Direct reprogramming of fibroblasts into diverse lineage cells by DNA demethylation followed by differentiating cultures

Direct reprogramming, also known as a trans-differentiation, is a technique to allow mature cells to be converted into other types of cells without inducing a pluripotent stage. It has been suggested as a major strategy to acquire the desired type of cells in cell-based therapies to repair damaged tissues. Studies related to switching the fate of cells through epigenetic modification have been progressing and they can bypass safety issues raised by the virus-based transfection methods.In this study, a protocol was established to directly convert fully differentiated fibroblasts into diverse mesenchymal-lineage cells, such as osteoblasts, adipocytes, chondrocytes, and ectodermal cells, including neurons, by means of DNA demethylation, immediately followed by culturing in various differentiating media. First, 24 h exposure of 5-azacytidine (5-aza-CN), a well-characterized DNA methyl transferase inhibitor, to NIH-3T3 murine fibroblast cells induced the expression of stem-cell markers, that is, increasing cell plasticity. Next, 5-aza-CN treated fibroblasts were cultured in osteogenic, adipogenic, chondrogenic, and neurogenic media with or without bone morphogenetic protein 2 for a designated period. Differentiation of each desired type of cell was verified by quantitative reverse transcriptase-polymerase chain reaction/western blot assays for appropriate marker expression and by various staining methods, such as alkaline phosphatase/alizarin red S/oil red O/alcian blue. These proposed procedures allowed easier acquisition of the desired cells without any transgenic modification, using direct reprogramming technology, and thus may help make it more available in the clinical fields of regenerative medicine.

Direct reprogramming of fibroblasts into diverse lineage cells by DNA demethylation followed by differentiating cultures

Direct reprogramming, also known as a trans-differentiation, is a technique to allow mature cells to be converted into other types of cells without inducing a pluripotent stage. It has been suggested as a major strategy to acquire the desired type of cells in cell-based therapies to repair damaged tissues. Studies related to switching the fate of cells through epigenetic modification have been progressing and they can bypass safety issues raised by the virus-based transfection methods.In this study, a protocol was established to directly convert fully differentiated fibroblasts into diverse mesenchymal-lineage cells, such as osteoblasts, adipocytes, chondrocytes, and ectodermal cells, including neurons, by means of DNA demethylation, immediately followed by culturing in various differentiating media. First, 24 h exposure of 5-azacytidine (5-aza-CN), a well-characterized DNA methyl transferase inhibitor, to NIH-3T3 murine fibroblast cells induced the expression of stem-cell markers, that is, increasing cell plasticity. Next, 5-aza-CN treated fibroblasts were cultured in osteogenic, adipogenic, chondrogenic, and neurogenic media with or without bone morphogenetic protein 2 for a designated period. Differentiation of each desired type of cell was verified by quantitative reverse transcriptase-polymerase chain reaction/western blot assays for appropriate marker expression and by various staining methods, such as alkaline phosphatase/alizarin red S/oil red O/alcian blue. These proposed procedures allowed easier acquisition of the desired cells without any transgenic modification, using direct reprogramming technology, and thus may help make it more available in the clinical fields of regenerative medicine.

Focal adhesion linker proteins expression of fibroblast related to adhesion in response to different transmucosal abutment surfaces

PURPOSE: To evaluate adherence of human gingival fibroblasts (HGFs) to transmucosal abutment of dental implant with different surface conditions with time and to investigate the roles of focal adhesion linker proteins (FALPs) involved in HGFs adhesion to abutment surfaces. MATERIALS AND METHODS: Morphologies of cultured HGFs on titanium and ceramic discs with different surface were observed by scanning electron microscopy. Biocompatibility and focal adhesion were evaluated by ultrasonic wave application and cell viability assay. FALPs expression levels were assessed by RT-PCR and western blot. RESULTS: There seemed to be little difference in biocompatibility and adhesion strength of HGFs depending on the surface conditions and materials. In all experimental groups, the number of cells remaining on the disc surface after ultrasonic wave application increased more than 2 times at 3 days after seeding compared to 1-day cultured cells and this continued until 7 days of culture. FALPs expression levels, especially of vinculin and paxillin, also increased in 5-day cultured cells compared to 1-day cultured fibroblasts on the disc surface. CONCLUSION: These results might suggest that the strength of adhesion of fibroblasts to transmucosal abutment surfaces increases with time and it seemed to be related to expressions of FALPs.

