ABSTRACT
Human breast milk stem cells (hBSCs) contain a population of cells with the ability to differentiate into various cell lineages for cell therapy applications. The current study examined the differentiation potential of hBSCs into hepatocytes- like cells. The cells were isolated from the breast milk and were treated with hepatogenic medium containing hepatocyte growth factor, insulin-like growth factor and dexamethasone for 7 days subsequently; Oncostatin M was added to the culture media. RT-PCR and immunocytochemistry were performed to detect the hepatogenic markers. The glycogen storage and the ability of the cells to absorb and release indocynanin green were also tested. The data showed that most of the differentiated cells formed cell aggregates after the 30th day, with more cells accumulated to form spheroids. RT-PCR revealed the expression of the hepatic nuclear factor, albumin, cytokeratin 18 and 19, cytochrome P2B6, glucose-6-phospahtase and claudin. The functional assays also showed glycogen storage and omission of indicynine green. Our study demonstrated hBSCs are novel population that can differentiate into hepatocyte-like cells.
Subject(s)
Humans , Breast , Cell Culture Techniques , Cell Lineage , Cell- and Tissue-Based Therapy , Culture Media , Cytochromes , Dexamethasone , Glycogen , Hepatocyte Growth Factor , Hepatocytes , Immunohistochemistry , Keratin-18 , Mesenchymal Stem Cells , Milk, Human , Oncostatin M , Stem CellsABSTRACT
Platelet-rich plasma (PRP) as a source of growth factors may induce tissue repairing and improve fibrosis. This study aimed to assess the effects of PRP on kidney regeneration and fibrosis in gentamicin (GM)-induced nephrotoxicity rat model by stereological study. Thirty-two male rats were selected. Nephrotoxicity was induced in animals by administration of GM (80 mg/kg/daily, intraperitoneally [IP], 8 day) and animals were treated by PRP (100 µL, intra-cortical injection using surgical microscopy, single dose). Blood samples were collected for determine blood urea nitrogen (BUN) and creatinine (Cr) before and after PRP therapy. At the end of experiment, right kidneys were sectioned by Isotropic Uniform Random (IUR) method and stained with H & E and Masson's Trichrome. The stereological methods were used for estimating the changes in different structures of kidney. PRP increased the number of epithelial cells in convoluted tubules, and decreased the volume of connective tissue, renal corpuscles and glomeruli in GM-treated animals (P < 0.05). Our findings indicate that PRP had beneficial effects on proliferation of epithelial cells in convoluted tubules and ameliorated GM-induced fibrosis.
Subject(s)
Animals , Humans , Male , Rats , Blood Urea Nitrogen , Connective Tissue , Creatinine , Epithelial Cells , Fibrosis , Gentamicins , Intercellular Signaling Peptides and Proteins , Kidney , Methods , Microscopy , Models, Animal , Platelet-Rich Plasma , RegenerationABSTRACT
Background: Protracted and repeated exposure to chronic variable stress [CVS] may lead to reproductive dysfunction. It is a basic cause of male infertility. Curcumin [CUR] is an active fraction of turmeric that used in traditional Chinese medicine. CUR represents various pharmacological activities
Objective: The purpose of this study was to determining the effects of CUR on testis and testosterone, follicle stimulating hormone [FSH] and luteinizing hormone [LH] in rats with establishing chronic variable stress
Materials and Methods: Twenty-one adult male Sprague-Dawley rats were divided into three groups: 1] control, 2] CVS and 3] CVS+ CUR [100 mg/kg/day dissolved in 0.5 mL of olive oil]. All of the animals in control, CVS, and CVS+CUR groups were sacrificed after 15 days. Testosterone, FSH, LH, and testis damage were evaluated
Results: Significant changes in the normal range of testosterone, FSH, LH serum levels and seminiferous tubule apoptotic cells were detected in CVS group compared to the control rats [p=0.02]. These parameters changed to a less extent in CVS+CUR animals compared to the CVS rats [p=0.02]
Conclusion: Our findings propose that curcumin might have curative potential on the reproductive system function and its impairment. It's regulated by stress and reproductive-related hormones
Subject(s)
Animals, Laboratory , Stress, Psychological , Testis , Rats, Sprague-Dawley , Testosterone , Follicle Stimulating Hormone , Luteinizing HormoneABSTRACT
Objectives: Adipose tissue as an appropriate source of Mesenchymal Stem Cells [MSCs] has the potential to differentiate into multiple lineages. Glycoconjugates content of the MSCs can be considered as biomarkers in self-renewal, pluripotency and differentiation processes. In this study, the lectin profile of MSCs isolated from adipose tissue was detected and according to that, a subpopulation was determined
