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Screening targeting agents and their cell surface biomarkers for high specificity and rapid internalization via cell death and fluorescence
Ancheta L, Bouajram R, Lappi DA (2018) Screening targeting agents and their cell surface biomarkers for high specificity and rapid internalization via cell death and fluorescence. Neuroscience 2018 Abstracts 128.20 / M17. Society for Neuroscience, San Diego, CA.
Summary: Some of the most recent successes in the treatment of cancers or research into passive immunotherapies for neurodegenerative diseases, employ the use of antibodies. These treatments utilize antibodies that either: 1) interfere with cell surface proteins responsible for tumor cell proliferation, 2) act as immune checkpoint inhibitors, or 3) are re-engineered to allow transport of other molecules across the blood-brain barrier (BBB). There are a growing number of antibody and small molecule therapeutic candidates and this demands a quick and efficient technique to screen for biomarkers that internalize effectively upon binding. The method described provides for the efficient determination of internalization of cell surface biomarkers upon binding of antibodies or peptides. This one-step, robust method uses a targeting agent combined with both a fluorescent reporter and a cytotoxic payload. The construct that makes this method effective was formed by cross-linking a fluorescent reporter, in this case fluorescein (FITC) and streptavidin to the ribosome-inactivating protein, Saporin. The conjugate used in screening potential therapeutics is a mixture of a biotinylated targeting agent mixed in a 1:1 molar ratio with FITC-labeled Streptavidinylated-Saporin. The method provides a definitive assay readout: fluorescence within 1 hour and cell death in 72 hours. This method is designed for rapid screening, in a quick and reproducible manner, for specificity and internalization in various cell types to explore suitability of candidates as therapeutics.
Related Products: Streptavidin-ZAP (Cat. #IT-27), FITC-Streptavidin-ZAP (Cat. #IT-85)
See Also:
- Tan HL et al. Conservation of oncofetal antigens on human embryonic stem cells enables discovery of monoclonal antibodies against cancer. Sci Rep 8:11608, 2018.
- Yuan X et al. Characterization of the first fully macropinocytosing human antibodies human anti-TEM1 scFv in models of solid tumor imaging and immunotoxin-based therapy. Cancer Immunol Immunother 66:367-378, 2017.
- Forsyth PA et al. p75 neurotrophin receptor cleavage by α- and γ-secretases is required for neurotrophin-mediated proliferation of brain tumor-initiating cells. J Biol Chem 289(12):8067-8085, 2014.
- Thakkar JP et al. Epidemiologic and molecular prognostic review of glioblastoma. Cancer Epidemiol Biomarkers Prev 23(10):1985-1996, 2014.
- Kohls M Evaluate Potential Targeting Molecules. Nature Methods , 2006.
Sonic hedgehog signalling pathway during regenerative processes in a mouse model of spinal motoneuronal loss
Gulisano M, Vicario N, Costantino A, Giunta MAS, Spitale FM, Parenti R, Gulino R (2018) Sonic hedgehog signalling pathway during regenerative processes in a mouse model of spinal motoneuronal loss. Neuroscience 2018 Abstracts 379.29 / L1. Society for Neuroscience, San Diego, CA.
Summary: Background – Neuronal loss represents the consequence of direct or indirect insults to neurons, as well as one of the major factors mediating persistent disability. Gliosis, neuroinflammation and neurodegeneration are processes in which different cell populations have an interplay mediating both hostile microenvironment and self-repairing mechanisms. Sonic hedgehog (Shh) signaling, which has been indicated as an important pathway in central nervous system development and neural stem cells (NSCs) function, may have a role in prompting the repairing and modulating actions of endogenous and/or exogenous NSCs in neurodegenerative conditions. Methodology – Somatic NSCs were obtained from the subventricular zone (SVZ) of 8 week old female 129/Sv mice. We studied the Shh pathway on NSCs both in vitro and in a mouse model of spinal motorneuronal degeneration induced by cholera toxin B conjugated to saporin (CTB-Sap) injection into the gastrocnemius muscle. Results – NSCs were derived, expanded and characterized in vitro. We analyzed the effects of Shh signaling pathway modulation on NSCs in vitro, finding a significant increase of the NSCs growth rate (2.98±0.58 vs. 5.26±0.57, p<0.05) and neurospheres diameters (109.9±2.4μm vs. 129.6±3.7μm, p<0.01) upon Shh pathway activation. We then characterized Shh signaling activation in CTB-Sap mice analyzing neuronal loss, gliosis, inflammation and compensatory self-repairing mechanisms, compared to intact control mice. Conclusions - Our results suggest a crucial role of Shh signaling during regenerative processes and NSCs as a potential strategy to support recovery after spinal motoneuronal degeneration, thus representing a promising approach for neurodegenerative disorders.
