CCL25-Supplemented Hyaluronic Acid Attenuates Cartilage Degeneration in a Guinea Pig Model of Knee Osteoarthritis.

There is evidence that the application of mesenchymal stromal cells (MSCs) counteracts osteoarthritis (OA) progression. However, the prospect of extracting and expanding these cells might be limited. The aim of this study was to investigate whether hyaluronic acid (HA) supplemented with MSC-recruiting chemokine C-C motif ligand 25 (CCL25) can influence the natural course of spontaneous OA in the guinea pig. CCL25 concentration in synovial fluid (SF) was quantified with enzyme-linked immunosorbent assay. Boyden chamber cell migration assay was used to test CCL25-mediated migration of guinea pig MSC. Forty-nine 11-month-old male guinea pigs were divided into seven groups. The main treatments consisted of five intra-articular injections of HA in pure form and in combination with three doses of CCL25 (63, 693, and 6,993 pg) given at a weekly interval. The severity of cartilage damage was assessed by using a modified Mankin score. The measured average physiological concentration of CCL25 in SF of animals is 85 ± 39 pg/ml. MSC showed a 3.2-fold increase in cell migration at 1,000 nM CCL25 in vitro demonstrating the biological migratory activity of CCL25 on these cells. In vivo, treatment with HA alone did not reduce OA progression. Similarly, OA scores were not found significantly reduced after treatment with 63 pg CCL25 + HA. However, when compared to pure HA, treatment with 693 pg CCL25 + HA and 6,993 pg CCL25 + HA significantly reduced the OA score from 10.1 to 7.4 (-28%) and 8.4 (-20%), respectively. These data suggest that intra-articular injections of HA supplemented with CCL25 attenuates OA. © 2019 Orthopaedic Research Society. Published by Wiley Periodicals, Inc. J Orthop Res 37:1723-1729, 2019.

Biomimetic sulfated polyethylene glycol hydrogel inhibits proteoglycan loss and tumor necrosis factor-α-induced expression pattern in an osteoarthritis in vitro model.

This study aimed to evaluate the potential of an anti-inflammatory polyethylene glycol (PEG) hydrogel for osteoarthritis (OA) management in an OA in vitro model. Freshly isolated porcine chondrocytes were maintained in high-density cultures to form cartilage-like three-dimensional micromasses. Recombinant porcine tumor necrosis factor-alpha (TNF-α) was used to induce OA-like changes. Normal and OA-like micromasses were treated with dendritic polyglycerol sulfate-based PEG hydrogel. Live/dead staining showed that all micromasses remained vital and presented similar morphological characteristics. Safranin-O staining demonstrated a typical depletion of glycosaminoglycans in TNF-α-treated micromasses but not in the presence of the hydrogel. There was no distinct difference in immunohistochemical detection of type II collagen. Microarray data showed that rheumatoid arthritis and TNF signaling pathways were down regulated in hydrogel-treated OA-like micromasses compared to nontreated OA-like micromasses. The hydrogel alone did not affect genes related to OA such as ANPEP, COMP, CXCL12, PTGS2, and TNFSF10, but it prevented their regulation caused by TNF-α. This study provides valuable insights toward a fully synthetic hydrogel for the intra-articular treatment of OA. The findings proved the potential of this hydrogel to prevent the development of TNF-α-induced OA with regard to proteoglycan loss and TNF-α-induced expression pattern without additional signs of differentiation and inflammation. © 2018 Wiley Periodicals, Inc. J Biomed Mater Res Part B: Appl Biomater, 2018. © 2018 Wiley Periodicals, Inc. J Biomed Mater Res Part B: Appl Biomater 107B: 490-500, 2019.

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.

CCL25 chemokine-guided stem cell attraction: an assessment of possible benefits and risks.

Due to its chemoattraction potential on mesenchymal stromal cells of the CCL25/CCR9 axis, local application of CCL25 to severely damaged tissues may be a promising approach for regenerative therapies. Analysis of the given data revealed that CCL25/CCR9 signaling has a crucial role in regulation of an adult immune homeostasis. CCR9 expression variations resulted in dysfunctional immune response in colitis, rheumatoid arthritis and endometriosis. Regarding oncology, different neoplastic tissues exploit CCL25-dependent CCR9 signaling for either local proliferation or migration processes. The CCR9 pathway likely can trigger crosstalk between the Akt and NOTCH pathway and thus participate in the regulation of the neoplastic behavior. In conclusion, the designated application-tissue requires precise molecular analysis of possible CCR9 expression due to its proto-oncogenic characteristics.