Materials and Methods: MSCs were isolated from adipose tissue by explanting of the tissue pieces. The FITC-conjugated lectins, WGA, UEA, PNA, BSA and PWM were used to detect the terminal sugar residues. The cells were then counterstained with DAPI. The intensity of the reaction was evaluated by ImageJ software. The cells were also stained with PAS method
Results: MSCs were reacted with all lectins with different intensity of the reactions. The cells reacted with WGA, UEA, and BSA "strongly" and with PWM "moderately" and with PNA with "weak" intensity. The morphological analysis of the isolated MSCs revealed the existence of the two different cell types in the cultures. Two types of cells were detected according to nucleus size and lectin reactivity. The cells with large nuclei constitute 20.62% of the total cells and stained significant more intensity by UEA and less intense with PWM [both P=0.014] and PNA [P=0.044]. Flow cytometry with CD34 shows that these large cells were not endothelial cells
Conclusion: The MSCs derived from adipose tissue seem to be a heterogeneous populations and lectin profile of the cells showed that they are different in the expression of the glycoconjugates
ABSTRACT
BACKGROUND: Human Wharton’s jelly mesenchymal stem cells (HWJMSCs) isolated from medical waste product can be considered as an accessible source of cells in regenerative medicine. Stem cell-derived hepatocytes have poor function and need appropriate niche to reconstruct the liver structure. Therefore, we attempted to find a novel approach in differentiating HWJMSCs into functional hepatic cells using 3D culture conditions and liver extract that recapitulates vital stage in liver development. MATERIALS AND METHODS: HWJMSCs were extracted from human Wharton’s jelly, characterized by flow cytometry, and differentiated towards osteogenic and adipogenic lineages. HWJMSCs were co-cultured with HUVECs in 3D matrigel/collagen scaffolds in the presence of fetal liver extract for 14 days. The expression of specific liver genes were evaluated by lectins, PAS and immunocytochemistry. RESULTS: According to flow cytometry data, isolated cells from HWJMSCs were shown to express MSC markers. HWJMSCs co-cultured with HUVECs in matrigel/collagen scaffold with extract expressed albumin, lectins UEA and PNA. Immunohistochemistry of the cells in matrigel/collagen scaffold with or without extract exhibited a positive reaction for CK19. CONCLUSIONS: Co-culturing of the HWJMSC/HUVEC in 3D matrigel/collagen scaffold is bimimicary of in vivo cell condition. The results showed that administration of the liver extract in 3D matrigel/collagen culture of HWJMSC/HUVEC can induce hepatocyte marker expression.
Subject(s)
Humans , Collagen , Endothelial Cells , Flow Cytometry , Hepatocytes , Immunohistochemistry , Lectins , Liver , Medical Waste , Mesenchymal Stem Cells , Regenerative MedicineABSTRACT
Both mature and stem cell-derived hepatocytes lost their phenotype and functionality under conventional culture conditions. However, the 3D scaffolds containing the main extracellular matrix constitutions, such as heparin, may provide appropriate microenvironment for hepatocytes to be functional. The current study aimed to investigate the efficacy of the differentiation capability of hepatocytes derived from human Wharton's jelly mesenchymal stem cells (WJ-MSCs) in 3D heparinized scaffold. In this case, the human WJ-MSCs were cultured on the heparinized and non-heparinized 2D collagen gels or within 3D scaffolds in the presence of hepatogenic medium. Immunostaining was performed for anti-alpha fetoprotein, cytokeratin-18 and -19 antibodies. RT-PCR was performed for detection of hepatic nuclear factor-4 (HNF-4), albumin, cytokeratin-18 and -19, glucose-6-phosphatase (G6P), c-met and Cyp2B. The results indicated that hepatogenic media induced the cells to express early liver-specific markers including HNF4, albumin, cytokeratin-18 and 19 in all conditions. The cells cultured on both heparinized culture conditions expressed late liver-specific markers such as G6P and Cyp2B as well. Besides, the hepatocytes differentiated in 3D heparinized scaffolds stored more glycogen that indicated they were more functional. Non-heparinized 2D gel was the superior condition for cholangiocyte differentiation as indicated by higher levels of cytokeratin 19 expression. In conclusion, the heparinized 3D scaffolds provided a microenvironment to mimic Disse space. Therefore, 3D heparinized collagen scaffold can be suggested as a good vehicle for hepatocyte differentiation.
