifferentiation of adipogenic-differentiated cells into osteogenic- or chondrogenic-differentiated cells proceeds via dedifferentiation and correlates with cell cycle arresting and deriving genes. Regarding clinical use, the knowledge of potency and underlying mechanisms are prerequisites.

Downregulation of ErbB3 by Wnt3a contributes to wnt-induced osteoblast differentiation in mesenchymal cells.

Mesenchymal stem cells (MSC) can differentiate into osteoblasts upon activation of Wnt signaling. Identifying targets of Wnt signaling in MSC may help promote MSC osteoblast differentiation for bone regeneration. In this study, using microarray analysis we found that Wnt3a upregulates neuregulin 1 (NRG-1) during Wnt3a-induced osteoblast differentiation in primary human MSC and murine C3H10T1/2 mesenchymal cells. Western blot and qPCR analyses confirmed that NRG-1 is upregulated by Wnt3a, and that this effect was counterbalanced by decreased expression of the NRG-1 receptor ErbB3. Consistently, exogenous NRG-1 had no effect on alkaline phosphatase (ALP) activity, an early marker of osteoblast differentiation. In contrast, small interfering RNA-mediated silencing of endogenous NRG-1 increased basal and Wnt3a-induced ALP activity in MSC. We showed that short hairpin (sh) ErbB3 and Wnt3a additively increased ß-catenin transcriptional activity and ALP activity in MSC. These effects were abrogated by DKK1, indicating that cross-talk between Wnt3a and ErbB3 control MSC osteoblast differentiation via Wnt/ß-catenin signaling. Furthermore, ErbB3 silencing decreased Src expression. Pharmacological inhibition of Src signaling promoted ErbB3- and Wnt-induced ALP activity, suggestive of a role of Src signaling in the modulation of osteoblast differentiation by ErbB3 and Wnt3a. The results indicate that downregulation of ErbB3 induced by Wnt3a contributes to Wnt3a-induced early osteoblast differentiation of MSCs through increased canonical Wnt/ß-catenin signaling and decreased Src signaling.

Increased EFG- and PDGFalpha-receptor signaling by mutant FGF-receptor 2 contributes to osteoblast dysfunction in Apert craniosynostosis.

Dysregulations of osteoblast function induced by gain-of-function genetic mutations in fibroblast growth factor receptors (FGFRs) cause premature fusion of cranial sutures in syndromic craniosynostosis. The pathogenic signaling mechanisms induced by FGFR genetic mutations in human craniosynostosis remain largely unknown. In this study, we have used microarray analysis to investigate the signaling pathways that are activated by FGFR2 mutations in Apert craniosynostosis. Transcriptomic analysis revealed that EGFR and PDGFRalpha expression is abnormally increased in human Apert calvaria osteoblasts compared with wild-type cells. Quantitative RT-PCR and western blot analyses in Apert osteoblasts and immunohistochemical analysis of Apert sutures confirmed the increased EGFR and PDGFRalpha expression in vitro and in vivo. We demonstrate that pharmacological inhibition of EGFR and PDGFR reduces the pathological upregulation of phenotypic osteoblast genes and in vitro matrix mineralization in Apert osteoblasts. Investigation of the underlying molecular mechanisms revealed that activated FGFR2 enhances EGFR and PDGFRalpha mRNA expression via activation of PKCalpha-dependent AP-1 transcriptional activity. We also show that the increased EGFR protein expression in Apert osteoblasts results in part from a post-transcriptional mechanism involving increased Sprouty2-Cbl interaction, leading to Cbl sequestration and reduced EGFR ubiquitination. These data reveal novel molecular crosstalks between activated FGFR2, EGFR and PDGFRalpha that functionally contribute to the osteoblastic dysfunction in Apert craniosynostosis, which may provide a molecular basis for novel therapeutic approaches in this severe skeletal disorder.

A method to screen and evaluate tissue adhesives for joint repair applications.

