Neurogenese ved hjælp af P19 embryonal carcinoma celler
Summary
Den P19 mus embryonale karcinom cellelinje (P19 cellelinje) er almindeligt anvendt til at studere den molekylære mekanisme af neurogenese med stor forenkling i forhold til in vivo-analyse. Her præsenterer vi en protokol for retinoinsyre-induceret neurogenese i P19 cellelinje.
Abstract
Den P19 cellelinje afledt af en mus embryo-afledte teratocarcinoma har evnen til at differentiere sig i de tre kimlag. I nærværelse af retinsyre (RA), affjedring kultiveret P19 cellelinje er induceret til at differentiere sig til neuroner. Dette fænomen er grundigt undersøgt som en Neuro Genesis model in vitro. Derfor, den P19 cellelinje er meget nyttigt for molekylære og cellulære undersøgelser forbundet med Neurogenesis. Men, protokoller for neuronal differentiering af P19 cellelinje, der er beskrevet i litteraturen er meget komplekse. Den metode, der er udviklet i denne undersøgelse er enkel og vil spille en rolle i belyse de molekylære mekanismer i neuroudviklingsmæssige abnormiteter og neurodegenerative sygdomme.
Introduction
Under embryonal udvikling omdannes et enkelt celle lag til tre forskellige kimlag1,2,3. For at øge forskningsmulighederne for fænomener, der forekommer in vivo, er generering af tredimensionale aggregater (embryonale organer) blevet udviklet som en bekvem model. Cellulære aggregater dannet på denne måde kan blive udsat for forskellige betingelser, der forårsager Celledifferentiering, som afspejler udviklingen af embryonet4,5. Den P19 murine embryonale karcinom cellelinje (P19 cellelinje) er almindeligt anvendt som en cellulær model for Neuro Genesis undersøgelser in vitro6,7,8. Den P19 cellelinje udviser typiske pluripotente stamcelle funktioner og kan differentiere sig til neuroner i nærværelse af retinsyre (RA) under celle sammenlægning efterfulgt af neurite udvækst under vedtagtige betingelser. Desuden, den udifferentierede P19 cellelinje er også i stand til at danne muskler-og kardiomyocyte-lignende celler under påvirkning af dimethylsulfoxid (DMSO)9,10,11,12.
Mange metoder13,14,15,16 er blevet rapporteret for neuronal differentiering, men metoden er undertiden kompliceret og ikke let at forstå ved kun at læse beskrivelserne. For eksempel kræver protokoller nogle gange en kombination af Dulbecco’s modificerede Eagle medium (DMEM) medium suppleret med en blanding af kalveserum (CS) og føtal bovint serum (FBS)13. Desuden, medier, der anvendes til neuronal udvikling er ofte sammensat af neurobasal og B27 kosttilskud13,14,15,16. Som sådan, eksisterende metoder indeholder kompleksitet i deres forberedelse og vores mål her er at forenkle protokollerne. I denne undersøgelse, vi viste, at DMEM med FBS kan udnyttes til at opretholde P19 cellelinje (DMEM + 10% FBS) samt for neuronal udvikling (DMEM + 5% FBS + RA). Denne forenklede metode til neurogenese ved hjælp af P19 cellelinje giver os mulighed for at studere den molekylære mekanisme af, hvordan neuroner er udviklet. Desuden er forskning i neurodegenerative sygdomme som Alzheimers sygdom også gennemført ved hjælp af P19 cellelinje17,18, og vi mener, at den metode, der er udviklet i denne undersøgelse vil spille en rolle i at belyse molekylære mekanismer i neuroudviklingsmæssige abnormiteter og neurodegenerative sygdomme.
Protocol
Representative Results
Discussion
Her beskriver vi en simpel protokol for neurogenese ved hjælp af P19 Cell line. Selv om mange rapporter er blevet offentliggjort i denne henseende, en detaljeret metodologi for Neuro Genesis induktion ved hjælp af P19 cellelinje forbliver uklar. Desuden har vi udnyttet en simpel høj glukose (4.500 mg/L) DMEM medium med 10% FBS for hele eksperimentet. Dette tillod os at udføre den neurogene eksperiment i en brugervenlig måde og udvide brugen af denne metode for fremtiden.
De mest kritiske …
Divulgations
The authors have nothing to disclose.
