Development of a Rabbit Monoclonal Antibody Group Against Smads and Immunocytochemical Study of Human and Mouse Embryonic Stem Cells

Huang, Yun-jian; Gu, Bin; Wu, Rongrong; Zhang, Jiarong; Liu, Ying; Zhang, Ming

doi:10.1089/hyb.2007.0517

Cited by 17 publications

(18 citation statements)

References 10 publications

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“…Western blotting was performed as previously described [43]. ES cells were harvested in lysis buffer consisting of …”

Section: Western Blottingmentioning

confidence: 99%

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Aire Promotes the Self-Renewal of Embryonic Stem Cells Through Lin28

Zhang²,

Wang³

et al. 2012

Stem Cells and Development

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Autoimmune regulator (Aire) is one of the most well-characterized molecules in autoimmunity, but its function outside the immune system is largely unknown. The recent discovery of Aire expression in stem cells and early embryonic cells and its function in the self-renewal of embryonic stem (ES) cells highlight the importance of Aire in these cells. In this study, we present evidence that Aire promotes the expression of the pluripotent factor Lin28 and the self-renewal of ES cells. We presented the first evidence that the let-7 microRNA family contributed to the self-renewal promoting effect of Aire on ES cells. Moreover, we showed that Aire and Lin28 are co-expressed in the genital ridge, oocytes, and cleavage-stage embryos, and the expression level of Lin28 is correlated with the expression level of Aire. Although it is widely considered to be a promiscuous gene expression activator, these results indicated that Aire promotes the self-renewal of ES cells through a specific pathway (i.e., the activation of Lin28 and the inhibition of the let-7 microRNA family). The correlation between Aire and Lin28 expression in germ cells and early embryos indicated an in vivo function for Aire in toti-and pluripotent stem cells. This study presents the first molecular pathway that incorporates Aire into the pluripotency network. Moreover, it presents the first evidence that microRNAs contribute to the regulatory function of Aire and highlights a novel function of Aire in stem cell biology and reproduction. These functions reveal novel perspectives for studying the molecular mechanisms behind the establishment and sustenance of pluripotent identity.

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“…Western blotting was performed as previously described [43]. ES cells were harvested in lysis buffer consisting of …”

Section: Western Blottingmentioning

confidence: 99%

“…Immunofluorescence was performed as previously described [43]. The subjects were observed, and images were acquired under an LSM 710 Confocal Microscope (Zeiss).…”

Section: Immunofluorescencementioning

confidence: 99%

Aire Promotes the Self-Renewal of Embryonic Stem Cells Through Lin28

Zhang²,

Wang³

et al. 2012

Stem Cells and Development

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“…Compared with ESCs, EGCs express higher levels of BMP pathway constituents BMPR1A (alk3), ACVR1 (alk2), and ACVRL1 (alk1) as well as downstream activators ID1, SMAD1P, and SMAD5P. Although others have also shown that human ESCs express Smad1 and Smad5 mRNA, it was not determined whether the pathways were activated [54]. Here, we show that both are phosphorylated in EGCs but not in ESCs, consistent with previous work that suggests that this pathway is not involved in the pluripotent nature of human ESCs.…”

Section: Figmentioning

confidence: 99%

Bone Morphogenetic Protein 4 Mediates Human Embryonic Germ Cell Derivation

Hiller

Liu

Blumenthal

et al. 2011

Stem Cells and Development

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Human primordial germ cells (PGCs) have proven to be a source of pluripotent stem cells called embryonic germ cells (EGCs). Unlike embryonic stem cells, virtually little is known regarding the factors that regulate EGC survival and maintenance. In mice, the growth factor bone morphogenetic protein 4 (BMP4) has been shown to be required for maintaining mouse embryonic stem cells, and disruptions in this gene lead to defects in mouse PGC specification. Here, we sought to determine whether recombinant human BMP4 could influence EGC derivation and=or human PGC survival. We found that the addition of recombinant BMP4 increased the number of human PGCs after 1 week of culture in a dose-responsive manner. The efficiency of EGC derivation and maintenance in culture was also enhanced by the presence of recombinant BMP4 based on alkaline phosphatase and OCT4 staining. In addition, an antagonist of the BMP4 pathway, Noggin, decreased PGC proliferation and led to an increase in cystic embryoid body formation. Quantitative real-time (qRT)-polymerase chain reaction analyses and immunostaining confirmed that the constituents of the BMP4 pathway were upregulated in EGCs versus PGCs. Downstream activators of the BMP4 pathway such as ID1 and phosphorylated SMADs 1 and 5 were also expressed, suggesting a role of this growth factor in EGC pluripotency.

