Neural Differentiation Of P19 Cells Research Articles

Functionalized Exosomes Promote Neural Differentiation of P19 Cells by Activating the Wnt Signaling Pathway.

Global population growth and population aging continue to accelerate, and lead to a further increase in the risk of neurological diseases worldwide. Extracellular vesicles secreted by mesenchymal stem cells carry many proteins, lipids, and genetic material that mediate cell-to-cell communication and improve therapeutic outcomes for neurological disorders. Stem cells from human exfoliated deciduous teeth are considered a suitable cell source for tissue regeneration, which exerts therapeutic effects via the secretion of exosomes. This study was performed to assess the effect of functionalized exosomes on neural differentiation of embryonic carcinoma cell line P19. We stimulated stem cells from human exfoliated deciduous teeth with the glycogen synthase kinase-3β inhibitor TWS119 and then extracted its exosomes. P19 cells were induced to differentiate using functionalized exosomes, and the biological functions and involved signaling pathways of differentially expressed genes were analyzed by RNA-sequencing. Immunofluorescence techniques detected neuronal specific markers. It was found that TWS119 activated the Wnt signaling pathway in stem cells from human exfoliated deciduous teeth. RNA-sequencing showed that upregulated differentially expressed genes in the functionalized exosome-treated group were responsible for cell differentiation, neurofilament, and structural constituent of the synapse. Kyoto Encyclopedia of Genes and Genomes enrichment analysis revealed that the functionalized exosome-treated group activated the Wnt signaling pathway. Immunofluorescence showed that functionalized exosomes induced neurite outgrowth in P19 cells. Our results demonstrated that functionalized exosomes promoted neural differentiation of P19 cells by activating the Wnt signaling pathway.

Prdm12 regulates inhibitory neuron differentiation in mouse embryonal carcinoma cells.

The epigenetic regulatory system significant influences the fate determination of cells during developmental processes. Prdm12 is a transcriptional regulator that modulates gene expression epigenetically. The Prdm12 gene has been shown to be expressed in neural tissues, specifically during development, but its detailed function is not fully understood. This study investigated the function of the Prdm12 gene in P19 mouse embryonic tumor cells as a model for neural differentiation. A decrease in the expression of neuron-specific genes and the alterations of dendrites and axons morphology was confirmed in Prdm12-knockout P19 cells. In addition, almost no astrocytes were generated in Prdm12-knockout P19 cells. Comprehensive gene expression analysis revealed that there was a reduction in the expression of the inhibitory neuron-specific genes Gad1/2 and Glyt2, but not the excitatory neuron-specific gene VGLUT2, in Prdm12-knockout P19 cells. Furthermore, the expression of inhibitory neuron-related factors, Ptf1a, Dbx1, and Gsx1/2, decreased in Prdm12-knockout P19 cells. Gene expression analysis also revealed that the Ptf1a, Hic1, and Foxa1 genes were candidate targets of Prdm12 during neurogenesis. These results suggest that Prdm12 regulates the differentiation of inhibitory neurons and astrocytes by controlling the expression of these genes during the neural differentiation of P19 cells.

Fabrication of Graphene Oxide Aerogel to Repair Neural Tissue

: The neural tissue engineering has been designed as a subset of tissue engineering for treating congenital malformations and accident injuries, particularly for individuals requiring tissue grafting. Such transplants, usually performed as autografting, can often not meet the requirements of an effective scaffold used in nerve tissue engineering. A novel neural tissue scaffold was introduced here to solve the problem concerning the reduced graphene oxide. The three-dimensional graphene oxide in the neural canal restricts the formation of fibroglandular tissues and facilities neural stem cell proliferation and growth. In these techniques, graphene oxide aerogel was initially made. Then, the freeze-drying process was used to fix the geometry of reduced graphene oxide hydrogels prepared using graphene oxide dispersion and ethylenediamine and gain aerogels. The X-ray diffraction patterns, FTIR and morphological related to samples were examined, followed by conducting in-vitro micropropagation and 4, 6-diamidino-2-phenylindol (DAPI) staining in fibroblast and P19 cultures. The results from immunofluorescence staining demonstrated the neural differentiation of P19 cells. It can be concluded that most cells attached to and differentiated on the scaffold surface and axons can penetrate randomly through them. Finally, the three-dimensional graphene oxide was proposed as an ideal alternative to be used in neural tissue engineering.

