scholarly journals Retaining mTeSR1 Medium during Hepatic Differentiation Facilitates Hepatocyte-Like Cell Survival by Decreasing Apoptosis

2018 ◽  
Vol 51 (4) ◽  
pp. 1533-1543
Author(s):  
Jian Hou ◽  
Yan Long ◽  
Bo Hu ◽  
Shaojie Huang ◽  
Guangtao Xu ◽  
...  
Keyword(s):  
Flow Cytometry ◽  
Growth Factor ◽  
Ips Cells ◽  
Glycogen Storage ◽  
Hepatic Cell ◽  
Hepatic Differentiation ◽  
Differentiated Cells ◽  
Urea Production ◽  
Cell Induction ◽  
H1 Cells

Background/Aims: Hepatocyte-like cells derived from human pluripotent stem cells could be an important cell source for hepatocyte transplantation. The present study investigated the effect of retaining mTeSR1 medium during hepatic differentiation on hepatocyte-like cells in vitro. Methods: Human embryonic stem cell line H1 were treated with activin A and bone morphogenetic protein 4 (BMP4) for definitive endoderm (DE) cell induction and subsequently treated with BMP2 and fibroblast growth factor 4 (FGF4) for early hepatic cell induction. Hepatocyte growth factor (HGF) and fibroblast growth factor (KGF) were added for early hepatic cell expansion and then mixed with oncostatin-M for maturation. During DE induction, 0%, 25%, 50% and 75% concentrations of mTeSR1 medium were separately added for early hepatic induction and expansion. For optimization, the expression levels of SRY-related HMG-box 17 (SOX17) and forkhead box A2 (FOXA2) at day 4, alpha fetoprotein (AFP) and hepatocyte nuclear factor 4α (HNF4α) at day 15, and albumin (ALB) at day 25 were quantified in differentiated cells by qRT-PCR. The ALB-positive cell proportion was measured by flow cytometry. Functional tests including ALB secretion and indocyanine green (ICG) angiography uptake and release by ELISA, urea production by urea assay kit, and glycogen storage ability by periodic acid Schif reaction (PAS) staining were performed in the differentiated cells. The induced pluripotent stem (iPS) cells were used to examine whether the optimized method was suitable for differentiating iPS cells. DE and hepatic markers were detected by immunostaining, and functional testing was performed as described above. Flow cytometry with an Annexin V-FITC apoptosis detection kit and fluorescence microscopy with Hoechst 33258 were used to analyze apoptosis in differentiated cells derived from H1 cells. Results: All differentiated cells with retention of 0%, 25%, 50% and 75% mTeSR1 expressed SOX17, FOXA2, AFP, HNF4α, and ALB, while higher expression levels were observed in differentiated cells in the 0% and 25% groups. The flow cytometry results showed that the proportion of ALB-positive differentiated cells derived from H1 cells was higher in the 25% mTeSR1 group than in other groups. However, no significant difference in ALB secretion, urea production, ICG uptake and release and glycogen storage ability was detected between the 25% and 0% groups. The iPS cells could differentiate into hepatocyte-like cells with 25% mTeSR1 retention. The apoptosis ratio of differentiated cells was lower in the 25% mTeSR1 group than in the 0% mTeSR1 group. Conclusion: Retaining 25% mTeSR1 medium during hepatic differentiation has been proposed to increase the percentage of ALB-positive cells and cell survival by decreasing cell apoptosis.

scholarly journals Zebularine Promotes Hepatic Differentiation of Rabbit Bone Marrow Mesenchymal Stem Cells by Interfering with p38 MAPK Signaling

2018 ◽  
Vol 2018 ◽  
pp. 1-9 ◽  
Author(s):  
Yong-Heng Luo ◽  
Juan Chen ◽  
En-Hua Xiao ◽  
Qiu-Yun Li ◽  
Yong-Mei Luo
Keyword(s):  
Stem Cells ◽  
Bone Marrow ◽  
Mesenchymal Stem Cells ◽  
Growth Factor ◽  
Hepatocyte Growth Factor ◽  
Hepatic Differentiation ◽  
Urea Production ◽  
Marrow Mesenchymal Stem Cells ◽  
Hepatocyte Growth

