Publication Date: 2018
Journal of Tissue Engineering and Regenerative Medicine (19326254)(9)
Tissue engineering, as a novel transplantation therapy, aims to create biomaterial scaffolds resembling the extracellular matrix in order to regenerate the damaged tissues. Adding bioactive factors to the scaffold would improve cell–tissue interactions. In this study, the effect of chitosan polyvinyl alcohol nanofibres containing carbon nanotube scaffold with or without active bioglass (BG+/BG−), in combination with neonatal rat brain extract on cell viability, proliferation, and neural differentiation of P19 embryonic carcinoma stem cells was investigated. To induce differentiation, the cells were cultured in α-MEM supplemented with neonatal rat brain extract on the scaffolds. The expression of undifferentiated stem cell markers as well as neuroepithelial and neural-specific markers was evaluated and confirmed by real-time Reverse transcription polymerase chain reaction (RT-PCR) and immunofluorescence procedures. Finally, the three-dimensional (3D) cultured cells were implanted into the damaged neural tubes of chick embryos, and their fates were followed in ovo. Based on the histological and immunofluorescence observations, the transplanted cells were able to survive, migrate, and penetrate into the host embryonic tissues. Gene network analysis suggested the possible involvement of neurotransmitters as a downstream target of synaptophysin and tyrosine hydroxylase. Overall, the results of this study indicated that combining the effects of 3D cell culture and natural brain tissue extract can accelerate the differentiation of P19 embryonic carcinoma cells into neuronal phenotype cells. © 2018 John Wiley & Sons, Ltd.
Publication Date: 2017
Molecular and Cellular Probes (08908508)
Murine P19 embryonal carcinoma (EC) cells are convenient to differentiate into all germ layer derivatives. One of the advantages of P19 cells is that the exogenous DNA can be easily inserted into them. Here, at the first part of this study, we generated stable GFP-expressing P19 cells (P19-GFP+). FACS and western-blot analysis confirmed stable expression of GFP in the cells. We previously demonstrated the efficient induction of neuronal differentiation from mouse ES and EC cells by application of a neuroprotective drug, selegiline In the second part of this study selegiline was used to induce differentiation of P19-GFP+ into stable GFP-expressing neuron-like cells. Cresyl violet staining confirmed neuronal morphology of the differentiated cells. Furthermore, real-time PCR and immunoflourescence approved the expression of neuron specific markers. P19-GFP+ cells were able to survive, migrate and integrated into host tissues when transplanted to developing chick embryo CNS. The obtained live GFP-expressing cells can be used as an abundant source of developmentally pluripotent material for transplantation studies, investigating the cellular and molecular aspects of early differentiation. © 2016 Elsevier Ltd
Publication Date: 2016
Journal of Tissue Engineering and Regenerative Medicine (19326254)(7)
The ability of embryonal carcinoma)EC (stem cells to generate insulin-producing cells (IPCs) is still unknown. We examined the trophic effects of pancreas-conditioned medium (PCM) on in vitro production of IPCs. Initially, P19 EC cells were characterized by the expression of stem cell markers, Oct3/4, Sox-2 and Nanog. To direct differentiation, P19-derived embryoid bodies (EBs) were induced by selection of nestin-positive cells and treatment with different concentrations of PCM. Morphological studies documented the presence of islet-like cell IPCs clusters. The differentiated cells were immunoreactive for β cell-specific proteins, including insulin, proinsulin, C-peptide and insulin receptor-β. The expression of genes related to pancreatic β cell development and function (PDX-1, INS1, INS2, EP300 and CREB1) was confirmed by qPCR. During differentiation, the expression of EP300 and CREB1 increased by 2.5 and 3.1 times, respectively. In contrast, a sharp decrease in the expression of Oct3/4, Sox-2 and Nanog by 4, 1.5 and 1.5 times, respectively, was observed. The differentiated cells were functionally active, synthesizing and secreting insulin in a glucose-regulated manner. Network prediction highlighted crosstalk between PDX-1 transcription factor and INS2 ligand in IPC generation and revealed positive regulatory effects of EP300, CREB1, PPARA, EGR, KIT, GLP1R, and PKT2 on activation of PDX-1 and INS2. This is the first report of the induction of IPC differentiation from EC cells by using neonate mouse PCM. Since P19 EC cells are widely available, easily cultured without feeders and do not require special growth conditions, they would provide a valuable tool for studying pancreatic β cell differentiation and development. Copyright © 2016 John Wiley & Sons, Ltd. © 2014 John Wiley & Sons, Ltd.