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“…Rabbit hybridomas were generated, 37 and the antibody heavy chain (HC) and light chain cell (LC) genes were molecularly cloned from a culture of a hybridoma cell line secreting a rabbit mAb (91-2) that bound to IdeScleaved human IgG1 F(ab') 2 fragments. Following sequence analysis, the single HC and two LC variable region gene fragments were cloned into mammalian expression plasmids containing human IgG1 or human kappa constant region coding sequences, respectively, to generate chimerized rabbit/human full antibody protein.…”

Section: Generation Of Recombinant 2095-2mentioning

confidence: 99%

A monoclonal antibody against hinge-cleaved IgG restores effector function to proteolytically-inactivated IgGs in vitro and in vivo

Brezski

Kinder

Grugan

et al. 2014

mAbs

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We report a chimeric monoclonal antibody (mAb) directed to a neo-epitope that is exposed in the IgG lower hinge following proteolytic cleavage. The mAb, designated 2095-2, displays specificity for IdeS-generated F(ab')₂ fragments, but not for full-length IgG or for closely-related F(ab')₂ fragments generated with other proteases. A critical component of the specificity is provided by the C-terminal amino acid of the epitope corresponding to gly-236 in the IgG1 (also IgG4) hinge. By its ability to bind to IdeS-cleaved anti-CD20 mAb, mAb 2095-2 fully restored antibody-dependent cell-mediated cytotoxicity (ADCC) and complement-dependent cytotoxicity (CDC) against WIL2-S cells to the otherwise inactive anti-CD20 IgG1 F(ab')₂ fragment. Similarly, 2095-2 reinstated ADCC against MDA-MB-231 cells to an anti-CD142 IgG1 F(ab')₂ fragment. mAb 2095-2 was also capable of eliciting both CDC and ADCC to IgG4 F(ab')₂ fragments, an IgG subclass that has weaker ADCC and CDC when intact relative to intact IgG1. The in vitro cell-based efficacy of 2095-2 was extended to the in vivo setting using platelets as a cell clearance surrogate. In a canine model, the co-administration of 2095-2 together with IdeS-generated, platelet-targeting anti-CD41/61 F(ab')₂ fragment not only restored platelet clearance, but did so at a rate and extent of clearance that exceeded that of intact anti-CD41/61 IgG at comparable concentrations. To further explore this unexpected amplification effect, we conducted a rat study in which 2095-2 was administered at a series of doses in combination with a fixed dose of anti-CD41/61 F(ab')₂ fragments. Again, the combination, at ratios as low as 1:10 (w/w) 2095-2 to F(ab')₂, proved more effective than the anti-CD41/61 IgG1 alone. These findings suggest a novel mechanism for enhancing antibody-mediated cell-killing effector functions with potential applications in pathologic settings such as tumors and acute infections where protease activity is abundant.

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Development of a Rabbit Monoclonal Antibody Group Against Smads and Immunocytochemical Study of Human and Mouse Embryonic Stem Cells

Cited by 17 publications

References 10 publications

Aire Promotes the Self-Renewal of Embryonic Stem Cells Through Lin28

Aire Promotes the Self-Renewal of Embryonic Stem Cells Through Lin28

Bone Morphogenetic Protein 4 Mediates Human Embryonic Germ Cell Derivation

A monoclonal antibody against hinge-cleaved IgG restores effector function to proteolytically-inactivated IgGs in vitro and in vivo

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