Hertwig\u27s epithelial root sheath cells contribute to formation of periodontal ligament through epithelial-mesenchymal transition by TGF-β.

In tooth root development, periodontal ligament (PDL) and cementum are formed by the coordination with the fragmentation of Hertwig\u27s epithelial root sheath (HERS) and the differentiation of dental follicle mesenchymal cells. However, the function of the dental epithelial cells after HERS fragmentation in the PDL is not fully understood. Here, we found that TGF-β regulated HERS fragmentation via epithelial-mesenchymal transition (EMT), and the fragmented epithelial cells differentiated into PDL fibroblastic cells with expressing of PDL extracellular matrix (ECM). In the histochemical analysis, TGF-β was expressed in odontoblast layer adjacent of HERS during root development. Periostin expression was detected around fragmented epithelial cells on the root surface, but not in HERS. In the experiment using an established mouse HERS cell line (HERS01a), TGF-β1 treatment decreased E-cadherin and relatively increased N-cadherin expression. TGF-β1 treatment in HERS01a induced further expression of important ECM proteins for acellular cementum and PDL development such as fibronectin and periostin. Taken together, activation of TGF-βsignaling induces HERS fragmentation through EMT and the fragmented HERS cells contribute to formation of PDL and acellular cementum through periostin and fibronectin expression.福岡歯科大学2016年

Robust and highly efficient hiPSC generation from patient non-mobilized peripheral blood-derived CD34+ cells using the auto-erasable Sendai virus vector

Background: Disease modeling with patient-derived induced pluripotent stem cells (iPSCs) is a powerful tool forelucidating the mechanisms underlying disease pathogenesis and developing safe and effective treatments. Patientperipheral blood (PB) cells are used for iPSC generation in many cases since they can be collected with minimuminvasiveness. To derive iPSCs that lack immunoreceptor gene rearrangements, hematopoietic stem and progenitorcells (HSPCs) are often targeted as the reprogramming source. However, the current protocols generally requireHSPC mobilization and/or ex vivo expansion owing to their sparsity at the steady state and low reprogrammingefficiencies, making the overall procedure costly, laborious, and time-consuming.Methods: We have established a highly efficient method for generating iPSCs from non-mobilized PB-derivedCD34+ HSPCs. The source PB mononuclear cells were obtained from 1 healthy donor and 15 patients and werekept frozen until the scheduled iPSC generation. CD34+ HSPC enrichment was done using immunomagnetic beads,with no ex vivo expansion culture. To reprogram the CD34+-rich cells to pluripotency, the Sendai virus vectorSeVdp-302L was used to transfer four transcription factors: KLF4, OCT4, SOX2, and c-MYC. In this iPSC generationseries, the reprogramming efficiencies, success rates of iPSC line establishment, and progression time wererecorded. After generating the iPSC frozen stocks, the cell recovery and their residual transgenes, karyotypes, T cellreceptor gene rearrangement, pluripotency markers, and differentiation capability were examined.Results：We succeeded in establishing 223 iPSC lines with high reprogramming efficiencies from 15 patients with 8 different disease types. Our method allowed the rapid appearance of primary colonies (~ 8 days), all of which were expandable under feeder-free conditions, enabling robust establishment steps with less workload. After thawing, the established iPSC lines were verified to be pluripotency marker-positive and of non-T cell origin. A majority of the iPSC lines were confirmed to be transgene-free, with normal karyotypes. Their trilineage differentiation capability was also verified in a defined in vitro assay.Conclusion：This robust and highly efficient method enables the rapid and cost-effective establishment of transgene-free iPSC lines from a small volume of PB, thus facilitating the biobanking of patient-derived iPSCs and their use for the modeling of various diseases

