Establishment of conditional Wnt5a transgenic embryonic stem cell line and its application during neural differentiation process
One of the properties of embryonic stem (ES) cells is their differentiation potential including the capacity to form specific regional neural cells identity. However, limitation in getting homogenous neuronal specific subtype population from ES cells has resulted in the formation of teratoma upon...
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| Format: | Thesis |
| Language: | English |
| Published: |
2017
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| Online Access: | http://psasir.upm.edu.my/id/eprint/70563/1/FPSK%28M%29%202017%2055%20-%20IR.pdf http://psasir.upm.edu.my/id/eprint/70563/ |
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| Summary: | One of the properties of embryonic stem (ES) cells is their differentiation potential including the capacity to form specific regional neural cells identity. However, limitation in getting homogenous neuronal specific subtype population from ES cells has resulted in the formation of teratoma upon transplantation, thus has hindered their use in clinical applications. Hence, understanding the key regulators during differentiation of ES cells is essential. Among the regulators, Wnt5a signalling molecule has been shown to play important role in neural differentiation process of mouse ES cells in a stage-dependent manner. Therefore, a system that allows for a tight regulation of Wnt5a expression in undifferentiated ES cells and also upon differentiation is indispensable in order to evaluate the stage-dependency effect of Wnt5a during the process. This study aims to generate and characterize Wnt5a transgenic cell line that carries the inducible Wnt5a transgene construct through a binary Cre/loxP system and to preliminarily evaluate its application in understanding the stage dependency effect of Wnt5a during the neural differentiation process. Two clones of Wnt5a transgenic line were successfully generated by transfecting thepCAG-floxed-neopA-Wnt5a plasmid into a Cre expressing cell line, R26CT2S. Stable transfected cells were screened by dual antibiotic selections before and after exposure to 4’-hydroxytamoxifen (4’-OHT). The cell line was found to maintain its pluripotency. The expression of Wnt5a transgene was observed to be temporally controlled upon exposure to a non-detrimental dosage of 4’-OHT. High level of transgene expression was observed in clones induced with 4’-OHT both in ES cells and the embryoid bodies (EBs), clearly indicating the stability and inducibility of the Wnt5a construct. The generated inducible Wnt5a transgenic ES cell line was then applied to preliminarily understand the effects of Wnt5a activity at specific time points during neural differentiation process. The formation of multicellular aggregates, embryoid bodies (EBs) and the addition of retinoic acid in the presence of serum was chosen to differentiate ES cells into neural lineage. The expression of Wnt5a transgene was induced at three different time points: 1) early and 2) late stages of neural differentiation process and 3) constitutive expression (since the undifferentiated stage). The expression of selected specific neural markers for the formation of post-mitotic, mature and dopaminergic (DA) neurons and astroglial cells was qualitatively and quantitatively analysed. The effects of stimulating Wnt5a signalling pathway atthe specific time points were analysed at three different stages of the neural differentiation process; day 2, day 8 and day 16 post-plating neural culture. Interestingly without influence of any standard patterning factors, high and early detection of TH positive neuron, Class III β-tubulin and Map2 markers was observed when Wnt5a was induced at early stage of neural differentiation process. A dynamic expression pattern of the neural proteins generated, indicates the complex roles of Wnt5a during the process. This study, highlights the application of the conditional EScell system in elucidating stage-dependency effect of Wnt5a during neural differentiation process and exposes the potential role of this molecule in generating EScells-derived neural cells that are suitable for cell-based therapy for neurodegenerative diseases. |
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