Related Products: CTB-SAP (Cat. #IT-14)
Postnatal neural crest stem cells from hair follicle interact with nerve tissue in vitro and in vivo
Kosykh A, Beilin A, Sukhinich K, Vorotelyak E (2018) Postnatal neural crest stem cells from hair follicle interact with nerve tissue in vitro and in vivo. Tissue Cell 54:94-104. doi: 10.1016/j.tice.2018.08.005 PMID: 30309515
Objective: To investigate the interaction between Neural crest stem cells that located in the postnatal hair follicle (HF-NCSC) and a nerve tissue (embryonic and adult).
Summary: HF-NCSC maintain their NC potency in the interaction with the embryonic central nerve system tissue. The migration capacity of the HF NCSC corresponds to the developmental stage but not a type of a nerve tissue present in the co-culture. In contrast, an adult brain interaction changes the HF-NCSC status and reduces the proliferation.
Usage: immunohistochemistry (1:50)
Related Products: NGFR (mu p75) Rabbit Polyclonal, affinity-purified (Cat. #AB-N01AP)
Conservation of oncofetal antigens on human embryonic stem cells enables discovery of monoclonal antibodies against cancer
Tan HL, Yong C, Tan BZ, Fong WJ, Padmanabhan J, Chin A, Ding V, Lau A, Zheng L, Bi X, Yang Y, Choo A (2018) Conservation of oncofetal antigens on human embryonic stem cells enables discovery of monoclonal antibodies against cancer. Sci Rep 8:11608. doi: 10.1038/s41598-018-30070-z
Objective: To identify and characterize an antibody raised using human embryonic stem cells with potential as a cancer therapeutic.
Summary: Antibody A19 not only binds to undifferentiated hESCs by flow cytometry, it also reacts with ovarian and breast cancer cell lines with low or no binding to normal cells.
Usage: in vitro – Number of viable cells treated showed a decrease in cell number (Hum-ZAP mixed with A19; Streptavidin-ZAP mixed with biotinylated A19). To determine if there were off-target effects, Hum-ZAP and chA19 were incubated with a non-binding cell line OVCAR10; no apparent cytotoxicity was observed. invivo – 5 x 106 SKOV3 cells were implanted s.c. in NUDE mice and Biotinylated A19-Streptavidin-ZAP (ADC), administered ip. The controls were free Saporin and naked A19. By the end of 10 weeks, mice administered with the ADC saw a 60% reduction in tumor size compared to control groups.
Related Products: Hum-ZAP (Cat. #IT-22), Streptavidin-ZAP (Cat. #IT-27), Saporin (Cat. #PR-01)
The embryotoxic effects of harm reduction tobacco products on osteoblasts developing from human embryonic stem cells
Sparks NR (2018) The embryotoxic effects of harm reduction tobacco products on osteoblasts developing from human embryonic stem cells. University of California Riverside Thesis.
Objective: To investigate the effect of maternal smoking in the development of embryonic skeleton and the molecular mechanisms involved in skeletal embryotoxicity induced by conventional and harm-reduction tobacco.
Summary: An in vitro model based on human embryonic stem cells was developed, that mimics the development of the human embryo; their differentiation into osteoblasts in vitro can be used to study chemical toxicity and the molecular mechanisms thereof. Tobacco-induced oxidative stress disrupts osteogenic differentiation in human embryonic stem cells.
Usage: Immunocytochemistry
Related Products: NGFR (mu p75) Rabbit Polyclonal, affinity-purified (Cat. #AB-N01AP)
Direct conversion of mouse embryonic fibroblasts into neural crest cells
Motohashi T, Kunisada T (2019) Direct conversion of mouse embryonic fibroblasts into neural crest cells. (eds. Turksen K). In: Skin Stem Cells. Methods in Molecular Biology. 1879:307-321. Humana Press, New York, NY. doi: 10.1007/7651_2018_145 PMID: 29797008
Objective: To describe methods for the direct conversion of mouse embryonic fibroblasts into neural crest cells (NCCs).
Summary: Sox10-IRES-Venus mouse fibroblasts were used for the conversion and isolation of converted NCCs as SOX10-positive cells.
Usage: Immunostaining for Flow Cytometry
Related Products: NGFr (mu p75) Rabbit Polyclonal (Cat. #AB-N01)
Differentiation of adipose-derived stem cells into Schwann cell-like cells through intermittent induction: potential advantage of cellular transient memory function
Sun X, Zhu Y, Yin HY, Guo ZY, Xu F, Xiao B, Jiang WL, Guo WM, Meng HY, Lu SB, Wang Y, Peng J (2018) Differentiation of adipose-derived stem cells into Schwann cell-like cells through intermittent induction: potential advantage of cellular transient memory function. Stem Cell Res Ther 9:133. doi: 10.1186/s13287-018-0884-3 PMID: 29751848
Objective: To improve the traditional methods for inducing the differentiation of mesenchymal stromal cells (MSCs) into Schwann cell-like cells (SCLCs).
