In Vitro Cellular & Developmental Biology - Animal

Recently, valuable characteristics of menstrual blood stem cells (MenSCs) have impelled scientists to take its advantages for cell therapy of different diseases including liver disorders. In this study, we examined messenger RNA (mRNA) expression levels of phases I and II drug metabolizing enzymes including glutathione S-transferase (GST) and cytochrome P-450 (CYP) in differentiated hepatocyte-like cells from MenSCs. The isolated MenSCs were characterized and differentiated into hepatocyte-like cells using hepatocyte growth factor (HGF) and oncostatin M (OSM) in combination with other components in serum-free culture media. After primary characterization of hepatocyte markers, mRNA expression of GSTA1, GSTA2, GSTP1, CYP3A4, and CYP7A1 was assessed in differentiated cells in reference to undifferentiated cells using real-time PCR. Based on immunofluorescent staining and real-time PCR data, the differentiated MenSCs could express functional hepatocyte markers at mRNA and/or protein levels suggesting development of hepatocyte-like cells from MenSCs. Moreover, the expression levels of GSTA1, GSTA2, and CYP3A4 mRNA were upregulated in differentiated cells compared to undifferentiated cells. The expression of CYP7A1 gene was also remarkable on the last day of differentiation process. However, the expression level of GSTP1 did not exhibit statistically significant change during differentiation (P = 0.6). Based on accumulative data, MenSCs could be viewed as an accessible population of stem cells with differentiation ability into drug-metabolizing hepatocyte-like cells.

Bovine somatotropin was given to six lactating (230 day) cows (40 mg/day × 5-days) and excipient was given to six control cows. Mammary, liver, and adipose explants from somatotrophin and control cows were co-cultured at 37° C for 24 h with 0.5 µCi [14C]acetate/ml media with or without 0.5 µg/ml somatotrophin. Tissue lipids were extracted with chloroform/methanol and separated by thin layer chromatography. In vivo somatotrophin increased milk production 2.4 kg/day compared to a 0.9 kg/day decrease by controls. Mammary tissue from somatotrophin cows incorporated more [14C]acetate into total lipids (4417 vs. 3016 dpm/mg tissue) than controls. Adding somatotrophin to explant cultures from somatotrophin cows further increased incorporation into total lipids (4839 vs. 3994 dpm/mg tissue). In contrast, adipose tissue from somatotrophin cows incorporated less [14C]acetate into total lipids than controls (1524 vs. 2581 dpm/mg tissue). Serum IGF-I concentration correlated well (r=0.69) with milk output differences between Days 1 and 5 of treatment. Media IGF-I concentration correlated well (r=0.61) with the difference in total lipid synthesis between the in vitro control and somatotrophin groups. Results support the concept that somatotrophin increases milk production by partitioning nutrients away from adipose toward mammary tissue.

The trabecular meshwork is a specialized tissue in the anterior chamber of the eye that regulates the aqueous humor outflow and controls the intraocular pressure. Cells in the trabecular meshwork are believed to be essential for maintenance of the outflow system, and their malfunctioning may lead to elevation of intraocular pressure and development of glaucoma. These cells are avid phagocytes. Using an in vitro tissue culture system, we have previously shown that bovine trabecular meshwork cells exhibited a short-term loss of cell-matrix adhesiveness after exposure to latex microspheres. The current study showed that 4 h after phagocytosis, the cytoskeletal structure in trabecular meshwork cells was disrupted, the formation of focal contact formation was limited, and the cellular migratory activity was increased. These in vitro responses paralleled those that occur in vivo. By 24 h, all the changes demonstrated returned to normal. Our data suggest that the short-term loss in cell-matrix cohesiveness observed after phagocytic challenge may be related to the reorganization of cytoskeletal structures and the decline of focal contact formation. The altered cell migration may also be interlinked.

It has been reported that 50–60 Hz magnetic fields (MF) with flux densities ranging from microtesla to millitesla are able to induce heat shock factor or heat shock proteins in various cells. In this study, we investigated the effect of 60 Hz sinusoidal MF at 8 and 80 μT on the expression of the luciferase gene contained in a plasmid labeled as electromagnetic field-plasmid (pEMF). This gene construct contains the specific sequences previously described for the induction of hsp70 expression by MF, as well as the reporter for the luciferase gene. The pEMF vector was transfected into INER-37 and RMA E7 cell lines that were later exposed to either MF or thermal shock (TS). Cells that received the MF or TS treatments and their controls were processed according to the luciferase assay system for evaluate luciferase activity. An increased luciferase gene expression was observed in INER-37 cells exposed to MF and TS compared with controls (p < 0.05), but MF exposure had no effect on the RMA E7 cell line.

Immunosurgery is a useful technique for the isolation of inner cell masses from murine blastocysts. Conventionally, rabbit antisera made ad hoc against murine splenic or fetal cells or fibroblasts have been used as antibody sources. We investigated the feasibility of using commercially available rabbit antiserum to murine erythrocytes (anti-RBC) and compared it with rabbit antiserum generated ad hoc to murine L-cells (anti-L-cell). Our results indicate that anti-RBC is at least as effective as anti-L-cell serum for the immunosurgical isolation of inner cell masses, which became either miniblastocysts (later forming outgrowths) or embryoid bodies (undergoing ectoderm-endodermlike differentiation within 48 h). Because anti-RBC is commercially available, the technical modification described herein increases the accessibility of the immunosurgical protocol for the isolation of murine inner cell masses.

Prostate stromal cells may play binary roles in the process of prostate cancer development. As the first to be encountered by infiltrating prostate cancer cells, prostate stromal cells form the first defense line against prostate cancer progression and metastasis. However, interaction between prostate cancer and stromal cells may facilitate the formation of a tumor microenvironment favoring cancer cell growth and survival. To establish an experimental system for studying the interaction between cancer and stromal cells, we isolated three matched pairs of normal and cancer-associated human prostate stromal clones. In this report, we describe the morphologic and behavioral characteristics of these cells and their effect on LNCaP prostate cancer cells in co-culture. Unlike LNCaP prostate cancer cells, the isolated prostate stromal clones are large fibroblast-like cells with a slow proliferation rate. Growth and survival of these clones are not affected by androgens. The stromal cells display high resistance to serum starvation, while cancer-associated stromal clones have differentiated survival ability. In co-culture experiments, the stromal cells protected some LNCaP prostate cancer cells from death by serum starvation, and cancer-associated stromal clones showed more protection. This work thus established a panel of valuable human prostate stromal cell lines, which could be used in co-culture to study the interaction between prostate cancer and prostate stromal cells.

The mesothelial cells obtained from human omental adipose tissue showed a typical cobblestone monolayer and reacted strongly with keratin, but did not have Von Willebrand factor. Ultrastructurally these cells revealed the existence of desmosome-like cell junctions as well as intracellular canaliculi, tubular structures surrounded by microvilli, and tonofilament-like filaments. The mesothelial cells grew much faster in the medium containing epidermal growth factor, actively took up acetylated-low density lipoprotein into their cytoplasm, and released angiotensin-converting enzyme. They also released urokinase-type plasminogen activator, but only half as much as do human umbilical vein endothelial cells; release of tissue-type plasminogen activator was not observed. Inasmuch as the mesothelial cells also released plasminogen activator inhibitor-1, as do human umbilical vein endothelial cells, we could not detect u-PA activity in culture medium. u-PA may play a role in the protection against adhesion among visceral organs. These observations indicate that cultured human mesothelial cells have characteristics closely related to those found in human endothelial cells.