Murine Teratocarcinoma-Derived Neuronal Cultures

Datta, Prasun K.

doi:10.1007/978-1-0716-1437-2_4

Cited by 4 publications

(5 citation statements)

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“…The SH-SY5Y derived differentiated neurons, an in vitro model system, is crucial for studying several neuroscientific research areas, including AD, PD, and HD. 31–33 Herein, for the first time, we demonstrate one such example of bioimaging endocytic pathways by differentiating SH-SY5Y neuroblastoma cells into fully mature neurons. The changes in the morphology of the undifferentiated SH-SY5Y cells during the differentiation process and fully differentiated neurons were observed using FM 1–43 and actin staining depicting the differentiation process from epithelial shape morphology into branched, more neurites formation corresponding to neuronal appearance (ESI Appendix, Fig.…”

Section: Discussionmentioning

confidence: 99%

“…The earlier model system of the well-differentiated SH-SY5Y cells shows distinct features to mature neurons with appropriate localization of markers for axons, dendrites, and synapses. 31,34,35 Using the same markers, we show the features of mature neurons being exhibited by studying the expression of markers like microtubule-associated protein-MAP2 (for dendrites), major synaptic vesicle protein – Synaptophysin (for synapses), microtubule-associated protein-tau (for axons) (ESI Appendix, Fig. S1m–p†).…”

Section: Discussionmentioning

confidence: 99%

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Red emitting fluorescent carbon nanoparticles to track spatio-temporal dynamics of endocytic pathways in model neuroblastoma neurons

et al. 2023

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Section: Discussionmentioning

confidence: 99%

Section: Discussionmentioning

confidence: 99%

Red emitting fluorescent carbon nanoparticles to track spatio-temporal dynamics of endocytic pathways in model neuroblastoma neurons

et al. 2023

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“…In addition, genetic manipulation of these cells is very easy [9]. Several murine teratocarcinoma cell lines of different tissue origins are available, the most well-known being the F9 and P19 [10]. The P19 cell line was rst identi ed in 1982 [8].…”

Section: Introductionmentioning

confidence: 99%

Possible role of lncRNAs in amelioration symptoms in an animal model of Parkinson’s disease following transplantation of dopaminergic cells derived from stem cells

Esmaeili,

Amini,

Golpich

2023

Preprint

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Background Long non-coding RNAs (lncRNAs) are important biomarkers for the pathogenesis of Parkinson's disease (PD) discovery and treatment approaches. Since dopaminergic cell transplantation is a promising clinical method to treat PD, this study investigated the effect of dopaminergic cell therapy on the expression of some lncRNAs and genes related to PD in the brains of rat models. Methods Twenty-eight rats were randomly assigned to four experimental groups. The animals injected with the saline solution were considered as control (Sal group). In the Par group, the PD rat model was developed by injecting 6-hydroxydopamine into the right striatum (ST). The ST of the animals was then transplanted by undifferentiated P19 stem cells (Par-E group), and/or P19-derived dopaminergic cells (Par-N group). The effects of cell transplantation were evaluated using behavioral tests (cylinder, open field, and rotarod tests), and histological methods (H&E, Nissl staining, and immunohistochemistry). Moreover, the expression of lncRNAs MALAT1, MEG3, and SNHG1 genes, as well as specific neuronal (synaptophysin, SYP) and dopaminergic (tyrosine hydroxylase, TH) markers was measured by qRT-PCR. Results Behavioral tests and histopathological evaluations showed that cell transplantation could partially compensate for dopaminergic cell degeneration in ST and substantia nigra (SN) of PD rats. The expression of MALAT1, SNHG1, and MEG3 was decreased in the ST of the Par group, while MEG3 and SNHG1gene expression was increased in PBMC compared to the Sal group. In PBMC of the Par-N group, all three lncRNAs showed a reduction in their expression. However, in ST tissue the expression of MALAT1 and SNHG1 was increased, while MEG3 gene expression was decreased compared to the Sal group. Conclusions Our data suggest that variations in the expression of lncRNAs genes may be considered as a marker for the possible incidence of PD.

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“…Even if ECCs show plenty overlapping characteristics with ESCs (Dawud et al, 2012), different ECC lines feature varied capacities to differentiate (Andrews et al, 1984;Pera et al, 1989, Thompson et al, 1984. The two murine ECC lines P19 and F9 were isolated a couple of decades ago and became popular models for studies of neurogenesis (Datta, 2013;Kuff and Fewell, 1980;McBurney and Roggers, 1982).…”

Section: In Vitro Models Of Neurogenesismentioning

confidence: 99%

“…For example, the two murine embryonic carcinoma cell lines F9 and P19, both popular ECC lines to study cell proliferation, differentiation and self-renewal, show differences in their expression profiles and thus partly different requirements for the conditions (Kelly and Gatie et al, 2017). Thus, unlike P19 cells, F9 cells require cAMP in addition to RA under serum deprivation to generate a neuronal phenotype (Datta, 2013;Murtomäki et al, 1999).…”

