Screening of natural compounds with neuronal differentiation promoting effects in a cell-based model / 中国天然药物

The purpose of this study was to establish a drug screening method for small molecules extracted from traditional Chinese medicines (TCM) that have neuronal differentiation promoting effects, using P19 embryonic carcinoma cell as a cell-based model. First, the constructed plasmid (pTα1-Luc) was transfected into P19 cells to establish a screening model. Second, several TCMs were screened using the established model and all-trans-retinoic acid as a positive control. Finally, the underlying molecular mechanism was explored using immunofluorescence staining, qT-PCR, and Western blot analysis. Our results indicated that the drug screen model was established successfully and that both honokiol and hyperoside induced P19 differentiation into neurons, with the possible molecular mechanism being modulating the Wnt signaling pathway. In conclusion, the drug screening model developed in the present study provides a rapid, cell-based screening platform for identifying natural compounds with neuronal differentiation effects.

NgR1 Expressed in P19 Embryonal Carcinoma Cells Differentiated by Retinoic Acid Can Activate STAT3

NgR1, a Nogo receptor, is involved in inhibition of neurite outgrowth and axonal regeneration and regulation of synaptic plasticity. P19 embryonal carcinoma cells were induced to differentiate into neuron-like cells using all trans-retinoic acid and the presence and/or function of cellular molecules, such as NgR1, NMDA receptors and STAT3, were examined. Neuronally differentiated P19 cells expressed the mRNA and protein of NgR1, which could stimulate the phosphorylation of STAT3 when activated by Nogo-P4 peptide, an active segment of Nogo-66. During the whole period of differentiation, mRNAs of all of the NMDA receptor subtypes tested (NR1, NR2A-2D) were consistently expressed, which meant that neuronally differentiated P19 cells maintained some characteristics of neurons, especially central nervous system neurons. Our results suggests that neuronally differentiated P19 cells expressing NgR1 may be an efficient and convenient in vitro model for studying the molecular mechanism of cellular events that involve NgR1 and its binding partners, and for screening compounds that activate or inhibit NgR1.

Molecular mechanism of cardiac differentiation in P19 embryonal carcinoma cells regulated by Foxa2 / 中南大学学报(医学版)

OBJECTIVE@#To investigate the involvement of transcription factor Foxa2 in cardiac differentiation in P19 embryonal carcinoma cells and its molecular mechanism.@*METHODS@#P19 cells were induced to differentiate into cardiomyocytes by adding dimethyl sulfoxide (DMSO) into the culture medium of their embryoid bodies (EBs). The mRNA levels of pluripotency markers of embryonic pluripotent stem cells, cardiac differentiation related genes, and Foxa2 in the cell samples at different time points of cardiac differentiation were detected by reverse transcription PCR (RT-PCR). Differentiated and mature cardiomyocytes were identified by immunofluorescence. Eukaryotic expression plasmid pCMV-rFoxa2 (rat Foxa2) was transfected into P19 cells, and clonal populations of P19 cells that stably expressed green fluorescence protein (GFP)-rFoxa2 were isolated to enhance the expression levels of Foxa2 in P19 cells. The mRNA and protein levels of pluripotency markers and cardiac differentiation related genes in the above cell samples were detected by RT-PCR and Western blot. The mRNA levels of cardiac differentiation related genes in EBs differentiation system were also examined.@*RESULTS@#P19 cells differentiated into cardiomyocytes in the presence of DMSO, accompanied by stimulated expression of Foxa2. Transfection of pCMV-rFoxa2 plasmids into P19 cells upregulated rFoxa2 expression transiently and activated the transcription of its downstream cardiac inducer Cerberus1 (Cer1). The expression of pluripotency marker Nanog was suppressed and the expression of cardiac inducer Sonic Hedgehog (Shh) was elevated in GFP-rFoxa2 P19 cells. The expression of Cer1 and cardiac muscle marker actin, alpha cardiac muscle 1 (Actc1) was upregulated in EBs of GFP-rFoxa2 P19 cells.@*CONCLUSION@#Foxa2 participates in cardiac differentiation in P19 embryonal carcinoma cells. Foxa2 may inhibit Nanog expression and stimulate the expression of Cer1 and Shh directly during cardiac differentiation in P19 cells in the presence of DMSO.

