Human embryonic stem cells (hESCs) are cells with unlimited self-renewing property and differentiation potential mainly derived from the inner cell mass (ICM) of human blastocyst. Because of the remarkable developmental potential and proliferative capacity, human embryonic stem cells hold great promise for future use in various research areas, such as human developmental biology and cell-based therapy. This review expounds the culture system of human embryonic stem cells and the induced differentiation technology in vitro.
Cell Culture Techniques ; Cell Differentiation ; drug effects ; physiology ; Cells, Cultured ; Embryonic Stem Cells ; cytology ; Humans

OBJECTIVETo analyze the influence of retinoic acid (RA) on the undifferentiated state and EB formation abilities of human embryonic stem cells.

METHODSThe biological characteristics of H9 ESCs after RA treatment were characterized by real-time PCR, MTS proliferation assay and immunofluorescence staining. The expression of three germ layers markers, osteogenic differentiation markers and adipogenic differentiation markers in H9-differentiated embryoid bodies (EBs) with RA treatment were quantified by real time PCR.

RESULTSThe proliferation of H9 ESCs in the early logarithmic growth phase was accelerated by RA treatment. In addition, RA induced differentiation of H9 ESC coupled with morphology changes, decreased expression of undifferentiated markers Oct4, Nanog, Sox2 and OCT4 mRNA binding protein Lin28 at mRNA level, and reduced expression of Oct4 at protein level. RA induced formation of cavities in EBs. Real time PCR results showed that the expressions of ectodermal markers: NeuroD1, Noggin; mesodermal markers: Brachyury, Twist and endodermal markers: AFP, GATA-4 were significantly increased (P < 0.05), especially for AFP (P < 0.01), by RA treatment in a dose-dependent manner. In addition, the expression of adipogenic differentiation marker C/EBPalpha was increased while the osteogenic differentiation marker OPN was decreased in EBs after RA treatment for 5 days.

CONCLUSIONSHigh concentrations of RA induced the loss of stemness in H9 ESCs and excessive differentiation in EBs, and damaged the balance between osteogenic and adipogenic differentiation during early EB differentiation, which may be relevant to the congenital malformations.

Cell Differentiation ; drug effects ; Cell Proliferation ; drug effects ; Cells, Cultured ; Embryonic Stem Cells ; cytology ; drug effects ; Humans ; Tretinoin ; pharmacology

OBJECTIVETo explore the effects of trichloroethylene (TCE) on cardiac developmental differentiation in human embryonic stem cells.

METHODSIn this study, based on the human embryonic stem cells in vitro cardiac differentiation assay, we investigated the potential effect of TCE exposure on the cardiac toxicity in embryo development. Human embryonic stem cells were treated with TCE at different concentrations of 100 ppb, 1 ppm, and 10 ppm and dimethyl sulfoxide(DMSO) treated as control. The MTT assay was performed to examine the cytoplasmic toxicity of TCE exposure. The beating percentages were recorded and the expression of cardiac specific gene was evaluated by PCR or flow cytometry. Also, real time PCR was performed to verify the micro array analysis on the expression level changes of genes which were involved in the Ca2+ signal pathways.

RESULTSCompared with the control group, there was no significant difference in cell viability when cells were treated with TCE at the concentrations of 100 ppb, 1 ppm, and 10 ppm. However, TCE could inhibit the expression of cTnT protein in a concentration-dependant manner. And the most interestingly, TCE significantly inhibited the cardiac differentiation characterized by the decrease beating percentages. Genes involved in Ca2+ signaling pathway were severely disrupted by TCE.

CONCLUSIONTCE inhibited the cardiac specific differentiation of human embryonic stem cell and at the meanwhile the genes responsible for Ca2+ signaling pathway were severely disrupted, which could contribute the severe effects of TCE cardiotoxicity.

Calcium Signaling ; Cell Differentiation ; Cells, Cultured ; Embryonic Development ; Embryonic Stem Cells ; cytology ; drug effects ; Heart ; embryology ; Humans ; Trichloroethylene ; toxicity

Effects of lysophosphatidic acid (LPA), an extracellular phospholipid signal, on the proliferation of rat embryonic neural stem cells (NSCs) and their differentiation into microtubule-associated protein 2 (MAP2)-positive and choline acetyltransferase (ChAT)-positive, i.e. cholinergic-committed neurons, were observed in vitro by [(3)H]-thymidine incorporation, immunocytochemistry, Western blot and other techniques. The results showed that: (1) Lower concentrations of LPA (0.01~1.0 mumol/L) dose-dependently enhanced the uptake of [(3)H]-thymidine by NSCs cultured in specific serum-free medium, indicating a significant promotive action of LPA on the proliferation of NSCs. (2) After fetal bovine serum which induces and commences the differentiation of NSCs, was used in the medium, the lower concentrations of LPA increased the percentages of both MAP2- and ChAT-immunoreactive neurons, with a peak at 0.1 mumol/L LPA in two cases. (3) The promotive effects of LPA on the differentiation of MAP2- and ChAT-positive neurons were also supported by the up-regulation of the expressions of both MAP2 and ChAT proteins detected by Western blot. (4) At the early phase of differentiation of NSCs, the cell migration and neurite extension were enhanced significantly by lower dosages of LPA under phase-contrast microscope. These results suggest that LPA within certain lower range of concentrations promotes the proliferation of NSCs and their differentiation into unspecific MAP2-positive and specific cholinergic-committed neurons, and also strengthens the migration and neurite extension of the newly-generated neuronal (and also glial as reported elsewhere) progenitors.
Animals ; Cell Differentiation ; drug effects ; Cell Proliferation ; drug effects ; Cells, Cultured ; Cholinergic Neurons ; cytology ; Embryonic Stem Cells ; drug effects ; Lysophospholipids ; pharmacology ; Neural Stem Cells ; drug effects ; Rats

