Earticle

The ability to generate patient’s induced pluripotent stem cells (iPSCs) could provide tremendous promises for regenerative medicine. It has reported that choice of reprogramming tools is essential for the safety and the increasing efficiency of reprogramming process and transduction. Non-integrating techniques as like mRNA based reprogramming protocols are available for the production of genetically stable iPSCs while avoiding the risks of genomic integration. In this study, to derive brain cancer patient’s iPSCs by mRNA’s reprogramming, we obtained patient’s somatic tissues at brain tumor surgery that informed consent obtained according to institutionally- approved protocols. Patient's primary somatic cells were isolated, and they used as the cell source for iPSCs generation under xeno-free conditions. Reprogramming produced by non-integration methods utilized the combination of reprogramming mRNAs (OSKMNL) with evasion mRNAs (EKB) (Stemgent) during four days. Reprogrammed cells showed the colonies like iPSCs after six days of transduction, and the colonies were expanded until pick-up at day 14. These colonies were stained for pluripotency-associated genes using TRA-1-60 and TRA-1-81 by immunocytochemistry. Established colonies were also expanded without feeder condition and stained with Alkaline Phosphatase. In the further study, generated patient's iPSCs will be required to investigate their availability of the clinical application under xeno-free conditions which could provide a path to GMP applicability that should facilitate the clinical implementation of patient- or disease-specific iPSCs therapies.

Germ cells are alternative cell sources for deriving pluripotent stem cells. When cultured with feeder cells and adequate cytokines, migrating primordial germ cells (PGCs) can be reprogrammed into pluripotent stem cells, named embryonic germ cells (EGCs). In this study, we attempted to establish and characterize pig embryonic germ cells from fetal gonads. Consequently, EGC lines were derived from the genital ridges of a porcine dpc 30 fetuses in media containing LIF, FGF2 and SCF. After establishment, this cells were cultured and stabilized in LIF or FGF2 contained media. These cell lines were maintained in both condition over an extended time period and were able to spontaneously differentiate into the three germ layers in vitro. Interestingly, cell lines cultured in LIF or FGF2 expressed different pluripotency markers. While LIF-treated pig EGCs (LIF-pEGCs) expressed only few pluripotent markers including OCT4, SOX2 and NANOG, FGF2-treated pEGCs (FGF2-pEGCs) expressed pluripotency markers such as OCT4, SOX2, NANOG and SSEA4. As a result of molecular analysis, it was verified that LIF- and FGF2-EGCs represented intermediated state of EGCs and complete reprogrammed EGCs respectively, and reprogramming of pig gonadal PGCs is progressed in stepwise manner, from PGCs via intermediated EGCs to EGCs. In conclusion, it is verified that FGF2 signaling have important roles in reprogramming and maintaining pEGCs from fetal gonads.

Although nuclear transfer (NT) techniques are used to clone animals, its efficiency is very low. Moreover, nuclear transfer has resulted in offspring with severe developmental problems, probably due to incomplete nuclear reprogramming. Nuclear reprogramming is characterized by functional modification of the transferred nucleus to allow it to direct normal embryo development with the potential to grow to term. Although the nature of the reprogramming factor(s) in mammals is not clear, various nuclear as well as cytoplasmic components are involved in the processes. In this article we review recent data on factors involved in the nuclear reprogramming of cloned embryos.

Limited success of somatic cell nuclear transfer(SCNT) is attributed to incomplete reprogramming of transferred donor cell. Several approachs, such as histone deacetylase inhibitors and DNA methyltransferase inhibitors have been used to improve the efficiency of somatic cell nuclear transfer. Recently, it is reported that pre-treatment of somatic cells with undifferentiated cell extract, such as embryonic stem cell and mammalian oocytes is an attractive alternative ways to reprogramming control. The aim of this study was to evaluate the early development of porcine cloned embryos produced with porcine ear skin fibroblasts pre-treated with extract from porcine induced pluripotent stem cell (iPSC). For transport of porcine iPSC extract into cultured porcine ear skin fibroblasts, the ChariotTM reagent system was used. Treated cells were cultured for 3 days, and used for the analysis of histone H3K9 acetylation and SCNT The acetylation status of H3K9 was increased in cells treated with iPSC extract and cultured for 3 days compared with control. But, no significant difference was observed between the extract treated and control groups. After SCNT. no difference was observed in the rate of fusion (86.6% vs 86.2%) and embryo cleavage (86.6% vs 87.1%) between the extract treated and control groups. Also, no significant difference was noted in blastocyst rates (23.4% vs 28.4%) as well as cell numbers (43.8±10.8 vs 41.2±11.6) with extract treated group compared with control group. Overall apoptosis rate in blastocyst was not differences between the extract treated and control groups (4.6±3.5% vs 6.0± 5.8%). However, blastocyst rate with high apoptotic cells(>10% appototic cells) was significantly lower in extract treated group when compared with control group (7.1% vs 21.8%).. Our results demonstrated that pre-treatment of porcine ear skin fibroblasts using porcine iPSc extract had beneficial effect on the decreasing apoptosis in the blastocyst cultured in vitro, although there was no effect on the embryonic development.