Acknowledgements
Undersøgelsen blev finansielt støttet af National Science Centre, Polen (Grant nr. UMO-2017/25/N/NZ3/01886) og KNOW (førende nationale Forskningscenter) videnskabeligt konsortium “sunde dyr-sikre fødevarer”, afgørelse fra ministeriet for videnskab og videregående uddannelse nr. 05-1/KNOW2/2015
Materials
| 6x DNA Loading Dye | EURx | E0260-01 | |
| Agarose | Sigma- Aldrich | A9539 | |
| cDNA synthesis kit | EURx | E0801-02 | |
| DAPI (4′,6-Diamidine-2′-phenylindole dihydrochloride) | Sigma- Aldrich | 10236276001 | Working concentration: 1 μg/mL |
| DMEM high glucose (4.5 g/L) with L-glutamine | Lonza | BE12-604Q | |
| Ethanol 99.8% | Chempur | CHEM*613964202 | |
| Fetal Bovine Serum (FBS) | EURx | E5050-03 | |
| MAP2 antibody | Thermo Fisher Scientific | PA517646 | Dilution 1:100 |
| PCR reaction kit | EURx | E0411-03 | |
| Penicillin/Streptomycin 10K/10K | Lonza | DE17-602E | |
| Phosphate Buffered Saline (PBS), 1x concentrated without Ca2+, Mg2+ | Lonza | BE17- 517Q | |
| Retinoic acid | Sigma- Aldrich | R2625-50MG | dissolved in 99.8% ethanol; store in -20 °C up to 6 months |
| Secondary Antibody (Alexa Fluor 488) | Thermo Fisher Scientific | A11034 | Dilution 1:500 |
| Skim milk | Sigma- Aldrich | 1153630500 | |
| TBE Buffer | Thermo Fisher Scientific | B52 | |
| Triton-X 100 | Sigma- Aldrich | T8787-100ML | |
| Trypsin 0.25% – EDTA in HBSS, without Ca2+, Mg2+,with Phenol Red | biosera | LM-T1720/500 | |
| Cell Culture Plastics | |||
| 1 mL Serological Pipettes | Profilab | 515.01 | |
| 10 mL Serological Pipettes | Profilab | 515.10 | |
| 100 mm dish dedicated for suspension culture | Corning | C351029 | |
| 15 mL centrifuge tubes | Sigma- Aldrich | CLS430791-500EA | |
| 5 mL Serological Pipettes | Profilab | 515.05 | |
| 6-well plate | Corning | CLS3516 | |
| Cell culture flasks, surface area 25 cm2 | Sigma- Aldrich | CLS430639-200EA |
References
- Ramkumar, N., Anderson, K. V. SnapShot: mouse primitive streak. Cell. 146 (3), 488 (2011).
- Solnica-Krezel, L., Sepich, D. S. Gastrulation: making and shaping germ layers. Annual Review of Cell and Developmental Biology. 28, 687-717 (2012).
- Tam, P. P. L., Gad, J. M., Stern, C. D. Chapter 16: Gastrulation in the Mouse Embryo. Gastrulation: From Cells to Embryo. , 233-262 (2004).
- Sajini, A. A., Greder, L. V., Dutton, J. R., Slack, J. M. W. Loss of Oct4 expression during the development of murine embryoid bodies. Biologie du développement. 371 (2), 170-179 (2012).
- ten Berge, D., et al. Wnt Signaling Mediates Self-Organization and Axis Formation in Embryoid Bodies. Cell Stem Cell. 3 (5), 508-518 (2008).
- Bain, G., Ray, W. J., Yao, M., Gottlieb, D. I. From embryonal carcinoma cells to neurons: the P19 pathway. Bioessays. 16 (5), 343-348 (1994).
- Lin, Y. T., et al. YAP regulates neuronal differentiation through Sonic hedgehog signaling pathway. Experimental Cell Research. 318 (15), 1877-1888 (2012).
- Neo, W. H., et al. MicroRNA miR-124 controls the choice between neuronal and astrocyte differentiation by fine-tuning Ezh2 expression. Journal of Biological Chemistry. 289 (30), 20788-20801 (2014).
- Jones-Villeneuve, E., McBurney, M. W., Rogers, K. A., Kalnins, V. I. Retinoic acid induces embryonal carcinoma cells to differentiate into neurons and glial cells. The Journal of Cell Biology. 94 (2), 253-262 (1982).
- McBurney, M. W., Rogers, B. J. Isolation of male embryonal carcinoma cells and their chromosome replication patterns. Biologie du développement. 89 (2), 503-508 (1982).
- Jones-Villeneuve, E., Rudnicki, M. A., Harris, J. F., McBurney, M. Retinoic acid-induced neural differentiation of embryonal carcinoma cells. Molecular and Cellular Biology. 3 (12), 2271-2279 (1983).
- Jasmin, D. C., Spray, A. C., Campos de Carvalho, R., Mendez-Otero, Chemical induction of cardiac differentiation in P19 embryonal carcinoma stem cells. Stem Cells and Development. 19 (3), 403-412 (2010).
