Expression In Mesenchymal Stem Cells Research Articles

Effects of hRAMP1 modified mesenchymal stem cells on restenosis and heart function in rabbit model of carotid angioplasty and myocardial infarction

To observe the effects of mesenchymal stem cells (MSC) modified by human receptor activity-modifying protein 1 (hRAMP1) on restenosis and cardiac function post-myocardial infarction (MI) and explore the therapeutic safety for gene modification of MSC. The double-injury rabbit model with MI reperfusion and sacculus damaged atherosclerotic carotid were established according to the previous study. MSC were transfected with adenovirus vector with enhanced green fluorescence protein (EGFP) and then introduced into rabbit model. The animals were randomly divided into Ad-EGFP-hRAMP1-MSC transfection group (hRAMP1-MSC group, n = 24) and Ad-EGFP-MSC transfection group (MSC group, n = 24), and PBS transfection group (C group, n = 24). At Day 28 post-transplantation, the injured carotid arteries and infarction myocardium were harvested to detect the expression of EGFP-positive MSC and assess organization morphology by hematoxylin and eosin, triphenyltetrazolium chloride or immunohistochemical stains and heart function by echocardiography. On flow cytometry, most cells expressed CD29 and CD90 while few ones expressed CD45. MSC with EGFP and a continuous expression of CD31 were found in intima of damaged carotid of both hRAMP1-MSC and MSC, but the expression of EGFP was not found in the control group. At Day 28 post-transplantation, the improvement of heart function and the decrease of infarct size were found in the hRAMP1-MSC and MSC groups compared with that in the control group(EF: 60.6% ± 1.5%,50.8% ± 3.2% vs 38.2% ± 2.0%, infarct size: 20.7% ± 1.4%, 33.2% ± 3.7% vs 35.6% ± 2.7%, all P < 0.05), especially much higher in hRAMP1-MSC group. At Day 28 post-transplantation, the area of intima hyperplasia and the rate of neointima and media in the hRAMP1-MSC group were lower than those in the MSC and C groups (0.15 ± 0.05 and 0.33 ± 0.08 vs 0.77 ± 0.11, 0.24 ± 0.07 and 0.51 ± 0.11 vs 1.09 ± 0.23, all P < 0.05). Also the expression of α-SMA was found in the hyperplasia intima. The expression of proliferating cell nuclear antigen significantly decreased in the hRAMP1-MSC group than those of the MSC and control groups (0.120 ± 0.028 vs 0.366 ± 0.013 and 0.627 ± 0.049, both P < 0.05). And it was much lower in the MSC group than that in the control group. Compared with MSC alone, hRAMP1-modified MSC have the potency not only of more improvement on cardiac function and the recovery of damaged endothelium, but also of significant inhibition of the proliferation of vascular smooth muscle cells. The recombinant hRAMP1 adenovirus vectors do not affect the differentiation potential MSC into endothelial cells.

Comparison of in vitro hepatogenic differentiation potential between various placenta-derived stem cells and other adult stem cells as an alternative source of functional hepatocytes

Mesenchymal stem cells (MSCs) are powerful sources for cell therapy in regenerative medicine. The capability to obtain effective stem cell-derived hepatocytes would improve cell therapy for liver diseases. Recently, various placenta-derived stem cells (PDSCs) depending on the localization of placenta have been suggested as alternative sources of stem cells are similar to bone marrow-derived MSC (BM-MSCs) and adipose-derived MSC (AD-MSCs). However, comparative studies for the potentials of the hepatogenic differentiation among various MSCs largely lacking. Therefore, we investigated to compare the potentials for hepatogenic differentiation of PDSCs with BM-MSCs, AD-MSCs, and UCB-MSCs. Several MSCs were isolated from human term placenta, adipose tissue, and umbilical cord blood and characterized isolated MSCs and BM-MSCs was performed by quantitative reverse transcription-PCR (RT-PCR) and special stains after mesodermal differentiation. The hepatogenic potential of PDSCs was compared with AD-MSCs, UCB-MSCs, and BM-MSCs using RT-PCR, PAS stain, ICG up-take assays, albumin expression, urea production, and cytokine assays. MSCs isolated from different tissues all presented similar characteristics of MSCs. However, the proliferative potential of PDSCs and the expression of hepatogenic markers in differentiated PDSCs were higher than other MSCs. Interestingly, the expression of hepatocyte growth factor (HGF) increased in PDSCs after hepatogenic differentiation. Interestingly, stem cell factor (SCF) expression in chorionic plate-derived MSCs, one of the PDSCs, was significantly higher than in the other PDSCs. Taken together, the results of the present study suggest that MSCs isolated from various adult tissues can be induced to undergo hepatogenic differentiation in vitro, and that PDSCs may have the greatest potential for hepatogenic differentiation and proliferation. Therefore, PDSCs could be used as a stem cell source for cell therapy in liver diseases.