BACKGROUND: Tissue adhesives are useful means for various medical procedures. Since varying requirements cause that a single adhesive cannot meet all needs, bond strength testing remains one of the key applications used to screen for new products and study the influence of experimental variables. This study was conducted to develop an easy to use method to screen and evaluate tissue adhesives for tissue engineering applications. METHOD: Tissue grips were designed to facilitate the reproducible production of substrate tissue and adhesive strength measurements in universal testing machines. Porcine femoral condyles were used to generate osteochondral test tissue cylinders (substrates) of different shapes. Viability of substrates was tested using PI/FDA staining. Self-bonding properties were determined to examine reusability of substrates (n = 3). Serial measurements (n = 5) in different operation modes (OM) were performed to analyze the bonding strength of tissue adhesives in bone (OM-1) and cartilage tissue either in isolation (OM-2) or under specific requirements in joint repair such as filling cartilage defects with clinical applied fibrin/PLGA-cell-transplants (OM-3) or tissues (OM-4). The efficiency of the method was determined on the basis of adhesive properties of fibrin glue for different assembly times (30 s, 60 s). Seven randomly generated collagen formulations were analyzed to examine the potential of method to identify new tissue adhesives. RESULTS: Viability analysis of test tissue cylinders revealed vital cells (>80%) in cartilage components even 48 h post preparation. Reuse (n = 10) of test substrate did not significantly change adhesive characteristics. Adhesive strength of fibrin varied in different test settings (OM-1: 7.1 kPa, OM-2: 2.6 kPa, OM-3: 32.7 kPa, OM-4: 30.1 kPa) and was increasing with assembly time on average (2.4-fold). The screening of the different collagen formulations revealed a substance with significant higher adhesive strength on cartilage (14.8 kPa) and bone tissue (11.8 kPa) compared to fibrin and also considerable adhesive properties when filling defects with cartilage tissue (23.2 kPa). CONCLUSION: The method confirmed adhesive properties of fibrin and demonstrated the dependence of adhesive properties and applied settings. Furthermore the method was suitable to screen for potential adhesives and to identify a promising candidate for cartilage and bone applications. The method can offer simple, replicable and efficient evaluation of adhesive properties in ex vivo specimens and may be a useful supplement to existing methods in clinical relevant settings.

Priming integrin alpha5 promotes human mesenchymal stromal cell osteoblast differentiation and osteogenesis.

Adult human mesenchymal stromal cells (hMSCs) have the potential to differentiate into chondrogenic, adipogenic, or osteogenic lineages, providing a potential source for tissue regeneration. An important issue for efficient bone regeneration is to identify factors that can be targeted to promote the osteogenic potential of hMSCs. Using transcriptome analysis, we found that integrin alpha5 (ITGA5) expression is up-regulated during dexamethasone-induced osteoblast differentiation of hMSCs. Gain-of-function studies showed that ITGA5 promotes the expression of osteoblast phenotypic markers and in vitro osteogenesis of hMSCs. Down-regulation of endogenous ITGA5 using specific shRNAs blunted osteoblast marker gene expression and osteogenic differentiation. Molecular analyses showed that the enhanced osteoblast differentiation induced by ITGA5 was mediated by activation of focal adhesion kinase/ERK1/2-MAPKs and PI3K signaling pathways. Remarkably, activation of endogenous ITGA5 using agonists such as a specific antibody that primes the integrin or a peptide that specifically activates ITGA5 was sufficient to enhance ERK1/2-MAPKs and PI3K signaling and to promote osteoblast differentiation and osteogenic capacity of hMSCs. Importantly, we demonstrated that hMSCs engineered to overexpress ITGA5 exhibited a marked increase in their osteogenic potential in vivo. Taken together, these findings not only reveal that ITGA5 is required for osteoblast differentiation of adult hMSCs but also provide a targeted strategy using ITGA5 agonists to promote the osteogenic capacity of hMSCs. This may be used for tissue regeneration in bone disorders where the recruitment or capacity of hMSCs is compromised.

Differential gene expression profiling of human bone marrow-derived mesenchymal stem cells during adipogenic development.

