Cell cycle analysis of in vitro cultured goral (Naemorhedus caudatus) adult skin fibroblasts

2006 ◽  
Vol 30 (9) ◽  
pp. 698-703 ◽  
Author(s):  
M HASHEM ◽  
D BHANDARI ◽  
S KANG ◽  
B LEE ◽  
H SUK
Keyword(s):  
Cell Cycle ◽  
Skin Fibroblasts ◽  
Cell Cycle Analysis ◽  
Adult Skin

37 CELL CYCLE ANALYSIS IN IN VITRO-CULTURED ADULT SKIN FIBROBLASTS OF THE GORAL (NAEMORHEDUS CAUDATUS)

2006 ◽  
Vol 18 (2) ◽  
pp. 127
Author(s):  
M. A. Hashem ◽  
P. B. Dilip ◽  
S. K. Kang ◽  
B. C. Lee ◽  
W. S. Hwang
Keyword(s):  
Cell Cycle ◽  
Cytochalasin B ◽  
Positive Impact ◽  
Statistical Significance ◽  
Skin Fibroblasts ◽  
Nuclear Transplantation ◽  
Serum Starvation ◽  
Significant Difference ◽  
Adult Skin

The present study was undertaken to examine the cell cycle characteristics of goral adult skin fibroblasts under a variety of cell cycle-arresting treatments. Gorals are listed as an endangered animal in CITES Appendix I. Experimental findings suggest that G0/G1 stages of the cell cycle give better results in somatic cell nuclear transfer (SCNT) in terms of normal ploidy and development of reconstructed embryos. In this regard, synchronization of the cell cycle stages in G0/G1 phase is suggested to be one of the key factors for determining the success of nuclear transplantation. Serum deprivation, contact inhibition, chemical inhibitors such as protease, and antioxidant inhibitors are widely used for cell cycle synchronization and inhibition of apoptosis. Four experiments were performed, and each with one-way completely randomized design involving three replicates of all treatments using the general linear models procedure in the statistical analysis system (SAS Institute, Inc., Cary, SC, USA). Least significant difference was used to determine statistical significance (P < 0.05) among treatment groups. In Exp. 1, effects of cyclic confluency, serum starvation, or full confluency on goral cells were studied. In Exp. 2, treatments with different antioxidants such as 2 mM beta-mercaptoethanol (B-ME), cysteine, or glutathione for 24 h were examined in fully confluent cells without serum starvation. In Exp. 3, three protein inhibitors, 2 mM 6-dimethylaminopurine (6-DMAP), 7.5 �g/mL cycloheximide, or 7.5 �g/mL cytochalasin B, were treated as in Exp. 2. In Exp. 4, different concentrations (0, 0.5, 1.0, or 2.5%) of dimethyl sulfoxide (DMSO) were treated as in Exp. 2. In all experiments, cell cycle stages were analyzed by fluorescence-activated cell sorting (FACS) analysis. In Exp. 1, more cells (70.4%) were found in the G0/G1 stage in the serum starved medium compared to cells in the cyclic (66.1%) and fully confluent growth phase (65.5%). In Exp. 2, supplementing with B-ME (61.8%) or cysteine (62.0%) in the culture medium synchronized cell cycle to the G0/G1 stage better than glutathione (53.1%). In Exp. 3, more cells were synchronized to the G0/G1 stage in media supplemented with cyclohexamide (62.3%) than with 6-DMAP (5.7%) or cytochalasin B (13.3%). Incidence of apoptosis was higher in media containing 6-DMAP (91.8%) or cytochalasin B (76.6%). In Exp. 4, supplementing the medium with 0.5% (76.0%) or 1.0% (75.9%) DMSO synchronized the cells effectively to the G0/G1 stage compared to the untreated control medium (69.3%) or medium supplemented with 2.5% of DMSO (8.0%). In conclusion, cyclohexamide, B-ME, cysteine, or DMSO at the optimum concentration can synchronize the cell cycle effectively, which may have a positive impact on the outcome of SCNT in the goral. This study was supported by grants from the Korean MOST (Top Scientist Fellowship) and MAF (Biogreen 21 #20050301-034-443-026-04-00).

scholarly journals Cell Cycle Analysis of Chicken Lymphocytes Stimulated to Grow in vitro using PHA and Labelled with BrdU

1989 ◽  
Vol 60 (2) ◽  
pp. 178-184
Author(s):  
Shin OKAMOTO ◽  
Mie OHWAKI ◽  
Yoshizane MAEDA ◽  
Tsutomu HASHIGUCHI
Keyword(s):  
Cell Cycle ◽  
Cell Cycle Analysis

scholarly journals Hydroxytyrosol Promotes Proliferation of Human Schwann Cells: An In Vitro Study

2020 ◽  
Vol 17 (12) ◽  
pp. 4404
Author(s):  
Khidhir Kamil ◽  
Muhammad Dain Yazid ◽  
Ruszymah Bt Hj Idrus ◽  
Jaya Kumar
Keyword(s):  
Cell Cycle ◽  
Protein Expression ◽  
Schwann Cells ◽  
Growth Factor Receptor ◽  
In Vitro Study ◽  
Cell Cycle Analysis ◽  
Proliferation Index ◽  
Proliferation Markers ◽  
Diphenyltetrazolium Bromide