Somatic cell nuclear transfer (SCNT) is an efficient technique which has been successfully applied to developmental biology, and resulted in the production of offspring from various species. It offers many opportunities in basic and medical research as well as endangered species preservation. On the other hand, embryonic stem (ES) cells are useful research tools for genetic engineering and developing disease models. In previous study, we established bovine IVF embryo derived ES cell line which can be grow indefinitely as undifferentiated cell state. In this study, we compared the effect of two different age cells (bovine ES cell; JNU-ibES-05 or adult ear fibroblast cell) on in vitro developmental potential of bovine SCNT embryo. To produce SCNT embryos, the ES cells or somatic cells were dissociated and transferred into enucleated MⅡ oocytes, and cleaved reconstructed embryos were cultured in CR1aa medium containing 10% FBS, 1 ug/ml epidermal growth factor (EGF) and 1 ug/ml insulin growth factor (IGF) for 8 days. In the result, blastocyst development rate was similar between ES cell treatment group and somatic cell treatment group, 27.7% (10/36) and 28.9% (11/ 38), respectively. However, there was particular difference in development speed from day 5 post SCNT, blastocyst expanding was 1 day faster in ES cell group than in somatic cell group. This difference was analyzed by semi-quantitative RT-PCR using pluripotency, growth and cell cycle gene markers. These results demonstrated that SCNT embryo using ES cell as a donor cell has better growth potential than somatic cell, and it will be a useful tool for a transgenic animal production.

MicroRNAs (miRNAs) are approximately 22 nucleotides of small noncoding RNAs that control gene expression at the posttranscriptional level through translational inhibition and destabilization of their target mRNAs. The miRNAs are phylogenetically conserved and have been shown to be instrumental in a wide variety of key biological processes including cell cycle regulation, apoptosis, metabolism, imprinting, and differentiation. Recently, a paper has shown that expression of the miRNA-302/367 cluster expressed abundantly in mouse and human embryonic stem cells (ESCs) can directly reprogram mouse and human somatic cells to induced pluripotent stem cells (iPSCs) efficiently in the absence of any of the four factors, Oct4, Sox2, c-Myc, and Klf4. To apply this efficient method to porcine, we analyzed porcine genomic sequence containing predicted porcine miRNA-302/367 cluster through ENSEMBL database, generated a non-replicative episomal vector system including miRNA-302/367 cluster originated from porcine embryonic fibroblasts (PEF), and tried to make porcine iPSCs by transfection of the miRNA-302/367 cluster. Colonies expressing EGFP and forming compact shape were found, but they were not established as iPSC lines. Our data in this study show that pig miRNA-302/367 cluster could not satisfy requirement of PEF reprogramming conditions for pluripotency. To make pig iPSC lines by miRNA, further studies on the role of miRNAs in pluripotency and new trials of transfection with conventional reprogramming factors are needed.

위성의 임무 수행 도중에 발생하는 소프트웨어 버그는 치명적인 문제점을 야기하여 위성 전체의 임무 실패를 초래할 수도 있다. 이를 막기 위해 개발단계부터 수많은 테스트와 검증을 수행하여 비행소프트웨어가 높은 신뢰도를 지니도록 만들어 준다 [1]. 하지만 위성이 발사 후 궤도에 올라갔을 때 하드웨어 측면에서의 문제, 혹은 미 발견 버그 등의 예상치 못한 문제들이 발견될 수도 있다. 이 경우 비행소프트웨어를 궤도 상에서 수정해야만 위성이 지속적으로 임무를 수행할 수 있다. 본 논문에서는 저궤도 위성의 reprogramming capability를 확인하고 reprogramming 절차에 대해 알아보고 검증한다.

In satellites, even a small error in flight software could cause a failure of missions. Therefore, there are strict development and verification processes for a high reliability of flight software. However, satellites on orbits could meet unexpected situations including hardware malfunction. In this case, it is necessary for flight software to be updated to cope with the unexpected situations and to continue their missions. This paper reviews reprogramming capability of next generation LEO satellites.

