Regulation About Multiple-Directory Differentiation Of Chicken Embryonic Germ Cells

Although the chick embryo has long been used as a model for developmental biology, its potential use as an experiment model for the repair and regeneration of adult tissues is often overlooked. This model has several advantages over mammalian systems for in vivo studies, as it is cost-effective, easily manipulated, and can be used for transgenesis in conjunction with viral vectors or electroporation. The molecular mechanisms underlying the development of nearly all major organ systems have been elucidated in the chick, making data interpretation more complete within this knowledge landscape. In this study, embryonic germ (EG) cells were derived from primordial germ cells (PGCs) of genital ridges of 3.5-4-days-old chicken embryos. These cells satisfied the criteria previously used for defining chicken EG cells by using the expression of markers characteristic to ES cells. When injected subcutaneously, chicken EG cells could form teratoma that enable differentiation into a wide range of tissue types of all three primary cell lineages including neural cells, cartilage, forming bone, adipocytes, blood vessels, smooth muscle, striated muscle and secretory epithelia in recipient. Furthermore, cells in embryoid bodies (EBs) expressed lineage-specific markers of three germ layers and could be induced to differentiate into more advanced stages of various committed cell types, including dopamine and cholinergic neurons, astrocytes, oligodendrocytes, adipocytes, cardiomyocytes and hepatocytes, that were demonstrated by immunocytochemical staining or RT-PCR analysis. These findings support the multiple differentiation capability of chicken pluripotent EG cells, thus confirming the presumption that chicken embryos may used as a potential model for better understanding the mechanisms of tissue-specific differentiation and regeneration, that will help to devise strategies based on the transplantation of stem cell-derived tissues for restoring function to damaged or diseased tissues.1. Preparation of of chicken EG cells and establishment of the culture modelsGenital ridges were collected by dissection of chicken embryos at 3.5-4 days with a fine glass needle under a microsurgery scope. For primary culture, cell suspension containing both PGCs and somatic cells was seeded onto gelatin-treated 35 mm culture plates at a density of 1×106/well in DMEM supplemented with 5% fetal calf serum (FCS), 10ng/ml leukemia inhibitory factor (LIF), 10ng/ml human basic fibroblast growth factor (bFGF), 0.1mmol/L MEM nonessential amino acids, O.lmmol/L 2-mercaptoethanol,2mmol/L L-glutamine (Gln), 100U/ml penicillin and 100μg/ml streptomycin. The seeded cells were then maintained at 38.5℃in 5% CO2/95% air with 60%-70% relative humidity until the PGCs colonized as a primary culture. To trace the origin of the colonies, the primary formed colonies were picked up with a fine glass needle, dissociated with 0.25% trypsin-EDTA 5 days after plating and then subjected to RT-PCR analysis for expression of PGC-specific markers. For further subculture, colonies that were positive for PGC markers were picked up and treated with 0.25% trypsin-EDTA to achieve single cell suspension and reseeded onto 6-well dishes. After three passages, staining of periodic acid-Schiff regent (PAS), stage-specific embryonic antigens (SSEA-1, SSEA-3 and SSEA-4) immunocytochemistry, and the expression of the pluripotency-associated genes cPouV, cNanog and Sox2 analysis all confirmed the characteristics of cultured EG cells. The above results indicated that the primary and subculture models of PGCs could be used for obtaining EG cells which represents a source of EG cells for studies about differentiation of germline stem cells (GSC).2. Teratoma formation of chicken EG cells in vivoSeven-day-old chicks were obtained from a commercial hatchery and fed with cyclosporine (5 mg/kg BW) known as an immunosuppressive agent to dampen down the body's immune reaction. EG colonies were collected and treated with 0.25% trypsin-EDTA to achieve single cell suspension. About 1×106 cells in 200μl PBS were injected subcutaneously into axilla region of the chicks. After 5 weeks, chicks were sacrificed and emerging tissue materials were dissected. Tissues were fixed in 4% paraformaldehyde and embedded in paraffin. Sections (5μm) were placed on slides for HE and irnmunohistochemical staining. Results showed that chicken EG cells were capable of forming teratomas after subcutaneous injection into the immunosuppressed chicks. The amount of differentiated tissues varied among individual teratomas. Each teratoma contained a broad variety of tissues, including neural cells, cartilage, forming bone, adipocytes, blood vessels, smooth muscle, striated muscle and secretory epithelia. There was no teratoma formed in chicks without immunosuppression or no EG cells injection. The above results indicated that the immunodeficiency chick model could be made by fed with immunosuppressive agents cyclosporine, and the present study using this chick model showed that the chicken EG cells could form teratomas containing a variety of differentiated tissues and cell types, as well as some patches of undifferentiated cells in vivo. 3. Formation of EBs from chicken EG cells in vitroEG colonies in culture were picked up and dissociated into single cells, transferred onto gelatin-coated plates for a 30 min period to remove contaminating somatic cells and then transferred onto Ultra Low Attachment plates to allow their aggregation and prevent adherence to the plate. Usually about 106 EG cells were incubated in each 35 mm plate accompanied by the withdrawal of LIF and 2-mercaptoethanol from the medium. During this period, the cells grew into compact aggregates and became simple EBs within 2-5 days, especially more easily accompanied by 15% FCS and 2 mmol/L Gln, then turned to cystic EBs with the formation of a central cavity. Inner structure of EBs were observed by acridine orange staining or HE staining of the EBs sections on Day 7 and Day 17 of suspension