The effect of mouse embryonic fibroblast in direct differentiation of mouse embryonic stem cells

Background: Since embryonic stem (ES) cells have the dual ability to proliferate indefinitely and differentiate into multiple tissue types, ES cells could potentially provide an unlimited cell supply for human transplantation. Objective: In order to study the differentiation of mouse embryonic stem (mES) cells, they were cultured in suspension by using ES media without Leukemia Inhibitory Factor (LIF) to induce spontaneous differentiation. Cellular morphology of differentiated derivatives was then evaluated. Materials and Methods: Undifferentiated mES from our laboratory were cultured in three different settings by using ES media containing 0.1% / 1mM trypsin/EDTA and removing LIF; in the absence of murine embryonic fibroblast (MEF) feeder cells (group 1), in the presence of MEF feeder cells with a density of 0.5×105 cells/ml (group 2), and 0.5×106 cells/ml (group 3). Five days after the initiation of cell culture, and inducing mES cells to form embryoid bodies (EBs), they were removed from dish by centrifugation, and then they were cultured on collagen coated dishes for 20 days. The dishes were fixed and stained by Wright-Gimsa method at the end of the study period. Results: In group 1, mES cells showed spontaneous differentiation to all derivatives of three germ cells, including: epithelia like, fibroblast like and neron-like cells. In group 2, almost all ES cells were found to be differentiated into granular progenitor cells including hematopoietic cell lineages. In group 3, various morphologies including nerve cell lineages and fibroblast-like cells were detected. Conclusion: Differentiation of mES cells can be a dose response process, depending on the factors that may be released from MEF feeder layer to ES media in a coculture system. Our results indicated that in the presence of low numbers of MEF cells, mES cells can spontaneously differentiate into hematopoeitic cell lineages

Isolation and differentiation of mouse embryonic stem cells

Background: Recently, embryonic stem (ES) cells have become very important resources in basic medical researches. These cells can differetiate into derivatives of all primary germ layers. Objectives: In order to isolate embryonic stem cells in vitro, the blastocyst were cultured and the morphological aspects, population doubling time, alkalin phosphatse and differentiation properties of the cells were investigated. Materials and Methods: The balstocysts from NMRI mice were cultured for 3 days up to time that inner cell mass (ICM) reach to the outgrowth stage. The cells were disaggregated and trypsinized every 3 days until the appearance of the colonies of ES cells. The colony positive cells were fixed and stained for alkaline phosphatase. The ES cells were cultured in suspension state for 5 days, at the same time Leukaemia Inhibitory Factor (LIF) was removed from media to form embryoid bodies(EBs). The EBs were cultured for 8 - 20 days on collagen coated dish to induce the spontaneouse differentiation. Results: During the 6-9 days after the disaggregation of ICM in the expansion stage, the colony of ES cells appeared as a flat monolayer mass with strike boundaries and nondistinguish cytoplasm including a few nuclei. In colony formation stage, the morphology changed from flat monolayer to round multilayer with strike define boundaries. Undifferentiated cells were seen as intensely small cells attached together compactly with high nucleus/cytoplasm (N/C) ratio. The cells of colonies tend to differetiate by separation from each other and became larger and diffused on substrate by attaching to dish. The positive alkaline phosphatase cells were seen in typical morphology of ES colonies. The EBs cells were seen in culture after 5 days in suspension and began to spontaneously differentiate into various types of cells such as nerve and hematopoitic lineages. Conclusion: Despite strike morphology of ES colonies, it is difficult to distinguish the differentiated from undifferentiated cell colonies in the colony formation stage. New ES cells are capable to give rise into EBs and are susceptible of spontaneously differentiation in various type of cells

A study of some biochemistry changes of phenylpropanoid pathway in sugarcane (Saccharum officinarum) organs during developmental stages

Phenylpropanoid compounds as secondary metabolites are synthesized by plants during development and in response to stress. Phenylpropanoids contribute to the compounds of plant pigmentation, antioxidants and protective agents and also play a regulatory role in growth and morphogenesis. In this research, samples of blade and sheath of leaf, , stem, and root of Saccharum officinarum (cultivar CP69-1062) during the germination, tillering, grand growth, and maturation stages were prepared. In order to studying biochemical changes of phenylpropanoid compounds during development of sugarcane, PAL activity enzyme, phenolic, flavonoids and flavonols component in samples were analyzed. The study of changes of enzyme activity in different tissues during developmental stages showed high enzyme activity of PAL in leaves during all of developmental stages and in sheath during grand growth stage. Enzyme activity was increased in stem during grand growth stage and in root during germination stage. Results indicated that changes of concentration of total phenol and the maximum amount of total phenol were detected in leaf during germination and maturation stages, in sheath during germination stage, in stem during grand growth stage and in root during maturation stage (

Improvement of mesenchymal stem cell differentiation into the endoderm lineage by four step sequential method in biocompatible biomaterial