Standardisation of basal medium for reproducible culture of human annulus fibrosus and nucleus pulposus cells.

BACKGROUND: The lifetime prevalence of degenerative disc disease is dramatically high. Numerous investigations on disc degeneration have been performed on cells from annulus fibrosus (AF) and nucleus pulposus (NP) of the intervertebral disc (IVD) in cell culture experiments utilising a broad variety of basal culture media. Although the basal media differ in nutrient formulation, it is not known whether the choice of the basal media itself has an impact on the cell's behaviour in vitro. In this study, we evaluated the most common media used for monolayer expansion of AF and NP cells to set standards for disc cell culture. METHODS: Human AF and NP cells were isolated from cervical discs. Cells were expanded in monolayer until passage P2 using six different common culture media containing alpha-Minimal Essential Medium (alpha-MEM), Dulbecco's Modified Eagle's Medium (DMEM) or Ham's F-12 medium (Ham's F-12) as single medium or in a mixture of two media (alpha/F-12, DMEM/alpha, DMEM/F-12). Cell morphology, cell growth, glycosaminoglycan production and quantitative gene expression of cartilage- and IVD-related markers aggrecan, collagen type II, forkhead box F1 and keratin 18 were analysed. Statistical analysis was performed with two-way ANOVA testing and Bonferroni compensation. RESULTS: AF and NP cells were expandable in all tested media. Both cell types showed similar cell morphology and characteristics of dedifferentiation known for cultured disc cells independently from the media. However, proceeding culture in Ham's F-12 impeded cell growth of both AF and NP cells. Furthermore, the keratin 18 gene expression profile of NP cells was changed in alpha-MEM and Ham's F-12. CONCLUSION: The impact of the different media itself on disc cell's behaviour in vitro was low. However, AF and NP cells were only robust, when DMEM was used as single medium or in a mixture (DMEM/alpha, DMEM/F-12). Therefore, we recommend using these media as standard medium for disc cell culture. Our findings are valuable for the harmonisation of preclinical study results and thereby push the development of cell therapies for clinical treatment of disc degeneration.

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.

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.

Delayed release of chemokine CCL25 with bioresorbable microparticles for mobilization of human mesenchymal stem cells.

Chemokines are guiding cues for directional trafficking of mesenchymal stem cells (MSC) upon injury and local chemokine delivery at injury sites is an up-to-date strategy to potentiate and prolong recruitment of MSC. In this study we present the chemokine CCL25, also referred to as thymus-expressed chemokine, to mobilize human MSC along positive but not along negative gradients. We hence proceeded to design a biodegradable and injectable release device for CCL25 on the basis of poly(lactic-co-glycolic acid) (PLGA). The conducted studies had the objective to optimize PLGA microparticle fabrication by varying selected formulation parameters, such as polymer type, microparticle size and interior phase composition. We found that microparticles of DV,50∼75 µm and fabricated using end-capped polymers, BSA as carrier protein and vortex mixing to produce the primary emulsion yielded high chemokine loading and delayed CCL25 release. To determine bioactivity, we investigated CCL25 released during the microparticle erosion phase and showed that deacidification of the release medium was required to induce significant MSC mobilization. The designed PLGA microparticles represent an effective and convenient off-the-shelf delivery tool for the delayed release of CCL25. However, continuative in vivo proof-of-concept studies are required to demonstrate enhanced recruitment of MSC and/or therapeutical effects in response to CCL25 release microparticles. STATEMENT OF SIGNIFICANCE: With the discovery of chemokines, particularly CXCL12, as stimulators of stem cell migration, the development of devices that release CXCL12 has proceeded quickly in the last few years. In this manuscript we introduce CCL25 as chemokine to induce mobilization of human MSC. This study proceeds to demonstrate how selection of key formulation parameters of CCL25 loading into PLGA microparticles exerts considerable influence on CCL25 release. This is important for a broad range of efforts in in situ tissue engineering where the candidate chemokine and the delivery device need to be selected carefully. The use of such a cell-free CCL25 release device may provide a new therapeutic option in regenerative medicine.

Mesenchymal stromal cells inhibit NLRP3 inflammasome activation in a model of Coxsackievirus B3-induced inflammatory cardiomyopathy.

