ABSTRACT
Objective To evaluate the in vitro differentiation of human amniotic epithelial cells (hAECs ) into hepatocyte-like cells. Methods Combined approach of dexamethasone, HGF, IGF and other cytokines were used to induce the differentiation of hAECs into hepatocyte-like cells. The induction lasted 2 weeks. During the induction, the expression of albumin ALB, CYP1A1, CYP1A2, IGFR, c-met and key functional genes related to liver cells as well as transcription factors HNF3, HNF4 and C/EBPa were monitored by RT-PCR. Time dependent changes of the surface marker colony ALB, AFP and CK18 were analyzed by cell flow cytometry. Results After the 2 week induction, the expressions of liver hepatocyte-like cell functional genes such as albumin, CYP1A1, CYP1A2, c-met, and transcription factors such as HNF3, HNF4, C/EBPa and HNF1 were observed. Six days after the induction, hAECs mainly were stained AFP+, and the positive rate was (15.1±2.1)%. While 10 days after the induction, part of the hAECs showed AFP+/ALB+ (6.5±1.4)%; and on 14th day, hAECs only showed ALB+, and the rate was (13.9±2.3)%. ALB+ cell increase indicated a gradual functional maturation from the hAECs to hepatocyte-like cells. Similaritly, the number of CK18+ cells in the whole population was also increased: On 10th day, the rate was (16.1±1.2)%; on 14th day, that was (21.3±4.6)%, which proved the above hypothesis of the trandifferentiation. By extending the induction time, the expression of functional genes increased gradually, and a maturing process of hAECs was detected by cell surface markers. Conclusion The differentiation of hAECs induced in vitro has the characteristics of hepatocyte-like cells.
ABSTRACT
Objective To establish a new method for inducing the primordial germ cells to differentiate into hepatocyte-like in vitro.Methods The primordial germ cells(PGCs) from the gonadal ridges of the mouse embryos of 13 days postcoitum from Kunming pregnant mice were cultured in vitro.Then embryonic hepatocytes enclosed in microcapsule and liver tissue extract of newborn mice were added into medium to co-culture with PGCs for committed differentiation.Albumin(ALB) and ?-1-antitrypsin(AAT) were assayed by immunocytochemistry.Results The morphology of cells differentiated from PGCs likes star or ovum,the ALB and AAT immune positive expression were detected in those differentiated cells.The ratio of positive cells was above 70% in 2 weeks.Conclusion Microenvironment of embryonic hepatocyte microcapsules and liver tissue extract could effectively induce PGCs to differentiate into hepatocytes.
ABSTRACT
Objective To investigate the function of amnion endothelial cell in the differentiation of human umbilical cord blood stem cells into dopaminergic neurons.Methods Primary human amnion endothelial cells were separated and cultured in vitro;the conditioned medium(CM) was prepared through high speed centrifugation.The cord blood mesenchymal stem cells of P_1 passage were induced by the conditioned medium,and the mophology of cells was observed under the inverted phase contrast microscope.The expression of tyrosine hydroxylase(TH)and dopamine transportor(DAT) of the induced cord blood mesenchymal stem cells were detected by immunocytochemistry staining method and immunoblotting(Western blotting).Results The masculine rate of TH and DAT of the cord blood mesenchymal stem cells of P_1 passage which were induced by amnion endothelial cells conditioned medium was higher than that of the control group,with a significant difference(P
ABSTRACT
AIM: To investigate the inducing action of retinal cells of rat neonates on the adult rat bone marrow stromal cells (BMSCs).METHODS: The full marrow from adult Wistar rat were cultured and passaged repeatedly to harvest the pure BMSCs. In vitro, the BMSCs were induced by retinal cells of rat neonates in transwell.The morphological changes of the BMSCs were observed under phase contrast microscope, the specific markers of the induced cells were ~identified immunocytochemically with nestin, Map-2, neuron-specific enolase(NSE),neurofilament(NF), Thy 1.1 and glial fibrillary acidic protein(GFAP) antibodies. The mRNA of nestin, NF, NSE, Thy 1.1 and Ran were detected in the induced cells by RT-PCR.RESULTS: The BMSCs were induced by reinal cells of rat neonates into the retina-like neurons, which showed the typical neural morphology and expression of the specific retinal neural antigens.CONCLUSION: The BMSCs from the adult rat can be induced into retina-like cells by retinal cells of rat neonates. [
ABSTRACT
Objective To explore the optimal condition of direct differentiation into dopaminergic neurons of embryonic stem cells in serum-free and feeder layer cell free medium. Methods We used the method of phase induction to culture embryonic stem cells. At first, embryonic stem cells were cultured in the serum-free medium with bFGF and LIF so as to realize the direct differentiation from embryonic stem cells to neural precursors. Differentiated cells were determined by nestin immunocytochemical staining. On this basis, we transferred embryonic stem cells to the B27 serum-free medium with IL-1 so as to realize the direct differentiation from neural precursors to dopaminergic neurons. Differentiated cells were determined by TH immunocytochemical staining. Results Approximately 85 percent of cell masses were nestin immuno-positive. The differentiation ratio of dopaminergic neurons was 13%, which increased significantly in comparison with natural differentiation ratio of dopaminergic neurons.Conclusion Without serum and feeder layer cell, we can induce embryonic stem cells to differentiate into dopaminergic neurons effectively by adding different growth factors at different phases, which makes the inductive processes more easily.
ABSTRACT
Objective To explore the inductive effect of striatal tissue on mouse embryonic stem cells and further analyse the cell source and inductive pattern of this inductive effect. Methods We employed striatal extracts、conditioned medium of striatal astrocytes and conditioned medium of striatal neurons to induce embryonic stem cell to differentiate directly. The differentiated cells were evaluated by morphological observation and TH immunocytochemical staining method. We also performed a quantitative analysis of the results. Results Striatal extracts and conditioned medium of striatal astrocytes had obvious inductive effect on embryonic stem cell.Percentages of three groups were 15%,15.2% and 3% respectively. Conclusions The astrocytes in striatum might have an inductive effect on dopaminergic neuronal differentiation of embryonic stem cells.The determination of inductive factor will be our next research aim.;
ABSTRACT
control group.Conclusion Compared with other conditions,the striatum has a more distinct inductive effect on HUCB stem cells to differentiate into dopaminergic neurons,and the inductive effect mainly comes from the astrocytes in the striatum.