Tanshinone IIA and astragaloside IV promote the migration of mesenchymal stem cells by up-regulation of CXCR4

Mesenchymal stem cells (MSCs) have a therapeutic potential to treat cardiovascular diseases. However, a significant barrier to MSC therapy is insufficient MSC engraftment in ischemic myocardium after systemic administration. Here, we investigated the modulatory effects of tanshinone IIA and astragaloside IV on the migration of MSCs and further defined the underlying mechanisms. CXCR4 expression in MSCs was determined by using flow cytometry, real-time PCR, and western blotting. The results showed that CXCR4 expression was significantly higher in tanshinone IIA- and astragaloside IV-stimulated MSCs than that of the control. MSC migration toward stromal cell-derived factor-1α (SDF-1α) was studied using a transwell system. MSCs treated with tanshinone IIA and astragaloside IV showed stronger migration than that of the control. Moreover, this enhanced migration ability was abrogated by a CXCR4 inhibitor. In a rat acute myocardial infarction model, MSCs stimulated with tanshinone IIA and astragaloside IV were stained with Dio and injected into model rats via the tail vein. Dio-labeled cells in myocardium sections were observed by fluorescence microscopy. Tanshinone IIA- and astragaloside IV-stimulated MSCs showed enhanced capacities to home to ischemic myocardium sites. In addition, there was no significant difference in the SDF-1α expression among groups. These data suggest that tanshinone IIA and astragaloside IV regulate MSC mobilization, at least partially via modulation of the CXCR4 expression.

Comparison of Gene Expression Profiling in Sarcomas and Mesenchymal Stem Cells Identifies Tumorigenic Pathways in Chemically Induced Rat Sarcoma Model

Mesenchymal stem cells (MSCs) are believed to be the cell of origin for most sarcomas including osteosarcoma and malignant fibrous histiocytoma (MFH/UPS). To identify the signaling pathways involved in sarcoma pathogenesis, we compared gene expression profiles in rat osteosarcoma and MFH cells with those in syngeneic rat MSCs. Analysis of genes that characterize MSCs such as CD44, CD105, CD73, and CD90 showed higher expression in MSCs compared to sarcomas. Pathways involved in focal and cell adhesion, cytokine-cytokine receptors, extracellular matrix receptors, chemokines, and Wnt signaling were down-regulated in both sarcomas. Meanwhile, DNA replication, cell cycle, mismatch repair, Hedgehog signaling, and metabolic pathways were upregulated in both sarcomas. Downregulation of p21Cip1 and higher expression of CDK4-cyclinD1 and CDK2-cyclinE could accelerate cell cycle in sarcomas. The current study indicated that these rat sarcomas could be a good model for their human counterparts and will provide the further insights into the molecular pathways and mechanisms involved in sarcoma pathogenesis.

Sustained and promoter dependent bone morphogenetic protein expression by rat mesenchymal stem cells after BMP-2 transgene electrotransfer

Transplantation of mesenchymal stem cells (MSCs) with electrotransferred bone morphogenetic protein-2 (BMP-2) transgene is an attractive therapeutic modality for the treatment of large bone defects: it provides both stem cells with the ability to form bone and an effective bone inducer while avoiding viral gene transfer. The objective of the present study was to determine the influence of the promoter driving the human BMP-2 gene on the level and duration of BMP-2 expression after transgene electrotransfer into rat MSCs. Cytomegalovirus, elongation factor-1α, glyceraldehyde 3-phosphate dehydrogenase, and beta-actin promoters resulted in a BMP-2 secretion rate increase of 11-, 78-, 66- and 36-fold over respective controls, respectively. In contrast, the osteocalcin promoter had predictable weak activity in undifferentiated MSCs but induced the strongest BMP-2 secretion rates in osteoblastically-differentiated MSCs. Regardless of the promoter driving the transgene, a plateau of maximal BMP-2 secretion persisted for at least 21 d after the hBMP-2 gene electrotransfer. The present study demonstrates the feasibility of gene electrotransfer for efficient BMP-2 transgene delivery into MSCs and for a three-week sustained BMP-2 expression. It also provides the first in vitro evidence for a safe alternative to viral methods that permit efficient BMP-2 gene delivery and expression in MSCs but raise safety concerns that are critical when considering clinical applications.

Cell-Derived Matrix Coatings for Polymeric Scaffolds

Cells in culture deposit a complex extracellular matrix that remains intact following decellularization and possesses the capacity to modulate cell phenotype. The direct application of such decellularized matrices (DMs) to 3D substrates is problematic, as transport issues influence the homogeneous deposition, decellularization, and modification of DM surface coatings. In an attempt to address this shortcoming, we hypothesized that DMs deposited by human mesenchymal stem cells (MSCs) could be transferred to the surface of polymeric scaffolds while maintaining their capacity to direct cell fate. The ability of the transferred DM (tDM)-coated scaffolds to enhance the osteogenic differentiation of undifferentiated and osteogenically induced MSCs under osteogenic conditions in vitro was confirmed. tDM-coated scaffolds increased MSC expression of osteogenic marker genes (BGLAP, IBSP) and intracellular alkaline phosphatase production. In addition, undifferentiated MSCs deposited significantly more calcium when seeded onto tDM-coated scaffolds compared with control scaffolds. MSC-seeded tDM-coated scaffolds subcutaneously implanted in nude rats displayed significantly higher blood vessel density after 2 weeks compared with cells on uncoated scaffolds, but we did not observe significant differences in mineral deposition after 8 weeks. These data demonstrate that DM-coatings produced in 2D culture can be successfully transferred to 3D substrates and retain their capacity to modulate cell phenotype.