BACKGROUND: Adipogenesis is the developmental process by which mesenchymal stem cells (MSC) differentiate into pre-adipocytes and adipocytes. The aim of the study was to analyze the developmental strategies of human bone marrow MSC developing into adipocytes over a defined time scale. Here we were particularly interested in differentially expressed transcription factors and biochemical pathways. We studied genome-wide gene expression profiling of human MSC based on an adipogenic differentiation experiment with five different time points (day 0, 1, 3, 7 and 17), which was designed and performed in reference to human fat tissue. For data processing and selection of adipogenic candidate genes, we used the online database SiPaGene for Affymetrix microarray expression data. RESULTS: The mesenchymal stem cell character of human MSC cultures was proven by cell morphology, by flow cytometry analysis and by the ability of the cells to develop into the osteo-, chondro- and adipogenic lineage. Moreover we were able to detect 184 adipogenic candidate genes (85 with increased, 99 with decreased expression) that were differentially expressed during adipogenic development of MSC and/or between MSC and fat tissue in a highly significant way (p < 0.00001). Subsequently, groups of up- or down-regulated genes were formed and analyzed with biochemical and cluster tools. Among the 184 genes, we identified already known transcription factors such as PPARG, C/EBPA and RTXA. Several of the genes could be linked to corresponding biochemical pathways like the adipocyte differentiation, adipocytokine signalling, and lipogenesis pathways. We also identified new candidate genes possibly related to adipogenesis, such as SCARA5, coding for a receptor with a putative transmembrane domain and a collagen-like domain, and MRAP, encoding an endoplasmatic reticulum protein. CONCLUSIONS: Comparing differential gene expression profiles of human MSC and native fat cells or tissue allowed us to establish a comprehensive differential kinetic gene expression network of adipogenesis. Based on this, we identified known and unknown genes and biochemical pathways that may be relevant for adipogenic differentiation. Our results encourage further and more focused studies on the functional relevance of particular adipogenic candidate genes.

Mesenchymal stem cell-dependent formation of heterotopic tendon-bone insertions (osteotendinous junctions).

Ligament-to-bone and tendon-to-bone interfaces (entheses, osteotendinous junctions [OTJs]) serve to dissipate stress between soft tissue and bone. Surgical reconstruction of these interfaces is an issue of considerable importance as they are prone to injury and the integration of bone and tendon/ligament is in general not satisfactory. We report here the stem cell-dependent spontaneous formation of fibrocartilaginous and fibrous entheses in heterotopic locations of the mouse if progenitors possess a tenogenic and osteo-/chondrogenic capacity. This study followed the hypothesis that enhanced Bone Morphogenetic Protein (BMP)-signaling in adult mesenchymal stem cells that are induced for tendon formation may overcome the tendon-inherent interference with bone formation and may thus allow the stem cell-dependent formation of tendon-bone interfaces. The tenogenic and osteo-/chondrogenic competence was mediated by the adeno- and/or lentiviral expression of the biologically active Smad8 signaling mediator (Smad8ca) and of Bone Morphogenetic Protein 2 (BMP2). Modified mesenchymal progenitors were implanted in subcutaneous or intramuscular sites of the mouse. The stem cell-dependent enthesis formation was characterized histologically by immunohistological approaches and by in situ hybridization. Transplantation of modified murine stem cells resulted in the formation of tendinous and osseous structures exhibiting fibrocartilage-type OTJs, while, in contrast, the viral modification of primary human bone marrow-derived mesenchymal stromal/stem cells showed evidence of fibrous tendon-bone interface formation. Moreover, it could be demonstrated that Smad8ca expression alone was sufficient for the formation of tendon/ligament-like structures. These findings may contribute to the establishment of stem cell-dependent regenerative therapies involving tendon/ligaments and to the improvement of the insertion of tendon grafts at bony attachment sites, eventually.

In vivo effect of bone marrow-derived mesenchymal stem cells in a rat kidney transplantation model with prolonged cold ischemia.

