Stage-related capacity for limb chondrogenesis in cell culture

Ahrens, Patricia Buckley; Solursh, Michael; Reiter, Rebecca S.

doi:10.1016/0012-1606(77)90110-5

Cited by 591 publications

(377 citation statements)

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“…The concept of a simple and well-reproducible in vitro experimental model of hyaline cartilage formation was originally described by Ahrens and colleagues [27]. In these micromass cell cultures (HDC), chicken embryonic limb bud-derived chondroprogenitor cells spontaneously differentiate to chondrocytes on days 2 and 3 of culturing, and a welldetectable amount of hyaline cartilage ECM is produced by culturing day 6.…”

Section: Primary High Density Chondrifying Cell Culturesmentioning

confidence: 99%

“…In primary chicken chondrifying HDC, differentiation and final commitment of chondroprogenitor cells take place predominantly on days 2 and 3 of culturing [27]. To monitor the effects of intermittent cyclic mechanical load exerted by our custom-made bioreactor on the differentiation of chondroprogenitor cells, loading scheme (0.05 Hz, 600 Pa) was applied on these two days of culturing for 30 min.…”

Section: Mechanical Stimulation Enhances Cartilage Formation and Redumentioning

confidence: 99%

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Mechanical loading stimulates chondrogenesis via the PKA/CREB-Sox9 and PP2A pathways in chicken micromass cultures

Juhász

Matta

Somogyi

et al. 2014

Cellular Signalling

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Biomechanical stimuli play important roles in the formation of articular cartilage during early foetal life, and optimal mechanical load is a crucial regulatory factor of adult chondrocyte metabolism and function. In this study, we undertook to analyse mechanotransduction pathways during in vitro chondrogenesis. Chondroprogenitor cells isolated from limb buds of 4-day-old chicken embryos were cultivated as high density cell cultures for 6 days. Mechanical stimulation was carried out by a self-designed bioreactor that exerted uniaxial intermittent cyclic load transmitted by the culture medium as hydrostatic pressure and fluid shear to differentiating cells. The loading scheme (0.05 Hz, 600 Pa; for 30 min) was applied on culturing days 2 and 3, when final commitment and differentiation of chondroprogenitor cells occurred in this model. The applied mechanical load significantly augmented cartilage matrix production and elevated mRNA expression of several cartilage matrix constituents, including collagen type II and aggrecan core protein, as well as matrixproducing hyaluronan synthases through enhanced expression, phosphorylation and nuclear signals of the main chondrogenic transcription factor Sox9. Along with increased cAMP levels, a significantly enhanced protein kinase A (PKA) activity was also detected and CREB, the archetypal downstream transcription factor of PKA signalling, exhibited elevated phosphorylation levels and stronger nuclear signals in response to mechanical stimuli. All the above effects were diminished by the PKA-inhibitor H89. Inhibition of the PKA-independent cAMP-mediators Epac1 and Epac2 with HJC0197 resulted in enhanced cartilage formation, which was additive to that of the mechanical stimulation, implying that the chondrogenesispromoting effect of mechanical load was independent of Epac. At the same time, PP2A activity was reduced following mechanical load and treatments with the PP2A-inhibitor okadaic acid were able to mimic the effects of the intervention. Our results indicate that proper mechanical stimuli augment in vitro cartilage formation via promoting both Juhász and Matta et al. 3 differentiation and matrix production of chondrogenic cells, and the opposing regulation of the PKA/CREB-Sox9 and the PP2A signalling pathways is crucial in this phenomenon.

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Section: Primary High Density Chondrifying Cell Culturesmentioning

confidence: 99%

Section: Mechanical Stimulation Enhances Cartilage Formation and Redumentioning

confidence: 99%

Mechanical loading stimulates chondrogenesis via the PKA/CREB-Sox9 and PP2A pathways in chicken micromass cultures

Juhász

Matta

Somogyi

et al. 2014

Cellular Signalling

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“…Chondrogenic processes have been investigated in vivo by means of 2 well-known methods of culturing mesenchymal cells: so-called "chondrogenic pellet culture" (16,17) and "micromass culture" (18,19). Both methods efficiently produce cartilaginous structures within 2-3 weeks from undifferentiated mesenchymal cells isolated from embryonic limb buds or wing buds (16)(17)(18).…”

mentioning

confidence: 99%

“…Both methods efficiently produce cartilaginous structures within 2-3 weeks from undifferentiated mesenchymal cells isolated from embryonic limb buds or wing buds (16)(17)(18). The established murine cell line C3H10T1/2 has also been used in these analyses (19).…”

mentioning

confidence: 99%

Methylation status of CpG islands in the promoter regions of signature genes during chondrogenesis of human synovium–derived mesenchymal stem cells

