폐 오가노이드와 유전자 교정을 이용한 섬모운동이상 모델 구축에 관한 연구

학위논문(박사) -- 서울대학교대학원 : 의과대학 의학과, 2023. 8. 김종일.Lung is the organ that inhale external air for gas exchange, causing continuous exposure to foreign substances. Therefore, various defense mechanisms that remove them have been developed. Mucociliary clearance is one of the importance defense mechanisms to remove relatively large particles deposited on airway. The synchronized motion of cilia on apical side of ciliated cells on airway plays a key role on this mechanism. When genes involved in cilia composition or assembly have defects, primary ciliary dyskinesia (PCD) occurs. The acquired dyskinesia can also occur when ciliary movement decreases due to chronic inflammation or repeated infections. However, research on ciliary movement is limited, and genomic studies using genetic perturbation have been conducted only with animal models, primarily due to the fact that ciliated cells are fully differentiated cells, so that it cannot be studied using cell lines. Primary cell cultures, which cannot be cultured long-term, are also not suitable for gene editing study. In addition, there have been difficulties in measuring ciliary beating frequency (CBF) using the conventional methods since cilia move together and images of cilia can be easily overlapped for each other. To address these issues, during this doctoral program, (1) lung organoids that can be cultured long-term were established using patient samples, and it was confirmed that ciliated cells were induced well enough. (2) A novel CBF measurement technique was jointly developed to evaluate the spatial distribution of ciliary movement in the organoids quickly and accurately. (3) The variant with unknown significance discovered through whole-exome sequencing of a PCD family was introduced in normal airway organoids and it is confirmed that the variant causes ciliary dyskinesia. Through this study, the CRISPR/Cas9-mediated organoid model was established, enabling functional validation of various PCD-causing variants and laying the foundation for future correction through editing, as well as demonstrating the potential for extension to acquired dyskinesia.폐는 외부 공기를 흡입하여 가스 교환을 하는 장기이므로, 필연적으로 외부 물질에 노출된다. 이에 따라, 흡입된 이물질을 제거하기 위한 다양한 방어 기전이 존재한다. 그 중 하나인 핵심적인 세포 소기관인 섬모 (cilium)는 기도의 섬모 세포 (ciliated cell)에 존재하며 서로 동기화된 움직임을 통해 기도에 침착한 이물질을 외부로 밀어낸다. 따라서, 섬모를 구성하거나 조립에 관여하는 유전자 중 하나에 이상이 생기면 유전질환인 일차성 섬모운동장애 (primary ciliary dyskinesia; 이하 PCD)가 발생한다. 또한, 만성 염증이나 반복적인 감염으로 인해 기관지 상피 세포가 손상되면, 다양한 만성 폐질환에서 획득성 섬모운동장애 (acquired ciliary dyskinesia)가 발생하기도 한다. 그럼에도, 여러 가지 이유로 섬모 운동에 대한 연구는 제한적이었다. 우선, 섬모 세포는 분화된 세포이고 분열하지 않기 때문에, 세포주가 따로 존재하지 않아 이를 이용한 연구가 불가능하며 주로 동물 모델이 이용되어왔다. 사람 조직에서 얻은 섬모 세포를 일시적으로 배양하는 일차 세포 배양 (primary cell culture)이 섬모 연구에 널리 쓰이고 있으나, 장기간 배양이 불가능하므로, 유전자 편집 연구에는 적합하지 않다. 또한, 기존 섬모 운동을 측정하는 시간과 노력이 많이 들면서도 편향이 발생하기 쉬우면서 부정확할 때가 있는데, 섬모들은 함께 움직이며 섬모 이미지가 겹쳐보이기 쉽기 때문이다. 박사 학위 기간동안 이를 해결하기 위해 여러 가지 접근을 수행하였다. (1) 호흡기 환자 샘플, 특히 기관지 내시경 브러싱 샘플을 이용하여 사람 폐 오가노이드를 수립하였으며, 섬모 세포를 비롯하여 기관지를 구성하는 세포들이 잘 유도됨을 확인하였다. (2) 공동 연구를 통해 새로운 섬모 운동 측정하는 방법을 개발하였으며, 이를 통해 오가노이드 내 다양하게 존재하는 섬모 운동을 빠르고 정확하게 평가할 수 있게 되었다. (3) PCD 환자 가족의 전장 유전체 염기서열 분석 (whole genome sequencing)에서 발견된 기능을 정확히 알 수 없는 돌연변이를 CRISPR/Cas9 시스템으로 정상 폐 오가노이드에 도입하였고, 이 돌연변이가 섬모 운동 이상을 유발하는 것을 확인하였다. 이번 연구를 통해, CRISPR/Cas9 기반의 사람 폐 오가노이드 유래 섬모 운동 연구 모델이 확립되었다. 이를 통해 PCD 유발하는 다양한 변이들의 기능적 검증이 가능 해졌으며, 향후 돌연변이 교정을 위한 유전자 편집을 위한 기반을 마련하였고, 만성 폐질환으로 인한 획득성 섬모운동장애 연구로의 확장 가능성도 보여주었다.Chapter 1. Introduction 1 Chapter 2. Materials and Methods 11 1. Lung Organoids from Bronchoscopic Brushing Specimens 11 2. Immunofluorescent Staining of Organoids 12 3. Transmission Electron Microscopy 13 4. Single Cell RNA Sequencing of Lung Organoids 14 5. CRISPR/Cas9 Gene Editing of Lung Organoids 15 6. Ciliary Beating Frequency Measurement of Organoids 16 7. Whole Genome Sequencing of PCD Family 18 Chapter 3. Results 19 1. Motion-contrast based CBF Measurement 19 2. Candidate Gene Selection from a PCD Family 24 3. Lung Airway Organoid from Bronchoscopic Specimens 28 4. CRISPR/Cas9-mediated Lung Organoids Modeling of PCD 31 Chapter 4. Discussion 35 Chapter 5. Conclusion and Perspectives 37 References 39 Abstract (Korean) 43박