Effective, safe nonviral gene transfer to preserve the chondrogenic differentiation potential of human mesenchymal stem cells

Genetic modification of mesenchymal stem cells (MSCs) comprises a promising tool to generate cell- and gene-based platforms for regenerative approaches of articular cartilage repair. In the present study, we systematically screened a panel of 15 nonviral compounds for their ability to promote safe, efficient and durable gene expression in human bone marrow-derived MSCs (hMSCS) without impeding their commitment towards chondrogenic differentiation. Primary hMSCs were transfected with plasmid vectors carrying sequences for the Photinus pyralis luciferase Escherichia coli β-galactosidase, or human insulin-like growth factor I via 15 nonviral formulations. Transgene expression and transfection efficiencies were monitored for each component in parallel with the effects on cell viability and cytotoxicity. Upon optimization, the most promising reagent was then evaluated for a possible influence on the chondrogenic potential of hMSCs. Among all formulations tested, GeneJammer® gave the best results for transgene expression and transfection efficacy (25-14% from days 2-21 in monolayer cultures and 35% in 21-day aggregate cultures), allowing for high levels of viability (92-94%) and modest cytotoxicity (< 12%). Most notably, the application of this reagent did not affect the potential of the cells for chondrogenic differentiation when maintained in long-term (21 days) three-dimensional (aggregate) cultures. The data indicate that safe, efficient transgene expression can be achieved in hMSCs over time using the nonviral GeneJammer® compound, showing promise for future therapeutic settings aiming to treat human articular cartilage disorders.

Tissue Engineering Construct Prevents Disk Degeneration after Nucleotomy in a Rat Model

IntroductionBack pain, a significant source of morbidity in our society, is directly linked to the pathology of the intervertebral disk (IVD). An IVD is a complex structure that is separated into the following three tissue types: the annulus fibrosus, the nucleus pulposus (NP), and the endplates. Recently, IVD engineering strategies have been increasingly focused on the regeneration or repair of the AF to increase the potential of NP engineering strategies and to mechanically assist NP replacement therapies.1,2 We have developed a novel scaffold-free three-dimensional tissue-engineered construct (TEC) derived from cultured synovial mesenchymal stem cells (MSCs) as a unique approach for cartilage repair.3,4 Previous in vitro studies reveal that growth factors and serum can modify the collagen expression and glycosaminoglycan (GAG) content of chondrogenic-differentiated MSCs.5 Therefore although there is a difference between hyaline cartilage and fibrocartilage, TEC derived from MSCs could have a potential to differentiate into the AF, a fibrocartilage in IVD. This study explores the use of TEC derived from synovial MSCs as a cell-based therapy for IVD regeneration.Materials and MethodsSynovial MSCs were isolated enzymatically from rat synovial membranes as previously reported.3,4 For development of the TEC, cells were cultured at the density of 4.0 × 105/cm2 in HG-DMEM in the presence of 0.2 mM ascorbic acid 2-phosphate for 2 weeks. The monolayer cultured cell-matrix complex was then detached from cell-substratum interface by addition of shear stress to convert to suspension culture. The cell-matrix complex immediately starts active contraction to form the three dimensional (3D) tissue-engineered construct (TEC, Fig. 1). To explore the use of TEC in degenerative spine, rat caudal IVD were denucleated and treated with TEC (controls were denucleation only). Nine male Sprague-Dawley rats (300 to 400 g) were used, and two most cranial tail disks were denucleated in each rat, giving three disks per group per time point. At 2, 8, and 12 weeks after implantation, the animals were euthanized and disks were evaluated for disk height based on micro-CT analysis, histology including Safranin O staining, and disk grade based on a scoring system.6ResultsAt the 2-week time point, there were no significant differences in disk height and disk grade between two groups (3.57 ± 0.28 vs 3.76 ± 0.46 points, p > 0.05) (462 ± 39 vs. 602 ± 49 um, p > 0.05). At the 8-week time point, the TEC-treated group showed significantly better results in disk grade (5 ± 0 vs. 3.14 ± 0.28 points, P<0.05). At the 12-week time point, TEC-treated group showed the higher disk height (234 ± 19 vs 584 ± 45 um, P<0.05) and better disk grade (5 ± 0 vs. 2.86 ± 0.46 points, p < 0.05). Untreated group experienced severe disruption of the AF and end plate, whereas such degenerative changes were alleviated with TEC implantation (Fig. 2).ConclusionThis study shows that TEC may prevent postnucleotomy disk degeneration in vivo. Larger animals and longer time points will be necessary to further judge potential clinical impact.I confirm having declared any potential conflict of interest for all authors listed on this abstractYesDisclosure of InterestNone declaredAndersson GB, et al. Directions for future research. Journal of Joint and Bone Surgery 2006Wilke HJ, et al. Is a collagen scaffold for a tissue engineered nucleus replacement capable of restoring disc height and stability in an animal model? European Spine Journal 2006W Ando, et al. Cartilage repair using an in vitro generated scaffold-free tissue-engineered construct derived from porcine synovial mesenchymal stem cells. Biomaterials 2007K Shimomura, et al. The influence of skeletal maturity on allogenic synovial mesenchymal stem cell-based repair of cartilage in a large animal model. Biomaterials 2010S Lee, et al. Effect of serum and growth factors on chondrogenic differentiation of synovium-derived stromal cells. Tissue Engineering: Part A 2009AA Allon, et al. Structured coculture of stem cells and disc cells prevent disc degeneration in a rat model. The Spine Journal 2010