OBJECTIVETo investigate the effect of propofol on the proliferation and differentiation of rat embryonic neural stem cells in vitro.

METHODSEmbryonic neural stem cells of fetal Wistar rats (gestational age of 14-16 days) in primary culture, after identification for nestin expression, were divided into control group, introlipid group, and propofol groups (treated with propofol at the doses of 5, 25, 50, and 100 µmol/L). The changes in the proliferation of the embryonic neural stem cells after the treatments were observed using Brdu incorporation assay. In the course of induced differentiation of the embryonic neural stem cells, 50 µmol/L propofol was added in the cells to assess its impact on the differentiation of the cells by immunohistochemical detection of NeuN and GFAP expressions.

RESULTSMore than 95% of the embryonic neural stem cells in primary culture were Nestin-positive. The percentages of Brdu-positive cells showed no significant changes after treatment with different concentrations of propofol, whereas the addition of 50 µmol/L propofol resulted in a significant increase of NeuN-positive cell percentage to (23.1∓0.9)% as compared with that of (13.4∓0.8)% in the control group (P<0.05) without affecting the GFAP-positive cells.

CONCLUSIONClinically relevant doses of propofol have no obvious effect on the proliferation of rat neural stem cells cultured in vitro, but can induce their differentiation into neuron-like cells.

Animals ; Cell Differentiation ; drug effects ; Cell Proliferation ; drug effects ; Cells, Cultured ; Embryonic Stem Cells ; cytology ; Female ; Neural Stem Cells ; cytology ; Pregnancy ; Propofol ; pharmacology ; Rats ; Rats, Wistar

The objective of this study was to explore the effects of BMP-4 and VEGF on the development of primary hematopoietic stem cells during the differentiation of embryonic stem cells (ESCs) into embryoid body (EB). Murine E14 ESCs were seeded into semisolid methylcellulose-based medium for EB formation. According to added or not cytokines, experiments were divided into: (1) group of spontaneous differentiation without cytokine as control; (2) group of BMP-4 in different concentrations (0, 5, 15, 25 and 50 ng/ml); (3) group of BMP-4 combined with VEGF; (4) group of VEGF alone. EBs were collected on days 3, 6, 9, 12, 15, and the proportion of Flk-1(+) cells were assayed by flow cytometry. The results showed that in the different BMP-4 concentration groups, the proportions of Flk-1(+) cells were significantly different, and it reached the peak values in 25 ng/ml BMP-4 group as 6.51 +/- 1.02% at day 3 and 7.70 +/- 1.12% at day 6 respectively, which were statistically higher than those in control group without-BMP-4 and in 5 ng/ml BMP-4 group (p < 0.05). When BMP-4 was used in combination with VEGF, Flk-1(+) cells went to peak proportion value at day 9 as 27.53 +/- 8.14%, which was statistically higher than that in spontaneous differentiation group as 8.77 +/- 2.35% (p < 0.05) and VEGF treatment group as 11.21 +/- 2.23% (p < 0.05). It is concluded that BMP-4 in combination with VEGF can promote Flk-1(+) cells genesis during EB formation in vitro, which provides experimental evidence for researches on directed differentiation of ESCs into hematopoietic stem cells simulating the microenvironment in vivo.
Animals ; Bone Morphogenetic Protein 4 ; pharmacology ; Cell Differentiation ; drug effects ; Cells, Cultured ; Embryonic Stem Cells ; cytology ; drug effects ; Hematopoietic Stem Cells ; cytology ; Mice ; Vascular Endothelial Growth Factors ; pharmacology

BACKGROUNDHepatocyte transplantation has been proposed as an alternative to whole-organ transplantation to support many forms of hepatic insufficiency. Unfortunately, the lack of donor livers makes it difficult to obtain enough viable human hepatocytes for hepatocyte-based therapies. Therefore, it is urgent to find new ways to provide ample hepatocytes. Induced pluripotent stem (iPS) cells, a breakthrough in stem cell research, may terminate these hinders for cell transplantation. For the promise of iPS cells to be realized in liver diseases, it is necessary to determine if and how efficient they can be differentiated into functional hepatocytes.

