Blocking vascular endothelial growth factor with soluble Flt‐1 improves the chondrogenic potential of mouse skeletal muscle–derived stem cells

Objective

To investigate the effect of vascular endothelial growth factor (VEGF) stimulation and the effect of blocking VEGF with its antagonist, soluble Flt‐1 (sFlt‐1), on chondrogenesis, using muscle‐derived stem cells (MDSCs) isolated from mouse skeletal muscle.

Methods

The direct effect of VEGF on the in vitro chondrogenic ability of mouse MDSCs was tested using a pellet culture system, followed by real‐time quantitative polymerase chain reaction (PCR) and histologic analyses. Next, the effect of VEGF on chondrogenesis within the synovial joint was tested, using genetically engineered MDSCs implanted into rat osteochondral defects. In this model, MDSCs transduced with a retroviral vector to express bone morphogenetic protein 4 (BMP‐4) were coimplanted with MDSCs transduced to express either VEGF or sFlt‐1 (a VEGF antagonist) to provide a gain‐ and loss‐of‐function experimental design. Histologic scoring was used to compare cartilage formation among the treatment groups.

Results

Hyaline‐like cartilage matrix production was observed in both VEGF‐treated and VEGF‐blocked (sFlt‐1–treated) pellet cultures, but quantitative PCR revealed that sFlt‐1 treatment improved the expression of chondrogenic genes in MDSCs that were stimulated to undergo chondrogenic differentiation with BMP‐4 and transforming growth factor β3 (TGFβ3). In vivo testing of articular cartilage repair showed that VEGF‐transduced MDSCs caused an arthritic change in the knee joint, and sFlt‐1 improved the MDSC‐mediated repair of articular cartilage, compared with BMP‐4 alone.

Conclusion

Soluble Flt‐1 gene therapy improved the BMP‐4– and TGFβ3‐induced chondrogenic gene expression of MDSCs in vitro and improved the persistence of articular cartilage repair by preventing vascularization and bone invasion into the repaired articular cartilage.

     

Osteoarthritic chondrocyte–secreted morphogens induce chondrogenic differentiation of human mesenchymal stem cells

Objective

The potential of stem cells to repair compromised cartilage tissue, such as in osteoarthritis (OA), depends strongly on how transplanted cells respond to factors secreted from the residing OA chondrocytes. This study was undertaken to determine the effect of morphogenetic signals from OA chondrocytes on chondrogenic differentiation of human mesenchymal stem cells (MSCs).

Methods

The effect of OA chondrocyte–secreted morphogens on chondrogenic differentiation of human MSCs was evaluated using a coculture system involving both primary and passaged OA chondrocytes. The findings were compared against findings for human MSCs cultured in OA chondrocyte–conditioned medium. Gene expression analysis, biochemical assays, and immunofluorescence staining were used to characterize the chondrogenic differentiation of human MSCs. Mass spectrometry analysis was used to identify the soluble factors. Numerical analysis was carried out to model the concentration profile of soluble factors within the human MSC–laden hydrogels.

Results

The human MSCs cocultured with primary OA chondrocytes underwent chondrogenic differentiation even in the absence of growth factors; however, the same effect could not be mimicked using OA chondrocyte–conditioned medium or expanded cells. Additionally, the cocultured environment down‐regulated hypertrophic differentiation of human MSCs. Mass spectrometry analysis demonstrated cell–cell communication and chondrocyte phenotype–dependent effects on cell‐secreted morphogens.

Conclusion

The experimental findings, along with the results of the numerical analysis, suggest a crucial role of soluble morphogens and their local concentrations in the differentiation pattern of human MSCs in a 3‐dimensional environment. The concept of using a small number of chondrocytes to promote chondrogenic differentiation of human MSCs while preventing their hypertrophic differentiation could be of great importance in formulating effective stem cell–based cartilage repair.