Subject(s)
Humans , Antibodies , Collagen , Collagen Type I , Constitution and Bylaws , Extracellular Matrix , Fetal Proteins , Gels , Glucose-6-Phosphatase , Glycogen , Heparin , Hepatocytes , Keratin-18 , Keratin-19 , Mesenchymal Stem Cells , Phenotype , Wharton JellyABSTRACT
Background: Human Wharton's jelly mesenchymal stem cells [HWJMSCs] express liver-specific markers such as albumin, alpha-fetoprotein, cytokeratin-19, cytokeratin-18, and glucose-6-phosphatase. Therefore, they can be considered as a good source for cell replacement therapy for liver diseases. This study aimed to evaluate the effects of various culture systems on the hepatocyte-specific gene expression pattern of naive HWJMSCs
Methods: HWJMSCs were characterized as MSCs by detecting the surface CD markers and capability to differentiate toward osteoblast and adipocyte. HWJMSCs were cultured in 2D collagen films and 3D collagen scaffolds for 21 days and were compared to control cultures. Real time RT-PCR was used to evaluate the expression of liver-specific genes
Results: The HWJMSCs which were grown on non-coated culture plates expressed cytokeratin-18 and -19, alpha-fetoprotein, albumin, glucose-6-phosphatase, and claudin. The expression of the hepatic nuclear factor 4 [HNF4] was very low. The cells showed a significant increase in caludin expression when they cultured in 3D collagen scaffolds compared to the conventional monolayer culture and 2D collagen scaffold
Conclusion: Various culture systems did not influence on hepatocyte specific marker expression by HWJMSCs, except for claudin. The expression of claudin showed that 3D collagen scaffold provided the extracellular matrix for induction of the cells to interconnect with each other
Subject(s)
Humans , Wharton Jelly , Cells, Cultured , Cell Culture Techniques , Collagen , Tissue Scaffolds , Hepatocytes , Liver , GenesABSTRACT
The present study investigated the differentiation potential of human Umbilical Cord Mesenchymal Stem Cells [UCMSCs] into hepatic lineage through embryonic body-like aggregate formation in the presence of IGF-1. Cells derived from Wharton's jelly have been reported to display a wide multilineage differentiation potential, showing some similarities to both embryonic [ESC] and mesenchymal stem cells [MSCs]. Human MSCs isolated from the umbilical cord were plated in 20 microL micro drops. A two-step differentiation protocol was used and the cell aggregates were exposed to the media supplemented with IGF, HGF, oncostatin M, and dexamethasone for 21 days. Immunoperoxidase and immuno-fluorescence were performed for cyrokeratins 18, 19 and albumin. Functional assays were done by periodic acid Schiff [PAS] and indocyanine green. The expression of cytokeratin 19 was shown to be higher in the cells derived from 3D spheroids compared to those cultured in conventional protocol. They showed a polygonal shape after being exposed to hepatogenic media. Immunostaining demonstrated the expression of cytokeratin-18, 19 and albumin by the differentiated cells. Besides, PAS staining revealed glycogen storage in differentiated cells. Also, a greater number of large size differentiated cells were found at the periphery of the expanded cell aggregates. We established a protocol for UCMSC differentiation into hepatocytes and these cells were morphologically and functionally similar to hepatocytes. Thus, hepatocyte differentiation may be facilitated by the UCMSCs aggregate formation before administration of the differentiation protocols
ABSTRACT
Pomegranate juice has several antioxidant components such as flavenoids and mineral materials such as sodium and potassium. In this study the effects of pomegranate juice on bone calcium content and body weight of adult mice were survived. In this applied study two doses [3.3 and 6.6 ml/kg] of pomegranate juice [PJ] were gavaged to female mice for 30 days. Animals were weighed at days of 0, 5, 10, 15, 20, 25 and 30. Bone calcium contents were measured by flame photometer. Bone calcium content of PJE treated mice increased but it was not significant statistically. Pomegranate juice did not affect body weight. Pomegranate juice extracts even its high dose did not show any side effect on body weight and tissues of adult female mice
ABSTRACT
Background: The regenerative capacity of the mammalian heart is quite limited. Recent reports have focused on reprogramming mesenchymal stem cells into cardiomyocytes. We investigated whether fibroblasts could transdifferentiate into myocardium