Introduction: The goal of the study described here, was to investigate the potential of umbilical cord derived mesenchymal stem cell (UC-MSCs) into hepatocyte like cells in a sequential 2D and 3D differentiation protocols as alternative therapy. Methods: Mesenchymal stem cells (MSCs) were isolated from the umbilical cord (UC) and CD markers were analyzed by flow cytometry. For hepatic differentiation of UC-MSCs, cells were induced with a sequential 4-step protocol in 3D and 2D culture system. Urea concentration and albumin secretion into the culture medium was quantified by ELISA. Gene expression levels of AFP, ALB, and CK18 were determined by RT-PCR. Data were statistically analyzed by the SPSS software. The difference between the mean was considered significant when p < 0.05. Results: Growth factor dependent morphological changes from elongated fibroblast-like cells to round epithelial cell morphology were observed in 2D culture. Cell proliferation analysis showed round-shaped morphology with clear cytoplasm and nucleus on the alginate scaffold in 3D culture. The mean valuses of albumin production and urea secretion were significantly higher in the 3D Culture system when compared with the 2D culture (p = 0.005 vs p = 0.001), respectively. Treatment of cells with TSA in the final step of differentiation induced an increased expression of CK18 and a decreased expression of αFP in both the 3D and 2D cultures (p = 0.026), but led to a decreased albumin gene expression, and an increased expression in the 2D culture (p = 0.001). Conclusion: Findings of the present study indicated that sequential exposure of UC-MSCs with growth factors in 3D culture improves hepatic differentiation

COMPARISON OF CD46 EXPRESSION ON THE INNER ACROSOMAL MEMBRANE OF SPERMS FROM NORMOSPERMIC AND ASTHENOSPERMIC INDIVIDUALS

Introduction: CD46 is a membrane cofactor protein (MCP) of complement system wich is present on the membrane of all somatic cells except RBC. It is also present on the inner acrosmal membrane of human sperm. Thus, the aim of this study was to compare the expression of this prote, in on the inner acrosmal membrane of sperms from normospermic and asthenospermic individuals.&#13; Method: Semen from 6 normospermic and 17 asthenospermic individuals were examined for CD46 expression. After solublization of sperms in solublizing detergent, the solublized sperm membrane was separated from the rest of cell organelles by centrifugation. Solublized sperm membrane were divide to equal parts and SOS-PAGE gel was canied out in paired on the same gel for each sample. Western blot was carried out on half of the gel and then the nitrocellose papers were stained by a monocolonal Ab and HRP conjugate Ab. The other half were stained by silver stain for identification of MW.&#13; Results: After scoring the stained nitrocellose papen in each groups, no statistical significant difference was observed for C046 expression between the two groups. However, a significant Spearmen correlation was observed between CD46 expression and sperm motility (r=0.597, P=0.003). The MW of C046 was between 36 to 45 KD. with a mean of 42 KD.&#13; Discussion: This is the first report of a positive Spearmen correlation between sperm CD46 expression and sperm motility which suggest that there might be relation between CD46 expression and sperm motility

DETECTION AND ISOLATION OF CD59 FROM HUMAN SEMINAL PLASMA

Introduction. CD59 is one of the complement regulatory proteins (CRPs) which exert inhibitory function by blocking the formation of C5b-9 complex or Membrane Attacks complex (MAC). Regarding the therapeutic role of CD59 in treatment of pathological effects in uncontrolled activation of complements system and its efficiency to overcome the hyper-acute rejection, CD59 was suggested for maintenance of transplanted organ. In this study We determined and isolated CD59 from seminal plasma.&#13; Methods. Six normospermic sample according to WHO standards were chosen. Plasma of samples was separated and to remove the postasomes, the seminal plama was ultra centrifuged. CD59 was detected by Dot-Blot using CD59 mAb (MEM43). The molecular weight and purity of protein was detected by SOS-PAGE method follwed by Westerm Blot.&#13; Results. Protein was present in the 6.5 ml and 15ml of gel fitration fractions. Molecular weight based on marker size in these two fractions was 65 and 21KD respectively. Discussion. CD59 had previously beem purified by lysis of erythrocyte cell membrane. Because of use of detergent and preservative agents, this method decreased physiologic effects of the protein. In this study the isolation was performed from prostasome granules" without using of any detergent and preservative agents

Sonic hedgehog inhibition induces mouse embryonic stem cells to differentiate toward definitive endoderm

201-207In the experimental group (shh inhibited group), there were significant decreases in the expression of Oct4, Nanog, Shh, GATA4, Brachyury and Goosecoid, while increases were observed for TAT and Pdx1. The expression of Sox17 did not differ between two control and experimental groups. In experimental group, the amount of GSC positive cells was somehow lower but it seems that there was no difference for Sox17. Shh inhibition induces ESCs to differentiate toward definitive endoderm by committing mesendodermal lineages. </span