Thermoreversible Hyaluronan Hydrogels Support Mesenchymal Stem Cells Discogenic Differentiation in Vitro and Ex Vivo

Introduction Hyaluronan is a proteoglycan widely present in the nucleus pulposus (NP) of the intervertebral disk (IVD). Recently, thermoreversible hyaluronan (HA) based hydrogels, which are injectable, minimally invasive, allow for encapsulation and maintenance of viable cells and promote re-differentiation of NP cells, have been developed.1 While it is known that hyaluronan is recognized by mesenchymal stem cells (MSCs)2 and can induce anabolic pathways, growth factors may further support MSC differentiation in HA-based hydrogels. Therefore, the objectives of the present work were: (i) to assess whether the addition of growth factors3 could support MSC differentiation towards the disk cell phenotype in HA based hydrogels and (ii) to determine if MSC preconditioning (culture under “chondrogenic” conditions) would improve the survival rate and differentiation potential of MSC supplemented to an IVD cultured ex vivo. Materials and Methods Hydrogel Thermoreversible HA-based hydrogels were prepared by grafting a thermoreversible segment poly (N-isopropylacrylamide) (pNIPAM) on a functionalized HA backbone, as previously described.1 In vitro Human bone marrow-derived MSCs (P2-P4) were cultured for 7 days in HA-pNIPAM hydrogel under hypoxic conditions (5% O2) in chondrogenic medium, with or without recombinant human growth and differentiation factor-5 (rhGDF-5) supplementation. Cell viability, DNA, and glycosaminoglycans (GAG) content were quantified by Live-dead, Picogreen, and 1,9 dimethylmethylene blue assays, respectively. Gene expression analysis was performed to assess the disc-like differentiation of MSCs. The expression of collagen type I and II (COL1 and COL2) aggrecan (ACAN), cytokeratin-19, sox-9, cluster of differentiation 24 (CD24), hyaluronan synthase I and II (HAS1 and HAS2), forkhead box protein F1 (FOXF1) and carbonic anhydrase 12 (CA12) was analyzed with 18s as endogenous control. Ex Vivo Bovine caudal IVDs were excised and nucleotomy performed through the endplate (Fig. 1A,B). MSCs were either suspended in HA-pNIPAM and directly supplied to the IVDs or precultured (under hypoxic conditions and with supplementation of GDF-5) for 1 week in HA-pNIPAM and then supplied to the IVDs. After 1 week of culture, gene expression analyses were performed using the same markers as for the in vitro study. Statistics After confirming the normality of the data distribution, one-way analysis of variance with Bonferroni posthoc was performed ( p &lt; 0.05). Results MSC viability was maintained&gt;90% over 1 week of culture in the HA-pNIPAM hydrogel both in vitro and ex vivo. DNA and GAG did not show significant variation over the 7 days of in vitro culture. Gene expression revealed 10–100-fold upregulation of the transcription factor SOX9 and 100–1000, fold upregulation of COL2, while COL1 was only slightly enhanced. The expression of the potential NP cell marker KRT19 was also increased. With the addition of rhGDF-5, an improved COL2/COL1 expression ratio was found. No significant differences were found for the other genes. Ex vivo cultures of nucleotomized discs supplemented with MSCs in HA-pNIPAM confirmed the positive effect of the hydrogel and the IVD environment on MSC differentiation towards the disc-like phenotype (Fig. 1C), with upregulation of collagen type II ( p &lt; 0.05), CD24, and FOX-F1. No significant differences were found for the other genes. Preculture of MSCs before the supplementation in the IVD did not enhance MSC differentiation. Figure 1 Bovine IVD after nucleotomy: (A) top view and (B) view of the nucleotomized area. (C) Gene expression of human MSCs after 1 week of culture in bovine IVDs normalized to the mRNA levels at day 0 of culture: human MSCs were either suspended in HA-pNIPAM and directly supplied to bovine IVDs (white bars) or precultured for 1 week in HA-pNIPAM hydrogel and then supplied to bovine IVDs (grey bars);* p &lt; 0.05, outliers. Conclusion The thermoreversible hyaluronan-based hydrogel was able to induce human MSC differentiation toward a “disc-like” phenotype without the addition of growth factors, although GDF-5 supplementation resulted in an improved gene expression profile. Preculture of MSCs before the supplementation in the IVD is not required with the thermoreversible hyaluronan-based hydrogel. Therefore, this type of hydrogel could be an appropriate carrier for MSCs supplementation to compromised discs. Investigations are ongoing to evaluate its biomechanical behavior under loading. Acknowledgements This study was partially supported by a NASS Research Grant. I confirm having declared any potential conflict of interest for all authors listed on this abstract Yes Disclosure of Interest None declared Peroglio M, et al. European Spine Journal (Epub ahead of print) Zhu H et al. Stem cells 2006; 24:928–935 Gantenbein-Ritter B, et al. European Spine Journal 2011;20(6):962–971

Tissue Engineering Construct Prevents Disk Degeneration after Nucleotomy in a Rat Model