Inflammation in myocarditis induces cardiac injury and triggers disease progression to heart failure. NLRP3 inflammasome activation is a newly identified amplifying step in the pathogenesis of myocarditis. We previously have demonstrated that mesenchymal stromal cells (MSC) are cardioprotective in Coxsackievirus B3 (CVB3)-induced myocarditis. In this study, MSC markedly inhibited left ventricular (LV) NOD2, NLRP3, ASC, caspase-1, IL-1ß, and IL-18 mRNA expression in CVB3-infected mice. ASC protein expression, essential for NLRP3 inflammasome assembly, increased upon CVB3 infection and was abrogated in MSC-treated mice. Concomitantly, CVB3 infection in vitro induced NOD2 expression, NLRP3 inflammasome activation and IL-1ß secretion in HL-1 cells, which was abolished after MSC supplementation. The inhibitory effect of MSC on NLRP3 inflammasome activity in HL-1 cells was partly mediated via secretion of the anti-oxidative protein stanniocalcin-1. Furthermore, MSC application in CVB3-infected mice reduced the percentage of NOD2-, ASC-, p10- and/or IL-1ß-positive splenic macrophages, natural killer cells, and dendritic cells. The suppressive effect of MSC on inflammasome activation was associated with normalized expression of prominent regulators of myocardial contractility and fibrosis to levels comparable to control mice. In conclusion, MSC treatment in myocarditis could be a promising strategy limiting the adverse consequences of cardiac and systemic NLRP3 inflammasome activation.

The atrial appendage as a suitable source to generate cardiac-derived adherent proliferating cells for regenerative cell-based therapies.

Cardiac-derived adherent proliferating (CardAP) cells obtained from endomyocardial biopsies (EMBs) with known anti-fibrotic and pro-angiogenic properties are good candidates for the autologous therapy of end-stage cardiac diseases such as dilated cardiomyopathy. However, due to the limited number of CardAP cells that can be obtained from EMBs, our aim is to isolate cells with similar properties from other regions of the heart with comparable tissue architecture. Here, we introduce the atrial appendage as a candidate region. Atrial appendage-derived cells were sorted with CD90 microbeads to obtain a CD90low cell population, which were subsequently analysed for their surface marker and gene expression profiles via flow cytometry and micro array analysis. Enzyme-linked immunosorbent assays for vascular endothelial growth factor and interleukin-8 as well as tube formation assays were performed to investigate pro-angiogenic properties. Furthermore, growth kinetic assays were performed to estimate the cell numbers needed for cell-based products. Microarray analysis revealed the expression of numerous pro-angiogenic genes and strong similarities to CardAP cells with which they also share expression levels of defined surface antigens, that is, CD29+ , CD44+ , CD45- , CD73+ , CD90low , CD105+ , and CD166+ . High secretion levels of vascular endothelial growth factor and interleukin-8 as well as improved properties of vascular structures in vitro could be detected. Based on growth parameters, cell dosages for the treatment of more than 250 patients are possible using one appendage. These results lead to the conclusion that isolating cells with regenerative characteristics from atrial appendages is feasible and permits further investigations towards allogenic cell-based therapies.

Characterization of single cell derived cultures of periosteal progenitor cells to ensure the cell quality for clinical application.

For clinical applications of cells and tissue engineering products it is of importance to characterize the quality of the used cells in detail. Progenitor cells from the periosteum are already routinely applied in the clinics for the regeneration of the maxillary bone. Periosteal cells have, in addition to their potential to differentiate into bone, the ability to develop into cartilage and fat. However, the question arises whether all cells isolated from periosteal biopsies are able to differentiate into all three tissue types, or whether there are subpopulations. For an efficient and approved application in bone or cartilage regeneration the clarification of this question is of interest. Therefore, 83 different clonal cultures of freshly isolated human periosteal cells derived from mastoid periosteum biopsies of 4 donors were generated and growth rates calculated. Differentiation capacities of 51 clonal cultures towards the osteogenic, the chondrogenic, and the adipogenic lineage were investigated. Histological and immunochemical stainings showed that 100% of the clonal cultures differentiated towards the osteogenic lineage, while 94.1% demonstrated chondrogenesis, and 52.9% could be stimulated to adipogenesis. For osteogenesis real-time polymerase chain reaction (PCR) of BGLAP and RUNX2 and for adipogenesis of FABP4 and PPARG confirmed the results. Overall, 49% of the cells exhibited a tripotent potential, 45.1% showed a bipotent potential (without adipogenic differentiation), 3.9% bipotent (without chondrogenic differentiation), and 2% possessed a unipotent osteogenic potential. In FACS analyses, no differences in the marker profile of undifferentiated clonal cultures with bi- and tripotent differentiation capacity were found. Genome-wide microarray analysis revealed 52 differentially expressed genes for clonal subpopulations with or without chondrogenic differentiation capacity, among them DCN, NEDD9, TGFBR3, and TSLP. For clinical applications of periosteal cells in bone regeneration all cells were inducible. For a chondrogenic application a fraction of 6% of the mixed population could not be induced.