Effects of CoCl2 on Osteogenic Differentiation of Human Mesenchymal Stem Cells

OBJECTIVE: To investigate the effects of the hypoxia inducible factor-1 (HIF-1) activation-mimicking agent cobalt chloride (CoCl2) on the osteogenic differentiation of human mesenchymal stem cells (hMSCs) and elucidate the underlying molecular mechanisms. STUDY DESIGN: The dose and exposure periods for CoCl2 in hMSCs were optimized by cell viability assays. After confirmation of CoCl2-induced HIF-1alpha and vascular endothelial growth factor expression in these cells by RT-PCR, the effects of temporary preconditioning with CoCl2 on hMSC osteogenic differentiation were evaluated by RT-PCR analysis of osteogenic gene expression, an alkaline phosphatase (ALP) activity assay and by alizarin red S staining. RESULTS: Variable CoCl2 dosages (up to 500 microM) and exposure times (up to 7 days) on hMSC had little effect on hMSC survival. After CoCl2 treatment of hMSCs at 100 microM for 24 or 48 hours, followed by culture in osteogenic differentiating media, several osteogenic markers such as Runx-2, osteocalcin and osteopontin, bone sialoprotein mRNA expression level were found to be up-regulated. Moreover, ALP activity was increased in these treated cells in which an accelerated osteogenic capacity was also verified by alizarin red S staining. CONCLUSIONS: The osteogenic differentiation potential of hMSCs could be preserved and even enhanced by CoCl2 treatment.

Methionine Enhances the Contractile Activity of Human Colon Circular Smooth Muscle In Vitro

Effective drug to manage constipation has been unsatisfactory. We sought to determine whether methionine has effect on the human colon. Human colon tissues were obtained from the specimens of colon resection. Microelectrode recording was performed and contractile activity of muscle strips and the propagation of the contractions in the colon segment were measured. At 10 microM, methionine depolarized the resting membrane potential (RMP) of circular muscle (CM) cells. In the CM strip, methionine increased the amplitude and area under the curve (AUC) of contractions. In the whole segment of colon, methionine increased the amplitude and AUC of the high amplitude contractions in the CM. These effects on contraction were maximal at 10 microM and were not observed in longitudinal muscles in both the strip and the colon segment. Methionine reversed the effects of pretreatment with sodium nitroprusside, tetrodotoxin and Nw-oxide-L-arginine, resulting in depolarization of the RMP, and increased amplitude and AUC of contractions in the muscle strip. Methionine treatment affected the wave pattern of the colon segment by evoking small sized amplitude contractions superimposed on preexisting wave patterns. Our results indicate that a compound mimicking methionine may provide prokinetic functions in the human colon.

Polyacetylene Compound from Cirsium japonicum var. ussuriense Inhibited Caspase-1-mediated IL-1beta Expression

Our previous report showed that polyacetylene compound, 1-Heptadecene-11, 13-diyne-8, 9, 10-triol (PA) from the root of Cirsium japonicum var. ussuriense has anti-inflammatory activity. In this study we investigated the role of the PA as inhibitor of caspase-1, which converts prointerleukin-1beta (proIL-1beta) to active IL-1beta and is activated by inflammasome involved in the inflammatory process. We tested the effect of PA on the production of pro-inflammatory cytokines, IL-1beta in murine macrophage cell line, RAW264.7. PA inhibited lipopolysaccharide (LPS)-induced IL-1beta production by macrophages at a dose dependent manner. PA also suppressed the activation of caspase-1. The mRNA level of ASC (apoptosis-associated spec-like protein containing a CARD), an important adaptor protein of inflammasome, was decreased in the PA treated group. Therefore our results suggest that the anti-inflammatory effect of PA is due to inhibit the caspase-1 activation.