Summary: Results indicated that the intermittent induction method is more efficient than traditional methods for inducing adipose-derived stem cells to differentiate into SCLCs.
Usage: immunofluorescence (1:500)
Related Products: NGFr (mu p75) Rabbit Polyclonal, affinity-purified (Cat. #AB-N01AP)
Effect of placenta-derived mesenchymal stem cells in a dementia rat model via microglial mediation: A comparison between stem cell transplant methods
Cho JS, Lee J, Jeong DU, Kim HW, Chang WS, Moon J, Chang JW (2018) Effect of placenta-derived mesenchymal stem cells in a dementia rat model via microglial mediation: A comparison between stem cell transplant methods. Yonsei Med J 59:406-415. doi: 10.3349/ymj.2018.59.3.406
Objective: To study the therapeutic effects of human placenta-derived mesenchymal stem cells (pMSCs) in a dementia rat model using either intracerebroventricular (ICV) or intravenous (IV) injections and analyze their mechanisms of therapeutic action.
Summary: ICV and IV injections of pMSCs facilitate the recovery of cholinergic neuronal populations and cognitive behavior. This recovery likely occurs through paracrine effects that resemble microglia function rather than direct differentiation of injected pMSCs into cholinergic neurons.
Usage: Dementia modeling was established by bilateral ventricle infusion of 192 IgG-SAP to lesion cholinergic neurons. 8 μL of 192 IgG-saporin (0.63 μg/μL) were bilaterally injected into the lateral ventricle at a rate of 1 μL/min and was left to diffuse for 5 min after injection. Rats were subjected to the Morris water maze and subsequent immunostaining analyses.
Related Products: 192-IgG-SAP (Cat. #IT-01)
Myoepithelial cells of submucosal glands can function as reserve stem cells to regenerate airways after injury
Tata A, Kobayashi Y, Chow RD, Tran J, Desai A, Massri AJ, McCord TJ, Gunn MD, Tata PR (2018) Myoepithelial cells of submucosal glands can function as reserve stem cells to regenerate airways after injury. Cell Stem Cell 22:668-683. doi: 10.1016/j.stem.2018.03.018 PMID: 29656943
Objective: To show that myoepithelial cells (MECs) of the submucosal glands (SMGs) proliferate and migrate to repopulate the airway surface epithelium (SE) in multiple injury models.
Summary: SMG-like cells appear on the SE of extra-and intra-lobular airways of large animal lungs following severe injury. The transcription factor SOX9 is necessary for MEC plasticity in airway regeneration.
Usage: Immunostaining
Related Products: NGFr (mu p75) Rabbit Polyclonal (Cat. #AB-N01)
Submucosal gland myoepithelial cells are reserve stem cells that can regenerate mouse tracheal epithelium
Lynch TJ, Anderson PJ, Rotti PG, Tyler SR, Crooke AK, Choi SH, Montoro DT, Silverman CL, Shahin W, Zhao R, Jensen-Cody CW, Adamcakova-Dodd A, Evans TIA, Xie W, Zhang Y, Mou H, Herring BP, Thorne PS, Rajagopal J, Yeaman C, Parekh KR, Engelhardt JF (2018) Submucosal gland myoepithelial cells are reserve stem cells that can regenerate mouse tracheal epithelium. Cell Stem Cell 22(5):653-667.e5. doi: 10.1016/j.stem.2018.03.017 PMID: 29656941
Objective: To investigate whether a lineage relationship exists between two stem cell compartments that contribute to airway repair – basal cells in the surface airway epithelium (SAE) and an unknown submucosal gland (SMG) celltype.
Summary: Using lineage tracing of glandular myoepithelial cells (MECs), they demonstrate that MECs can give rise to seven cell types of the SAE and SMGs following severe airway injury. MECs progressively adopted a basal cell phenotype on the SAE and established asting progenitors capable of further regeneration following reinjury. MECs activate Wnt-regulated transcription factors (Lef-1/TCF7) following injury and Lef-1 induction in cultured MECs promoted transition to a basal cell phenotype. Surprisingly, dose-dependent MEC conditional activation of Lef-1in vivo promoted self-limited airway regeneration in the absence of injury.
Usage: immunofluorescence
Related Products: NGFR (mu p75) Rabbit Polyclonal, affinity-purified (Cat. #AB-N01AP)