Induction of Tyrosine Hydroxylase Gene Expression in Embryonal Carcinoma Stem Cells Using a Natural Tissue-Specific Inducer.

A large number of studies have focused on the generation of dopaminergic neurons from pluripotent cells. Differentiation of stem cells into distinct cell types is influenced by tissue-specific microenvironment. Since, central nervous system undergoes further development during postnatal life, in the present study neonatal rat brain tissue extract (NRBE) was applied to direct the differentiation of embryonal carcinoma stem cell line, P19 into dopaminergic (DA) phenotypes. Additionally, a neuroprotective drug, deprenyl was used alone or in combination with the extract. Results from morphological, immunofluorescence, and qPCR analyses showed that during a period of one to three weeks, a large percentage of stem cells were differentiated into neural cells. The results also indicated the greater effect of NRBE on the differentiation of the cells into tyrosine hydroxylase-expressing cells. MS analysis of NRBE showed the enrichment of gene ontology terms related to cell differentiation and neurogenesis. Network analysis of the studied genes and some DA markers resulted in the suggestion of potential regulatory candidates such as AVP, ACHE, LHFPL5, and DLK1 genes. In conclusion, NRBE as a natural native inducer was apparently able to simulate the brain microenvironment and support neural differentiation of P19 cells.

Directed differentiation of mouse P19 embryonal carcinoma cells to neural cells in a serum- and retinoic acid-free culture medium.

P19 embryonal carcinoma cells (EC-cells) provide a simple and robust culture system for studying neural development. Most protocols developed so far for directing neural differentiation of P19 cells depend on the use of culture medium supplemented with retinoic acid (RA) and serum, which has an undefined composition. Hence, such protocols are not suitable for many molecular studies. In this study, we achieved neural differentiation of P19 cells in a serum- and RA-free culture medium by employing the knockout serum replacement (KSR) supplement. In the KSR-containing medium, P19 cells underwent predominant differentiation into neural lineage and by day 12 of culture, neural cells were present in 100% of P19-derived embryoid bodies (EBs). This was consistently accompanied by the increased expression of various neural lineage-associated markers during the course of differentiation. P19-derived neural cells comprised of NES+ neural progenitors (~ 46%), TUBB3+ immature neurons (~ 6%), MAP2+ mature neurons (~ 2%), and GFAP+ astrocytes (~ 50%). A heterogeneous neuronal population consisting of glutamatergic, GABAergic, serotonergic, and dopaminergic neurons was generated. Taken together, our study shows that the KSR medium is suitable for the differentiation of P19 cells to neural lineage without requiring additional (serum and RA) supplements. This stem cell differentiation system could be utilized for gaining mechanistic insights into neural differentiation and for identifying potential neuroactive compounds.

Expression and Regulation of Tal2 during Neuronal Differentiation in P19 Cells.

T-cell acute lymphocytic leukemia 2 (Tal2) is a gene encoding a member of the basic helix-loop-helix transcription factor family, which is essential for the normal development of the mouse brain. We found that Tal2 was induced during neural differentiation in P19 cells, which are pluripotent mouse embryonal carcinoma cells that differentiate into the neural lineage upon both exposure to all-trans retinoic acid (atRA) and the formation of cell aggregation. Tal2 expression during neural differentiation in P19 cells was detected within 3 h after induction with atRA and retinoic acid receptor α (RARα). The atRA-RARα complex is known to bind to a characteristic retinoic acid response element (RARE) located in the promoter of target genes. We found a RARE-like element in the intron of Tal2. We also found a TATA-box-like element in the 5' region. The TATA-box-like element functioned as a core promoter, and TATA- box binding protein bound to this element upstream of Tal2 in P19 cells. The RARE-like element responded to atRA signaling that activated the transcription, and RARα was bound to this element in the intron of Tal2 in P19 cells. Furthermore, the interaction between these elements on Tal2 was confirmed in a chromatin immunoprecipitation assay. Because the neural differentiation of P19 cells mimics in part the development of the nervous system, P19 cells are useful for studying the mechanism underlying the role of Tal2 in neural differentiation. Further work is underway to clarify the function of Tal2 in neural differentiation using the differentiation system of P19 cells.