Publication Date: 2014
PLoS ONE (19326203)(3)
An attractive approach to replace the destroyed insulin-producing cells (IPCs) is the generation of functional β cells from stem cells. Embryonal carcinoma (EC) stem cells are pluripotent cells which can differentiate into all cell types. The present study was carried out to establish a simple nonselective inductive culture system for generation of IPCs from P19 EC cells by 1-2 weeks old mouse pancreas extract (MPE). Since, mouse pancreatic islets undergo further remodeling and maturation for 2-3 weeks after birth, we hypothesized that the mouse neonatal MPE contains essential factors to induce in vitro differentiation of pancreatic lineages. Pluripotency of P19 cells were first confirmed by expression analysis of stem cell markers, Oct3/4, Sox-2 and Nanog. In order to induce differentiation, the cells were cultured in a medium supplemented by different concentrations of MPE (50, 100, 200 and 300 μg/ml). The results showed that P19 cells could differentiate into IPCs and form dithizone-positive cell clusters. The generated P19-derived IPCs were immunoreactive to proinsulin, insulin and insulin receptor beta. The expression of pancreatic β cell genes including, PDX-1, INS1 and INS2 were also confirmed. The peak response at the 100 μg/ml MPE used for investigation of EP300 and CREB1 gene expression. When stimulated with glucose, these cells synthesized and secreted insulin. Network analysis of the key transcription factors (PDX-1, EP300, CREB1) during the generation of IPCs resulted in introduction of novel regulatory candidates such as MIR17, and VEZF1 transcription factors, as well as MORN1, DKFZp761P0212, and WAC proteins. Altogether, we demonstrated the possibility of generating IPCs from undifferentiated EC cells, with the characteristics of pancreatic β cells. The derivation of pancreatic cells from EC cells which are ES cell siblings would provide a valuable experimental tool in study of pancreatic development and function as well as rapid production of IPCs for transplantation. © 2014 Ebrahimie et al.
Publication Date: 2013
Iranian Journal of Endocrinology and Metabolism (16834844)(4)
Introduction: Type I diabetes mellitus results from the autoimmune destruction of the β cells in pancreatic islets. Currently, extensive research is being conducted on the generation of insulin-producing cells (IPCs) from stem cells. P19 embryonal carcinoma cells are multipotent and can differentiate into cell types of all three germ layers. In this study, the differentiation of P19 cells into IPCs by using mouse pancreas extract (MPE) was investigated. Materials and Methods: Embryoid bodies (EBs) obtained from P19 cells were cultured in medium containing 3% fetal bovine serum, supplemented by concentration of 50, 100, 200,300 μg/mL MPE for 7-14 days. Dithizone (DTZ) staining was used to detect IPCs derived from EBs in vitro. Mouse monoclonal insulin-proinsulin and monoclonal insulin receptor beta antibodies were used for immunoflourescence. Insulin content from the cells and insulin secreted by differentiated cells in response to concentrations of 5.5 and 25 mM glucose were measured using ELISA kits. Results: DTZ-positive cells showed purple-red clusters. immunoflourescence indicated expression of Beta cell markers (insulin-proinsulin and insulin receptor beta) in these cells. Increasing glucose concentration, caused more insulin to be secreted by differentiated cells. Conclusions: P19 cells can in the presence of pancreas extract differentiate to cell producing and secreting insulin cells. Differentiated cells can increase insulin secretion in response to increasing glucose medium.
Publication Date: 2011
In Vitro Cellular and Developmental Biology - Animal (10712690)(8)
Selegiline, the irreversible inhibitor of monoamine oxidase B (MAO-B), is currently used to treat Parkinson's disease. However, the mechanism of action of selegiline is complex and cannot be explained solely by its MAO-B inhibitory action. It stimulates gene expression, as well as expression of a number of mRNAs or proteins in nerve and glial cells. Direct neuroprotective and anti-apoptotic actions of selegiline have previously been observed in vitro. Previous studies showed that selegiline can induce neuronal phenotype in cultured bone marrow stem cells and embryonic stem cells. Embryonal carcinoma (EC) cells are developmentaly pluripotene cells which can be differentiated into all cell types under the appropriate conditions. The present study was carried out to examine the effects of selegiline on undifferentiated P19 EC cells. The results showed that selegiline treatment had a dramatic effect on neuronal morphology. It induced the differentiation of EC cells into neuron-like cells in a concentration-dependent manner. The peak response was in a dose of selegiline significantly lower than required for MAO-B inhibition. The differentiated cells were immunoreactive for neuron-specific proteins, synaptophysin, and β-III tubulin. Stem cell therapy has been considered as an ideal option for the treatment of neurodegenerative diseases. Generation of neurons from stem cells could serve as a source for potential cell therapy. This study suggests the potential use of combined selegiline and stem cell therapy to improve deficits in neurodegenerative diseases. © 2011 The Society for In Vitro Biology.