Methods: Mouse embryonic fibroblasts were treated with Trichostatin A [TSA] and 5-Aza-2-Deoxycytidine [5-aza-dC]. The treated cells were permeabilized with streptolysin O and exposed to the mouse cardiomyocyte extract and cultured for 1, 10, and 21 days. Cardiomyocyte markers were detected by immunohistochemistry. Alkaline phosphatase activity and OCT4 were also detected in cells treated by chromatinmodifying agents
Results: The cells exposed to a combination of 5-aza-dC and TSA and permeabilized in the presence of the cardiomyocyte extract showed morphological changes. The cells were unable to express cardiomyocyte markers after 24 h. Immunocytochemical assays showed a notable degree of myosin heavy chain and ?-actinin expressions after 10 days. The expression of the natriuretic factor and troponin T occurred after 21 days in these cells. The cells exposed to chromatin-modifying agents also expressed cardiomyocyte markers; however, the proportion of reprogrammed cells was clearly smaller than that in the cultures exposed to 5-aza-dC, TSA, and extract
Conclusion: It seems that the fibroblasts were able to eliminate the previous epigenetic markers and form new ones according to the factors existing in the extract. Since no beating was observed, at least up to 21 days, the cells may need an appropriate extracellular matrix for their function
ABSTRACT
The interaction between follicular cells and oocyte leads to a change in gene expression involved in oocyte maturation processes. The purpose of this study was to quantify the expression of more common genes involved in follicular growth and oocyte developmental competence. In this experimental study, the expression of genes was evaluated with qRT-PCR assay in female BALB/c mice pups at 3-day of pre-pubertal and 8 week old virgin adult ovaries. The tissue was prepared by H and E staining for normal morphological appearance. The data were calculated with the 2-delta Ct formula and assessed using non-parametric two-tailed Mann-Whitney test. The p<0.05 was considered as significant. The data showed a significant increase in the level of Stra8 and GDF9 in adult compared with newborn mice ovaries [p=0.049]. In contrast, a significant decrease in the level of Mvh, REC8, SCP1, SCP3, and ZP2 was observed in adult mice ovaries compared to those in the newborn mice ovaries [all p=0.049 except SCP1: p=0.046]. There was no significant difference in the level of OCT4 and Cx37 expression between adult and newborn mice ovaries. The modifications in gene expression patterns coordinate the follicular developmental processes. Furthermore, the findings showed higher expression level of premeiotic gene [Stra8] and lower level of meiotic entry markers [SCP1, SCP3, and REC8] in juvenile than newborn mouse ovaries
Subject(s)
Female , Animals, Laboratory , Gene Expression , Ovarian Follicle , Mice, Inbred BALB C , Ovary , Animals, NewbornABSTRACT
Background: The glycoconjugate content of sperms indicates their physiological and fertility properties. Lectin reactivity is indicative of intact, capacitated, and acrosome-reacted sperms. In the epididymis, sperms experience maturation, glycoconjugate modification, and simultaneously, higher L-carnitine [LC] concentrations. The aim of this project was to evaluate the effects of LC and Pentoxifylline [PF] on the integrity, capacitation, and acrosomal reaction of sperms by studying their lectin reactivity
Methods: Mouse testicular sperm samples were divided into three parts. Each sample was added Ham's F10 [control] or media containing 1.76 mM LC or PF. At 30 and 90 minutes after incubation, sperm motility was assessed. Peanut agglutinin [PNA], wheat germ agglutinin [WGA], and Concanavalin A [Con A] were used to detect non-acrosome-reacted, non-capacitated, and acrosome-reacted sperms, respectively and the frequency was evaluated by flow cytometry. Statistical analysis was performed using the ANOVA
Results: Sperm motility increased after 30 and 90 minutes of incubation in the LC- and PF-treated cultures [P=0.001]. LC administration created a significant increase in the percentage of the non-acrosome-reacted sperms compared to the control sperms after 30 and 90 minutes [P=0.02 and P=0.03, respectively]. The frequency of the non-capacitated sperms in the LC-treated group increased compared to the control sperms after 30 minutes significantly [P=0.01]
Conclusion: Although the administration of LC and PF enhanced sperm motility, LC also impacted glycoconjugates on the sperm surface. Glycoconjugates are involved in the interaction between the sperm and the zona pellucida and subsequently fertilization, thereby probably influencing the male fertility state
ABSTRACT