     

Potent induction of chondrocytic differentiation of human adipose‐derived adult stem cells by bone morphogenetic protein 6

Objective

Recent studies have identified an abundant source of multipotent progenitor cells in subcutaneous human adipose tissue, termed human adipose‐derived adult stem cells (ADAS cells). In response to specific media formulations, including transforming growth factor β1 (TGFβ1), these cells exhibit significant ability to differentiate into a chondrocyte‐like phenotype, expressing cartilage‐specific genes and proteins such as aggrecan and type II collagen. However, the influence of other growth factors on the chondrogenic differentiation of ADAS cells is not fully understood. This study was undertaken to investigate the effects of TGFβ1, TGFβ3, insulin‐like growth factor 1, bone morphogenetic protein 6 (BMP‐6), and dexamethasone, in various combinations, on the chondrogenic potential of ADAS cells in alginate beads.

Methods

The chondrogenic response of alginate‐encapsulated ADAS cells was measured by quantitative polymerase chain reaction, ³H‐proline and ³⁵S‐sulfate incorporation, and immunolabeling for specific extracellular matrix components.

Results

Significant differences in chondrogenesis were observed under the different culture conditions for all outcomes measured. Most notably, BMP‐6 up‐regulated AGC1 and COL2A1 expression by an average of 205‐fold and 38‐fold, respectively, over day‐0 controls, while down‐regulating COL10A1 expression by ∼2‐fold.

Conclusion

These findings suggest that BMP‐6 is a potent inducer of chondrogenesis in ADAS cells, in contrast to mesenchymal stem cells, which exhibit increased expression of type X collagen and a hypertrophic phenotype in response to BMP‐6. Combinations of growth factors containing BMP‐6 may provide a novel means of regulating the differentiation of ADAS cells for applications in the tissue‐engineered repair or regeneration of articular cartilage.

     

Cartilage repair using bone morphogenetic protein 4 and muscle‐derived stem cells

Objective

Muscle‐derived stem cells (MDSCs) isolated from mouse skeletal muscle exhibit long‐time proliferation, high self‐renewal, and multipotent differentiation. This study was undertaken to investigate the ability of MDSCs that were retrovirally transduced to express bone morphogenetic protein 4 (BMP‐4) to differentiate into chondrocytes in vitro and in vivo and enhance articular cartilage repair.

Methods

Using monolayer and micromass pellet culture systems, we evaluated the in vitro chondrogenic differentiation of LacZ‐ and BMP‐4–transduced MDSCs with or without transforming growth factor β1 (TGFβ1) stimulation. We used a nude rat model of a full‐thickness articular cartilage defect to assess the duration of LacZ transgene expression and evaluate the ability of transplanted cells to acquire a chondrocytic phenotype. We evaluated cartilage repair macroscopically and histologically 4, 8, 12, and 24 weeks after surgery, and performed histologic grading of the repaired tissues.

Results

BMP‐4–expressing MDSCs acquired a chondrocytic phenotype in vitro more effectively than did MDSCs expressing only LacZ; the addition of TGFβ1 did not alter chondrogenic differentiation of the BMP‐4–transduced MDSCs. LacZ expression within the repaired tissue continued for up to 12 weeks. Four weeks after surgery, we detected donor cells that coexpressed β‐galactosidase and type II collagen. Histologic scoring of the defect sites 24 weeks after transplantation revealed significantly better cartilage repair in animals that received BMP‐4–transduced MDSCs than in those that received MDSCs expressing only LacZ.

Conclusion

Local delivery of BMP‐4 by genetically engineered MDSCs enhanced chondrogenesis and significantly improved articular cartilage repair in rats.

     

Changes in surface markers of human mesenchymal stem cells during the chondrogenic differentiation and dedifferentiation processes in vitro

Objective

To investigate surface markers showing specific changes during the chondrogenic differentiation and dedifferentiation of human mesenchymal stem cells (MSCs).

Methods

Human MSCs from adult bone marrow were subjected to chondrogenic differentiation in 3‐dimensional (3‐D) alginate culture with or without transforming growth factor β3 (TGFβ3) for 2 weeks, followed by dedifferentiation in monolayer for 1 week. Surface antigens were selected from those previously reported to show changes in expression during dedifferentiation of human articular chondrocytes (HACs).

