Derivation Of Rat Embryonic Cell Lines And Their Potential For Neural Differentiation

Rat is a valuable model for pharmacological and physiological studies. So establishing a stable rat embryonic stem cells (rES) differentiation system is necessary for analysis of early development and gene function of rES, and production of animal models for human disease by gene targeting. Although germline-competent rat embryonic stem (rES) cell lines have been established successfully and the molecular networks that maintain the self-renewing, undifferentiated state of rES cells have also been well uncovered, still little is known about the methods and mechanisms during differentiation of these authentic pluripotent stem cells. Until now,there is no report about differentiation of rat neural stem cell (rNS) from rES cells. The aim of this study is to test the capacity of rES cells in differentiating to different subtypes of neural cells in vitro. It may provide a valuable platform for studying the neural lineage development of rat, pharmacological testing, as well as serving as a powerful tool for exploring the genetic factor-linked pathogenesis in some neurodegenerative disorders. The major findings are as follows:1) The brain tissue cells from E14.5DA-rat were cultured in serum-free conditions (N2B27with bFGF and mEGF) in monolayer culture and could formed floating spheres. The cells expressed multiple NS-spccific markers, including Nestin, Pax6, and Olig2. Furthermore, most rNS stained positive for proliferative cell marker-Mki-67, and about0.8%of rNS spontaneously differentiated to Doublecortin (Dcx) positive neural precursors.2) After about10days in vitro differentiation, Thecells stained positive for neuron specific marker-Tuj1, GFAP and04, representing the generation of neurons, astrocytes and oligodendrocytes. After a three-week in vitro differentiation, rES cell-derived neurons also expressed MAP2-another neuronal specific marker. In addition, we could observe Tuj1-positive neurons co-labeled with GABA or ChAT, suggesting that rES cell-derived NS cells could differentiate to different subtypes of neurons in vitro.3) Total of ten rES cell lines were derived from rat blastocysts.The rES cell colonies displayed typical embryonic stem cell-like morphologies and stained positive for alkaline phosphatase (AP). Immunostaining results further confirmed the expression of pluripotent stem cell markers Oct4, Sox2, and SSEA1. The rES cells had been expanded for over30passages without overt differentiation and Karyotype analysis was also carried out on rat ES cells, and results showed a predominantly normal diploid.4) rEBs were cultured in modified culturing medium. Abundant homogeneous rEBs emerged. A majority of rES cells died during the initial stage of differentiation under removal of LIF from normal ES cell medium. 5) rEBs expressed ectodermal, endodermal and mesodermal markers markers-Nestin, Afp, sox17, Gata6and Flkl. After adherent differentiation of6-15d, rEB could differentiate into regularity pulsating myocardial cells. It was suggested that rEB has capacity to differentiate into three germ layers in vitro.6) After about11days induction in N2B27medium, we carefully picked up the neural tube-like rosettes by mouth pipette.The small neuroepithelial cell aggregates rapidly attached to the surface, and proliferated in adherent monolayer cultures. The cells expressed multiple NS-specific markers, including Nestin, Sox2, Pax6, and Olig2. Furthermore, most rNS cells stained positive for proliferative cell marker-Mki-67, and about0.8%of rNS cells spontaneously differentiated to Doublecortin (Dcx) positive neural precursors.7) After about10d in vitro differentiation, the cells stained positive for neuron specific marker-Tujl, GFAP and O4-positive cells, representing the generation of neurons, astrocytes and oligodendrocytes. After a three-week in vitro differentiation, rES cell-derived neurons also expressed MAP2-another neuronal specific marker. In addition, we could observe Tuj1-positive neurons co-labeled with GABA or TH, suggesting that rES cell-derived rNS cells could differentiate to different subtypes of neurons in vitro.Based on the above, the differentiation system of neural stem cell of DA5-3rES cells was feasibility and effectiveness.