Migrating motor complex changes after side-to-side ileal bypass in mouse ileum ex-vivo: mechanism underlying the blind loop syndrome? / 대한외과학회지

PURPOSE: This study was intended to investigate the migrating motor complex (MMC) changes after ileal bypass in ex-vivo mouse models. METHODS: Partial (side-to-side) and total bypass (occlusion of proximal part of bypassed loop) were performed on ileums of female Institute of Cancer Research mice. After 2 and 4 weeks, the bypassed segments were harvested and MMCs were recorded at 4 different sites ex-vivo. Amplitude, duration, interval, direction of propagation, and the area under the curve (AUC) of MMCs were measured and compared to those of the controls. RESULTS: In control mice (n = 7), most MMCs propagated aborally (91.1%). After 2 weeks of partial bypass (n = 4), there was a significant decrease in both amplitude and AUC, and orally-propagating MMCs increased significantly (45%, P = 0.002). Bidirectional MMCs (originating in the bypassed loop and propagating in both directions) were also observed (10%). The amplitude of the MMCs remained decreased at 4 weeks after partial bypass (n = 4), and neither the AUC nor the direction of propagation showed significant changes compared to 2 weeks. Similarly, in the total bypass model, both the amplitude and AUC of the MMCs decreased significantly compared to controls. In contrast to partial bypass, 95% of the MMCs within the bypassed loop propagated aborally after 2 weeks (n = 6), which was similar to the control state. After 4 weeks (n = 5), however, MMCs either lost their temporal relationship or completely disappeared. CONCLUSION: The changes in propagation direction of the MMCs in the partially bypassed loop may contribute to stagnation of bowel contents and the development of blind loop syndrome.

The Epithelial-Mesenchymal Transition During Tooth Root Development

Hertwig's epithelial root sheath (HERS) consists of bilayered cells derived from the inner and outer dental epithelia and plays important roles in tooth root formation as well as in the maintenance and regeneration of periodontal tissues. With regards to the fate of HERS, and although previous reports have suggested that this entails the formation of epithelial rests of Malassez, apoptosis or an epithelialmesenchymal transformation (EMT), it is unclear what changes occur in the epithelial cells in this structure. This study examined whether HERS cells undergo EMT using a keratin-14 (K14) cre:ROSA 26 transgenic reporter mouse. The K14 transgene is expressed by many epithelial tissues, including the oral epithelium and the enamel organ. A distinct K14 expression pattern was found in the continuous HERS bi-layer and the epithelial diaphragm were visualized by detecting the beta-galactosidase (lacZ) activity in 1 week postnatal mice. The 2 and 4 week old mice showed a fragmented HERS with cell aggregation along the root surface. However, some of the lacZ-positive dissociated cells along the root surface were not positive for pan-cytokeratin. These results suggest that the K14 transgene is a valuable marker of HERS. In addition, the current data suggest that some of the HERS cells may lose their epithelial properties after fragmentation and subsequently undergo EMT.

Apoptosis during Rat Tooth Development

Teeth develop via a reciprocal induction between the ectomesenchyme originating from the neural crest and the ectodermal epithelium. During complete formation of the tooth morphology and structure, many cells proliferate, differentiate, and can be replaced with other structures. Apoptosis is a type of genetically-controlled cell death and a biological process arising at the cellular level during development. To determine if apoptosis is an effective mechanism for eliminating cells during tooth development, this process was examined in the rat mandible including the developing molar teeth using the transferase-mediated dUTP-biotin nick labeling (TUNEL) method. The tooth germ of the mandibular first molar in the postnatal rat showed a variety of morphological appearances from the bell stage to the crown stage. Strong TUNEL-positive reactivity was observed in the ameloblasts and cells of the stellate reticulum. Odontoblasts near the prospective cusp area also showed a TUNEL positive reaction and several cells in the dental papilla, which are the forming pulp, were also stained intensively in this assay. Our results thus show that apoptosis may take place not only in epithelial-derived dental organs but also in the mesenchyme-derived dental papilla. Hence, apoptosis may be an essential biological process in tooth development.