Section: Induction Of Neuronal Differentiation In Vitromentioning

confidence: 99%

Metabolically induced neuronal differentiation

Maurer¹

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In recent years, several neuronal differentiation protocols were published that circumvent the requirement of embryoid body (EB) formation under serum-deprivation and simplified medium conditions. But a neuronal default model to establish an approach that works efficiently for all pluripotent cells and neuronal precursors is still lacking. Whether such a default neural mechanism exist and how this is implemented across a broad spectrum of cell source, is addressed in several studies and still controversially discussed. It was proposed that the default neuronal fate is initiated in the absence of extrinsic signals and is achieved by eliminating extracellular inhibitors of neuroectodermal fate and suppressing cell-cell signalling through limited cell density. Previous studies reported that ESC and ECC grown at low density and in absence of exogenous factors or feeder layers die within 24 h but acquire a neural identity as indicated by expression of the neural marker Nestin. Thus, this application is not suitable for generating neural cultures. Furthermore, it was reported that P19 cells survive and express neuroectodermal marker genes in serum-free DMEM/F12 medium containing transferrin, insulin, and selenite, although no neurites were identified. Based on this background, in this study, a novel approach to induce neuronal differentiation in vitro was developed that implements a nutrient-poor environment, which, in contrast to previous studies, ensures the survival of neuronally differentiated cells over a long period of time and allows normal formation of neurites. Neither the formation of free-floating aggregates nor supplementation of growth factors or known inducers was required to establish a reliable neuronal differentiation protocol. A simple medium, consisting of DMEM/F12+N2 that was highly diluted in salt solution, was sufficient to drive a fast neuronal differentiation in monolayer cultures. Serum deprivation and strong dilution of DMEM/F12+N2 medium cause a nutrient-poor environment in which the influence of growth factors and inducers is minimized. This medium creates a metabolically defined environment that is presumably free of extrinsic signals that prevent the decision of neuronal fate. Analysis of the medium components discovered no actual inducer. Hence, it was suggested that the metabolic composition of the medium exclusively covers specific cell requirements of neurons, therefore ensures their survival, and drives the switch from pluripotent cells to neurons. The self-developed method was established by usage of the murine embryonal carcinoma cell line P19 and could be transferred to murine ESC. Consequently, the method could provide a feasible protocol for a generally valid neuronal default model. The established protocol provides several advantages such as the possibility to generate stable pure neuronal cultures by a fast, simple, and highly reproducible one-step induction under defined medium conditions with a minimum of exogen effectors. The method is characterised by clear and steady medium conditions that makes the investigation of specific cell requirements during differentiation accessible. It is therefore expected to be a useful tool to investigate the molecular basis of neuronal differentiation as well as for high throughput screenings. The phenotype of mature postmitotic neurons was arising within one week and cultures were shown to stay stable at least for three weeks. The neuronal identity was confirmed by expression of neuronal markers through immunofluorescence staining and mass spectrometry analysis. Furthermore, increased levels of axon markers were detected in early neuronal differentiation and functionality of the synapses of the P19-derived neurons was ascertained by detection of calcium activity. Axonal laser ablation, immediately followed by fast regrowth of connections in the neuronal network, revealed a strong regeneration potential under the given conditions. Furthermore, the generated neurons showed a morphologically distinct phenotype and the formation of neural rosettes. Immunofluorescence staining demonstrated the generation of pure and homogeneous neuronal cultures, free of glial cells. Retinoic acid (RA) plays an essential role in cell signalling during embryogenesis and efficiently induces neuronal differentiation in vitro in a concentration dependent manner. Neither retinol nor retinoic acid was included in any of the components of the self-prepared medium in this work. However, I observed, dependence on RARβ- and/or RARγ-regulated RA signalling in serum-free monolayer cultures. Nevertheless, neuronal differentiation in serum-free monolayer cultures was assumed to be RARα-independent because (i) RARα was slightly downregulated after neuronal induction, (ii) the truncated RARα of the RAC65 mutant had no effect on induction efficiency, and (iii) a pan-RAR inhibitor suppressed neuronal differentiation. In contrast to serum-free monolayer cultures, the truncated RARα prevented neuronal differentiation by application of the conventional protocol where cells are grown in free floating cell aggregates in serum-containing medium. Proteome analysis of P19 cells, treated by the self-developed differentiation protocol over five days showed increased levels of cellular RA binding proteins that mediate the cellular RA transport and are involved in canonical as well as non-canonical RA signalling. ...

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Murine Teratocarcinoma-Derived Neuronal Cultures

Cited by 4 publications

References 22 publications

Red emitting fluorescent carbon nanoparticles to track spatio-temporal dynamics of endocytic pathways in model neuroblastoma neurons

Red emitting fluorescent carbon nanoparticles to track spatio-temporal dynamics of endocytic pathways in model neuroblastoma neurons

Possible role of lncRNAs in amelioration symptoms in an animal model of Parkinson’s disease following transplantation of dopaminergic cells derived from stem cells

Metabolically induced neuronal differentiation

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