- Solari, M., Paquin, J., Ducharme, P., Boily, M. P19 neuronal differentiation and retinoic acid metabolism as criteria to investigate atrazine, nitrite, and nitrate developmental toxicity. Toxicological Sciences. 113 (1), 116-126 (2010).
- Babuska, V., et al. Characterization of P19 cells during retinoic acid induced differentiation. Prague Medical Report. 111 (4), 289-299 (2010).
- Monzo, H. J., et al. A method for generating high-yield enriched neuronal cultures from P19 embryonal carcinoma cells. Journal of Neuroscience Methods. 204 (1), 87-103 (2012).
- Popova, D., Karlsson, J., Jacobsson, S. O. P. Comparison of neurons derived from mouse P19, rat PC12 and human SH-SY5Y cells in the assessment of chemical- and toxin-induced neurotoxicity. BMC Pharmacology and Toxicology. 18 (1), 42 (2017).
- Woodgate, A., MacGibbon, G., Walton, M., Dragunow, M. The toxicity of 6-hydroxydopamine on PC12 and P19 cells. Molecular Brain Research. 69 (1), 84-92 (1999).
- Tsukane, M., Yamauchi, T. Ca2+/calmodulin-dependent protein kinase II mediates apoptosis of P19 cells expressing human tau during neural differentiation with retinoic acid treatment. Journal of Enzyme Inhibition and Medicinal Chemistry. 24 (2), 365-371 (2009).
- Adler, S., Pellizzer, C., Paparella, M., Hartung, T., Bremer, S. The effects of solvents on embryonic stem cell differentiation. Toxicology in Vitro. 20 (3), 265-271 (2006).
- Jones-Villeneuve, E. M., McBurney, M. W., Rogers, K. A., Kalnins, V. I. Retinoic acid induces embryonal carcinoma cells to differentiate into neurons and glial cells. The Journal of Cell Biology. 94 (2), 253-262 (1982).
- Roy, B., Taneja, R., Chambon, P. Synergistic activation of retinoic acid (RA)-responsive genes and induction of embryonal carcinoma cell differentiation by an RA receptor alpha (RAR alpha)-, RAR beta-, or RAR gamma-selective ligand in combination with a retinoid X receptor-specific ligand. Molecular and Cellular Biology. 15 (12), 6481-6487 (1995).
- Hamada-Kanazawa, M., et al. Sox6 overexpression causes cellular aggregation and the neuronal differentiation of P19 embryonic carcinoma cells in the absence of retinoic acid. FEBS Letters. 560 (1-3), 192-198 (2004).
- Tangsaengvit, N., Kitphati, W., Tadtong, S., Bunyapraphatsara, N., Nukoolkarn, V. Neurite Outgrowth and Neuroprotective Effects of Quercetin from Caesalpinia mimosoides Lamk on Cultured P19-Derived Neurons. Evidence-Based Complementary and Alternative. , 838051 (2013).
- Magnuson, D. S., Morassutti, D. J., McBurney, M. W., Marshall, K. C. Neurons derived from P19 embryonal carcinoma cells develop responses to excitatory and inhibitory neurotransmitters. Developmental Brain Research. 90 (1-2), 141-150 (1995).
- MacPherson, P., Jones, S., Pawson, P., Marshall, K., McBurney, M. P19 cells differentiate into glutamatergic and glutamate-responsive neurons in vitro. Neurosciences. 80 (2), 487-499 (1997).
- Hong, S., et al. Methyltransferase-inhibition interferes with neuronal differentiation of P19 embryonal carcinoma cells. Biochemical and Biophysical Research Communications. 377 (3), 935-940 (2008).
- Wenzel, M., et al. Identification of a classic nuclear localization signal at the N terminus that regulates the subcellular localization of Rbfox2 isoforms during differentiation of NMuMG and P19 cells. FEBS Letters. 590 (24), 4453-4460 (2016).
- Harada, Y., et al. Overexpression of Cathepsin E Interferes with Neuronal Differentiation of P19 Embryonal Teratocarcinoma Cells by Degradation of N-cadherin. Cellular and Molecular Neurobiology. 37 (3), 437-443 (2017).
- Morassutti, D. J., Staines, W. A., Magnuson, D. S., Marshall, K. C., McBurney, M. W. Murine embryonal carcinoma-derived neurons survive and mature following transplantation into adult rat striatum. Neurosciences. 58 (4), 753-763 (1994).
- Magnuson, D. S., Morassutti, D. J., Staines, W. A., McBurney, M. W., Marshall, K. C. In vivo electrophysiological maturation of neurons derived from a multipotent precursor (embryonal carcinoma) cell line. Developmental Brain Research. 84 (1), 130-141 (1995).