culture respectively. HE staining analysis of EB sections showed heterogeneous structure with the ability to differentiate into multiple cell types, including neural tube like structure, vascular structures, connective tissues, and glandular-like cells. Furthermore, expression of the lineage-specific markers of three germ layers, including endoderm specific gene characterized by AFP, mesoblast specific gene characterized by Gata6, ectoderm specific gene characterized by Sox3 and the trophectoderm specific gene characterized by Cdx2 was detected in EBs, suggesting their differentiation potential in vitro. In addition, we demonstrated the expression profiles of pluripotency-associated markers, including chicken Oct4 homologue PouV (cPouV), chicken Nanog (cNanog) and Sox2 genes during the formation of EBs, which were required for the maintenance of pluripotency in ES cells. All of these genes were expressed in undifferentiated EG cells, and down regulated during the formation of EBs, suggesting their presumable effects in the inhibition of chicken EG cells differentiation. The above results demonstrate the formation potential of EG cells into EBs, which will facilitate the establishment of the differentiation model of EG cells in vitro.3. Multipledifferentiation regulation of chicken EG cells in vitroFor the generation of the neural cells, EBs were plated onto gelatin coated tissue culture plates at Day 4. Neural progenitors were induced in serum-free medium containing 10-6 M RA,2 mmol/L glutamine,5 mmol/L HEPES,25μg/ml insulin,100μg/ml transferrin, and 30 nmol/L sodium selenite for 2 days. Results showed that, RA treatment together with serum free culture condition significantly increased levels of neural specific gene expression. After additional 7 days in culture, cells possessing typical neuronal morphology were fixed and proved to be NSE, TH, ChAT, GFAP and CNP positive by immunocytochemical staining. RT-PCR analysis also showed that after induction, nestin was down-regulated, whereas NSE,β-tubulinⅢ, ChAT, TH, GFAP and CNP became positive, which displayed mature neuron, astrocyte and oligodendrocyte differentiation. To induce mature neural cells, these progenitors were continually cultured for 7 days. Adipogenic differentiation was induced by culture 7-day-old EBs in medium supplemented with 10% FCS,10μg/ml insulin,1μmol/L dexamethasone,0.2 mmol/L indomethacin for 10 days. The accumulation of lipid vacuoles in the cells was first detected 5 days after the addition of adipogenic induction medium and determined by staining for Oil Red O 10 days after induction. As differentiation progressed, expression of PPARγ, GLUT1 were significantly increased. Mature adipocytes marker gene lipoprotein lipase (LPL) was detected 10 days after induction, thereby confirming the mature adiogenic differentiation. Fetal chicken liver-derived cells were prepared as adherent cells from 7-day-old chicken embryos, after which 14-day-old EBs were plated onto the gelatin-coated culture dishes and incubated in the liver cells-conditioned medium. The AFP mRNA expression was detected in 14-day-old EBs. However, the expression of cytochrome P450 7a1 (CYP7A1), hepatocyte nuclear factor la (HNFla) and hepatocyte nuclear factor 4a (HNF4a), possible markers for embryonic endoderm-derived mature hepatocytes, were only observed after a co-culture with fetal liver cell derived conditioned medium. Immunocytochemical staining together with histochemical results demonstrated a high ratio of ALB and PAS positive cells 10 days after induction of hepatocyte differentiation, thus showing that co-culture of EG cells with liver cells-derived conditioned medium guided the differentiation toward mature and functional hepatocytes. We also investigated the effect of 5-azacytidine (5-aza) on EG cells to differentiate into cardiomyocytes. EBs were formed after 10 days with 5-aza in suspension culture, beating cell clusters were observed. Results showed that 5-aza (0～10μmol/L) promoted the production of beating cell clusters of EG cells in a dose-dependent manner. In response to 5μmol/L of 5-aza, EG cells expressed cardiac markers such as GATA-6, Nkx2.5, cardiac troponin T, a-actinin, MHC6, Desmin, ANF, CASQ, SMHC and SMA were up-regulated in a time-dependent manner after induction. Immunocytochemistry revealed the expression of smooth muscle SMA, sarcomeric a-actinin, cardiac troponin T and connexin 43 respectively.Through these studies we successfully isolated positive cells for chicken PGC-specific markers from genital ridges. The positive cells proliferated to form colonies on somatic cells during primary culture. Firmly packed colonies with ES cell-like morphology could be observed after subculture. These cells showed important features of pluripotent stem cells especially the expression of the pluripotency-associated genes cPouV, cNanog and Sox2 and form teratoma in vivo. After suspension culture, simple and cystic EBs were formed from single EG cells and 15% FCS and 2 mmol/L Gln could promote their formation. The expression of the lineage-specific markers of three germ layers and the expression profiles of cPouV, cNanog and Sox2 genes during the EBs development in vitro were also examined. Results showed that cPouV, cNanog and Sox2 were down-Tregulated during the formation of EBs, and the the lineage-specific markers were expressed in 14-day-old EBs, suggesting their presumable effects in their regulatory effects in maintaining the pluripotency during differentiation of chicken EG cells. The differentiation potency into mature neuron, glial cells, adipocytes, hepatocytes and cardiomyocytes of EBs fully provide evidence that, chicken PGC-derived EG cells possess the pluripotency to differentiate into precursor cell lineages of all three germ layers and then into mature cells after the directed induction methods in vitro, thus making them a potential experimented model for further study the cellular mechanisms of tissue-specific differentiation and regeneration, that will help to devise strategies for restoring function to damaged or diseased tissues in humans.