METHODSIn this study, we directly compared the hepatic-differentiation capacity of mouse iPS cells and embryonic stem (ES) cells with three different induction approaches: conditions via embryonic body (EB) formation plus cytokines, conditions by combination of dimethyl sulfoxide and sodium butyrate and chemically defined, serum free monolayer conditions. Among these three induction conditions, more homogenous populations can be promoted under chemically defined, serum free conditions. The cells generated under these conditions exhibited hepatic functions in vitro, including glycogen storage, indocynine green (ICG) uptake and release as well as urea secretion. Although efficient hepatocytes differentiation from mouse iPS cells were observed, mouse iPS cells showed relatively lower hepatic induction efficiency compared with mouse ES cells.

RESULTSMouse iPS cells would be efficiently differentiated into functional hepatocytes in vitro, which may be helpful in facilitating the development of hepatocytes for transplantation and for research on drug discovery.

CONCLUSIONWe demonstrate that mouse iPS cells retain full potential for fetal liver development and describe procedures that facilitates the efficient generation of highly differentiated human hepatocyte-like cells from iPS cells in vitro.

Animals ; Butyrates ; pharmacology ; Cell Differentiation ; drug effects ; Cells, Cultured ; Cytokines ; pharmacology ; Embryonic Stem Cells ; cytology ; drug effects ; Hepatocytes ; cytology ; drug effects ; metabolism ; Induced Pluripotent Stem Cells ; cytology ; drug effects ; Mice ; Reverse Transcriptase Polymerase Chain Reaction

OBJECTIVETo set up a platform for phenotype-based primary screening of drug candidates promoting neuronal subtype differentiation in embryonic stem cells (ES) with light microscope.

METHODSHanging drop culture 4-/4+ method was employed to harvest the cells around embryoid body (EB) at differentiation endpoint. Morphological evaluation for neuron-like cells was performed with light microscope. Axons for more than three times of the length of the cell body were considered as neuron-like cells. The compound(s) that promote neuron-like cells was further evaluated. Icariin (ICA, 10(-6)mol/L) and Isobavachin (IBA, 10(-7)mol/L) were selected to screen the differentiation-promoting activity on ES cells. Immunofluorescence staining with specific antibodies (ChAT, GABA) was used to evaluate the neuron subtypes.

RESULTSThe cells treated with IBA showed neuron-like phenotype, but the cells treated with ICA did not exhibit the morphological changes. ES cells treated with IBA was further confirmed to be cholinergic and GABAergic neurons.

CONCLUSIONPhenotypic screening with light microscope for molecules promoting neuronal differentiation is an effective method with advantages of less labor and material consuming and time saving, and false-positive results derived from immunofluorescence can be avoided. The method confirms that IBA is able to facilitate ES cells differentiating into neuronal cells, including cholinergic neurons and GABAergic neurons.

Animals ; Cell Differentiation ; drug effects ; physiology ; Cell Line ; Drug Evaluation, Preclinical ; methods ; Embryoid Bodies ; cytology ; Embryonic Stem Cells ; cytology ; Mice ; Nerve Regeneration ; drug effects ; Neurons ; cytology ; Phenotype

The aim of this research is to find an effective cardiomyocyte-induced method derived from porcine amniotic fluid stem cells (pAFS). For cardiac differentiation, the cells were formed embryoid bodies (EBs) firstly, then cultured in induced-medium including 5-azacytidine (5-aza) and vitamin C (Vc). We detected the specific markers of cardiomyocyte by immunocytochemistry, RT-PCR and transmission electron microscope. The results showed that some embryoid bodies beat rhythmically after 10 days of induction. Furthermore, analysis of t test revealed that the percentage of beating cardiomyocyte-like cell clusters was highest (33%) when induction using 0.1 mmol/L Vc and 5 micromol/L 5-aza. Immunocytochemistry analysis demonstrated that cardiomyocyte-like cell clusters expressed alpha-actin, Tnni3. RT-PCR analysis also illustrated that TbX5, Gata4, alpha-MHC and Tnni3 were expressed positive in cardiomyocyte-like cell clusters. Especially, we observed basic structures of myocardium, such as myofilament, glycogen granule and so on by transmission electron microscope. In conclusion, 5-azacytidine and vitamin C could promote differentiation of pAFS into myocardium.
Amniotic Fluid ; cytology ; Animals ; Ascorbic Acid ; pharmacology ; Azacitidine ; pharmacology ; Cell Differentiation ; drug effects ; Cells, Cultured ; Embryoid Bodies ; Embryonic Stem Cells ; cytology ; Female ; Myocytes, Cardiac ; cytology ; Swine

Animals ; Cell Differentiation ; drug effects ; Cell Transplantation ; Cells, Cultured ; Disease Models, Animal ; Drug Evaluation, Preclinical ; Embryonic Stem Cells ; cytology ; Humans ; Regeneration ; drug effects