ABSTRACT
To establish a method of directional differentiation and efficient production of neurons from embryonic stem cells (ES cells) in vitro, based on the 4-/4+ protocol described by Bain, a new method was established to induce ES cells differentiating into neurons by means of three-step differentiation using all-trans retinoic acid (ATRA) combined with astrocyte-conditioned medium (ACM) in Vitro. The totipotency of ES cells was identified by observation of cells' morphology and formations of teratoma in immunocompromised mice. The cells' differentiation was evaluated continuously by the detection of the specific cellular markers of neural stem cells, neurons and astrocytes, including nestin, NSE and GFAP using immunohistochemistry assay. The NSE positive cells' ratio of the differentiated cells was determined by flow cytometry. It was found that the transparent circular clusters surrounding embryoid bodies induced with combining induction protocol formed just after 24 h and gradually enlarged later. This phenomenon could not be observed in EBs induced only by ATRA. The NSE positive cells' ratio in the cells induced with ATRA and ACM was higher than that of the cells induced by ATRA at different time points of differentiation, and finally reached up to 73.5% among the total differentiated population. It was concluded that ES cells could be induced into neurons with high purity and yield by means of inducing method combining with ATRA and ACM.
Subject(s)
Astrocytes/cytology , Cell Differentiation , Cells, Cultured , Embryo, Mammalian , Neurons/cytology , Stem Cells/cytology , Tretinoin/pharmacologyABSTRACT
To establish a method of directional differentiation and efficient production of neurons from embryonic stem cells (ES cells) in vitro, based on the 4-/4+ protocol described by Bain, a new method was established to induce ES cells differentiating into neurons by means of three-step differentiation using all-trans retinoic acid (ATRA) combined with astrocyte-conditioned medium (ACM) in Vitro. The totipotency of ES cells was identified by observation of cells' morphology and formations of teratoma in immunocompromised mice. The cells' differentiation was evaluated continuously by the detection of the specific cellular markers of neural stem ceils, neurons and astrocytes,including nestin, NSE and GFAP using immunohistochemistry assay. The NSE positive cells' ratio of the differentiated cells was determined by flow cytometry. It was found that the transparent circular clusters surrounding embryoid bodies induced with combining induction protocol formed just after 24 h and gradually enlarged later. This phenomenon could not be observed in EBs induced only by ATRA. The NSE positive cells' ratio irthe cells induced with ATRA and ACM was higher than that of the cells induced by ATRA at different time points of differentiation, and finally reached up to 73.5 % among the total differentiated population. It was concluded that ES cells could be induced into neurons with high purity and yield by means of inducing method combining with ATRA and ACM.
ABSTRACT
Objective To study the effects of the mistura of leech,peach pit,huangqi and angelica for liver fibrosis of mice .Methods The mice (Kunming) were divided into four groups: ① the fibrosis model group: infected with Schistosoma japonicum without treatment (n=8);② mistura treated group: the infected mice treated with the mistura above mentioned (n=12);③ colchicine group: the infected mice treated with colchicine (n=7) and ④ normal control group(n=7). Results ① SOD increased and MDA decreased (P