Results

Flow cytometry was used to identify 3 groups of surface antigens with differential expression patterns that were quite different from those previously reported on HACs. Two groups of antigens were expressed at high levels on human MSCs. The expression of the first group of antigens (CD44, CD58, CD81, CD90, CD105, and CD166) was decreased reversibly by the 3‐D alginate culture and irreversibly in the presence of TGFβ3, except for CD81, which showed reversible changes regardless of TGFβ3. The expression of the second group of antigens (CD49c, CD49e, and CD151) was decreased during chondrogenic differentiation only in the presence of TGFβ3. During all experimental stages, the expression of the third group of antigens (CD14, CD26, CD49f, CD54, CD106, CD119, and CD140a) was maintained at low levels (expressed on <30% of cells), although with some fluctuations.

Conclusion

We speculate that the second group of surface antigens could be negative markers for chondrogenic differentiation of human MSCs.

     

Cartilage repair in a rat model of osteoarthritis through intraarticular transplantation of muscle‐derived stem cells expressing bone morphogenetic protein 4 and soluble flt‐1

Objective

The control of angiogenesis during chondrogenic differentiation is an important issue affecting the use of stem cells in cartilage repair, especially with regard to the persistence of regenerated cartilage. This study was undertaken to investigate the effect of vascular endothelial growth factor (VEGF) stimulation and the blocking of VEGF with its antagonist, soluble Flt‐1 (sFlt‐1), on the chondrogenesis of skeletal muscle‐derived stem cells (MDSCs) in a rat model of osteoarthritis (OA).

Methods

We investigated the effect of VEGF on cartilage repair in an immunodeficiency rat model of OA after intraarticular injection of murine MDSCs expressing bone morphogenetic protein 4 (BMP‐4) in combination with MDSCs expressing VEGF or sFlt‐1.

Results

In vivo, a combination of sFlt‐1– and BMP‐4–transduced MDSCs demonstrated better repair without osteophyte formation macroscopically and histologically following OA induction, when compared with the other groups. Higher differentiation/proliferation and lower levels of chondrocyte apoptosis were also observed in sFlt‐1– and BMP‐4–transduced MDSCs compared with a combination of VEGF‐ and BMP‐4–transduced MDSCs or with BMP‐4–transduced MDSCs alone. In vitro experiments with mixed pellet coculture of MDSCs and OA chondrocytes revealed that BMP‐4–transduced MDSCs produced the largest pellets, which had the highest gene expression of not only type II collagen and SOX9 but also type X collagen, suggesting formation of hypertrophic chondrocytes.

Conclusion

Our results demonstrate that MDSC‐based therapy involving sFlt‐1 and BMP‐4 repairs articular cartilage in OA mainly by having a beneficial effect on chondrogenesis by the donor and host cells as well as by preventing angiogenesis, which eventually prevents cartilage resorption, resulting in persistent cartilage regeneration and repair.

     

Human articular chondrocytes secrete parathyroid hormone–related protein and inhibit hypertrophy of mesenchymal stem cells in coculture during chondrogenesis

Objective

The use of bone marrow–derived mesenchymal stem cells (MSCs) has shown promise in cell‐based cartilage regeneration. A yet‐unsolved problem, however, is the unwanted up‐regulation of markers of hypertrophy, such as alkaline phosphatase (AP) and type X collagen, during in vitro chondrogenesis and the formation of unstable calcifying cartilage at heterotopic sites. In contrast, articular chondrocytes produce stable, nonmineralizing cartilage. The aim of this study was to address whether coculture of MSCs with human articular chondrocytes (HACs) can suppress the undesired hypertrophy in differentiating MSCs.

Methods

MSCs were differentiated in chondrogenic medium that had or had not been conditioned by parallel culture with HAC pellets, or MSCs were mixed in the same pellet with the HACs (1:1 or 1:2 ratio) and cultured for 6 weeks. Following in vitro differentiation, the pellets were transplanted into SCID mice.

Results

The gene expression ratio of COL10A1 to COL2A1 and of Indian hedgehog (IHH) to COL2A1 was significantly reduced by differentiation in HAC‐conditioned medium, and less type X collagen protein was deposited relative to type II collagen. AP activity was significantly lower (P < 0.05) in the cells that had been differentiated in conditioned medium, and transplants showed significantly reduced calcification in vivo. In mixed HAC/MSC pellets, suppression of AP was dose‐dependent, and in vivo calcification was fully inhibited. Chondrocytes secreted parathyroid hormone–related protein (PTHrP) throughout the culture period, whereas PTHrP was down‐regulated in favor of IHH up‐regulation in control MSCs after 2–3 weeks of chondrogenesis. The main inhibitory effects seen with HAC‐conditioned medium were reproducible by PTHrP supplementation of unconditioned medium.