エナメル質の横紋形成メカニズムの解明

エナメル質の基本構造をなすエナメル小柱には横紋が観察される.この横紋は概日リズムを刻んだ成長線のひとつとして知られているが,形成メカニズムについては様々な説がある.非脱灰凍結切片で基質形成期のエナメル基質を抗アメロゲニン抗体で免疫染色すると横紋様パターンを示した.この結果から,横紋は基質形成期のアメロゲニンのタンパク量に依存した石灰化パターンであり,アメロゲニンの発現は概日的に変動するのではないかと推測した.そこで,アメロゲニンの発現に周期があるか観察するために,アメロゲニンプロモーターの下流にルシフェラーゼを繋いだコンストラクト(pGL3-1730-luc)をラットエナメル芽細胞株HAT7に遺伝子導入してアメロゲニンの転写活性を計測したところ,一定の周期をもって変動することが認められた.次に,プロモーター領域のDeletion-mutant (pGL3-464, -74, -48-luc)を作製して周期性の制御に関わる領域を検索した結果, C/EBPαのbindingモチーフが有力な候補と考えられた.その転写はMsx2によって制御を受けることが知られていることから, Msx2の発現ベクターを用いた強制発現の影響を調べたところ,そのリズムが消失した.またMsx2の遺伝子欠損マウスには基質形成の横紋様パターンが見られなかった.以上の結果から, Msx2がアメロゲニンの発現周期に影響していることが推測され,さらにアメロゲニンの発現量の周期的変化によって生じた基質形成パターンが,横紋形成に関与していると考えられた.In enamel rods, periodic bands referred to as \u27cross-striations\u27 are observed, and known as incremental lines of circadian rhythm. Though it is considered that the cross-striation is involved in the biological circadian rhythm during the secretion of enamel matrix protein by ameloblasts, the developmental mechanism involved has not been examined in detail. By immunostaining amelogenin in fresh frozen sections of mouse lower incisor, we could observe fluorescent periodic bands in the enamel matrix, which were identical to the pattern of these cross-striations. Accordingly, we focused on the biological rhythm in the section of amelogenin. We examined amelogenin mRNA transcriptional activity in an ameloblast cell line (HAT7) by using real-time luminescence microscopy. The results showed that amelogenin mRNA transcriptional activity exhibited periodic rhythmicity and that Msx2 over-expression led to the disappearance of the periodic change. Further, in the lower incisors of Msx2-deficient mice, the periodic bands were not observed. Taken together, our findings suggest that the formation of cross-striations in the enamel rods was associated with the expression of amelogenin regulated by the transcriptional activity

Design and fabrication of 2.4 GHz pre-biased rectifier

A 2.4 GHz rectifier operating in a region of low RF input power was developed. The rectifier has a cross-coupled bridge configuration and is driven by a differential RF input signal. Since a rectifier needs an RF signal higher than the threshold voltage of transistors, we introduced a pre-biasing circuit to compensate for the threshold voltage. A low-voltage digital circuit, subthreshold voltage regulator, and low-power level shifter were introduced for reducing the power consumption of the pre-biasing circuit and increasing the driving voltage for the switches at the same time. The circuit simulations revealed that the pre-biasing circuit was effective in a low RF input power region. However, the output voltage was degraded in a high power region. Then, we combined the pre-biased rectifier in parallel with a non-biased rectifier. Three types of rectifiers consisting of LC matching circuits, three-stage rectifier cells, and biasing circuits were designed and fabricated using a 0.18-mu m mixed signal/RF CMOS process with one poly and six metal layers. The fabricated pre-biased rectifier operated in a region of RF input power of less than -15 dBm, while the non-biased rectifier could not operate in this region. The parallel combination of pre-biased and non-biased rectifiers effectively solved the drawback of the pre-biased rectifier in a high RF input power region

Low-power, small-size transmitter module with metamaterial antenna

Development of a low-power, small-size transmitter is needed for wireless sensor networks. An effective way to reduce power consumption is to reduce the operating time in a voltage-controlled oscillator. In this study, a 2.4 GHz on-off keying transmitter circuit is designed and implemented with an electrically small antenna using a left-handed transmission line. The transmitter circuit was fabricated with a standard 0.18 mu m CMOS technology, while the antenna was fabricated with a 3.0 x 4.5 cm printed circuit board, chip capacitors, and chip inductors. Measured output power was -6.8 dBm with a power consumption of 3.59 mW when the baseband signal was always "high". The power consumption was reduced to 1.96 mW for the baseband signal with a mark ratio of 0.5