본 논문에서는 차량 ECU(Electronic Control Unit)의 리프로그래밍 시간 단축을 위한 차량 게이트웨이 개선 방안을 제안한다. 리프로그래밍 시간 단축을 위해 게이트웨이는 리프로그래밍을 진행하는 동안 리프로그래밍하는 ECU가 연결된 통신 채널에 리프로그래밍 이외의 메시지 전송을 금지하여야 하며, 이때 메시지 전송 금지로 인해 특정 ECU가 CAN 통신 수신 불가로 인한 고장이 발생하지 않도록 할 수 있어야 한다. 또한, 게이트웨이 내의 버퍼 오버플로우를 방지하 기 위해 연속된 통신 메시지 전송 시 추가하는 지연 시간(STmin)을 최소로 할 수 있어야 한다. 이를 위해 본 논문에서는 UDS(Unified Diagnostic Services)의 링크 제어 명령 및 최신 MCU(Micro Controller Unit)에서 제공되는 HW 게이트 웨이 기능을 이용한 개선 방안을 제안한다. 제안한 개선 방안은 차량에서 널리 사용되고 있는 인피니언사의 TC275 기반 임베디드시스템을 이용하여 구현하였으며, 개선된 실험 결과를 제시한다.

This paper proposes the method of an in-vehicle gateway to reduce the reprogramming time for the ECU (Electronic Control Unit). In order to reduce the reprogramming time, the gateway must prohibit transmitting messages, that are not related to reprogramming, to the destination CAN network, and no ECU should diagnose the DTC(Diagnostic Trouble Code) that indicates CAN communication error caused by prohibiting CAN messages by the gateway. Moreover, STmin, which are the minimum time between two consecutive CAN messages, should be minimized. In order to do this, this paper proposes the method that uses the link control command specified in UDS(Unified Diagnostic Services) and hardware based gateway functionality that are supported by the latest MCU(Micro Controller Unit). The proposed method is developed using TC275 based embedded system, and its results are presented.

Canine induced Pluripotent Stem Cells (iPSCs) can provide great potential for regenerative veterinary medicine and may assist in the development of new therapeutics and pre-clinical studies for both dogs and human. To date, there have been several reports on the generation of canine iPSCs using retroviral or lentiviral transduction of Yamanaka’s factors. However, there is no report of canine iPSCs generated by genomic integration-free methods. According to previous studies, a polycistronic and synthetic self-replicating RNA system was developed for generating human iPSCs by the RNA replicon of Venezuelan Equine Encephalitis (VEE) virus. The VEE replicon is a positive-sense and single-stranded RNA which is similar with cellular mRNA containing a 5’ cap and poly (A) tail. It is no potential for genomic DNA integration problems because it does not use a DNA intermediate. Here, we investigated to generate canine iPSCs by a single transfection of the VEE-reprogramming factor (VEE-RF) RNA. To generate integration- free canine iPSCs, the VEE-OKS-iG RNA that expresses four reprogramming ORFs (hOct4, hKlf4, hSox2 and hGlis1) was transfected. Also, B18R mRNA was co-transfected to reduce immune response by VEE replicon. Putative canine iPSC colonies first appeared between day 15-25. Interestingly, they have two distinct types of initial canine iPSC colonies. They were identified by immunohistochemistry of live cells using TRA-1-60 antibody and also showed clear alkaline phosphatase (AP) activity. Further study will be required to analyze the characterization for a clinical application, and the non-integrating and self-replicating VEE RNA replicon system has the potential to make a great contribution to generating clinically applicable canine iPSCs.

Embryonic stem cell-preconditioned microenvironment is important for cancer cells properitities by change cell morphology and proliferation. This microenvironment induces cancer cell reprogramming and results in a change in cancer cell properties such as differentiation and migration. The cancer microenvironment affects cancer cell proliferation and growth. However, the mechanism has not been clarified yet. Using the ES-preconditioned 3-D microenvironment model, we provide evidence showing that the ES microenvironment inhibits proliferation and reduces oncogenic gene expression. But ES microenvironment has no effect on telomerase activity, cell viability, cellular senescence, and methylation on Oct4 promoter region. Furthermore, methylation of Nanog was increase on ES-preconditioned microenvironment and supports results that no difference on RNA expression levels. Taken together, these results demonstrated that in the ES-preconditioned 3-D microenvironment is a crucial role for cancer cell proliferation not senescence.