Relationship between Mecp2 and NFκb signaling during neural differentiation of P19 cells

The pluripotent P19 embryo carcinoma cell line was studied to determine a signaling pathway regulating MeCP2 expression. P19 cells were induced to differentiate into neurons by RA and express β-III tubulin at one day after induction and synaptophysin by 7 days. MeCP2 was first observed after β-III tubulin expression was detected and continued to rise over the course of differentiation. Both Mecp2 e1 and e2 mRNA forms progressively increased in differentiating cells. MeCP2 expression was increased by tumor necrosis factor (TNF) in early differentiating cells, which was blocked by NFκB inhibitors. TNF did not increase MeCP2 expression in naïve cells. Moreover, TNF did not increase NFκB reporter gene activity in naïve cells even though increases were observed in early differentiating cells. The protein kinase C activator phorbol 12-myristate 13-acetate (PMA) increased MeCP2 expression in naïve P19 cells, which was also blocked by NFκB inhibitors. Interestingly, PMA increased NFκB reporter gene activity in naïve cells. Finally, PMA, but not TNF, induced IκBα degradation in naïve P19 cells. Taken together, our data indicates that MeCP2 expression is regulated in part by signaling pathways involving NFκB.

Regulation of neurogenesis and gliogenesis of retinoic acid-induced P19 embryonal carcinoma cells by P2X2 and P2X7 receptors studied by RNA interference

Embryonic carcinoma cells are widely used models for studying the mechanisms of proliferation and differentiation occurring during early embryogenesis. We have now investigated how down-regulation of P2X2 and P2X7 receptor expression by RNA interference (RNAi) affects neural differentiation and phenotype specification of P19 embryonal carcinoma cells. Wild-type P19 embryonal carcinoma cells or cells stably expressing shRNAs targeting P2X2 or P2X7 receptor expression were induced to differentiate into neurons and glial cells in the presence of retinoic acid. Silencing of P2X2 receptor expression along differentiation promoted cell proliferation and an increase in the percentage of cells expressing glial-specific GFAP, while the presence of beta-3 tubulin-positive cells diminished at the same time. Proliferation induction in the presence of stable anti-P2X2 receptor RNAi points at a mechanism where glial proliferation is favored over growth arrest of progenitor cells which would allow neuronal maturation. Differently from the P2X2 receptor, inhibition of P2X7 receptor expression during neural differentiation of P19 cells resulted in a decrease in cell proliferation and GFAP expression, suggesting the need of functional P2X7 receptors for the progress of gliogenesis. The results obtained in this study indicate the importance of purinergic signaling for cell fate determination during neural differentiation, with P2X2 and P2X7 receptors promoting neurogenesis and gliogenesis, respectively. The shRNAs down-regulating P2X2 or P2X7 receptor gene expression, developed during this work, present useful tools for studying mechanisms of neural differentiation in other stem cell models.

Retinoic acid treatment and cell aggregation independently regulate alternative splicing in P19 cells during neural differentiation

To induce neural differentiation of P19 cells, two different treatments, RA (retinoic acid) and cell aggregation, are required. However, there has been no report that RA treatment alone or cell aggregation alone could control alternative splicing regulation in P19 cells. Therefore, we focused on alternative splicing effects by neural induction (RA treatment and/or cell aggregation) in P19 cells. We analysed the splicing patterns of several genes, including 5-HT3R-A (5-hydroxytryptamine receptor), Actn1 (actinin alpha1), CUGBP2 (CUG-binding protein) and PTB (polypyrimidine track-binding protein), which showed different responses during the early neural induction of P19 cells. We show here that RA treatment alone changes the alternative splice mechanism of 5-HT3R-A. Cell aggregation alone controls alternative splicing regulation of Actn1. Both treatments (RA and cell aggregation) compensate and regulate the alternative splicing mechanism of CUGBP2. However, PTB is independent of RA and cell aggregation. Taken together, our results suggest that RA treatment and cell aggregation independently regulate the alternative splicing mechanism in the early stage of P19 cells during neural differentiation.

