Retinoic acid as the stimulating factor for differentiation of wharton's jelly-mesenchymal stem cells into hepatocyte-like cells

Wharton's Jelly-Mesenchymal Stem Cells [WJ-MSCs] are pluripotent cells with differentiation capability into most cell lineages. The aim of the current work was to examine the role of Retinoic Acid [RA] in differentiation process of these cells into hepatocyte-like cells and determine the morphological and functional patterns. Human WJ-MSCs were extracted, cultured and expanded; after approximately 95% of confluence, the cells were treated with hepatogenic media containing RA. The cells were subsequently analyzed for morphological changes, glycogen storage, albumin production, and specific gene expression. WJ-MSCs expressed high levels of CD90 [93.6%] and CD105 [90.7%], but low levels of CD34 [0.3%] and CD45 [0.8%]. Albumin production had significant difference in the two groups [p

Co-culture of spermatogonial stem cells with sertoli cells in the presence of testosterone and FSH improved differentiation via up-regulation of post meiotic genes

Spermatogonial stem cells [SSCs] maintain spermatogenesis throughout life in the male. Maintenance of SSCs and induction of spermiogenesis in vitro may provide a therapeutic strategy to treat male infertility. This study investigated in vitro differentiation of mouse SSCs in presence or absence of Sertoli cells, hormones and vitamins. Spermatogonial populations were enriched from testes of 4-6 week old males by magnetic activated cell sorting and anti-Thy-1 antibody. Sertoli cells isolated from 6-8 week old testes were enriched using lectin-DSA-coated plates. Isolated SSCs were cultured in the presence of Leukemia inhibitory factor [LIF] for 7 days in gelatin-coated dishes, then dissociated and cultured for 7 days in media lacking LIF in the presence or absence of Sertoli cells, with or without FSH, testosterone and vitamins. After one week, the effects of Sertoli cells +/- supplementary media on SSC differentiation was evaluated by microscopy and expression of meiotic and postmeiotic transcripts using RT-PCR. SSC colonies had limited development after LIF removal alone, exhibiting low expression of meiotic [Scp3, Th2b] but not postmeiotic transcript, and loss of Stra8 and Dazl expression. SSCs co-cultured with Sertoli cells, hormones and vitamins developed spermatid-like cells expressing postmeiotic markers [TP1, TP2, Prm1] at levels over 2-fold higher than Sertoli cells or hormone/vitamins alone. Our present SSC-Sertoli co-culture provides conditions that may allow efficient in vitro differentiation of SSCs for the treatment of male infertility

Effect of trolox addition to cryopreservation media on human sperm motility

Sperm parameters and motion kinetics are affected by cryopreservation. The main purpose of the current study was to determine the effect of different concentrations of Trolox as an antioxidant to freezing-thawing procedure on human sperm kinematic parameter. Semen was collected from 20 normal donors and divided into five aliquots prior to cryopreservation. The first aliquot was analyzed by computer-assisted sperm analysis [CASA]. Other aliquots were mixed with cryo-protective agent containing 0, 20, 40, and 80 micro mol Trolox and treated samples were cryopreserved in liquid nitrogen. After two weeks samples were thawed and sperm motion kinematics was measured by CASA. Percent motility [Mot], curvilinear velocity [VCL], straight-line velocity [VSL], average path velocity [VAP], linearity [LIN], and amplitude of lateral head displacement [ALH] were compared before and after freeze. Addition of 40 micro mol Trolox resulted in significantly higher [p<0.05] post thaw VCL, VSL and VAP compared to other groups. Therefore the percentage of post thaw motile spermatozoa were significantly higher [p<0.01]. The supplementation of Trolox significantly improved the post-thawed human semen quality, especially progressive motility and average path velocity

Melatonin influences the proliferative and differentiative activity of rat adipose-derived stem cells

This study was performed to determine whether melatonin at physiological concentrations [0.01-10nM] could affect the proliferation and osteogenic differentiation of Rat ADSCs in vitro. ADSCs were isolated from the fat of adult rats. After cell expansion in culture media and through three passages, osteogenesis was induced on a monolayer culture with osteogenic medium with or without melatonin at physiological concentrations [0.01-10nM]. After 4 weeks cultures were examined for mineralization by Alizarin Red S and von Kossa staining and for alkaline phosphatase [ALP] activity by ALP kit. Cell viability and apoptosis were also assayed by 3-[4, 5-dimethylthiazol-2-yl]-5-[3-carboxymethoxyphenlyl]-2-[4-ulfophenyl]-2H-tetrazolium assay and flowcytometry, respectively. All assays were performed in triplicate. The results indicated that at physiological concentrations, melatonin suppressed proliferation and differentiation of ADSCs. These data indicate that ADSCs exposed to melatonin, had a lower ALP activity in contrast to the cells exposed to the osteogenic medium alone. Similarly, the mineral deposition [calcium level] also decreased. The flow cytometry proved that the cell growth decreased and the apoptotic cells increased. These results suggest that physiological concentration of melatonin has a negative effect on ADSCs osteogenesis