Mesenchymal Stromal Cells Modulate Monocytes Trafficking in Coxsackievirus B3-Induced Myocarditis.

Mesenchymal stromal cell (MSC) application in Coxsackievirus B3 (CVB3)-induced myocarditis reduces myocardial inflammation and fibrosis, exerts prominent extra-cardiac immunomodulation, and improves heart function. Although the abovementioned findings demonstrate the benefit of MSC application, the mechanism of the MSC immunomodulatory effects leading to a final cardioprotective outcome in viral myocarditis remains poorly understood. Monocytes are known to be a trigger of myocardial tissue inflammation. The present study aims at investigating the direct effect of MSC on the mobilization and trafficking of monocytes to the heart in CVB3-induced myocarditis. One day post CVB3 infection, C57BL/6 mice were intravenously injected with 1 x 106 MSC and sacrificed 6 days later for molecular biology and flow cytometry analysis. MSC application reduced the severity of myocarditis, and heart and blood pro-inflammatory Ly6Chigh and Ly6Cmiddle monocytes, while those were retained in the spleen. Anti-inflammatory Ly6Clow monocytes increased in the blood, heart, and spleen of MSC-treated CVB3 mice. CVB3 infection induced splenic myelopoiesis, while MSC application slightly diminished the spleen myelopoietic activity in CVB3 mice. Left ventricular (LV) mRNA expression of the chemokines monocyte chemotactic protein-1 (MCP)-1, MCP-3, CCL5, the adhesion molecules intercellular adhesion molecule-1, vascular cell adhesion molecule-1, the pro-inflammatory cytokines interleukin-6, interleukin-12, tumor necrosis factor-α, the pro-fibrotic transforming growth factorß1, and circulating MCP-1 and MCP-3 levels decreased in CVB3 MSC mice, while LV stromal cell-derived factor-1α RNA expression and systemic levels of fractalkine were increased in CVB3 MSC mice. MSC application in CVB3-induced myocarditis modulates monocytes trafficking to the heart and could be a promising strategy for the resolution of cardiac inflammation and prevention of the disease progression. Stem Cells Translational Medicine 2017;6:1249-1261.

Human Endomyocardial Biopsy Specimen-Derived Stromal Cells Modulate Angiotensin II-Induced Cardiac Remodeling.

: Cardiac-derived adherent proliferating cells (CardAPs) are cells derived from human endomyocardial biopsy specimens; they share several properties with mesenchymal stromal cells. The aims of this study were to evaluate whether intramyocardial injection of CardAPs modulates cardiac fibrosis and hypertrophy in a mouse model of angiotensin II (Ang II)-induced systolic heart failure and to analyze underlying mechanisms. Intramyocardial application of 200,000 CardAPs improved left ventricular function. This was paralleled by a decline in left ventricular remodeling, as indicated by a reduction in cardiac fibrosis and hypertrophy. CardAPs reduced the ratio of the left ventricle to body weight and cardiac myosin expression (heavy chain), and decreased the Ang II-induced phosphorylation state of the cardiomyocyte hypertrophy mediators Akt, extracellular-signal regulated kinase (ERK) 1, and ERK2. In accordance with the antifibrotic and antihypertrophic effects of CardAPs shown in vivo, CardAP supplementation with cardiac fibroblasts decreased the Ang II-induced reactive oxygen species production, α-SMA expression, fibroblast proliferation, and collagen production. Coculture of CardAPs with HL-1 cardiomyocytes downregulated the Ang II-induced expression of myosin in HL-1. All antifibrotic and antihypertrophic features of CardAPs were mediated in a nitric oxide- and interleukin (IL)-10-dependent manner. Moreover, CardAPs induced a systemic immunomodulation, as indicated by a decrease in the activity of splenic mononuclear cells and an increase in splenic CD4CD25FoxP3, CD4-IL-10, and CD8-IL-10 T-regulatory cells in Ang II mice. Concomitantly, splenocytes from Ang II CardAPs mice induced less collagen in fibroblasts compared with splenocytes from Ang II mice. We conclude that CardAPs improve Ang II-induced cardiac remodeling involving antifibrotic and antihypertrophic effects via paracrine actions and immunomodulatory properties. SIGNIFICANCE: Despite effective pharmacological treatment with angiotensin II type I receptor antagonists or angiotensin II-converting enzyme inhibitors, morbidity and mortality associated with heart failure are still substantial, prompting the search of novel therapeutic strategies. There is accumulating evidence supporting the use of cell therapy for cardiac repair. This study demonstrates that cells derived from human endomyocardial biopsies, cardiac-derived adherent proliferating cells (CardAPs), have the potential to reduce angiotensin II-induced cardiac remodeling and improve left ventricular function in angiotensin II mice. The mechanism involves antifibrotic and antihypertrophic effects via paracrine actions and immunomodulatory properties. These findings support the potential of CardAPs for the treatment of heart failure.