Alternative splicing ofMef2cpromoted by Fox‐1 during neural differentiation in P19 cells

Mef2c protein is one of the MADS-box type transcription factors involved in muscular differentiation and synaptic formation. Previously, it has been reported that the Mef2c gene is responsible for three alternative splicing regulations. Here, we investigated the alternative splicing variants of Mef2c during neural differentiation of P19 cells and during cardio muscular differentiation of P19 clone 6 (P19CL6). We detected that two Mef2c mRNA isoforms, using exon α1 with and without the γ region at exon 10, are mainly produced in immature P19 cells. Remarkably, Mef2c isoforms containing exon β specifically appeared in the neural cell stage. Because most transcripts contain exon β in the neural cell stage and in the brain, this suggests that the alternative splicing of exon β is highly regulated. Among known regulators, Fox-1 was specifically expressed in the neural cell stage in correlation with Mef2c exon β. Fox-1 promoted exon β inclusion in transfection experiments using Mef2cβ minigene. Moreover, we found that the promotion required RNA-binding activity of Fox-1 and GCAUG sequence located in adjacent intron of exon β. Taken together, our results suggest that Fox-1, expressed specifically in the neural cell stage, promoted Mef2c exon β inclusion via the GCAUG.

Expression analysis of Fgf8a & Fgf8b in early stage of P19 cells during neural differentiation

Fgf8 is a member of the fibroblast growth factor (FGF) family that plays an important role in early neural development. Cellular aggregation and retinoic acid (RA) are needed for mouse embryonic carcinoma (EC) P19 cell neural differentiation. We have examined the Fgf8 gene in P19 cells during neural differentiation and identified 2 alternatively spliced Fgf8 isoforms, Fgf8a and Fgf8b, among the 8 known splicing isoforms in mammals. The expression of Fgf8a and Fgf8b mRNAs transiently and rapidly increased in the early stage of P19 cells during RA-induced neural differentiation, followed by a decline in expression. The relative amount of Fgf8b was clearly higher than that of Fgf8a at different time-points measured within 24h after RA treatment. Increased Fgf8b mRNA expression was cellular-aggregation dependent. The results demonstrated that cellular-aggregation-induced Fgf8b, but not Fgf8a, may play a pivotal role in early neural differentiation of P19 cells.

Involvement of the Mouse Prp19 Gene in Neuronal/Astroglial Cell Fate Decisions

The molecular mechanisms involved in neuronal/astroglial cell fate decisions during the development of the mammalian central nervous system are poorly understood. Here, we report that PRP19beta, a splice variant of mouse PRP19alpha corresponding to the yeast PRP19 protein, can function as a neuron-astroglial switch during the retinoic acid-primed neural differentiation of P19 cells. The beta-variant possesses an additional 19 amino acid residues in-frame in the N-terminal region of the alpha-variant. The forced expression of the alpha-variant RNA caused the down-regulation of oct-3/4 and nanog mRNA expression during the 12-48 h of the late-early stages of neural differentiation and was sufficient to convert P19 cells into neurons (but not glial cells) when the cells were cultured in aggregated form without retinoic acid. In contrast, the forced expression of the beta-variant RNA suppressed neuronal differentiation and conversely stimulated astroglial cell differentiation in retinoic acid-primed P19 cells. Based on yeast two-hybrid screening, cyclophilin A was identified as a specific binding partner of the beta-variant. Luciferase reporter assay mediated by the oct-3/4 promoter revealed that cyclophilin A could act as a transcriptional activator and that its activity was suppressed by the beta-variant, suggesting that cyclophilin A takes part in the induction of oct-3/4 gene expression, which might lead to neuroectodermal otx2 expression within 12 h of the immediate-early stages of retinoic acid-primed neural differentiation. These results show that the alpha-variant gene plays a pivotal role in neural differentiation and that the beta-variant participates in neuronal/astroglial cell fate decisions.

Classification of neural differentiation-associated genes in P19 embryonal carcinoma cells by their expression patterns induced after cell aggregation and/or retinoic acid treatment

Expression of neural differentiation-associated genes was examined by RT-PCR and macroarray analyses during neural differentiation of P19 embryonal carcinoma cells induced by cell aggregation and/or retinoic acid (RA) treatment. Results revealed that the neural genes examined could be classified into 4 groups based on their expression patterns. The 1st group included the Wnt-1, Id-1, Id-3 and cdc42 genes, expression of which was altered by cell aggregation alone, but not by RA treatment alone. The 2nd group included the alphaN-catenin, Neuro D and GDNFRbeta genes, expression of which was altered by RA treatment alone, but not by cell aggregation. The 3rd group consisted of the Brn-2, TrkA, bcl-X, N-cadherin, E-cadherin and Otx-2 genes, expression of which was altered by either treatment. The 4th group included the ACTH, D4DR, NGC and Oct-3 genes, the expression of which changed only when both treatments were applied simultaneously. Expression of the Ets-1 and Fli-1 transcription factor genes was up-regulated by either treatment alone at initial stages of neural differentiation of P19 cells, although overexpression of these genes alone could not induce cell differentiation. Our results suggest that although both treatments are required for complete neural differentiation of P19 cells, cell aggregation or RA treatment alone drive differentiation to a certain extent at the gene expression level.

