Retroviral Gene Transfer in Chondrogenic Limb Bud Micromass Cultures

Stott, N. Susan; Lee, Y.-S.; Chuong, Cheng‐Ming

doi:10.2144/98244rr03

Cited by 5 publications

(10 citation statements)

References 41 publications

(43 reference statements)

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“…Retrovirus was prepared as described by Morgan and Fekete (1996) and Stott et al (1998). Briefly, dorsal hypodermal fibroblasts from SPAFAS chicken E8 embryos were used as retrovirus-producing cells.…”

Section: Retrovirus Transduction In Micromass Culturesmentioning

confidence: 99%

“…Retroviral infection of micromass cultures was performed as described in Stott et al (1998). Dissociated mesenchymal cells were prepared from the distal one third of stage 23/24 leg buds.…”

Section: Retrovirus Transduction In Micromass Culturesmentioning

confidence: 99%

“…To expand further the usefulness of the micromass culture system, we have recently developed a new method to apply RCAS retroviral technology (Morgan and Fekete, 1996) to micromass cultures (Stott et al, 1998). We have used this technique to show that the hedgehog signaling pathway induces characteristics of hypertrophic cartilage in chondrogenic cell culture (Stott and Chuong, 1997) as well as inhibits cartilage differentiation in vivo indirectly via PTHrP (Vortkamp et al, 1996).…”

mentioning

confidence: 99%

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Successive formative stages of precartilaginous mesenchymal condensations in vitro: Modulation of cell adhesion by Wnt-7A and BMP-2

1999

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High-density chick limb bud cell culture is a useful model to study mesenchymal condensatifons and chondrogenesis. Most previous studies have focused on the effects of soluble reagents on terminal chondrogenic differentiation and have not defined the early cellular processes and signaling events. In this study, we defined five successive stages in the differentiation process: 1) dissociated cells, 2) small aggregates, 3) formation of cell clusters, 4) precartilaginous condensations, and 5) cartilage nodule. We used RCAS retrovirus-mediated Wnt-7a gene transduction to test the effect of Wnt-7a on the differentiation process. We found that Wnt-7a suppressed chondrogenic differentiation. Wnt-7a did not inhibit the initiation of condensation formation but blocked the progression of precartilaginous condensations to cartilage nodules. The Wnt-7a-transduced cultures showed characteristics of a less mature culture with persistent expression of NCAM, N-cadherin, wider distribution of integrin beta1 and fibronectin, and suppression of tenascin-C. BMP-2 is known to enhance chondrogenic differentiation in these cultures by promoting cell clusters to form continuous sheet-like precartilaginous condensations. However, cultures exposed to both BMP-2 and Wnt-7a showed inhibition of chondrogenic differentiation. Different signaling molecules such as Wnt-7a and BMP-2 may have antagonistic effects on cartilage differentiation and the gradient of the two molecules may be involved in defining the boundaries of the initial precartilaginous condensation. We propose that the shape of the precartilaginous condensations may be modulated by local concentrations of signaling molecules, such as Wnt-7a and BMP-2, which act to alter cell-substrate and cell-cell adhesions.

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Section: Retrovirus Transduction In Micromass Culturesmentioning

confidence: 99%

“…Retroviral infection of micromass cultures was performed as described in Stott et al (1998). Dissociated mesenchymal cells were prepared from the distal one third of stage 23/24 leg buds.…”

Section: Retrovirus Transduction In Micromass Culturesmentioning

confidence: 99%

mentioning

confidence: 99%

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Successive formative stages of precartilaginous mesenchymal condensations in vitro: Modulation of cell adhesion by Wnt-7A and BMP-2

1999

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“…1G). We further performed micromass assays to test whether keratinocytes at the periphery of the micromass foci, which we assumed to be less differentiated than those in the center (Bedal et al, 2014; Stott et al, 1998), generated H 2 O 2 similar to scratch margin cells. This showed elevated H 2 O 2 levels at the outer edges compared with confluent regions where less H 2 O 2 /HPF signal was measured (Fig.…”

Section: Resultsmentioning

confidence: 99%

IKKα regulates human keratinocyte migration through surveillance of the redox environment

Lisse

Rieger

2017

Journal of Cell Science

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Although the functions of H2O2 in epidermal wound repair are conserved throughout evolution, the underlying signaling mechanisms are largely unknown. In this study we used human keratinocytes (HEK001) to investigate H2O2-dependent wound repair mechanisms. Scratch wounding led to H2O2 production in two or three cell layers at the wound margin within ∼30 min and subsequent cysteine modification of proteins via sulfenylation. Intriguingly, exogenous H2O2 treatment resulted in preferential sulfenylation of keratinocytes that adopted a migratory phenotype and detached from neighboring cells, suggesting that one of the primary functions of H2O2 is to stimulate signaling factors involved in cell migration. Based on previous findings that revealed epidermal growth factor receptor (EGFR) involvement in H2O2-dependent cell migration, we analyzed oxidation of a candidate upstream target, the inhibitor of κB kinase α (IKKα; encoded by CHUK), as a mechanism of action. We show that IKKα is sulfenylated at a conserved cysteine residue in the kinase domain, which correlates with de-repression of EGF promoter activity and increased EGF expression. Thus, this indicates that IKKα promotes migration through dynamic interactions with the EGF promoter depending on the redox state within cells.

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“…Amaxa nucleofection (Lonza) and the transfection reagent SAINT-MIX (Synvolux Therapeutics) achieved much higher efficiencies, 97% and 55% respectively; however, both of these methods simultaneously reduced proliferation and increased the presence of early apoptotic cells (5). Viral transduction of plasmid DNA into dissociated embryonic chick limb mesenchyme is characterized by high efficiency and low cytotoxicity (6). Unfortunately, the time required for retroviral integration renders transduction less useful for studying the early events of chondrogenesis in this model as cellular condensation and the beginnings of overt chondrocyte differentiation occur in the first 24 h of micromass culture (reviewed in 7).…”

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confidence: 99%

High Efficiency Transfection of Embryonic Limb Mesenchyme With Plasmid DNA Using Square Wave Pulse Electroporation and Sucrose Buffer

2014

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Micromass cultures of primary embryonic limb mesenchyme are a valuable model system for studying cartilage formation in vitro. However, high efficiency introduction of plasmid DNA into this hard-to-transfect cell type typically results in considerable cell death and significantly impeded chondrogenesis when the cells are subsequently plated in high density micromass. Here, we describe a novel method in which square wave pulse electroporation of chick embryo wing bud mesenchyme suspended in protective sucrose buffer results in high efficiency transfection without substantially affecting micromass culture cell viability or chondrogenic differentiation potential. Furthermore, we show that this protocol can be employed, along with effector gene expression vectors, to generate observable changes in the amount of cartilage tissue formed in micromass, unlike lower efficiency, higher cytotoxicity techniques that require co-transfection of reporter plasmids to monitor changes in the extent of chondrogenesis and correct for differences in cell viability.

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Retroviral Gene Transfer in Chondrogenic Limb Bud Micromass Cultures

Abstract: We report development of a model of retroviral gene transduction in high-

Cited by 5 publications

References 41 publications

Successive formative stages of precartilaginous mesenchymal condensations in vitro: Modulation of cell adhesion by Wnt-7A and BMP-2

Successive formative stages of precartilaginous mesenchymal condensations in vitro: Modulation of cell adhesion by Wnt-7A and BMP-2

IKKα regulates human keratinocyte migration through surveillance of the redox environment

High Efficiency Transfection of Embryonic Limb Mesenchyme With Plasmid DNA Using Square Wave Pulse Electroporation and Sucrose Buffer

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