Human embryonic stem (hES) cells require cooperative interactions with each other for their survival. Previously, the size of hES cell clumps has been reported to be an important factor in determining their viability during routine serial passage. However, the effects of seeding density of the hES cell clumps per se have not yet been investigated. Therefore, this study attempted to compare the level of spontaneous differentiation of hES colonies passaged at two different split ratios (1:3 and 1:8) of a single confluent well of a six-well dish. After 7 d of in vitro culture following serial passage, hES colonies were assigned into three grades according to their degree of spontaneous differentiation: (1) Grade A, which was completely or mostly undifferentiated; (2) grade B, which was partially differentiated; and (3) grade C, which was mostly differentiated. Assessment of the degree of spontaneous differentiation was based on morphological observations under bright-field and phase-contrast microscopy, as well as on immunocytochemical staining for the pluripotency markers SSEA-3 and TRA-1–81. We observed that, at a split ratio of 1:3, the percentages of grade A, B, and C colonies were 89.5, 8.8, and 1.7%, respectively. This was significantly different from the corresponding values of 52.7, 31.3, and 16.0%, respectively, obtained at a split ratio of 1:8. Hence, our results indicated that a lower passage density led to a higher degree of spontaneous differentiation of hES colonies.

To increase the efficiency of stable cell line establishment from primary ovarian cancer specimens, we simultaneously initiated cultures under multiple conditions, varying extracellular matrices and the inclusion of supplements (e.g., serum or serum albumin), while minimizing exposure to xenogeneic antigens (e.g., fetal calf serum). Primary cultures were initiated from 30 specimens; cell lines were established from 10 of these for a success rate of 33%. In some instances, multiple cell lines were established from the same specimen. Five lines were characterized extensively with respect to growth properties, antigen expression, and genomic alterations. Although these lines are all low-passage, marked heterogeneity was observed, even between lines derived from the same specimen. The culture approach outlined herein will facilitate generation of reagents useful for many aspects of ovarian cancer biology.

We have developed a simple method for rapid detection of mycoplasma contamination in cell cultures using SYBR Green-based real-time polymerase chain reaction (PCR). To detect eight common contaminant mollicutes, including Mycoplasma (M. arginini, M. fermentans, M. orale, M. hyorhinis, M. hominis, M. salivarium, M. pirum) and Acholeplasma laidlawii, four primers were prepared based on the 23S rRNA regions. Using these primers and a minimum of 100 fg of mycoplasma genomic DNA, the 23S rRNA regions of these eight mycoplasma species were consistently amplified by real-time PCR. In contrast, no specific amplification product was observed using DNA templates prepared from various mammalian cell lines. Frozen and cultured samples of several cell lines were tested for mycoplasma contamination to evaluate the utility of this method. Of 25 samples that tested positive for mycoplasma by Hoechst staining, which requires two passages of cell cultures started from frozen samples, mycoplasma was detected by real-time PCR in 24 samples of cell extracts prepared directly from frozen samples. When cultured samples were used for this assay, the accuracy of the diagnoses was further improved. Thus, this technique, which is simple, rapid, and sensitive enough for practical application, is suitable for handling many samples and for routine screening for mycoplasma contamination of cell cultures.

Fibroblasts are important cellular components in wound healing, scar formation, and fibrotic disorders; and the fibroblast-populated collagen-gel (FPCG) model allows examination of fibroblast behavior in an in vitro three-dimensional environment similar to that in vivo. Contraction of free-floating FPCGs depends on an active and dynamic cytoskeleton, and the contraction dynamics are highly influenced by cell density. We investigated mechanistic differences between high– and low–cell density FPCG contraction by evaluating contraction dynamics in detail, using specific cytoskeletal disruptors. Collagen gels were seeded with human lung fibroblasts at either high (HD) or low (LD) density, and incubated with or without cytoskeletal disruptors colchicine (microtubules) or cytochalasin D (microfilaments). Gel area was measured daily. FPCG contraction curves were essentially sigmoidal, featuring an initial period of no contraction (lag phase), followed by a period of rapid contraction (log phase). Contraction curves of HD-FPCGs were distinct from those of LD-FPCGs. For example, HD-FPCGs had a negligible lag phase (compared with 3 d for LD-FPCGs) and exhibited a higher rate of log-phase contraction. Both colchicine and cytochalasin dose-dependently inhibited contraction but specifically affected different phases of contraction in HD- and LD-FPCGs; and colchicine inhibited LD-FPCGs much more than HD-FPCGs. The data indicate that LD- and HD-FPCGs contract through different primary mechanisms. Microtubules and microfilaments are both complementarily and dynamically involved in the contraction of FPCGs, and cell density influences primary cytoskeletal mechanisms. These results provide valuable information about fibroblast behavior in healing and fibrosis, and may suggest novel treatment options.

Previously, we have engineered three-dimensional (3-D) skeletal muscle constructs that generate force and display a myosin heavy-chain (MHC) composition of fetal muscle. The purpose of this study was to evaluate the functional characteristics of 3-D skeletal muscle constructs cocultured with fetal nerve explants. We hypothesized that coculture of muscle constructs with neural cells would produce constructs with increased force and adult MHC isoforms. Following introduction of embryonic spinal cord explants to a layer of confluent muscle cells, the neural tissue integrated with the cultured muscle cells to form 3-D muscle constructs with extensions. Immunohistochemical labeling indicated that the extensions were neural tissue and that the junctions between the nerve extensions and the muscle constructs contained clusters of acetylcholine receptors. Compared to muscles cultured without nerve explants, constructs formed from nerve– muscle coculture showed spontaneous contractions with an increase in frequency and force. Upon field stimulation, both twitch (2-fold) and tetanus (1.7-fold) were greater in the nerve–muscle coculture system. Contractions could be elicited by electrically stimulating the neural extensions, although smaller forces are produced than with field stimulation. Severing the extension eliminated the response to electrical stimulation, excluding field stimulation as a contributing factor. Nerve– muscle constructs showed a tendency to have higher contents of adult and lower contents of fetal MHC isoforms, but the differences were not significant. In conclusion, we have successfully engineered a 3-D nerve–muscle construct that displays functional neuromuscular junctions and can be electrically stimulated to contract via the neural extensions projecting from the construct.