Establishment of optimized neuronal differentiation-promoting model derived from P19 embryonic carcinoma cells / 浙江大学学报·医学版

<p><b>OBJECTIVE</b>To establish an optimized primary drug screen model of neuronal differentiation using P19 embryonal carcinoma cells.</p><p><b>METHODS</b>The final concentration of retinoid acid (RA), days of suspension culture, manner of adherent culture, suitable cell density and adherent culture medium were tested, respectively. Two stages of neuronal differentiation were examined based on morphological changes and immunocytochemistry analysis of neuronal specific protein β-tubulin III.</p><p><b>RESULTS</b>On d 8 of differentiation culture, neuron-like cells were observed with final concentration of 1 μmol/L RA. Neuron-like network was formed on d 16 of neuronal differentiation. β-tubulin III was positively stained on both stages, indicating P19 cells were differentiated into neurons.</p><p><b>CONCLUSION</b>The model using RA to induce P19 embryonic carcinoma cells to differentiate into neuron-like cells has been successfully established, which may provide a rapid, phenotypic cell-based platform for primary screening of neurogenesis-promoting drugs.</p>

CCN2/CTGF silencing blocks cell aggregation in embryonal carcinoma P19 cell

Connective tissue growth factor (CCN2/CTGF) is a matricellular-secreted protein involved in extracellular matrix remodeling. The P19 cell line is an embryonic carcinoma line widely used as a cellular model for differentiation and migration studies. In the present study, we employed an exogenous source of CCN2 and small interference RNA to address the role of CCN2 in the P19 cell aggregation phenomenon. Our data showed that increasing CCN2 protein concentrations from 0.1 to 20 nM decreased the number of cell clusters and dramatically increased cluster size without changing proliferation or cell survival, suggesting that CCN2 induced aggregation. In addition, CCN2 specific silencing inhibited typical P19 cell aggregation, which could be partially rescued by 20 nM CCN2. The present study demonstrates that CCN2 is a key molecule for cell aggregation of embryonic P19 cells.

Enhanced Cardiomyogenic Differentiation of P19 Embryonal Carcinoma Stem Cells

BACKGROUND AND OBJECTIVES: We investigated the effects of different concentrations of serum, 5-azacytidine, and culture time on the cardiomyogenic differentiation of P19 embryonal carcinoma stem cells in the course of developing an efficient protocol for generating the cardiomyogenic lineage. MATERIALS AND METHODS: P19 cells were plated at a density of 1x10(6) cells on 10-cm bacterial dishes for 96 hours in the presence of 1% dimethyl sulfoxide to form embryoid bodies. The embryoid bodies were cultured in medium with 2% or 10% fetal bovine serum for an additional 10 or 15 consecutive days in the presence of 0, 1, or 3 microM 5-azacytidine. RESULTS: Quantitative real-time polymerase chain reaction (PCR) analysis showed that the messenger ribonucleic acid (mRNA) expression of cardiac muscle-specific genes, such as GATA4, alpha-actin, alpha-myosin heavy chain, and cardiac troponin T, were significantly higher in the 15-day culture groups than in the 10-day culture groups. Furthermore, the cardiac muscle-specific genes were expressed more in the high-serum groups compared to the low-serum groups regardless of the culture time. Cardiomyogenic differentiation of the P19 cells was most effective in 1 microM 5-azacytidine regardless of the serum concentrations. In addition, the stimulation effects of 5-azacytidine on cardiomyogenic differentiation were more significant under low-serum culture conditions compared to high-serum culture conditions. Cardiomyogenic differentiation of P19 cells was further confirmed by immunostaining with cardiac muscle-specific antibodies. CONCLUSION:Taken together, these results demonstrated that cardiomyogenic differentiation of P19 cells was enhanced by a combination of different experimental factors.

Role of Cardiac Transcription Factor Nkx2.5 on Cardiomyoplasty Model in vitro / 대한해부학회지

Despite therapeutic advance, the prevalence of ischemic heart disease continues to increase. Recently, cell transplantation of stem cell has been proposed as a strategy for cardiac repair following myocardial damage. However, low differentiation efficiency into cardiomyocyte and poor cell viability associated with transplantation have limited the reparative capacity of these cell. In this study, we engineered P19 embryonal carcinoma cells using plasmid vector to overexpress the transcription factor MEF2c, Nkx2.5 involved in cardiomyogenesis. We investigated 1) formation of intercellular junction of P19 in mono-culture and co-culture with cardiomyocyte for functional and structural synchronous contraction after transplantation, 2) differentiation into cardiomyocyte, 3) resistance to hypoxic condition. An P19 embryonal carcinoma cell line expressing GFP, MEF2c, Nkx2.5 was generated by gene transfection and clonal selection. Nkx2.5 overexpression induced connexin43 expression level decrease. Electron microscopy revealed myofibril organization and immunostaining with cTnT showed positive staining in P19-Nkx2.5, consistent with early stage cardiomyocyte. Connexin43 and N-cadherin was expressed between P19-MEF2c and cardiomyocyte, P19- Nkx2.5 and cardiomyocyte in co-culture. And beating rate of cardiomyocyte co-cultured with P19-Nkx2.5 increased much more than other group, even if P19-Nkx2.5 did not have synchronous contraction with cardiomyocyte. Additionally, P19-Nkx2.5 had a resistance against hypoxia. These result suggest that overexpression of Nkx2.5 induced differentiation of P19 into cardiomyocyte and would be electro-mechanical coupling with cardiomyocyte after transplantation. Futhermore, Nkx2.5 overexpression had protection potential to hypoxic injury. Therefore, P19 cell overexpressed Nkx2.5 would be promising cell source for further study of new therapy of myocardial disease and building up in vitro model.