User:Juitunl/P19 cells

P19 cells P19 cells are embryonic carcinoma cell lines derived from an embryo-derived teratocarcinoma in mice. The cell line is multipotent cells which can differentiate into all three germ layers cell types. Also, it is the most characterized embryonic carcinoma (EC) cells that can be specific induced into cardiac muscle cells and neuronal cells by different specific treatment. Exposing aggregated P19 cells to dimethyl sulfoxid (DMSO) can let differentiate into cardiac and skeletal muscle. Also, exposing P19 cells to retinoic acid (RA) can let differentiate into neuronal cells.

Origin of P19 cells
The cancer cells in human are always a big problem which may cause death if the aggressive cancer cells grow and spread out in human. However, recent biological scientists use those cells in research very well to study the development of cancer cells and try to find the therapy of cancers. For developmental biologist, embryonal carcinoma, which is derived from teratocarcinoma, is a good object for developmental study. In 1982, McBurney and Rogers transplanted 7.5 day mouse embryo into the testis to induce tumor growth. The cell cultures containing undifferentiatied stem cells were isolated from the primary tumor which which have a euploid karyotype. Those stem cells called embryonal carcinoma P19 cells (2). This derived P19 cells can grew rapidly without feeder cells and easy to maintain. Moreover, the multipotency of P19 cells was then confirmed by injecting the cells into blastocysts of another strain. They found that there were tissues from all three germ layers growing in the mouse (3). Based on their continuous studies, they further derived subtype cell lines from original P19 cells: P19S18, P19 D3, P19 RAC65 and P19 C16. The difference between theses subtype cell lines are the ability to differentiate into neuronal cell or muscle cells in response to retinoic acid or DMSO (3-6). More recently, lots of studies generate cell lines that were derived from differentiated P19 cells. Because of the multipotency of P19 cells, those new derived cell lines can be ectoderm, mesoderm and endoderm-like cells (7).

Differentiation of P19 cells
P19 cells can be maintained exponential growth because of the stable chromosomal composition. Since embryonal carcinoma can differentiate into cells of all three germ layers, P19 cells can also differentiate into those ectoderm, mesoderm and endoderm-like cells. When embryonal carcinoma are cultured at high density, they would start differentiation process (8). By aggregating the cells into embryonic body, EC cells can also process differentiation (9). In P19 cells, using non-toxic concentration of drugs to aggregated embryoid body cells can induce P19 cells differentiating into specific cell lines based on different drugs (1). The most two common and effective drugs are retinoic acid (RA) and dimethyl sulfoxide (DMSO). Studies have shown that at certain concentration of RA can induce P19 cells differentiating to neuronal cells including neuron and glial cells (10), while 0.5% - 1% DMSO led P19 cells differentiate to cardiac or skeletal muscle cells (11). In RA treatment method, neurons, astroglia and fibroblasts can be indentified after aggregation. Differentiated cells also have choline acetyl transferase and acetyl cholinesterase activities (12). When treated with DMSO, Cardiac muscle cells developed after 5 days of exposure and skeletal muscle cells appeared after 8 days of exposure (11). Those studies revealed that the different drug exposure can lead the multipotent P19 cells to different layers of cells. Since the concentration for both drugs are not toxicant to cells, the drug specific differentiation is not due to selection but induction of cells. Mutants of P19 cells were further generated in order to investigate the mechanism of drug specific differentiation (11-12). Moreover, signaling pathways related to neurogenesis and myogenesis were also investigated by studying gene expression or generating mutants of P19 cells.

Neurogenesis in P19 cells.
Using retinoic acid to treat undifferentiated P19 cells can specifically induced cells into neuronal cells. Using the dose between 1 μM to 3 μM of RA can generate the most abundant cell type of neurons (5). Neurons in this treatment performed the most population between six days and nine days. Several neurons markers such as neurofilament proteins, HNK-1 antigen and tetanus toxin binding sites are expressed the most during these days (13). After six to nine days of treatment, the neurons population declined probably because the other non neuron cells continue to proliferate. After 10 days of exposure, astroglial cells appeared by detecting the glial fibrillar acidic protein (GFAP), which is the specific marker of glial cells. Rather than neurons and astrocytes, P19 cells can also differentiate to oligodendrocytes by detecting the specific markers, markers, myelin associated glycoprotein and 2', 3'-cyclic nucleotide 3'-phosphodiesterase (14). Moreover, oligodendrocytes also developed and migrated in to fiber bundles in mice when the RA-induced cells were transplanted into the brains (14). Retinoic acid can induce not only P19 cells but also other progenitor cells or embryonic stem cells to differentiation. Since cells after retinoic acid treatment didn’t express neuron marker genes immediately, RA must initiate some pathway to process cellular differentiation. Lots of studies used P19 cells to investigate RA induced mechanism, including generating the mutant allele of retinoic acid receptor genes and studying the expression of receptor genes, Hox genes and retinol binding proteins while exposing to RA (15-17). All those studies indicate that P19 cell is a good in vitro model system for investigating the mechanism of drugs that interfere with the specific cellular pathway. What is more, by using the ability of RA-induced neurogenesis in P19 cell, lots of researchers started to identify the in vitro differentiation mechanism of neurogenesis. Several related pathways or including Wnt/β-catenin pathway, Notch pathway and hedgehog pathway are investigated either using gene expression or generating alleles for related genes (18-20).

Myogenesis in P19 cells
Same as retinoic acid, DMSO induced differentiation is not specific to P19 cells (7). It could also induce neuroblastoma cells, lung cancer cells and mouse ES cells (21-23). At concentration of 0.5%-1% DMSO induced P19 cells to aggregate and process mesodermal and endoermal cell types (1, 11, 24). After 2 days of exposure, the endoderm—like cells appeared and resembled primitive extraembryonic endoderm. After 6 days of exposure, the cardiac muscle appeared in the interior of the aggregates. The content of cardiac muscle cells were 25% of the cells. After 10 days of exposure, skeletal muscle cells appeared around the embryo body (11).

The cellular mechanism that occurs during aggregation and differentiation is still not fully studied. However, some studies showed that the cellular communication plays an important role in muscle differentiation in P19 cells which might explain why cells need to aggregate first to process the muscle differentiation (7). In order to elucidate the mechanism of myogenesis in P19 cells, several cardiac specific transcription factors including GATA-4, MEF2c, Msx-1, Nkx2.5, MHox, Msx-2 and MLP are found to change during differentiation (7). Reports have shown that GATA-4, NKx2.5 and MEF2c were all upregulated after DMSO induction (25-26). In recent years, P19 cells were also used in studying the mechanism of cardiac differentiation and myogenesis. The main affected signaling pathway, bone morphogenetic proteins (BMPs) pathway is the most strongly studied signaling in P19 cells (27). By generating the P19CL6noggin cell line, which overexpresses the BMP antagonist noggin, they found that the mutant cells didn’t differentiate into cariomyocytes when treated with 1% of DMSO, suggesting that the BMPs were indispensible for cardiomyocyte differentiation in this system. They also provided the evidence showing that TAK1, Nkx-2.5, and GATA-4 are important in cardiogenic BMP signaling pathway (27).

Future directions
P19 cells give us a valuable formation of both neuronal cells and muscle cells in vitro. Since the P19 cells are easy to maintain and culture comparing to other embryonic stem cells, it is convenient do the developmental studies in vitro. Based on the technique to manipulate this cell line to express or knock out certain gene, we can investigate the detail of the signaling pathway, functional studies and the regulation proteins expression of myogenesis and neurogenesis. The extended research can also look up the later stage of heart or brain development and maturation.