Evaluation of human breast adenocarcinoma [MCF-7] cells proliferation in co-culture with human adipocytes in three dimensional collagen gel matrix: norepinephrine as a lipolytic factor

Norepinephrine plays a trophic role in the control of cell replication and differentiation in target cells that express adrenergic receptors. In this study, we have tested the influence of infraphysiological, physiological and supraphysiological concentrations [0.0001 nM, 1 nM, 10000 nM] of human norepinephrine on the proliferation of breast cancer cells [human breast adenocarcinoma [MCF-7]] in co-culture with human adipocytes in three-dimensional collagen gel matrix culture. Cell proliferation and lipolysis rate were measured by 3-[4, 5-dimethylthiazolyl]-2, 5-diphenyl-tetrazolium bromide [MTT] and Oil red O colorimetric assay in second, 7[th] and 14[th] days of culture experiments. Our results showed a direct correlation between lipolysis rate of adipocytes and proliferation rate of MCF-7 cells. Both physiological and supraphysiological concentrations of human norepinephrine significantly [P<0.05] increased the proliferation of MCF-7 cells synchronously with progress of adipocyte lipolysis. The proliferations of MCF-7 cells were significantly decreased after conversion of adipocytes to fibroblast-like cells by supraphysiological concentration of norepinephrine. There was no statistical difference in lipolysis of adipocytes and proliferation of MCF-7 cells in response to infraphysiological concentration of norepinephrine. These findings indicated that norepinephrine stimulated the proliferation of MCF-7 cells in co-culture with human adipocytes as a lipolytic factor and that norepinephrine effect was suppressed by conversion of adipocytes to fibroblast-like cells, suggesting adipocytes as another target for prevention and therapy of breast cancer

Ultrastructural study of neutrophils in fetal rat spleen following lead intoxication

Introduction: Lead is one of the heavy metals that have adverse effects on blood cells and hemopoiesis. In this study the ultrastructure of neutrophils in fetal rat spleen were investigated following lead intoxication

Material and Methods: Thirty female and 6 male Sprague-Dawley rats were chosen by simple random sampling. After mating the pregnant rats were classified into test and control groups. From the first day of pregnancy the test group was provided ad lib with water containing 0.13% lead acetate and the control group had access to distilled water. After birth 10 newborn in each group were chosen by systematic random sampling. The spleens of the newborn rats were fixed in a solution of 2% glutaraldehyde, and after processing, sections were studied by a transmission electron microscope

Results: The ultrastructural changes included: irregular nuclei with deep invagination, plasma membrane pockets, presence of vacuoles with a heterogeneous material and an increasing incidence of rough endoplasmic reticulum with dilated cisternae. No differences between the groups were observed in the mitochondrial morphology and pattern of cytoplasmic granules [primary granules with electron dense appearance and specific or secondary granules with less electron density and heterogeneous appearance]

Conclusion: Lead transmitted via the placenta can affect the ultrastructure, and most probably the function, of fetal neutrophils. More attention must be given to the dangers of lead pollution of the environment and the need to eliminate exposure to lead in work places

role of L-arginine in control of apoptosis in preimplantation mouse embryos cultured in high glucose media

Maternal hyperglycemia causes delay in early stages of embryonic growth and development, higher incidence of congenital malformations and spontaneous miscarriage compared with those of non-diabetic conditions. High glucosis tratogenicity seems to be related to reduction of Nitric Oxide production [NO] in hyperglycemic condition. In order to test this hypothesis, 2-cell stage embryos of normal mice were cultured with high concentration of glucose [30mM] and different concentrations of L-arginine [5,10,20 mM] or L-NAME, an NO syntase [NOS] inhibitor. In the end of culture, blastocysts were stained by by terminal deoxynucleotidyl transferase-mediated dUTP nick end-labeling [TUNEL] technique and apoptotic cells were detected by using a Fluorescence microscope. Finally the amount of nitrite in the cultured media was assayed by Griess method. The results indicated that high glucose reduces Nitric Oxide production by preimplantation embryos and increases apoptosis of embryonic cells, but 5-20mM of L-arginine significantly increases Nitric Oxide production and decreases apoptosis. On the contrary L-NAME significantly inhibits the development of pre-implantation embryos. In conclusion, this study indicated that reduced nitric oxide production in high glucosis condition is a main factor for embryonic damage, and supplementation of high glucose media with L-arginine has an important role in prevention of high glucosis embryotoxicity