Recent advances in phytomedicine have explored some potential candidates for nerve regeneration, including hydroxytyrosol (HT). This study was undertaken to explore the potential effects of HT on human Schwann cells’ proliferation. Methods: The primary human Schwann cell (hSC) was characterized, and the proliferation rate of hSC supplemented with various concentrations of HT was determined via 3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide (MTT) assay. Cell cycle analysis and protein expression of glial fibrillary acidic protein (GFAP) and p75 nerve growth factor receptor (p75 NGFR) were evaluated via the immunofluorescence technique. Results: In vitro culture of hSCs revealed spindle-like, bipolar morphology with the expression of specific markers of hSC. Hydroxytyrosol at 10 and 20 ng/mL significantly increased the proliferation of hSCs by 30.12 ± 5.9% and 47.8 ± 6.7% compared to control (p < 0.05). Cell cycle analysis showed that HT-treated hSCs have a higher proliferation index (16.2 ± 0.2%) than the control (12.4 ± 0.4%) (p < 0.01). In addition, HT significantly increased the protein expression of GFAP and p75NGFR (p < 0.05). Conclusion: HT stimulates the proliferation of hSCs in vitro, indicated by a significant increase in the hSC proliferation index and protein expression of hSCs’ proliferation markers, namely p75 NGFR and GFAP.

Long Term Maintenance of Myeloid Leukemia Stem Cell-Like Populations Cultured with Mesenchymal Stromal Cells (MSC)

Blood ◽  
2012 ◽  
Vol 120 (21) ◽  
pp. 3546-3546
Author(s):  
Sawa Ito ◽  
A. John Barrett ◽  
Andre Larochelle ◽  
Nancy F. Hensel ◽  
Keyvan Keyvanfar ◽  
...  
Keyword(s):  
Cell Cycle ◽  
Stem Cells ◽  
Stem Cell ◽  
Culture Conditions ◽  
Myelogenous Leukemia ◽  
Cell Cycle Analysis ◽  
Remission Induction ◽  
Leukemia Stem Cell

Abstract Abstract 3546 Because MSC support the growth and the differentiation of normal hematopoietic stem cells we hypothesized that MSC might also support leukemia cells, in particular leukemia stem cells (LSC) in vitro. We cultured blast cells from patients with acute myelogenous leukemia (AML) in liquid medium to study persistence of stem-cell-like and differentiated leukemia cell populations by flow cytometry, with and without MSC and additional growth factors. Cryopresrerved peripheral blood mononuclear cells (PBMC) were obtained from 6 AML patients (mean Age 47, range 23–74). Leukemia blasts were isolated by sorting live (propidium iodide (PI)-negative) CD34+ lineage (CD2+, CD3+, CD14+ and CD19+) -negative cells using a FACS ARIA II cell sorter (BD). Sorted blasts (2.5 ×105 cells) were co-cultured with an equal number of irradiated MSC derived from healthy donor bone marrow in RPMI medium supplemented with 10% human serum, with or without a human cytokine (CYTO) mixture (50 ng/ml interleukin 3, 150 ng/ml stem cell factor, and 150ng/ml Flt-3 ligand). MSC were replenished every two weeks. The phenotype of cultured cells was analyzed weekly using fluorescently-conjugated monoclonal antibodies against CD34, CD38, and CD45, plus the lineage panel and a dead cell exclusion dye Cell cycle analysis with Hoeschst 33342 and Pyronin Y was performed on cells co-stained with CD34, CD45 and PI. Primary leukemia samples were phenotypically heterogeneous with respect to proportions of cells (co-)staining for CD34 and CD38 as previously reported: three samples showed CD34+CD38- predominance (LSC-like leukemia), and three were CD34+CD38+ (common myeloid progenitor (CMP)-like leukemia). LSC-like leukemia maintained viable CD34+CD38- cells for at least 6 weeks when co-cultured with MSC alone, in contrast to cultures with cytokines or medium only which showed rapid decline in the LSC populations and no prolonged maintenance of viable cells (p=0.0005) (Figure, left panel). CMP-like leukemia maintained their CD34+CD38+ phenotype when co-cultured with MSC alone but persistence of this subset was not significantly different from the other culture conditions (p=0.5) and no culture remained viable after 4 weeks (Figure, right panel). Cell cycle analysis showed that co-culture with MSC maintained CD34+ blasts in G0 significantly more than other culture conditions (P<0.0001). We conclude that MSC support the maintenance of a leukemia stem cell phenotype in a long- term (6 week) in vitro culture system. The differential capacity of MSC to support LSC- like and CMP- like leukemia may be associated with the different frequency of leukemia initiating cells within each leukemic blast population. NSG mice xenotranplant model experiments are ongoing to confirm this hypothesis. Co-culture of LSC with MSC represents a simple approach to maintain LSC in vitro and could be utilized to screen the drug targeting LSCs. Further study of the effect of MSC on LSC would elucidate a potential mechanism whereby the marrow microenvironment serves as a reservoir of persisting leukemia after remission induction chemotherapy. Disclosures: No relevant conflicts of interest to declare.

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