Electrophysiological Characteristics of Human Colon Circular Muscle

PURPOSE: Interstitial cells of Cajal (ICC) play a core function in colonic motility as a pacemaker by regulating the slow wave. We intended to investigate the electrophysiological characteristics of the circular smooth muscle in the human colon, especially in Koreans. METHODS: Colon tissues were obtained from patients undergoing an elective colectomy for non-obstructive lesions. Tissues were immediately stored in oxygenated Krebs-Ringer's bicarbonate solution, and conventional microelectrode recordings of the colonic circular muscle were done from muscle cells. RESULTS: The trans-membrane potentials from 100 tissues were recorded. Regular slow-wave patterns were observed in 36 patients, and 64 patients showed irregular wave patterns. In the tissues exhibiting regular slow-wave patterns, 20 tissues were recorded from the proximal colon and 16 from the distal colon. The frequency of the slow wave from the proximal part was significantly higher than that from the distal part. The resting membrane potential, the upstroke amplitude, the spike amplitude, and the maximal rate of rise showed no significant difference between the two parts, but the time to reach half amplitude did show a significant difference between the two parts. No significant differences in parameters were observed based on the patients' demographics such as sex and age (younger or older than 60). We were able to divide the irregular waves into 3 groups. Irregular slow waves were observed in 35 tissues, irregular slow waves mixed with spike waves were observed in 26 tissues, and spike waves alone were observed in 3 tissues. CONCLUSION: We analyzed the electrophysiological characteristics of the human colonic circular smooth muscle, especially those of the slow wave originating from ICC of patients who had not been pretreated with drugs.

Changes in Migrating Motor Complex after Bowel Obstruction in the Murine Ileum

PURPOSE: Partial obstruction of the small bowel causes hypertrophy of smooth muscle cells and enteric neurons. After small bowel obstruction, slow waves have also been reported to disappear or to be greatly reduced at the oral site of the obstruction in the murine ileum. The purpose of this research was to study the changes in migrating motor complexes (MMCs) after partial obstruction in order to compensate for the attenuated function of slow waves. METHODS: A ring of film (6 mm in length, 4 mm in internal diameter) was placed over the small intestine 5-6 cm oral to the ileocecal valve in 8-10 wk old female ICR mice. These rings resulted in a partial obstruction of the intestine after 2 wk. The mechanical activities of the small intestine were recorded and the amplitude, interval, and half-duration of the MMCs were analyzed. RESULTS: The MMCs from a partially obstructed ileum occurred every 1.58+/-1.06 min and had a half-duration of 6.90+/-5.54 sec. The interval and the half-duration of the control MMCs were 3.60+/-1.11 min and 31.5+/-11.4 sec, respectively. The difference in interval and the half-duration of the MMCs reached statistical significance (P=0.03; P=0.00). The amplitude and the area under the curve (AUC) of the MMCs of the obstructed ileum were much higher than those of the control (31.3+/-8.86 vs. 6.05+/-1.92 mN; 161.18+/-44.09 vs. 72.95+/-2.45 mN . sec/MMC wave; P=0.00, 0.02). CONCLUSION: The MMCs with higher amplitude and AUC, with shorter interval, and with shorter half-duration, compared with those of the control, were recorded from the partially obstructed murine ileum, reflecting efforts to overcome the effect of obstruction by increasing the power of contractions.