ABSTRACT
OBJECTIVE: This study was to examine the in vitro neural cell differentiation patterns of human embryonic stem (hES) cells following treatment of various neurotrophic factors [basic fibroblast growth factor (bFGF), retinoic acid (RA), brain derived neurotrophic factor (BDNF) and transforming growth factor (TGF)-alpha], particulary in dopaminergic neuron formation. METHODS: The hES cells were induced to differentiate by bFGF and RA. Group I) In bFGF induction method, embryoid bodies (EBs, for 4 days) derived from hES were plated onto gelatin dish, selected for 8 days in ITSFn medium and expanded at the presence of bFGF (10 ng/ml) for another 6 days followed by a final differentiation in N2 medium for 7, 14 and 21 days. Group II) For RA induction, EBs were exposed of RA (10-6 M) for 4 days and allowed to differentiate in N2 medium for 7, 14 and 21 days. Group III) To examine the effects of additional neurotrophic factors, bFGF or RA induced cells were exposed to either BDNF (10 ng/ml) or TGF-alpha (10 ng/ml) during the 21 days of final differentiation. Neuron differentiation and dopamine secretion were examined by indirect immunocytochemistry and HPLC, respectively. RESULTS: The bFGF or RA treated hES cells were resulted in similar neural cell differentiation patterns at the terminal differentiation stage, specifically, 75% neurons and 11% glial cells. Additionally, treatment of hES cells with BDNF or TGF-alpha during the terminal differentiation stage led to significantly increased tyrosine hydroxylase (TH) expression of a dopaminergic neuron marker, compared to control (p<0.05). In contrast, no effect was observed on the rate of mature neuron (NF-200) or glutamic acid decarboxylase-positive neurons. Immunocytochemistry and HPLC analyses revealed the higher levels of TH expression (20.3%) and dopamine secretion (265.5+/-62.8 pmol/mg) in bFGF and TGF-alpha sequentially treated hES cells than those in RA or BDNF treated hES cells. CONCLUSION: These results indicate that the generation of dopamine secretory neurons from in vitro differentiated hES cells can be improved by TGF-alpha addition in the bFGF induction protocol.
Subject(s)
Humans , Brain-Derived Neurotrophic Factor , Cell Differentiation , Chromatography, High Pressure Liquid , Dopamine , Dopaminergic Neurons , Embryoid Bodies , Embryonic Stem Cells , Fibroblast Growth Factor 2 , Fibroblast Growth Factors , Gelatin , Glutamic Acid , Immunohistochemistry , Nerve Growth Factors , Neuroglia , Neurons , Transforming Growth Factor alpha , Transforming Growth Factors , Tretinoin , Tyrosine 3-MonooxygenaseABSTRACT
No abstract available.
Subject(s)
Humans , Cryopreservation , Embryonic Stem Cells , VitrificationABSTRACT
OBJECTIVE: This study was to examine in vitro neural cell differentiation pattern of the genetically modified human embryonic stem cells expressing tyrosine hydroxylase (TH). MATERIALS AND METHODS: Human embryonic stem (hES, MB03) cell was transfected with cDNAs cording for TH. Successful transfection was confirmed by western immunoblotting. Newly transfected cell line (TH#2/MB03) was induced to differentiate by two neurogenic factors retinoic acid (RA) and b-FGF. Exp. I) Upon differentiation using RA, embryoid bodies (EB, for 4 days) derived from TH#2/MB03 cells were exposed to RA (10-6 M)/AA (5x10-2 mM) for 4 days, and were allowed to differentiate in N2 medium for 7, 14 or 21 days. Exp. II) When b-FGF was used, neuronal precursor cells were expanded at the presence of b-FGF (10 ng/ml) for 6 days followed by a final differentiation in N2 medium for 7, 14 or 21 days. Neuron differentiation was examined by indirect immunocytochemistry using neuron markers (NF160 & NF200). RESULTS: After 7 days in N2 medium, approximately 80% and 20% of the RA or b-FGF induced Th#2/MB03 cells were immunoreactive to anti-NF160 and anti-NF200 antibodies, respectively. As differentiation continued, NF200 in RA treated cells significantly increased to 73.0% on 14 days compared to that in b-FGF treated cells (53.0%, p<0.05), while the proportion of cells expressing NF160 was similarly decreased between two groups. However, throughout the differentiation, expression of TH was maintained (~90%). HPLC analyses indicated the increased levels of L-DOPA in RA treated genetically modified hES cells with longer differentiation time. CONCLUSION: These results suggested that a genetically modified hES cells (TH#2/MB03) could be efficiently differentiated in vitro into mature neurons by RA induction method.