Epigenetic reprogramming of differentiated cells can modify somatic cells into pluripotential state. Pluripotency can be induced in somatic cells by several approaches. One of the easy ways to induce pluripotency is the exposure of the somatic cells to the embryonic stem cell [ESC] extract. The objective of this study was to increase the efficiency of reprogramming of granulosa cell as a differentiated cell into pluripotential state by using epigenetic modifier agents and extract. The human granulosa cells were cultured in the medium containing 5-Aza-Deoxycytidine and trichostatin A. Then, the cells were exposed to mouse ESCs extract and co-cultured with mouse embryonic fibroblast in the presence of leukemia inhibitory factor [LIF]. Alkaline phosphatase test and also immunohistochemistery staining for Oct4, Sox2 and Nanog were performed after 24 and 72 hours and 1 week. The granulosa cells showed the alkaline phosphatase activity after 24 hours and the enzyme activity maintained for 72 hours. They also expressed Oct4 after 24 hours. The cells also expressed Sox2 and Nanog, 72 hours after exposure to the ESCs extract. The expression of the pluripotency markers decreased after 1 week. It seems that the extract can induce dedifferentiation in granulosa cells and they can express the stem cell markers. It seems that the inhibitors of the methyl transferase [5-Aza-Deoxycytidine] and histone deacetylase [trichostatin A] could delete the epigenetic markers and prepare the cells for reprogramming by administration of the extract
Subject(s)
Humans , Granulosa Cells , Cellular Reprogramming , Azacitidine/analogs & derivatives , Hydroxamic Acids , Epigenesis, GeneticABSTRACT
Sperm cells extracted from testes [TESE] have poor chromatin quality and motility. Various substances are used in the laboratory to increase sperm motility and improve the ART outcomes; however, there are few research which considered improving both sperm motility and chromatin quality. The aim of this investigation was to evaluate the improvement of the testicular sperm motility and chromatin quality exposed to L-carnitine [LC] and L-acetyl-carnitine [LAC], which are normally concentrated in testis and epididymis, compared with Pentoxifylline [PF], which used for sperm motility enhancement in IVF procedures. TESE samples from 30 male mice divided into four parts. The sperm samples were added to Ham' F10 [control] or the media contained 1.76mM of LC, LAC or PF], then, the samples were kept in the room temperature for 30, 90 and 180 min. At each time step, sperm motility and chromatin quality were assessed. Chromatin quality was evaluated by chromomycin A3 and aniline blue. Statistical analysis was performed using one way analysis of variance [ANOVA]. A p-value less than 0.05 were accepted as a statistically significant difference. The results showed LC, LAC and PF significantly increased the sperm motility. However, sperm chromatin quality only improved significantly by administration of LC and LAC. Administration of LC and LAC to the testicular sperm samples can lead to improve both sperm motility and chromatin quality. It may be because they can mimic in vivo sperm condition during late spermatogenesis
Subject(s)
Male , Animals, Laboratory , Carnitine/pharmacology , Acetylcarnitine/pharmacology , Testis , Chromatin , Epididymis , Pentoxifylline , Mice , Chromomycin A3 , Aniline CompoundsABSTRACT
Recurrent spontaneous abortion impacts almost 1% of couples. The sera from women with unexplained recurrent spontaneous abortion [URSA] have toxic effects on embryos that grow in the uterus. Therefore, the abnormal condition of the uterus may also affect sperm qualities. The objectives of this study were to search if these sera could induce DNA denaturation in sperm nuclei and also it could reduce sperm motility. Sera of 20 women with URSA history and sera from 20 women with at least two healthy children were added to the sperms samples from 20 healthy men for 2 hours. The sperm motility was assessed after incubation with sera. The samples were stained with Tdt mediated dUTP nick end labeling [TUNEL] assay for DNA fragmentation. The samples were analyzed with flow cytometry and the percentage of the TUNEL positive sperms were calculated. The data were analyzed by t-test. The incubation of the sperm samples in sera with URSA lead to a decrease in the percentage of the motile sperm from 55% in control to 41% in the treated group, significantly [p=0.038]. The percentage of the sperm with abnormal fragmented DNA increased after incubation with URSA [26.6%] compare to the control [21.2%]; however, it was not significant. It seems that sera from URSA patients could not induce a significant increase in the percentage of the sperms with nuclei contain DNA fragmentation. However, the sera of women with URSA could affect the fertility rate by reduction of the sperm motility