Conclusion

HAC‐derived soluble factors and direct coculture are potent means of improving chondrogenesis and suppressing the hypertrophic development of MSCs. PTHrP is an important candidate soluble factor involved in this effect.

     

Screening of chondrogenic factors with a real‐time fluorescence‐monitoring cell line ATDC5‐C2ER: Identification of sorting nexin 19 as a novel factor

Objective

To establish a cell culture system for noninvasive and real‐time monitoring of chondrogenic differentiation in order to screen for chondrogenic factors.

Methods

The optimum reporter construct transfected into chondrogenic ATDC5 cells was selected by a luciferase reporter assay and fluorescence analysis during cultures with insulin. The established cell line was validated according to its fluorescence following stimulation with SOX proteins, bone morphogenetic protein 2 (BMP‐2), or transforming growth factor β (TGFβ) and was compared with the level of messenger RNA for COL2A1 as well as with the degree of Alcian blue staining. Screening of chondrogenic factors was performed by expression cloning using a retroviral expression library prepared from human tracheal cartilage. The expression pattern of the identified molecule was examined by in situ hybridization and immunohistochemistry. Functional analysis was performed by transfection of the identified gene, the small interfering RNA, and the mutated gene.

Results

We established an ATDC5 cell line with 4 repeats of a highly conserved enhancer ligated to a COL2A1 basal promoter and the DsRed2 reporter (ATDC5‐C2ER). Fluorescence was induced under the stimulations with SOX proteins, BMP‐2, or TGFβ, showing good correspondence to the chondrogenic markers. Screening using the ATDC5‐C2ER system identified several chondrogenic factors, including sorting nexin 19 (SNX19). SNX19 was expressed in the limb cartilage of mouse embryos and in the degraded cartilage of adult mouse knee joints during osteoarthritis progression. The gain‐of‐function and loss‐of‐function analyses revealed a potent chondrogenic activity of SNX19.

Conclusion

We established the ATDC5‐C2ER system for efficient monitoring of chondrogenic differentiation by fluorescence analysis, and we identified a novel chondrogenic factor (SNX19) using this system. This system will be useful for elucidating the molecular network of chondrogenic differentiation.

     

Chondrogenic differentiation of bovine synovium: Bone morphogenetic proteins 2 and 7 and transforming growth factor β1 induce the formation of different types of cartilaginous tissue

Objective

To compare the potential of bone morphogenetic proteins 2 and 7 (BMP‐2 and BMP‐7) and transforming growth factor β1 (TGFβ1) to effect the chondrogenic differentiation of synovial explants by analyzing the histologic, biochemical, and gene expression characteristics of the cartilaginous tissues formed.

Methods

Synovial explants derived from the metacarpal joints of calves were cultured in agarose. Initially, BMP‐2 was used to evaluate the chondrogenic potential of the synovial explants under different culturing conditions. Under appropriate conditions, the chondrogenic effects of BMP‐2, BMP‐7, and TGFβ1 were then compared. The differentiated tissue was characterized histologically, histomorphometrically, immunohistochemically, biochemically, and at the gene expression level.

Results

BMP‐2 induced the chondrogenic differentiation of synovial explants in a dose‐ and time‐dependent manner under serum‐ and dexamethasone‐free conditions. The expression levels of cartilage‐related genes increased in a time‐dependent manner. BMP‐7 was more potent than BMP‐2 in inducing chondrogenesis, but the properties of the differentiated tissue were similar in each case. The type of cartilaginous tissue formed under the influence of TGFβ1 differed in terms of both cell phenotype and gene expression profiles.

Conclusion

The 3 tested members of the TGFβ superfamily have different chondrogenic potentials and induce the formation of different types of cartilaginous tissue. To effect the full differentiation of synovial explants into a typically hyaline type of articular cartilage, further refinement of the stimulation conditions is required. This might be achieved by the simultaneous application of several growth factors.