Introduction Back pain, a significant source of morbidity in our society, is directly linked to the pathology of the intervertebral disk (IVD). An IVD is a complex structure that is separated into the following three tissue types: the annulus fibrosus, the nucleus pulposus (NP), and the endplates. Recently, IVD engineering strategies have been increasingly focused on the regeneration or repair of the AF to increase the potential of NP engineering strategies and to mechanically assist NP replacement therapies.1,2 We have developed a novel scaffold-free three-dimensional tissue-engineered construct (TEC) derived from cultured synovial mesenchymal stem cells (MSCs) as a unique approach for cartilage repair.3,4 Previous in vitro studies reveal that growth factors and serum can modify the collagen expression and glycosaminoglycan (GAG) content of chondrogenic-differentiated MSCs.5 Therefore although there is a difference between hyaline cartilage and fibrocartilage, TEC derived from MSCs could have a potential to differentiate into the AF, a fibrocartilage in IVD. This study explores the use of TEC derived from synovial MSCs as a cell-based therapy for IVD regeneration. Materials and Methods Synovial MSCs were isolated enzymatically from rat synovial membranes as previously reported.3,4 For development of the TEC, cells were cultured at the density of 4.0 × 105/cm2 in HG-DMEM in the presence of 0.2 mM ascorbic acid 2-phosphate for 2 weeks. The monolayer cultured cell-matrix complex was then detached from cell-substratum interface by addition of shear stress to convert to suspension culture. The cell-matrix complex immediately starts active contraction to form the three dimensional (3D) tissue-engineered construct (TEC, Fig. 1). To explore the use of TEC in degenerative spine, rat caudal IVD were denucleated and treated with TEC (controls were denucleation only). Nine male Sprague-Dawley rats (300 to 400 g) were used, and two most cranial tail disks were denucleated in each rat, giving three disks per group per time point. At 2, 8, and 12 weeks after implantation, the animals were euthanized and disks were evaluated for disk height based on micro-CT analysis, histology including Safranin O staining, and disk grade based on a scoring system.6 Results At the 2-week time point, there were no significant differences in disk height and disk grade between two groups (3.57 ± 0.28 vs 3.76 ± 0.46 points, p &gt; 0.05) (462 ± 39 vs. 602 ± 49 um, p &gt; 0.05). At the 8-week time point, the TEC-treated group showed significantly better results in disk grade (5 ± 0 vs. 3.14 ± 0.28 points, P&lt;0.05). At the 12-week time point, TEC-treated group showed the higher disk height (234 ± 19 vs 584 ± 45 um, P&lt;0.05) and better disk grade (5 ± 0 vs. 2.86 ± 0.46 points, p &lt; 0.05). Untreated group experienced severe disruption of the AF and end plate, whereas such degenerative changes were alleviated with TEC implantation (Fig. 2). Conclusion This study shows that TEC may prevent postnucleotomy disk degeneration in vivo. Larger animals and longer time points will be necessary to further judge potential clinical impact. I confirm having declared any potential conflict of interest for all authors listed on this abstract Yes Disclosure of Interest None declared Andersson GB, et al. Directions for future research. Journal of Joint and Bone Surgery 2006 Wilke HJ, et al. Is a collagen scaffold for a tissue engineered nucleus replacement capable of restoring disc height and stability in an animal model? European Spine Journal 2006 W Ando, et al. Cartilage repair using an in vitro generated scaffold-free tissue-engineered construct derived from porcine synovial mesenchymal stem cells. Biomaterials 2007 K Shimomura, et al. The influence of skeletal maturity on allogenic synovial mesenchymal stem cell-based repair of cartilage in a large animal model. Biomaterials 2010 S Lee, et al. Effect of serum and growth factors on chondrogenic differentiation of synovium-derived stromal cells. Tissue Engineering: Part A 2009 AA Allon, et al. Structured coculture of stem cells and disc cells prevent disc degeneration in a rat model. The Spine Journal 2010