Brain death and prolonged cold ischemia are major contributors to the poorer long-term outcome of transplants from deceased donor kidney transplants, with an even higher impact if expanded criteria donors ('marginal organs') are used. Targeting ischemia-reperfusion injury-related intragraft inflammation is an attractive concept to improve the outcome of those grafts. As mesenchymal stem cells (MSCs) express both immunomodulatory and tissue repair properties, we evaluated their therapeutic efficacy in a rat kidney transplant model of prolonged cold ischemia. The in vitro immunomodulatory capacity of bone marrow-derived rat MSCs was tested in co-cultures with rat lymph node cells. For in vivo studies, Dark Agouti rat kidneys were cold preserved and transplanted into Lewis rats. Syngeneic Lewis MSCs were administered intravenously. Transplants were harvested on day 3, and inflammation was examined by quantitative RT-PCR and histology. Similarly to MSCs from other species, rat MSCs in vitro also showed a dose-dependent immunomodulatory capacity. Most importantly, in vivo administration of MSCs reduced the intragraft gene expression of different pro-inflammatory cytokines, chemokines, and intercellular adhesion molecule-1. In addition, fewer antigen-presenting cells were recruited into the renal allograft. In conclusion, rat MSCs ameliorate inflammation induced by prolonged cold ischemia in kidney transplantation.

Novel markers of mesenchymal stem cells defined by genome-wide gene expression analysis of stromal cells from different sources.

Mesenchymal stem cells (MSC) represent a mixture of different cell types, of which only a minority is therapeutically relevant. Surface markers specifically identifying non-differentiated MSC from their differentiated progeny have not been described in sufficient detail. We here compare the gene expression profile of the in vivo bone-forming bone marrow-derived MSC (BM-MSC) with non-bone-forming umbilical vein stromal cells (UVSC) and other non-MSC. Clustering analysis shows that UVSC are a lineage homogeneous cell population, clearly distinct from MSC, other mesenchymal lineages and hematopoietic cells. We find that 89 transcripts of membrane-associated proteins are represented more in cultured BM-MSC than in UVSC. These include previously identified molecules, but also novel markers like NOTCH3, JAG1, and ITGA11. We show that the latter three molecules are also expressed on fibroblast colony-forming units (CFU-F). Both NOTCH3 and ITGA11, but not JAG1, further enrich for CFU-F when combined with CD146, a known marker of cells with MSC activity in vivo. Differentiation studies show that NOTCH3+ and CD146+ NOTCH3+ cells sorted from cultured BM-MSC are capable of adipogenic and osteogenic progeny, while ITGA11-expressing cells mainly show an osteogenic differentiation profile with limited adipogenic differentiation. Our observations may facilitate the study of lineage relationships in MSC as well as facilitate the development of more homogeneous cell populations for mesenchymal cell therapy.

Crosstalks between integrin alpha 5 and IGF2/IGFBP2 signalling trigger human bone marrow-derived mesenchymal stromal osteogenic differentiation.

BACKGROUND: The potential of mesenchymal stromal cells (MSCs) to differentiate into functional bone forming cells provides an important tool for bone regeneration. The identification of factors that trigger osteoblast differentiation in MSCs is therefore critical to promote the osteogenic potential of human MSCs. In this study, we used microarray analysis to identify signalling molecules that promote osteogenic differentiation in human bone marrow stroma derived MSCs. RESULTS: Microarray analysis and validation experiments showed that the expression of IGF2 and IGFBP2 was increased together with integrin alpha5 (ITGA5) during dexamethasone-induced osteoblast differentiation in human MSCs. This effect was functional since we found that IGF2 and IGFBP2 enhanced the expression of osteoblast phenotypic markers and in vitro osteogenic capacity of hMSCs. Interestingly, we showed that downregulation of endogenous ITGA5 using specific shRNA decreased IGF2 and IGFBP2 expression in hMSCs. Conversely, ITGA5 overexpression upregulated IGF2 and IGFBP2 expression in hMSCs, which indicates tight crosstalks between these molecules. Consistent with this concept, activation of endogenous ITGA5 using a specific antibody that primes the integrin, or a peptide that specifically activates ITGA5 increased IGF2 and IGFBP2 expression in hMSCs. Finally, we showed that pharmacological inhibition of FAK/ERK1/2-MAPKs or PI3K signalling pathways that are enhanced by ITGA5 activation, blunted IGF2 and IGFBP2 expression in hMSCs. CONCLUSION: The results show that ITGA5 is a key mediator of IGF2 and IGFBP2 expression that promotes osteoblast differentiation in human MSCs, and reveal that crosstalks between ITGA5 and IGF2/IGFBP2 signalling are important mechanisms that trigger osteogenic differentiation in human bone marrow derived mesenchymal stromal cells.