     

Matrix fixed‐charge density as determined by magnetic resonance microscopy of bioreactor‐derived hyaline cartilage correlates with biochemical and biomechanical properties

Objective

To use noninvasive magnetic resonance imaging (MRI), biochemical analyses, and mechanical testing of engineered neocartilage grown in a hollow‐ fiber bioreactor (HFBR) to establish tissue properties, and to test the hypothesis that MRI can be used to monitor biochemical and biomechanical properties of neocartilage.

Methods

Chondrocytes from day 16 embryonic chick sterna were inoculated into an HFBR and maintained for up to 4 weeks with and without exposure to chondroitinase ABC. The fixed‐charge density (FCD) of the cartilage was determined using the MRI gadolinium exclusion method. The sulfated glycosaminoglycan (S‐GAG), hydroxyproline, and DNA contents were determined using biochemical procedures, while dynamic and equilibrium moduli were determined from mechanical indentation tests.

Results

S‐GAG content, tissue cross‐sectional area, and equilibrium modulus of the neocartilage increased with development time. There was a gradient of S‐GAG content across the length of control neocartilage at the 4‐week time point, with higher values being found toward the inflow region. Exposure to chondroitinase ABC resulted in a decrease in tissue area, negative FCD, proteoglycan content, and equilibrium and dynamic moduli. The treated bioreactors displayed a lengthwise variation in S‐GAG content, with higher values toward the outflow end. Linear correlations were established among FCD, proteoglycan content, and biomechanical properties.

Conclusion

HFBR‐derived neocartilage showed regional variation in S‐GAG content under control conditions, and in the decrease of S‐GAG in response to enzyme treatment. In addition, the results support the hypothesis that tissue parameters derived from MRI can be used to noninvasively monitor focal neocartilage formation and biochemical and biomechanical properties.

     

Estradiol inhibits chondrogenic differentiation of mesenchymal stem cells via nonclassic signaling

Objective

We undertook this study to examine the effects of estradiol on chondrogenesis of human bone marrow–derived mesenchymal stem cells (MSCs), with consideration of sex‐dependent differences in cartilage repair.

Methods

Bone marrow was obtained from the iliac crest of young men. Density‐gradient centrifugation–separated human MSCs proliferated as a monolayer in serum‐containing medium. After confluence was achieved, aggregates were created and cultured in a serum‐free differentiation medium. We added different concentrations of 17β‐estradiol (E2) with or without the specific estrogen receptor inhibitor ICI 182.780, membrane‐impermeable E2–bovine serum albumin (E2‐BSA), ICI 182.780 alone, G‐1 (an agonist of G protein–coupled receptor 30 [GPR‐30]), and G15 (a GPR‐30 antagonist). After 21 days, the aggregates were analyzed histologically and immunohistochemically; we quantified synthesized type II collagen, DNA content, sulfated glycosaminoglycan (sGAG) concentrations, and type X collagen and matrix metalloproteinase 13 (MMP‐13) expression.

Results

The existence of intracellular and membrane‐associated E2 receptors was shown at various stages of chondrogenesis. Smaller aggregates and significantly lower type II collagen and sGAG content were detected after treatment with E2 and E2‐BSA in a dose‐dependent manner. Furthermore, E2 enhanced type X collagen and MMP‐13 expression. Compared with estradiol alone, the coincubation of ICI 182.780 with estradiol enhanced suppression of chondrogenesis. Treatment with specific GPR‐30 agonists alone (G‐1 and ICI 182.780) resulted in a considerable inhibition of chondrogenesis. In addition, we found an enhancement of hypertrophy by G‐1. Furthermore, the specific GPR‐30 antagonist G15 reversed the GPR‐30–mediated inhibition of chondrogenesis and up‐regulation of hypertrophic gene expression.

Conclusion

The experiments revealed a suppression of chondrogenesis by estradiol via membrane receptors (GPR‐30). The study opens new perspectives for influencing chondrogenesis on the basis of classic and nonclassic estradiol signaling.