Demethylating agent zebularine is reported to be capable of inducing differentiation of stem cells by activation of methylated genes, though its function in hepatocyte differentiation is unclear. p38 signal pathway is involved in differentiation of hepatocytes and regulating of DNA methyltransferases 1 (DNMT1) expression. However, little is known about the impact of zebularine on bone marrow mesenchymal stem cells (BMMSCs) and p38 signaling during hepatic differentiation. The present study investigated the effects of zebularine on hepatic differentiation of rabbit BMMSCs, as well as the role of p38 on DNMT1 and hepatic differentiation, with the aim of developing a novel strategy for improving derivation of hepatocytes. BMMSCs were treated with zebularine at concentrations of 10, 20, 50, and 100 μM in the presence of hepatocyte growth factor; changes in the levels of hepatic-specific alpha-fetoprotein and albumin were detected and determined by RT-PCR, WB, and immunofluorescence staining. Expression of DNMT1 and phosphorylated p38 as well as urea production and ICG metabolism was also analyzed. Zebularine at concentrations of 10, 20, and 50 μM could not affect cell viability after 48 h. Zebularine treatment leads to an inhibition of DNMT activity and increase of hepatic-specific proteins alpha-fetoprotein and albumin in BMMSCs in vitro; zebularine addition also induced expression of urea production of and ICG metabolism. p38 signal was activated in BMMSCs simulated with HGF; inhibition of p38 facilitated the synthesis of DNMT1 and albumin in cells. Zebularine restrained DNMT1 and phosphorylated p38 which were induced by HGF. Therefore, this study demonstrated that treatment with zebularine exhibited terminal hepatic differentiation of BMMSCs in vitro in association with hepatocyte growth factor; p38 pathway at least partially participates in zebularine-induced hepatic differentiation of rabbit BMMSCs.

Development of Simple Protocol for Generation of Functionally Active Hepatocyte-like Cells from Human Adipose Tissue-derived Stem Cells

2019 ◽  
Author(s):  
Sahere Rouzbehan ◽  
Nahid Davoodian ◽  
Ali Jamshidi ◽  
Ali Atashabparvar ◽  
Najmeh Davoodian
Keyword(s):  
Stem Cells ◽  
Adipose Tissue ◽  
Growth Factor ◽  
Human Adipose Tissue ◽  
Culture Conditions ◽  
Alpha Fetoprotein ◽  
Oncostatin M ◽  
Hepatic Differentiation ◽  
Differentiated Cells ◽  
Simple Protocol

Background and Aims: Human adipose tissue-derived stem cells (hASCs) are considered as an attractive source of regenerative stem cells, mainly because of their higher proliferation rate, more accessibility and hepatocyte like properties as compared to mesenchymal stem cells isolated from other tissues. Numerous studies have described the beneficial use of adipose tissue-derived stem cells for generating hepatocyte-like cells. However, due to the lack of appropriate culture conditions, most of the produced cells exhibit poor functionality. The aim of the present study was to establish a new protocol for the efficient hepatic differentiation of hASCs. Materials and Methods: hASCs were cultured in hepatic differentiation medium containing fibroblast growth factor 4, hepatocyte growth factor, dexamethasone and oncostatin M using a three-step protocol up to 21 days. Then, the functionality of the treated cells was evaluated by analyzing specific hepatocyte genes and biochemical markers at various time points. Results: A significant upregulation in albumin, alpha-fetoprotein, cytokeratin 18 and hepatocyte nuclear factor-4α expressions was observed in differentiated cells relative to day 1 of differentiation protocol. Moreover, the finding of glycogen deposits increased urea production and positive immunofluorescence staining for albumin and alpha-fetoprotein in hepatocyte-like cells suggesting that most of the cells differentiate into hepatocyte-like cells. Conclusions: Our report has provided a simple protocol for differentiation of hASCs into more functional hepatocyte-like cells.