Subject(s)
Humans , Antibodies , Blotting, Western , Cell Differentiation , Cell Line , Chromatography, High Pressure Liquid , DNA, Complementary , Embryoid Bodies , Embryonic Stem Cells , Immunohistochemistry , Levodopa , Neurons , Transfection , Tretinoin , Tyrosine 3-Monooxygenase , TyrosineABSTRACT
OBJECTIVE: This study was to establish a reproducible differentiation system from the parthenogenetic mouse embryonic stem (P-mES02) cells into functional cardiomyocytes like as in vitro fertilization mouse embryonic stem (mES01) cells. MATERIALS AND METHODS: To induce differentiation, P-mES02 cells were dissociated and aggregated in suspension culture environment for embryoid (EB) formation. For differentiation into cardiomyocytes, day 4 EBs were treated with 0.75% dimethyl sulfoxide (DMSO) for another 4 days (4-/4+) and then were plated onto gelatin-coated dish. Cultured cells were observed daily using an inverted light microscope to determine the day of contraction onset and total duration of continuous contractile activity for each contracting focus. This frequency was compared with the results of DMSO not treated P-mES02 group (4-/4-) and mES01 groups (4-/4+ or 4-/4-). For confirm the generation of cardiomyocytes, beating cell masses were treated with trypsin-EDTA, dispersed cells were plated onto glass coverslips and incubated for 48 h. Attached cells were fixed using 4% paraformaldehyde and incubated with specific antibodies (Abs) to defect cardiomyocytes (anti-sarcomeric alpha-actinin Ab, 1: 100; anti-cardiac troponin I Ab, 1: 2000) for 1 h. And the cells were finally treated with FITC or TRITC labelled 2nd Abs, respectively, then they were examined under fluorescence microscopy. RESULTS: Rhythmically contracting areas in mES01 or P-mES02 cells were firstly appeared at 9 or 10 days after EBs plating, respectively. The highest cumulative frequency of beating EBs was not different in both treatment groups (mES01 and P-mES02, 4-/4+) with the results of 61.3% at 13 days and 69.8% at 15 days, respectively. Also the contracting duration of individual beating EBs was different from minimal 7 days to maximal 53 days. However, DMSO not treated groups (mES01 and mES02,4-/4-) also had contracting characteristics although their frequency was a few compared to those of DMSO treated groups (6.0% and 4.0%). Cells recovered from the spontaneously contracting areas within EBs in both treated groups were stained positively with muscle specific anti-sarcomeric alpha-actinin Ab and cardiac specific anti-cardiac troponin I Ab. CONCLUSION: This study demonstrated that the P-mES02 cell-derived cardiomyocytes displayed similarly structural properties to mES01 cell-derived cardiomyocytes and that the DMSO treatment enhanced the cardiomyocytes differentiation in vitro.
Subject(s)
Animals , Mice , Actinin , Antibodies , Cells, Cultured , Dimethyl Sulfoxide , Embryonic Stem Cells , Fertilization in Vitro , Fluorescein-5-isothiocyanate , Glass , Microscopy, Fluorescence , Myocytes, Cardiac , Troponin IABSTRACT
No abstract available.
Subject(s)
Animals , Mice , Coculture Techniques , Fertilization in VitroABSTRACT
OBJECTIVE: This study was to establish the human embryonic stem (ES) cells derived from frozenthawed blastocyst stage embryo that were destined to be discarded after five years in routine human IVF-ET program. METHODS: Frozen-thawed and survived human blastocysts were treated by immunosurgery, and recovered ICM cells were cultured onto STO feeder cell layer and ICM colony was subcultured by mechanical dissociation into clumps. To identify ES cell, alkaline phosphatase staining and expression of Oct4 in replated ICM colonies were examined. Also, to examine the possibility of ES cell differentiation, retinoic acid (RA), basic fibroblast growth factor (b-FGF), nerve growth factor (NGF) were added in culture medium. In addition, to classify the specific cell type, differentiated cells were stained by indirect immunocytochemistry. RESULTS: One ICM colony recovered from frozen-thawed six blastocysts was subcultured, continuously replated during 40 passage culture duration without differentiation. Subcultured colonies were strong positively stained by alkaline phophatase. When the expression of Oct4 in cultured ES colony was examined, Oct4b type is more clearly indicated than Oct4a one although there was not detected in embryoid body or differentiated cells. In differentiated cardiomyocytes from ES colony, cells were beaten regularly (60 times/min). In differentiated neural cells from ES colony, neurofilament (NF) 200 kDa protein, microtubule associated protein (MAP) 2 and beta-tubulin of specific marker in neurons, glial fibrillary acidic protein (GFAP) of specific marker in astrocytes and galactocelebrocide (GalC) of specific marker in oligodendrocytes were confirmed by indirect immunocytochemistry. Also, muscle cells were detected by indirect immunocytochemistry. In addition, ES colonies can be successfully cryopreserved. CONCLUSION: This study suggested that establishment of human ES cells can be successfully derived from frozen-thawed blastocysts that were destined to be discarded, and obtained specific cell types (cardiomyocytes, neurons and muscle cells) through the in vitro differentiation procedures of ES cells.