Endomyocardial biopsy derived adherent proliferating cells - a potential cell source for cardiac tissue engineering.

Heart diseases are a leading cause of morbidity and mortality. Cardiac stem cells (CSC) are considered as candidates for cardiac-directed cell therapies. However, clinical translation is hampered since their isolation and expansion is complex. We describe a population of human cardiac derived adherent proliferating (CAP) cells that can be reliably and efficiently isolated and expanded from endomyocardial biopsies (0.1 cm(3)). Growth kinetics revealed a mean cell doubling time of 49.9 h and a high number of 2.54 x 10(7) cells in passage 3. Microarray analysis directed at investigating the gene expression profile of human CAP cells demonstrated the absence of the hematopoietic cell markers CD34 and CD45, and of CD90, which is expressed on mesenchymal stem cells (MSC) and fibroblasts. These data were confirmed by flow cytometry analysis. CAP cells could not be differentiated into adipocytes, osteoblasts, chondrocytes, or myoblasts, demonstrating the absence of multilineage potential. Moreover, despite the expression of heart muscle markers like alpha-sarcomeric actin and cardiac myosin, CAP cells cannot be differentiated into cardiomyocytes. Regarding functionality, CAP cells were especially positive for many genes involved in angiogenesis like angiopoietin-1, VEGF, KDR, and neuropilins. Globally, principal component and hierarchical clustering analysis and comparison with microarray data from many undifferentiated and differentiated reference cell types, revealed a unique identity of CAP cells. In conclusion, we have identified a unique cardiac tissue derived cell type that can be isolated and expanded from endomyocardial biopsies and which presents a potential cell source for cardiac repair. Results indicate that these cells rather support angiogenesis than cardiomyocyte differentiation.

Specific lineage-priming of bone marrow mesenchymal stem cells provides the molecular framework for their plasticity.

Lineage-priming is a molecular model of stem cell (SC) differentiation in which proliferating SCs express a subset of genes associated to the differentiation pathways to which they can commit. This concept has been developed for hematopoietic SCs, but has been poorly studied for other SC populations. Because the differentiation potential of human bone marrow mesenchymal stem cells (BM MSCs) remains controversial, we have explored the theory of lineage-priming applied to these cells. We show that proliferating primary layers and clones of BM MSCs have precise priming to the osteoblastic (O), chondrocytic (C), adipocytic (A), and the vascular smooth muscle (V) lineages, but not to skeletal muscle, cardiac muscle, hematopoietic, hepatocytic, or neural lineages. Priming was shown both at the mRNA (300 transcripts were evaluated) and the protein level. In particular, the master transactivator proteins PPARG, RUNX2, and SOX9 were coexpressed before differentiation induction in all cells from incipient clones. We further show that MSCs cultured in the presence of inducers differentiate into the lineages for which they are primed. Our data point out to a number of signaling pathways that might be activated in proliferating MSCs and would be responsible for the differentiation and proliferation potential of these cells. Our results extend the notion of lineage-priming and provide the molecular framework for inter-A, -O, -C, -V plasticity of BM MSCs. Our data highlight the use of BM MSCs for the cell therapy of skeletal or vascular disorders, but provide a word of caution about their use in other clinical indications.