Publication Date: 2010
In Vitro Cellular and Developmental Biology - Animal (10712690)(1)
In healthy individuals, skin integrity is maintained by epidermal stem cells which self-renew and generate daughter cells that undergo terminal differentiation. Epidermal stem cells represent a promising source of stem cells, and their culture has great potential in scientific research and clinical application. However, no single method has been universally adopted for identifying and isolating epidermal stem cells. Here, we reported the isolation and characterization of putative epidermal stem cells from newborn mouse skin. The keratinocytes were separated enzymatically. Putative epidermal stem cells were selected by rapid adherence on a composite matrix made of type I collagen and fibronectin. Unattached cells were discarded after 10 min, and the attached cells were cultured in a defined culture medium. The isolated cells showed the typical epidermal stem cell morphology. Immunofluorescence indicated that the cells were strongly stained for β1 integrin family of extracellular matrix receptors. In conclusion, mouse putative epidermal stem cells were successfully isolated from newborn mouse epidermis on the basis of high rapid adhesion to extracellular matrix proteins and cultured in vitro. © 2009 The Society for In Vitro Biology.
Publication Date: 2010
In Vitro Cellular and Developmental Biology - Animal (10712690)(10)
RNA interference (RNAi) can induce gene silencing via two pathways: post-transcriptional gene silencing (PTGS) and transcriptional gene silencing (TGS). The mediators of gene inactivation in both pathways are 21-bp small interfering RNAs (siRNAs) generated from longer double-stranded RNA (dsRNA). PTGS involves siRNA-mediated targeting and degradation of mRNA. However, siRNAs induce TGS via DNA methylation at the targeted promoter. Synthetic siRNAs can induce loss of gene activity comparable to long dsRNA. The limitation of this method is that the transfected synthetic siRNA works for only a few days. In this study, we tested the RNAi response to siRNA (PTGS pathway) by using a plasmid containing an enhanced green fluorescent protein (eGFP) gene as a target as well as a plasmid creates siRNA transcript, in a form of a hairpin, against eGFP gene. To investigate TGS pathway via RNAi, we also used a plasmid creates hairpin siRNA transcript against pgk-1 promoter. The data presented here indicated long-lasting inhibition in expression of eGFP and puromycin genes, both under the control of the murine Pgk-1 promoter. However, Southern blot analysis showed no methylation in pgk-1 promoter. © 2010 The Society for In Vitro Biology.
Publication Date: 2009
Iranian Biomedical Journal (1028852X)(1)
Background: RNA interference (RNAi) is a phenomenon uses double-stranded RNA (dsRNA) to specifically inhibit gene expression. The non-specific silencing caused by interferon response to dsRNA in mammalian cells limits the potential of utilizing RNAi to study gene function. Duplexes of 21-nucleotide short interfering dsRNA (siRNA) inhibit gene expression by RNAi. In some organisms, siRNA can also function as a primer converting mRNA into dsRNA that are further cleaved to produce more siRNA. This activity involves the enzyme RNA-dependent RNA polymerase (RdRP). There are no known RdRP involved in RNAi in mammals. By using an RdRP from Caenorhabditis elegance named ego-1, investigators intend to enhance RNAi effect in mammalian cells. The aims of this project were: 1) to investigate the efficiency of siRNA to enhanced green fluorescent protein (eGFP) gene silencing and 2) to enhance the RNAi effect. Methods: We used a vector-based siRNA to target eGFP. Also we used a vector expressing ego-1 to test for a possible amplification effect of RNAi. The expression of eGFP in the cells was detected by using fluorescent microscopy, flowcytometry and Western-blotting. Results: Transfection of the plasmid into P19 cells significantly decreased eGFP fluorescence. In addition, eGFP protein was reduced. Preliminary data suggested that the presence of ego-1 enhanced the RNAi effect. Conclusion: The results indicated that use of hairpin siRNA expression vectors for RNAi is a promising method to inhibition of gene expression in mammalian cells. Also, introducing RdRP enzyme to mammalian cells might amplify the RNAi effect in the cells.
Publication Date: 2006
Rejuvenation Research (15491684)(4)
The antiaging effect of selegiline was reported by several investigators; therefore, there is a growing interest in the potential use of stern cell therapy in aging. In this investigation, selegiline was used to induce neuronal differentiation in undifferentiated pluripotent embryonic stem cells (ESCs). The results show that selegiline can induce neuronal phenotype associated with neurotrophic factor expression. Morphologic and immunohistochemical techniques were used to evaluate the differentiation of the CCE cells, Cresyl violet for the morphologic study, anti-synaptophysin and antityrosine hydroxylase antibodies for characterizing the neuronal phenotype of ESCs, and RT-PCR to study the neurotrophins. The results showed that selegiline can induce dose-dependent ESC differentiation into neurons. Moreover, selegiline can induce neurotrophin expression. This study suggests the potential use of combined selegiline and stem cell therapy to improve deficits in neurodegenerative diseases in aging. © Mary Ann Liebert, Inc.