scholarly journals Wound-inducible ANAC071 and ANAC096 transcription factors promote cambial cell formation in incised Arabidopsis flowering stems

2021 ◽  
Vol 4 (1) ◽  
Author(s):  
Keita Matsuoka ◽  
Ryosuke Sato ◽  
Yuki Matsukura ◽  
Yoshiki Kawajiri ◽  
Hiromi Iino ◽  
...  
Keyword(s):  
Transcription Factors ◽  
Culture System ◽  
Cell Formation ◽  
Related Gene ◽  
Mesophyll Cells ◽  
Cambial Cell ◽  
Differentiated Cells ◽  
Cell Induction ◽  
Cambial Cells ◽  
Wound Tissue

AbstractANAC071 and its homolog ANAC096 are plant-specific transcription factors required for the initiation of cell division during wound healing in incised Arabidopsis flowering stems and Arabidopsis hypocotyl grafts; however, the mechanism remains mostly unknown. In this study, we showed that wound-induced cambium formation involved cell proliferation and the promoter activity of TDR/PXY (cambium-related gene) in the incised stem. Prior to the wound-induced cambium formation, both ANAC071 and ANAC096 were expressed at these sites. anac-multiple mutants significantly decreased wound-induced cambium formation in the incised stems and suppressed the conversion from mesophyll cells to cambial cells in an ectopic vascular cell induction culture system (VISUAL). Our results suggest that ANAC071 and ANAC096 are redundantly involved in the process of “cambialization”, the conversion from differentiated cells to cambial cells, and these cambium-like cells proliferate and provide cells in wound tissue during the tissue-reunion process.

scholarly journals Control by fibroblast growth factor of differentiation in the BC3H1 muscle cell line.

1985 ◽  
Vol 100 (5) ◽  
pp. 1540-1547 ◽  
Author(s):  
B Lathrop ◽  
E Olson ◽  
L Glaser
Keyword(s):  
Growth Factor ◽  
Cell Growth ◽  
Fibroblast Growth Factor ◽  
Cell Line ◽  
Calf Serum ◽  
Specific Protein ◽  
Fibroblast Growth ◽  
Mouse Muscle ◽  
Differentiated Cells ◽  
High Concentrations

The regulation of creatine phosphokinase (CPK) expression by polypeptide growth factors has been examined in the clonal mouse muscle BC3H1 cell line. After arrest of cell growth by exposure to low concentrations of serum, BC3H1 cells accumulate high levels of muscle-specific proteins including CPK. The induction of this enzyme is reversible in the presence of high concentrations of fetal calf serum, which cause quiescent, differentiated cells to reenter the cell cycle. Under these conditions, the rate of CPK synthesis is drastically reduced. We show in the present communication that either pituitary-derived fibroblast growth factor (FGF) or brain-derived FGF are as effective as serum in repressing the synthesis of CPK when added to quiescent, differentiated cells. The decrease in the rate of synthesis of CPK occurs within 22 h after the addition of pituitary FGF to the cells. Pituitary FGF had very little effect, if any, on the rate CPK degradation. The overall rate of protein synthesis and the pattern of synthesis of the major polypeptides made by these cells was not altered by the addition of FGF. Although pituitary FGF was mitogenic for BC3H1 cells, the rate of cell growth was not absolutely correlated with the extent of repression of CPK. Brain-derived FGF fully repressed CPK induction under conditions where it showed no significant mitogenic activity. These results show that the expression of a muscle-specific protein, CPK, can be controlled by a single defined polypeptide growth factor in fully differentiated cultures, and that initiation of cell division is not required for their regulation to take place.

scholarly journals Effect of Placental Extract on Omentum Mesenchymal Stem Cells Differentiation in NMRI Mice

