Hepatogenic Differentiation Capacity of Human Wharton’s Jelly Mesenchymal Stem Cell in a Co-culturing System with Endothelial Cells in Matrigel/collagen Scaffold in the Presence of Fetal Liver Extract

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.

Comparison of the expression of hepatic genes by human Wharton's jelly mesenchymal stem cells cultured in 2D and 3D collagen culture systems

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

in vitro model for hepatocyte-like cell differentiation from Wharton's jelly derived-mesenchymal stem cells by cell-base aggregates

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

Cardiomyocyte marker expression in mouse embryonic fibroblasts by cell- free cardiomyocyte extract and epigenetic manipulation

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

Expression of pluripotency markers in human granulosa cells after embryonic stem cell extract exposure and epigenetic modification

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

Effects of high-fat diet on the numerical density and number of neuronal cells and the volume of the mouse hypothalamus: a stereological study / 대한해부학회지

It has been demonstrated that the type of diet affects the brain structure and function. Consumption of fat-rich food is one of the most important factors that lead to increase in the prevalence of cardiovascular and neurological diseases. High-fat diet may change the volume and neuronal number or density in the hypothalamus, which is the center of energy control. Therefore, this study was designed to study the effect of high-fat diet on the density and number of neurons, and also the volume of hypothalamus in adult male mice. Forty male mice were divided into the control and experimental groups. The control group were fed with standard and the experimental groups, with high-fat diet for 4 (short-term) or 8 (long-term) weeks. The animals were perfused and brains were immediately removed, post-fixed and cut coronally and serially using cryostat at 30-microm thickness. Every 6th sections were stained by cresyl violet. The numerical density and number of neuron and the volume of hypothalamus were estimated by using unbiased stereological methods. Data analysis showed that both short and long time consumption of high-fat diet decreased the neuronal cell density of the hypothalamus. Interestingly, despite a decrease in the neuronal cell density, long time consumption of high-fat diet could significantly increase the volume of hypothalamus (P<0.05). High fat diet decreased the neuronal cell density and increased the volume of the hypothalamus, but it did not significantly change its total neurons. These changes might be due to an increase in the extracellular space through inflammation or gliosis in the hypothalamus.

Effects of Carthamus tinctorius on Semen Quality and Gonadal Hormone Levels in Partially Sterile Male Rats

PURPOSE: Traditional herbal medicine is just one of the many different approaches using plants in the remedy of diseases. Carthamus tinctorius (CT) or safflower is a popular plant that is used for coloring and flavoring in food industries. The effect of CT on spermatogenesis and sperm parameters has been reported in traditional medicine but has not yet been confirmed scientifically. Therefore, this study was designed to determine the effects of CT on spermatogenesis and the male reproductive system in an animal model. MATERIALS AND METHODS: Sixty male rats were divided into five groups. Four groups were injected with 5 mg/kg of busulfan as a model of partial infertility. Then, the experimental groups were treated with 10 mg/kg, 25 mg/kg, or 50 mg/kg of CT extract for 35 days. The control was treated with busulfan (infertile control) or distilled water only. After this period, the animals were sacrificed and blood samples were taken for hormonal assay. The semen was collected from the epididymis and the reproductive organs were assessed. Sperm count and motility were measured and smears were prepared for assessment of the other parameters. RESULTS: The results indicated that the percentage of sperm with good morphology, motility, and count increased significantly in the group treated with 10 mg/kg CT (p=0.002, p=0.03, and p=0.00001, respectively). The effects on hormonal changes and genital organ weights were also positive. CONCLUSIONS: It is probable that the CT extract affects spermatogenesis and as a result sperm quality. Further studies are needed.