The Effect of Link N on the Differentiation of Human Mesenchymal Stem Cells

Introduction The degeneration of intervertebral disk (IVD) can cause low back pain, and invoking the regeneration of degenerated IVD can relieve the pain.1,2 Mesenchymal stem cells (MSCs) are multipotent cells that can differentiate into chondrocytes and can be employed in cartilage regeneration.3,4 Link N (DHLSDNYTLDHDRAIH) is the amino terminal peptide of link protein in the extracellular matrix of cartilage. It can stimulate the biosynthesis of collagens II and IX and aggrecan in IVD cells in vitro as well as in vivo.5–7 We hypothesized that Link N can enhance the chondrogenic differentiation of MSCs, and can be applied with MSCs in bio-regeneration of IVDs. The specific aim of this research is to determine the effect of Link N on gene expression of MSCs. Materials and Methods MSCs were isolated from the bone marrow of osteoarthritis (OA) patients. The cells were cultured in 24-well plates (3000 cells/well) in chondrogenesis differentiation medium (Invitrogen, Canada) according to the manufacturer's instructions. Link N was dissolved in the media with a final concentration of 0.1 µg/mL and 1 µg/mL, respectively. Medium without Link N was applied as a control. The media were changed every 3 days, and the used media were collected and stored at −20°C for GAG analysis. For gene expression analysis, the cells were cultured for 7, 14, and 21 days, then were washed twice with PBS and dissolved in Trizol (Invitrogen, Canada). Total RNA was isolated and cDNA was synthesized by reverse transcription. Gene expression was analyzed by real-time PCR and relative expression was calculated using GAPDH as a reference gene. MSCs from three OA patients were analyzed and statistical significance was calculated using analysis of variance followed by Fisher protected least significant difference (PLSD) comparison test. Results With the concentrations of 0.1 and 1.0 µg/mL, no toxic effect of Link N on MSCs was observed. After MSCs were cultured for 7 days and 14 days, the expression of aggrecan (ACAN) (Fig. 1), collagen II (COL2A1) (Fig. 2) and the transcription regulator SOX9 increased significantly in the media with 0.1 µg/mL or 1.0 µg/mL Link N. No significant differences were observed at day 21 (Figs. 1 and 2). When cells were cultured with 0.1 or 1.0 µg/mL Link N, the quantity of GAG in the media increased significantly at day 9, 12, and 15, but no major difference was observed at day 3 and 6 and at day 18 and 21. When MSCs were cultured for 7, 14, and 21 days, no significant effect of Link N on the expression of alkaline phosphatase (ALP) was observed. Furthermore, Link N significantly inhibited osteocalcin (OC) gene expression after culturing MSCs for 21 days. Conclusion Link N can improve the chondrogenic differentiation of MSCs and inhibit their osteogenic differentiation. It can therefore be used in conjunction with MSCs in the repair of IVD degeneration. I confirm having declared any potential conflict of interest for all authors listed on this abstract Yes Disclosure of Interest None declared Luoma K, Riihimaki H, Luukkonen R, Raininko R, Viikari-Juntura E, Lamminen A. Low back pain in relation to lumbar disk degeneration. Spine 2000;25:487–492 Roughley PJ. Biology of intervertebral disk aging and degeneration: involvement of the extracellular matrix. Spine 2004;29:2691–2699 Xian CJ, Foster BK. Repair of injured articular and growth plate cartilage using mesenchymal stem cells and chondrogenic gene therapy. Current Stem Cell Research Therapy 2006;1:213–229 Koga H, Engebretsen L, Brinchmann JE, Muneta T, Sekiya I. Mesenchymal stem cell-based therapy for cartilage repair: a review. Knee Surgery Sports Traumatol Arthroscopy 2009;7:1289–1297 Mwale F, Demers CN, Petit A et al. A synthetic peptide of link protein stimulates the biosynthesis of collagens II, IX and proteoglycan by cells of the intervertebral disc. Journal of Cell Biochemistry 2003;88:1202–1213 Petit A, Yao G, Rowas SA, et al. Effect of synthetic Link N peptide on the type I and type II collagens in human intervertebral discs cells. Tissue Engeneering Part A 2011;17:899–904

Stem cell-based delivery of Hypoxamir-210 to the infarcted heart: implications on stem cell survival and preservation of infarcted heart function

This study seeks to test our hypothesis that transgenic induction of miR-210 in mesenchymal stem cells (MSC) simulates the pro-survival effects of ischemic preconditioning (IPC) and that engraftment of (PC)MSC helps in the functional recovery of ischemic heart by miR-210 transfer to host cardiomyocytes through gap junctions. miR-210 expression in MSC was achieved by IPC or nanoparticle-based transfection of miR-210 plasmid ((miR)MSC) and functional recovery of the infarcted heart of rat transplanted with (PC)MSC or (miR)MSC was evaluated. Both (PC)MSC and (miR)MSC showed higher survival under lethal anoxia as compared to (non-PC)MSC and scramble-transfected MSC ((Sc)MSC) controls with concomitantly lower CASP8AP2 expression. Similarly, both (PC)MSC and (miR)MSC survived better and accelerated functional recovery of ischemic heart post-transplantation. To validate our hypothesis that MSC deliver miR-210 to host cardiomyocytes, in vitro co-culture between cardiomyocytes and (PC)MSC or (miR)MSC (using (non-PC)MSC or (Sc)MSC as controls) showed co-localization of miR-210 with gap-junctional connexin-43. miR-210 transfer to cardiomyocytes was blocked by heptanol pretreatment. Moreover, higher survival of cardiomyocytes co-cultured with (PC)MSC was observed with concomitant expression of CASP8AP2 as compared to cardiomyocytes co-cultured with (non-PC)MSC thus suggesting that miR-210 was translocated from MSC to protect host cardiomyocytes. Induction of miR-210 in MSC promoted their survival post-engraftment in the infarcted heart. Moreover, direct transfer of pro-survival miR-210 from (miR)MSC to host cardiomyocytes led to functional recovery of the ischemic heart.

Unsaturated fatty acids induce mesenchymal stem cells to increase secretion of angiogenic mediators

Mesenchymal stem cells (MSC) represent emerging cell-based therapies for diabetes and associated complications. Ongoing clinical trials are using exogenous MSC to treat type 1 and 2 diabetes, cardiovascular disease and non-healing wounds due to diabetes. The majority of these trials are aimed at exploiting the ability of these multipotent mesenchymal stromal cells to release soluble mediators that reduce inflammation and promote both angiogenesis and cell survival at sites of tissue damage. Growing evidence suggests that MSC secretion of soluble factors is dependent on tissue microenvironment. Despite the contribution of fatty acids to the metabolic environment of type 2 diabetes, almost nothing is known about their effects on MSC secretion of growth factors and cytokines. In this study, human bone marrow-derived MSC were exposed to linoleic acid, an omega-6 polyunsaturated fatty acid, or oleic acid, a monounsaturated fatty acid, for seven days in the presence of 5.38 mM glucose. Outcomes measured included MSC proliferation, gene expression, protein secretion and chemotaxis. Linoleic and oleic acids inhibited MSC proliferation and altered MSC expression and secretion of known mediators of angiogenesis. Both unsaturated fatty acids induced MSC to increase secretion of interleukin-6, VEGF and nitric oxide. In addition, linoleic acid but not oleic acid induced MSC to increase production of interleukin-8. Collectively these data suggest that exposure to fatty acids may have functional consequences for MSC therapy. Fatty acids may affect MSC engraftment to injured tissue and MSC secretion of cytokines and growth factors that regulate local cellular responses to injury.