Embryonic stem (ES) cells can replicate indefinitely and differentiate into all cell types, including hepatocytes. Research using primate ES cells is considered to be important for studies of potential cell therapies. Recently, we established cynomolgus monkey ES cells designated as CMK6. The CMK6 cell line is a useful tool for investigating the mechanism of differentiation in primate ES cells and developing cell therapies, because of its biological similarity to human ES cells. To examine whether cynomolgus monkey ES cells differentiate into hepatocytes, CMK6 cells were cultured with or without acidic fibroblast growth factor (aFGF). Evaluation of the hepatic differentiation was performed by analysis of the mRNA expression in early hepatic marker genes using the reverse transcriptase–polymerase chain reaction (RT-PCR). The protein expression of albumin (ALB) was also studied by immunocytochemistry. RT-PCR analyses revealed mRNA expressions of alpha-fetoprotein, transthyretin, and ALB in the presence of aFGF at 3 wk of differentiation, whereas no mRNA expression of these genes was detected in cells without aFGF. The protein expression of ALB in the presence of aFGF at 3 wk of differentiation was also confirmed by immunocytochemistry. However, tyrosine aminotransferase, which is a mature hepatic marker, was not detected in the presence or absence of aFGF at any stage of differentiation. These results suggested that aFGF successfully promoted in vitro differentiation of cynomolgus monkey ES cells to an early hepatic lineage.

The γ-irradiation of normal cells causes an increased synthesis of specific proteins. However, few studies have described the effects of high doses of irradiation on the expression of cell surface antigens in tumor cells. This study analyzed the effects of high doses of γ-irradiation on the surface antigen expression of Major Histocompatability Complex (MHC) class I/II and intercellular adhesion molecule-1 (ICAM-I) in human multiple myeloma (MM) cell lines ARP-1, ARK-RS, and 10 MM primary tumors. The expression of surface antigens was evaluated by fluorescence-activated cell sorter analysis at different time points, following the exposure to high doses of γ-irradiation. Doses of 10,000 and 15,000 cGy were not sufficient to totally block cell replication in both cell lines and primary tumors; cell replication was able to be inhibited completely only at 18,000 cGy. Lower doses (10,000 cGy) and lethal doses of irradiation (i.e., 15,000 and 18,000 cGy) increased the expression of all surface antigens present on the cells before irradiation. Essentially, such upregulation was shown to be dose dependent, with higher radiation doses resulting in higher antigen expression. Furthermore, when the kinetics of this upregulation were studied 3 and 6 d after irradiation, there was a constant increase in antigen expression in MM cells. These findings suggest that upregulation of costimulatory molecules, such as of MHC class I/II antigens and ICAM-I molecules in MM patients treated by γ-radiation, can increase the immunogenicity of the tumor cells. In light of these findings, radiotherapy combined with immunotherapy might be considered in relapsing patients after receiving the standard treatment.

Microgravity induces stress, and the brain is one of the targets that is more influenced in this environment. Alteration in transcription factors can have enormous effect because of discrepancy in the signaling process of the cells. Activator protein-1 (AP-1) is a stress-regulated transcription factor and is involved in the regulation of physiological and pathological stimuli that include cytokines, growth factors, and stress signals. In the present study, an attempt has been made to observe the effect of a microgravity environment on the activation of AP-1 in the mouse brain. Our results show that AP-1 transcription factor is activated in simulated microgravity conditions in different regions of the brain. The activation of the AP-1 is dependent upon the increased kinase activity of c-Jun NH-terminal₂ kinase-1. These results suggest that microgravity stress in the brain can elicit AP-1 activity.

Unossified horn or pilose antler cut from deer, which belong to the Cervidae generally is termed Nokyong. Nokyong is one of the most famous Korean traditional medicines and has been considered to possess sexual-reinforcing and antiaging actions. In this study, water extract of deer antler extract (DAA) prepared from the growing antler of Cervus korean TEMMINCK var. mantchuricus Swinhoe was used to investigate the efficacy of the DAA on the development of type II collagen (CII)–induced arthritis (CIA) in rats. Male rats were immunized with an emulsion of 200 μg of CII and complete Freund's adjuvant (CFA). The rats then were administered by injection a suspension of DAA or phosphate-buffered saline. The effect of DAA on cellular responses to CII was examined. The injection of DAA suppressed the CII-specific secretion of interferon (IFN)-γ from splenocytes ex vivo. The influence of DAA also was evaluated on the incidence and development of arthritis in rat CIA. Rats were immunized twice at a 3-wk interval with bovine CII, with DAA being given by injection once a d for 14 d with four different regimens. A 14-d course of DAA treatment at a daily dose of 100 μg/kg, which began on the d of the first CII immunization, suppressed the development of arthritis, as well as antibody formation and delayed-type hypersensitivity to CII. Treatment with DAA resulted in inhibition of development of arthritis and immune responses to CII.