     

Induction of chondrogenesis from human embryonic stem cells without embryoid body formation by bone morphogenetic protein 7 and transforming growth factor β1

Objective

Human embryonic stem cells (ESCs) provide an unlimited supply of pluripotent cells for articular cartilage tissue engineering and regenerative medicine applications. Articular cartilage is an avascular tissue with precise polarity and organization comprising 3 distinct functional zones: surface, middle, and deep. To date, attempts at differentiating human ESCs into articular chondrocytes have been unsuccessful. The majority of studies have focused on chondrogenic (but not specifically articular cartilage) differentiation. Furthermore, previous investigations of induction of chondrogenesis by human ESCs required embryoid body formation; however, embryoid body formation often results in heterogeneous differentiation. The present study was undertaken to determine the in vitro chondrogenic potential of bone morphogenetic protein 7 (BMP‐7) and transforming growth factor β1 (TGFβ1)–induced human ESC differentiation toward the articular cartilage phenotype.

Methods

Dissociated single human ESCs were cultured and passaged on a gelatin‐coated flask. The human ESCs were cultured as an aggregate in a pellet culture system for 14 days in basal chondrogenic medium (CM), CM with TGFβ1, CM with BMP‐7, or CM with both TGFβ1 and BMP‐7.

Results

The size and wet weight of the cartilage pellets and glycosaminoglycan levels increased, with the smallest, intermediate, and greatest increases, respectively, observed with CM plus TGFβ1 treatment, CM plus BMP‐7 treatment, and CM plus TGFβ1 and BMP‐7 treatment (compared with CM treatment alone). The largest size and highest weight of the pellet was in the group in which TGFβ1 and BMP‐7 were added to the medium. However, expression of the genes for cartilage‐specific aggrecan and type II collagen II, as assessed by determination of messenger RNA levels, was highest in the BMP‐7–treated group. Superficial zone protein (SZP)/lubricin, a marker of the superficial zone articular chondrocyte, was not detectable under identical culture conditions.

Conclusion

These results demonstrate an efficient and reproducible model system of human ESC‐induced chondrogenesis, using a novel direct plating method in which intervening embryoid body formation does not occur. Further work is needed for optimization of conditions to obtain the articular cartilage phenotype that includes the superficial zone marker as demonstrated by SZP/lubricin synthesis.

     

MicroRNA‐140 is expressed in differentiated human articular chondrocytes and modulates interleukin‐1 responses

Objective

MicroRNA (miRNA) are a class of noncoding small RNAs that act as negative regulators of gene expression. MiRNA exhibit tissue‐specific expression patterns, and changes in their expression may contribute to pathogenesis. The objectives of this study were to identify miRNA expressed in articular chondrocytes, to determine changes in osteoarthritic (OA) cartilage, and to address the function of miRNA‐140 (miR‐140).

Methods

To identify miRNA specifically expressed in chondrocytes, we performed gene expression profiling using miRNA microarrays and quantitative polymerase chain reaction with human articular chondrocytes compared with human mesenchymal stem cells (MSCs). The expression pattern of miR‐140 was monitored during chondrogenic differentiation of human MSCs in pellet cultures and in human articular cartilage from normal and OA knee joints. We tested the effects of interleukin‐1β (IL‐1β) on miR‐140 expression. Double‐stranded miR‐140 (ds–miR‐140) was transfected into chondrocytes to analyze changes in the expression of genes associated with OA.

Results

Microarray analysis showed that miR‐140 had the largest difference in expression between chondrocytes and MSCs. During chondrogenesis, miR‐140 expression in MSC cultures increased in parallel with the expression of SOX9 and COL2A1. Normal human articular cartilage expressed miR‐140, and this expression was significantly reduced in OA tissue. In vitro treatment of chondrocytes with IL‐1β suppressed miR‐140 expression. Transfection of chondrocytes with ds–miR‐140 down‐regulated IL‐1β–induced ADAMTS5 expression and rescued the IL‐1β–dependent repression of AGGRECAN gene expression.

Conclusion

This study shows that miR‐140 has a chondrocyte differentiation–related expression pattern. The reduction in miR‐140 expression in OA cartilage and in response to IL‐1β may contribute to the abnormal gene expression pattern characteristic of OA.