Regulation of aggrecanases from the ADAMTS family and aggrecan neoepitope formation during in vitro chondrogenesis of human mesenchymal stem cells

Aggrecanases from the ADAMTS (A Disintegrin And Metalloproteinase with ThromboSpondin motifs) family are important therapeutic targets due to their essential role in aggrecan depletion in arthritic diseases. Whether their function is also important for matrix rearrangements during chondrogenesis and thus, cartilage regeneration, is however so far unknown. The aim of this study was to analyse the expression and function of ADAMTS with aggrecanase activity during chondrogenic differentiation of human mesenchymal stem cells (MSCs). Chondrogenic differentiation was induced in bone marrow-derived MSC pellets and expression of COL2A1, aggrecan, ADAMTS1, 4, 5, 9, 16 and furin was followed by quantitative RT-PCR. Formation of the NITEGE (ADAMTS-cleaved) and DIPEN (MMP-cleaved) aggrecan neoepitopes was detected by immunohistochemistry. While the expression of ADAMTS4, 9, 16 and furin was up-regulated during chondrogenesis, ADAMTS1 and 5 were down-regulated. Despite this regulation of ADAMTS, no formation of NITEGE neoepitopes occurred in MSC pellets, indicating no ADAMTS-induced cleavage of aggrecan. In contrast, MMP-induced cleavage of aggrecan appeared at 14 d after induction of chondrogenesis. Submission of differentiated MSC pellets to IL1β treatment for 3 d resulted in strong upregulation of ADAMTS1, 4 and 5, rapid proteoglycan depletion, and stimulation of ADAMTS-induced but not MMP-induced cleavage of aggrecan. Thus, there is no evidence for ADAMTS-induced aggrecan cleavage during chondrogenesis, but proteoglycan turnover is rapidly inducible under inflammatory signals. Therapeutic aggrecanase inhibition for treatment of arthritic disease may thus not impede regenerative self-healing pathways based on chondrogenesis of local progenitor cells in the joint.

Derivation of islet‐like cells from mesenchymal stem cells using PDX1‐transducing lentiviruses

Pancreatic duodenum homeobox protein-1 (PDX1) is a master regulatory gene in pancreatic development. Reprogramming of mesenchymal stem cells (MSCs) is a promising tool for producing insulin-producing cells. In this study, lentivirus harboring PDX1 (LV-PDX1) has been used for persistence gene expression in MSCs. The objective of this study was to evaluate the potential of lentivirus to introduce the PDX1 gene into MSCs to produce insulin-secreting cells and apply it for treatment of hyperglycemia in diabetic rats. MSCs were isolated from rat bone marrow, characterized, and transduced by LV-PDX1. Significant expressions of PDX1, neurogenin3, glucagon, glucose transporter2 (Glut2), and insulin were detected by quantitative reverse transcription-polymerase chain reaction (P < 0.05). PDX1 and insulin were detected at the protein level by immunofluorescence analysis. PDX1 could trigger a gene expression cascade that involved pancreatic endocrine differentiation and also revealed the glucose sensing ability by expressing Glut2 in high-glucose medium. The insulin secretion of MSCs(PDX1+) in the high-glucose medium was 1.75-fold higher than that secreted in the low-glucose medium (P < 0.05). MSCs(PDX1+) implanted into diabetic rats could decrease the blood glucose level from 485 mg/dL to the normal level in 3 days. This study showed MSCs(PDX1+) have the potential to be used as a viable resource in cell-based gene therapy of type 1 diabetes.

Atmospheric Oxygen Inhibits Growth and Differentiation of Marrow‐Derived Mouse Mesenchymal Stem Cells via a p53‐Dependent Mechanism: Implications for Long‐Term Culture Expansion

Large scale expansion of human mesenchymal stem cells (MSCs) is routinely performed for clinical therapy. In contrast, developing protocols for large scale expansion of primary mouse MSCs has been more difficult due to unique aspects of rodent biology. Currently, established methods to isolate mouse MSCs select for rapidly dividing subpopulations that emerge from bone marrow cultures following long-term (months) expansion in atmospheric oxygen. Herein, we demonstrate that exposure to atmospheric oxygen rapidly induced p53, TOP2A, and BCL2-associated X protein (BAX) expression and mitochondrial reactive oxygen species (ROS) generation in primary mouse MSCs resulting in oxidative stress, reduced cell viability, and inhibition of cell proliferation. Alternatively, procurement and culture in 5% oxygen supported more prolific expansion of the CD45(-ve) /CD44(+ve) cell fraction in marrow, produced increased MSC yields following immunodepletion, and supported sustained MSC growth resulting in a 2,300-fold increase in cumulative cell yield by fourth passage. MSCs cultured in 5% oxygen also exhibited enhanced trilineage differentiation. The oxygen-induced stress response was dependent upon p53 since siRNA-mediated knockdown of p53 in wild-type cells or exposure of p53(-/-) MSCs to atmospheric oxygen failed to induce ROS generation, reduce viability, or arrest cell growth. These data indicate that long-term culture expansion of mouse MSCs in atmospheric oxygen selects for clones with absent or impaired p53 function, which allows cells to escape oxygen-induced growth inhibition. In contrast, expansion in 5% oxygen generates large numbers of primary mouse MSCs that retain sensitivity to atmospheric oxygen, and therefore a functional p53 protein, even after long-term expansion in vitro.