Differential display of proteins involved in the neural differentiation of mouse embryonic carcinoma P19 cells by comparative proteomic analysis

Mouse embryonic carcinoma P19 cell has been used extensively as a model to study molecular mechanisms of neural differentiation in vitro. After retinoic acid (RA) treatment and aggregation, P19 cells can differentiate into neural cells including neurons and glial cells. In this study, comparative proteomic analysis is utilized to approach the protein profiles associated with the RA-induced neural differentiation of P19 cells. Image analysis of silver stained two-dimensional gels indicated that 28 protein spots had significantly differential expression patterns in both quantity and quality. With mass spectrometry analysis and protein functional exploration, many proteins demonstrated an association with distinct aspects of neural differentiation. These proteins were gag polyprotein, rod cGMP-specific 3',5'-cyclic phosphodiesterase, 53 kDa BRG1-associated factor A, N-myc downstream regulated 1, Vitamin D receptor associated factor 1, stromal cell derived factor receptor 1, phosphoglycerate mutase, Ran-specific GTPase-activating protein, and retinoic acid (RA)-binding protein. While some cytoskeleton-related proteins such as beta cytoskeletal actin, gamma-actin, actin-related protein 1, tropomyosin 1, and cofilin 1 are related to cell migration and aggregation, other proteins have shown a relationship with distinct aspects of neural differentiation including energy production and utilization, protein synthesis and folding, cell signaling transduction, and self-protection. The differential expression patterns of these 28 proteins indicate their different roles during the neural differentiation of P19 cells. As an initial step toward unveiling the regulations involved in the commitment of pluripotent cells to a neural fate, information from this study may be helpful to uncover the molecular mechanisms of neural differentiation.

Tra2? Promotes RA Induced Neural Differentiation of P19 Cells

The present study demonstrates a high level of the nuclear Transformer 2alpha (Tra2alpha) protein in adult mouse brain relative to other tissues, including muscle, heart, liver, lungs, kidney and small intestine, suggesting the potential importance of Tra2alpha in neural function. The level of Tra2alpha in mouse cerebrum is developmentally regulated, peaking at neonate stage. In P19 carcinoma cells, Tra2alpha is transiently up-regulated upon retinoic acid (RA) treatment. Although over-expression of Tra2alpha protein alone does not elicit P19 differentiation, under these conditions the response of P19 cells to RA is significantly increased. The results suggest that Tra2alpha proteins may act as a mediator in the signal pathway associated with RA-induced differentiation in P19 cells.

DNA rearrangement activity during retinoic acid-induced neural differentiation of P19 mouse embryonal carcinoma cells.

Because of the many superficial similarities between the immune system and the central nervous system, it has long been speculated that somatic DNA recombination is, like the immune system, involved in brain development and function. To examine whether or not the V(D)J recombination signals of the immune system work in an in vitro neural differentiation model, the P19 mouse embryonal carcinoma cell line was transfected with a reporter gene that is designed, when rearranged, to express bacterial beta-galactosidase, which was previously reported to exhibit somatic DNA recombination in the transgenic mouse brain. The cloned cells were then induced into neural cells by retinoic acid treatment. This neural induction treatment resulted in the cloning of a P19 cell line that showed a high incidence of beta-galactosidase-positive cells. Most of these beta-galactosidase-positive cells were immunocytochemically identified as either neurons, neuroepithelial cells, or astrocytes. The 5'-end sequences of the beta-galactosidase transcripts expressed in the induced cells were analyzed, and sequences were found that seemed to reflect DNA rearrangement through re-integration of the reporter gene into the host genome. However, the V(D)J recombination signals did not work in the in vitro model. These results suggested that DNA rearrangement activity though integration increased during neural differentiation of P19 cells.