Experimental Tracheal Replacement Using 3-dimensional Bioprinted Artificial Trachea with Autologous Epithelial Cells and Chondrocytes

Various treatment methods for tracheal defects have been attempted, such as artificial implants, allografts, autogenous grafts, and tissue engineering; however, no perfect method has been established. We attempted to create an effective artificial trachea via a tissue engineering method using 3D bio-printing. A multi-layered scaffold was fabricated using a 3D printer. Polycaprolactone (PCL) and hydrogel were used with nasal epithelial and auricular cartilage cells in the printing process. An artificial trachea was transplanted into 15 rabbits and a PCL scaffold without the addition of cells was transplanted into 6 rabbits (controls). All animals were followed up with radiography, CT, and endoscopy at 3, 6, and 12 months. In the control group, 3 out of 6 rabbits died from respiratory symptoms. Surviving rabbits in control group had narrowed tracheas due to the formation of granulation tissue and absence of epithelium regeneration. In the experimental group, 13 of 15 animals survived, and the histologic examination confirmed the regeneration of epithelial cells. Neonatal cartilage was also confirmed at 6 and 12 months. Our artificial trachea was effective in the regeneration of respiratory epithelium, but not in cartilage regeneration. Additional studies are needed to promote cartilage regeneration and improve implant stability.ope

Direct Control of Stem Cell Behavior Using Biomaterials and Genetic Factors

Stem cells have recently emerged as an important candidate for cell therapy. However, some major limitations still exist such as a small quantity of cell supply, senescence, and insufficient differentiation efficiency. Therefore, there is an unmet need to control stem cell behavior for better clinical performance. Since native microenvironment factors including stem cell niche, genetic factors, and growth factors direct stem cell fate cooperatively, user-specified in vitro settings are required to understand the regulatory roles and effects of each factor, thereby applying the factors for improved cell therapy. Among others, various types of biomaterials and transfection method have been employed as key tools for development of the in vitro settings. This review focuses on the current strategies to improve stemness maintenance, direct differentiation, and reprogramming using biomaterials and genetic factors without any aids from additional biochemicals and growth factors.ope

Association Between Impairment of DNA Double Strand Break Repair and Decreased Ovarian Reserve in Patients With Endometriosis