Mesenchymal stem cells isolated from bone marrow and compact bone express both VEGF and PLGF

Chronic obstructive pulmonary disease (COPD) is associated with dysregulation of vascular endothelial growth factor (VEGF) family protein levels (decreased VEGF and/or increased placental growth factor, PLGF). We hypothesized that mesenchymal stem cells (MSC) could enhance maintenance and repair of lung vasculature in COPD through VEGF family signaling. Toward this goal, we characterized VEGF and PLGF expression in MSC isolated from mouse bone marrow and compact bone. MSC were cultured in 5% O2, 10% CO2 and 85% N2 and characterized by flow cytometry. MSC from both sources highly expressed the MSC marker CD44 and moderately expressed MSC markers CD105 and CD73. MSC were cultured up to passage 2. Media was collected from primary MSC and p1–2 MSC for ELISA of VEGF and PLGF expression. Fresh media (DMEM + 15% FBS) contained neither PLGF nor VEGF. ELISA of conditioned media from MSC showed that MSC express both VEGF and PLGF, with VEGF&gt;PLGF. VEGF expression increased with passage number, whereas PLGF expression decreased with passage number. Pericytes, cells found in association with the vascular wall, are very similar structurally and functionally to MSC. MSC have also been reported to share similarity with vascular smooth muscle cells (SMC). Our findings suggest that MSCs have a SMC‐like pattern of VEGF and PLGF expression, consistent with a role in vascular maintenance and repair. Funding: OCASCR, NIH R01 HL084494 (PL).

T3 effect through SCR2 on chondrogenesis in vitro

T3 effect through SCR2 on chondrogenesis in vitro

Smad3 and Snail show circadian expression in human gingival fibroblasts, human mesenchymal stem cell, and in mouse liver

Smads are intracellular signaling mediators. Complexes of Smad2 and Smad3 with Smad4 transmit transforming growth factor-beta (TGF-β) receptor-induced signaling. Snail plays important roles in mesoderm formation, gastrulation, neural crest development, and epithelial mesenchymal transition. However, it remains unknown whether Smad3 and Snail expression is circadian rhythm-dependent. Here, we showed for the first time that Smad3 and Snail show circadian expression in human gingival fibroblasts (HGF-1) and human mesenchymal stem cells (MSC) after serum shock. They also showed circadian expression in the mouse liver. We confirmed that BMAL1/2, DEC1/2, VEGF, and PER1/2/3 also show circadian expression in both HGF-1 and MSC. The mRNA peaks and phases in circadian expression of these genes differed between HGF-1 and MSC. In a luciferase assay, Smad3 promoter activity was upregulated by CLOCK/BMAL1. These findings suggest that Smad3 and Snail have circadian rhythm in vitro and vivo, and that circadian expression of Smad3 depends on CLOCK/BMAL1.

Differential Signalling Through ALK-1 and ALK-5 Regulates Leptin Expression in Mesenchymal Stem Cells

Leptin plays a central role in maintaining energy balance, with multiple other systemic effects. Despite leptin importance in peripheral regulation of mesenchymal stem cells (MSC) differentiation, little is known about its expression mechanism. Leptin is often described as adipokine, while it is expressed by other cell types. We have recently shown an in vitro leptin expression, enhanced by glucocorticoids in synovial fibroblasts (SVF). Here, we investigated leptin expression in MSC from bone marrow (BM-MSC) and umbilical cord matrix (UMSC). Results showed that BM-MSC, but not UMSC, expressed leptin that was strongly enhanced by glucocorticoids. Transforming growth factor β1 (TGF-β1) markedly inhibited the endogenous- and glucocorticoid-induced leptin expression in BM-MSC. Since TGF-β1 was shown to signal via ALK-5-Smad2/3 and/or ALK-1-Smad1/5 pathways, we analyzed the expression of proteins from both pathways. In BM-MSC, TGF-β1 increased phosphorylated Smad2 (p-Smad2) expression, while ALK-5 inhibitor (SB431542) induced leptin expression and significantly restored TGF-β1-induced leptin inhibition. In addition, both prednisolone and SB431542 increased p-Smad1/5 expression. These results suggested the ALK-5-Smad2 pathway as an inhibitor of leptin expression, while ALK-1-Smad1/5 as an activator. Indeed, Smad1 expression silencing induced leptin expression inhibition. Furthermore, prednisolone enhanced the expression of TGF-βRII while decreasing p-Smad2 in BM-MSC and SVF but not in UMSC. In vitro differentiation revealed differential osteogenic potential in SVF, BM-MSC, and UMSC that was correlated to their leptin expression potential. Our results suggest that ALK-1/ALK-5 balance regulates leptin expression in MSC. It also underlines UMSC as leptin nonproducer MSC for cell therapy protocols where leptin expression is not suitable.