Ezura

Sekiya

Koga

et al. 2009

Arthritis & Rheumatism

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Objective. Human synovium-derived mesenchymal stem cells (MSCs) can efficiently differentiate into mature chondrocytes. It has been suggested that DNA methylation is one mechanism that regulates human chondrogenesis; however, the methylation status of genes related to chondrogenic differentiation is not known. The purpose of this study was to investigate the CpG methylation status in human synovium-derived MSCs during experimental chondrogenesis, with a view toward potential therapeutic use in osteoarthritis.Methods. Human synovium-derived MSCs were subjected to chondrogenic pellet culture for 3 weeks. The methylation status of 12 regions in the promoters of 10 candidate genes (SOX9, RUNX2, CHM1, FGFR3, CHAD, MATN4, SOX4, GREM1, GPR39, and SDF1) was analyzed by bisulfite sequencing before and after differentiation. The expression levels of these genes were analyzed by real-time reverse transcription-polymerase chain reaction. Methylation status was also examined in human articular cartilage.Results. Bisulfite sequencing analysis indicated that 10 of the 11 CpG-rich regions analyzed were hypomethylated in human progenitor cells before and after 3 weeks of pellet culture, regardless of the expression levels of the genes. The methylation status was consistently low in SOX9, RUNX2, CHM1, CHAD, and FGFR3 following an increase in expression upon differentiation and was low in GREM1 and GPR39 following a decrease in expression upon chondrogenesis. One exceptional instance of a differentially methylated CpGrich region was in a 1-kb upstream sequence of SDF1, the expression of which decreased upon differentiation. Paradoxically, the hypermethylation status of this region was reduced after 3 weeks of pellet culture.Conclusion. The DNA methylation levels of CpGrich promoters of genes related to chondrocyte phenotypes are largely kept low during chondrogenesis in human synovium-derived MSCs.

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“…Stage 20-22 chicken embryo limb bud mesoderm and ectoderm were cultured according to Ahrens et al (1977). In some cases, ectoderm was removed before dissociating the mesoderm for culture by use of 1% ethylenediaminetetraacetic acid in double strength calcium and magnesium free Tyrode's solution (Carrington and Fallon, 1984).…”

Section: Limb Bud Culturesmentioning

confidence: 99%

Selective expression of the chicken platelet‐derived growth factor α (PDGFα) receptor during limb bud development

Potts

Carrington

1993

Developmental Dynamics

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Platelet-derived growth factor (PDGF) affects proliferation and differentiation of chicken limb bud mesoderm in vitro. However, no PDGF receptor has been characterized in the chicken wing bud in vivo. In this study, we used reverse transcription PCR (rtPCR), Northern blot analysis, and Western blot analysis to identify a molecule, in the developing wing bud, which represents the chicken homolog of the PDGFa receptor. The chicken PDGFa receptor mRNA was present in both mesoderm and ectoderm and all stages of the developing limb bud examined. Cultured limb bud mesoderm also expressed the PDGFa receptor transcript. In addition, the PDGFa receptor protein was present in whole limb buds and cultured limb bud mesoderm. Expression of the PDGFa receptor in cultured mesoderm was independent of the presence of ectoderm cells. The relative sizes of both the mRNA and protein for the PDGFa receptor in the chicken limb bud were similar to mammalian counterparts. Using similar approaches, neither the mRNA nor protein representing the chicken homolog of the PDGFp receptor was detected. These data demonstrate for the first time that a PDGFa receptor is present in the embryonic chicken limb bud and may help regulate growth and differentiation of the embryonic limb. 0 1993 Wiley-Liss, Inc.*

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Stage-related capacity for limb chondrogenesis in cell culture

Cited by 591 publications

References 20 publications

Mechanical loading stimulates chondrogenesis via the PKA/CREB-Sox9 and PP2A pathways in chicken micromass cultures

Mechanical loading stimulates chondrogenesis via the PKA/CREB-Sox9 and PP2A pathways in chicken micromass cultures

Methylation status of CpG islands in the promoter regions of signature genes during chondrogenesis of human synovium–derived mesenchymal stem cells

Selective expression of the chicken platelet‐derived growth factor α (PDGFα) receptor during limb bud development

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