Tra2βl regulates P19 neuronal differentiation and the splicing of FGF‐2R and GluR‐B minigenes

The present study demonstrates that the expression of Tra2beta1 (Transformer 2-beta1) proteins, an SR (serine/arginine rich) protein, is developmentally up-regulated in a neural-specific pattern. The up-regulation is also observed in RA (retinoic acid) induced neural differentiation of P19 cells. Tra2betal proteins are located in the nuclei of P19 cells, which are consistent with its functional site as an SR protein. The over-expression of Tra2betal proteins promotes RA induced neuronal differentiation of P19 cells. In P19 cells, the splicing of FGF-2R (fibroblast growth factor receptor 2) minigene produces the BEK form, while the alternative splicing of GluR-B (glutamate receptor subunit B) minigene generates two products, the Flop and the Truncated isoforms. Tra2betal inhibits the BEK splicing, but it promotes the Flop splicing. The results therefore suggest that Tra2betal is involved in the regulation of alternative splicing processes during neural development, peculiarly the splicing of FGF-2R and GluR-B genes. Both FGF-2R and GluR-B genes are known to play important roles in neural differentiation.

Activation Mechanism of c-Jun Amino-terminal Kinase in the Course of Neural Differentiation of P19 Embryonic Carcinoma Cells

P19 embryonic carcinoma cells, a model system for studying early development and differentiation, can differentiate into neurons and primitive endoderm-like cells depending on the culture conditions. We have previously reported that the activation of c-Jun amino-terminal kinase (JNK) is required for the retinoic acid-induced neural differentiation of P19 cells. However, the signaling pathway(s) responsible for the activation of JNK has not been known. In this study, we demonstrated that activities of MAPK kinase 4 (MKK4) and TAK1, one of the upstream kinases of MKK4, were enhanced in the neurally differentiating cells. Inhibition of the neural differentiation by an overexpression of protein phosphatase 2Cepsilon, an inactivator of TAK1, suggested a critical role of the TAK1 signaling pathway during the differentiation. Confocal microscopic analysis indicated that TAK1, phospho-MKK4, and phospho-JNK were colocalized with tubulin in the neurites and localized also in the nuclei of the differentiating cells. In contrast, two TAK1-binding proteins, TAB1 and TAB2, which are involved in the activation of TAK1, were localized in the neurites and the nuclei of the differentiating cells, respectively. These results suggest that two distinct TAK1-MKK4-JNK signaling pathways are independently activated at the different intracellular locations and may participate in the regulation of the neural differentiation of P19 cells.

Cyclophilin A is required for retinoic acid-induced neuronal differentiation in p19 cells.

Stable transfectants with expression of small interfering RNA for targeting cyclophilin A (CypA) in p19 cells lose their potential for retinoic acid (RA)-induced neuronal differentiation but not Me(2)SO-induced mesodermal differentiation. This difference suggests that CypA is specifically required for the RA-induced neuronal pathway. In addition to the loss of RA-induced RA receptor beta expression and retinoic acid response element (RARE)-binding activity, a dramatic reduction in RA-induced RARE-mediated luciferase activity in the CypA knockdown cell line suggests that CypA affects RARE-mediated regulation of gene expression. Silent mutation of target sequences confirms the specificity of RNA interference in p19 embryonal carcinoma cells. Collectively, our data reveal that a novel function of CypA is required in the processing of RA-induced neuronal differentiation in p19 embryonal carcinoma cells.

HB-GAM/Pleiotrophin and Midkine are Differently Expressed and Distributed During Retinoic Acid-induced Neural Differentiation of P19 Cells

HB-GAM/Pleiotrophin and Midkine (MK) are developmentally-regulated proteins with putative functions during cell growth and differentiation. Using the P19 cell which is a model to study the events associated with early development, we examined the expression and cellular localization of HB-GAM and MK during neural differentiation of P19 cells induced by retinoic acid (RA). The temporal expressions of HB-GAM and MK transcripts and both the levels and cellular localizations of the corresponding proteins appeared dramatically different. MK mRNA, already expressed in untreated P19 cells, was transiently increased by exposure to RA and then largely down regulated. More interestingly, HB-GAM which was not detected in untreated P19 cells was strongly expressed after 2 days of RA treatment and this expression persists throughout the duration of the culture suggesting that it could be involved in different aspects of this differentiation process.