Background: Repair of DNA double strand break (DSB) is an important mechanism for maintaining genetic stability during a DNA damage event. Although, a growing body of recent evidence suggests that DNA DSBs and related repair mechanisms may be important in ovarian aging and in various cancers, there are few reports in endometriosis. We, therefore, examined expression levels of genes pertaining to DNA DSB repair in patients with endometriosis to assess the potential effects on ovarian reserves. Materials and methods: A total of 69 women undergoing laparoscopic surgery for endometriosis and other benign conditions was included; endometriosis group (n = 38) vs. controls (n = 31). DNA DSBs in endometrial and ovarian tissues of both groups were compared via immunohistochemistry, aimed at γ-H2AX expression. To gauge genotoxin-induced DNA DSBs in endometrial stromal cells, γ-H2AX expression was determined by western blot after H2O2 treatment of cultured endometrial stromal cells (endometriosis group and controls) and Ishikawa cell-line cultures. Endometrial and ovarian tissue levels of BRCA1, BRCA2, Rad51, and ATM (ataxia-telangiectasia mutated) mRNA expression were also compared. Correlations between expression levels of genes of interest and serum anti-müllerian hormone (AMH) levels were assessed as well. Results: Expression of γ-H2AX in immunostained endometrial and ovarian tissue preparations was greater in the endometriosis group, compared with controls. After H2O2 treatment, γ-H2AX expression levels were also significantly greater in cultured stromal cells of the endometriosis group and in the Ishikawa cell line than in controls. Endometrial expression of BRCA1 and Rad51 mRNA proved significantly lower in the endometriosis group (vs. controls), as did ovarian expression of BRCA1 and BRCA2 mRNA. Serum AMH concentration showed a significant correlation with ovarian BRCA1 mRNA expression in women with endometriosis (p = 0.03). Conclusions: In women with endometriosis, expression levels of various genes implicated in DSB repair are decreased and ovarian BRCA1 expression correlates with.ope