2017 ◽  
Vol 7 (1) ◽  
pp. 176
Author(s):  
Maryam Sadat Nezhadfazel ◽  
Kazem Parivar ◽  
Nasim Hayati Roodbari ◽  
Mitra Heydari Nasrabadi
Keyword(s):  
Cell Cycle ◽  
Stem Cells ◽  
Flow Cytometry ◽  
Endothelial Cells ◽  
Mesenchymal Stem Cells ◽  
Control Group ◽  
Fat Cell ◽  
Nmri Mice ◽  
Differentiated Cells ◽  
Placental Extract

Omentum mesenchymal stem cells (OMSCs) could be induced to differentiate into cell varieties under certain conditions. We studied differentiation of OMSCs induced by using placenta extract in NMRI mice. Mesenchymal stem cells (MSCs) were isolated from omentum and cultured with mice placenta extract. MSCs, were assessed after three passages by flow cytometry for CD90, CD44, CD73, CD105, CD34 markers and were recognized their ability to differentiate into bone and fat cell lines. Placenta extract dose was determined with IC50 test then OMSCs were cultured in DMEM and 20% placenta extract.The cell cycle was checked. OMSCs were assayed on 21 days after culture and differentiated cells were determined by flow cytometry and again processed for flow cytometry. CD90, CD44, CD73, CD105 markers were not expressed, only CD34 was their marker. OMSCs were morphologically observed. Differentiated cells are similar to the endothelial cells. Therefore, to identify differentiated cells, CD31 and FLK1 expression were measured. This was confirmed by its expression. G1 phase of the cell cycle shows that OMSCs compared to the control group, were in the differentiation phase. The reason for the differentiation of MSCs into endothelial cells was the sign of presence of VEGF factor in the medium too high value of as a VEGF secreting source.

The effect of nerve growth factor on the development of sodium channels in PC12 cells

1986 ◽  
Vol 64 (11) ◽  
pp. 1153-1159 ◽  
Author(s):  
Juta K. Reed ◽  
Diane England
Keyword(s):  
Nerve Growth Factor ◽  
Growth Factor ◽  
Pc12 Cells ◽  
Specific Binding ◽  
Neuronal Cell ◽  
Scorpion Toxin ◽  
Excitable Cells ◽  
Response Curves ◽  
Nerve Growth ◽  
Differentiated Cells

We have studied the development of the action potential Na+ channels in PC12 cells, an established line that has been useful as a model for neuronal differentiation. In continuous culture PC12 cells, although electrically inexcitable, nevertheless have a low level of Na+ channels as judged by the increase in 22Na+ uptake in the presence of veratridine and scorpion toxin. These two neurotoxins have been shown to promote activation of Na+ channels in a variety of electrically excitable cells. Following treatment with nerve growth factor (NGF), conditions which induce differentiation to an electrically excitably neuronal-cell type, the neurotoxin-activated 22Na+ uptake increases approximately 12-fold, on a per cell basis, reaching a maximum in 12–16 days. The dose–response curves for veratridine and scorpion toxin are unchanged by NGF treatment (K0.5 for veratridine, 18–14 μM; K0.5 for scorpion toxin, 120–96 nM). Na+ channels in both undifferentiated and differentiated cells are tetrodotoxin sensitive and NGF treatment has no effect on the inhibition constant (Ki, 10–12 nM). Na+ channel sites were measured directly by the specific binding of [3H]saxitoxin. In NGF-treated cells, the saxitoxin receptor density reaches 154 fmol/mg protein (Kd, 1.3 nM), a level comparable to other excitable cells. Levels in control cells were too low to measure accurately. These findings show that NGF treatment of PC12 cells leads to a substantial increase in the expression of neurotoxin-sensitive Na+ channels. Furthermore, these channels are pharmacologically similar, if not identical, to those which exist in undifferentiated cells and therefore do not appear to result from the conversion of preexisting channels.

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