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.

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.

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.

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 adipogenically differentiated cells into osteogenically or chondrogenically differentiated cells: phenotype switching via dedifferentiation.

Reprogramming is a new wave in cellular therapies to achieve the vital goals of regenerative medicine. Transdifferentiation, whereas the differentiated state of cells could be reprogrammed into other cell types, meaning cells are no more locked in their differentiated circle. Hence, cells of choice from abundant and easily available sources such as fibroblast and adipose tissue could be converted into cells of demand, to restore the diseased tissues. Before diverting this new approach into effective clinical use, transdifferentiation could not be simply overlooked, as it challenges the normal paradigms of biological laws, where mature cells transdifferentiate not only within same germ layers, but even across the lineage boundaries. How unipotent differentiated cells reprogram into another, and whether transdifferentiation proceeds via a direct cell-to-cell conversion or needs dedifferentiation. To address such questions, MSC were adipogenically differentiated followed by direct transdifferentiation, and subsequently examined by histology, immunohistochemistry, qPCR and single cell analysis. Direct cellular conversion of adipogenic lineage cells into osteogenic or chondrogenic resulted in mixed culture of both lineage cells (adipogenic and new acquiring osteogenic/chondrogenic phenotypes). On molecular level, such conversion was confirmed by significantly upregulated expression of PPARG, FABP4, SPP1 and RUNX2. Chondrogenic transdifferentiation was verified by significantly upregulated expression of PPARG, FABP4, SOX9 and COL2A1. Single cell analysis did not support the direct cell-to-cell conversion, rather described the involvement of dedifferentiation. Moreover, some differentiated single cells did not change their phenotype and were resistant to transdifferentiation, suggesting that differentiated cells behave differently during cellular conversion. An obvious characterization of differentiated cells could be helpful to understand the process of transdifferentiation.

Mesenchymal stem cells and their chondrogenic differentiated and dedifferentiated progeny express chemokine receptor CCR9 and chemotactically migrate toward CCL25 or serum.

INTRODUCTION: Guided migration of chondrogenically differentiated cells has not been well studied, even though it may be critical for growth, repair, and regenerative processes. The chemokine CCL25 is believed to play a critical role in the directional migration of leukocytes and stem cells. To investigate the motility effect of serum- or CCL25-mediated chemotaxis on chondrogenically differentiated cells, mesenchymal stem cells (MSCs) were induced to chondrogenic lineage cells. METHODS: MSC-derived chondrogenically differentiated cells were characterized for morphology, histology, immunohistochemistry, quantitative polymerase chain reaction (qPCR), surface profile, and serum- or CCL25-mediated cell migration. Additionally, the chemokine receptor, CCR9, was examined in different states of MSCs. RESULTS: The chondrogenic differentiated state of MSCs was positive for collagen type II and Alcian blue staining, and showed significantly upregulated expression of COL2A1and SOX9, and downregulated expression of CD44, CD73, CD90, CD105 and CD166, in contrast to the undifferentiated and dedifferentiated states of MSCs. For the chondrogenic differentiated, undifferentiated, and dedifferentiated states of MSCs, the serum-mediated chemotaxis was in a percentage ratio of 33%:84%:85%, and CCL25-mediated chemotaxis was in percentage ratio of 12%:14%:13%, respectively. On the protein level, CCR9, receptor of CCL25, was expressed in the form of extracellular and intracellular domains. On the gene level, qPCR confirmed the expression of CCR9 in different states of MSCs. CONCLUSIONS: CCL25 is an effective cue to guide migration in a directional way. In CCL25-mediated chemotaxis, the cell-migration rate was almost the same for different states of MSCs. In serum-mediated chemotaxis, the cell-migration rate of chondrogenically differentiated cells was significantly lower than that in undifferentiated or dedifferentiated cells. Current knowledge of the surface CD profile and cell migration could be beneficial for regenerative cellular therapies.