압전탄성 배양표면에 의해 분화가 촉진된 중간엽 줄기세포의 심근 및 골격근 재생효과

학위논문 (박사)-- 서울대학교 대학원 공과대학 화학생물공학부, 2017. 8. 김병수.In native muscle microenvironment, electrical and mechanical stimuli exist in the form of action potential and muscle contraction. Here we developed a cell culture system that can mimic the in vivo microenvironment and provide these stimuli to cultured cells and investigated whether the stimulation can promote myogenic differentiation of human mesenchymal stem cells (hMSCs). Ex vivo induction of myogenic differentiation of MSCs prior to implantation would potentiate therapeutic efficacy of stem cell therapies for muscle diseases, since MSCs rarely undergo myogenic differentiation following implantation. In muscle microenvironments, electric pulse and cyclic mechanical strain are sequentially produced. However, no study has applied the pulsatile mechanoelectric cues (PMEC) to stimulate myogenic differentiation of MSCs ex vivo. Stretchable and piezoelectric substrate (SPS) was fabricated by polydimethylsiloxane spin-coating on aligned ZnO nanorods. PMEC were provided to hMSCs cultured on SPS by subjecting SPS to cyclic stretching and bending, resulting in significantly promoted myogenic differentiation of hMSCs as well as intracellular signaling related to the differentiation. There are three types of muscle in human body: cardiac muscle, skeletal muscle, and smooth muscle. In the present study, we have focused on hMSCs differentiation into cardiac muscle cells and skeletal muscle cells in part 3 and 4, respectively. In part 3, bone marrow-derived hMSCs were induced to differentiate into cardiomyocytes to confirm the efficiency of PMEC for myogenic differentiation. Furthermore, in part 4, human umbilical cord blood MSCs were induced to differentiate into skeletal myocyte on pNIPAAm-engrafted thermosensitive SPS (TSPS). Following differentiation ex vivo, the cells were detached from TSPS in the form of cell-sheet fragments by changing the temperature to 4°C. The injection of cell-sheet fragments of differentiated cells into injured skeletal muscle in mice showed improved cell retention and muscle regeneration compared to injection of either undifferentiated cells or differentiated/dissociated cells. Our system may provide a tool for study of electrical and mechanical regulation of stem cells and be utilized to potentiate stem cell therapies.Table of contents Abstract ………………………………………………………………… I Table of contents ……………………………………………………… III List of figures ………………………………………………………… VII Abbreviations …………………………………………………………… IX Chapter 1. Research backgrounds and objective …………………… 1 1.1. Cardiovascular diseases and stem cell therapy …………………………… 3 1.2. Skeletal muscle disorders and stem cell therapy ………………………… 5 1.3. Cardiomyogenic and skeletal myogenic differentiation of MSCs ……… 6 1.4. Piezoelectricity …………………………………………………………… 8 1.5. Thermosensitivity and cell sheet formation …………………………… 10 1.6. Research objective of thesis …………………………………………… 11 Chapter 2. Experimental methods …………………………………… 13 2.1. Fabrication and characterization of SPS, TSPS ………………………… 15 2.1.1. Chemicals …………………………………………………………… 15 2.1.2. Synthesis of bi-axially grown ZnO NRs …………………………… 15 2.1.3. Fabrication of SPS …………………………………………………… 16 2.1.4. pNIPAAm grafting …………………………………………………… 17 2.1.5. Piezoelectric properties of SPS and TSPS …………………………… 18 2.1.6. Thermosensitivity of TSPS ………………………………………… 19 2.2. In vitro assays …………………………………………………………… 20 2.2.1. hBMSC culture and cardiomyogenic differentiation ………………… 20 2.2.2. hUCBMSC culture and skeletal myogenic differentiation ………… 21 2.2.3. Immunocytochemistry ……………………………………………… 22 2.2.4. Reverse transcription polymerase chain reaction (RT-PCR) ………… 23 2.2.5. Phalloidin staining …………………………………………………… 24 2.2.6. Western blot analysis ………………………………………………… 25 2.2.7. Quantitative polymerase chain reaction (qPCR) …………………… 26 2.2.8. Transmission electron microscope (TEM) …………………………… 26 2.2.9. Fragmentation of cell sheet ………………………………………… 27 2.3. In vivo assays …………………………………………………………… 28 2.3.1. Inducing skeletal muscle injury and cell transplantation …………… 28 2.3.2. Histological examinations …………………………………………… 28 2.3.3. In vivo live imaging of injected cells ……………………………… 29 2.4. Statistical analysis ……………………………………………………… 29   Chapter 3. A stretchable piezoelectric substrate providing pulsatile mechanoelectric cues for cardiomyogenic differentiation of mesenchymal stem cells ……………………………………………………………………… 31 3.1. Introduction ……………………………………………………………… 33 3.2. Results and discussion …………………………………………………… 37 3.2.1. Piezoelectric characteristics of SPS ………………………………… 37 3.2.2. Cytotoxicity of SPS, bending, stretching, and EP …………………… 40 3.2.3. Cell alignment induced by cyclic stretching ………………………… 43 3.2.4. Enhanced cardiomyogenic differentiation by EP and cyclic stretching ………………………………………………………………………… 45 3.2.5. Cardiomyogenic differentiation-related autocrine growth factor expression and intracellular signaling enhanced by EP and cyclic stretching …………………………………………………………… 49 Chapter 4. Thermosensitive, stretchable, and piezoelectric substrate for generation of myogenic cell sheet fragments from human mesenchymal stem cells …………………………………………………………………………… 53 4.1. Introduction ……………………………………………………………… 55 4.2. Results and discussion …………………………………………………… 59 4.2.1. Piezoelectric characteristics of TSPS ……………………………… 59 4.2.2. Simulation of the electrical effects of TSPS on hUCBMSCs ……… 62 4.2.3. Cell sheet formation using thermosensitive pNIPAAm ……………… 67 4.2.4. Enhanced myogenic differentiation of hUCBMSCs by PMEC …… 69 4.2.5. Intracellular signaling related to myogenic differentiation ………… 73 4.2.6. Characteristics of myogenic differentiated cell sheet fragments …… 77 4.2.7. Improved skeletal muscle regeneration by injecting myogenic differentiated hUCBMSC sheet fragments …………………………… 79 4.2.8. Enhanced in vivo retention of hUCBMSC sheet fragments ………… 83 Chapter 5. Conclusions ……………………………………………………… 85 References …………………………………………………………………… 89 요약(국문초록) …………………………………………………………… 109Docto

Capabilities-Based Query Rewriting with Permissible Queries of External Query Processor

Maste

그래핀의 재생의학적 이용

-Graphene is a one-atom-thick sheet composed of carbon atoms only. It has a two-dimensional honeycomb structure with sp2 orbital bonding, which presents some unique properties. Due to large Youngs modulus, good electrical conductivity, ability to immobilize several kinds of small molecules and proteins, and biocompatibility of graphene, it has attracted interests inits ability to enhance cell growth and differentiation, followed by recent several studies. We reviewed about the osteogenic differentiation of mesenchymal stem cells, and neurogenic differentiation of neuron stem cells,and the ectodermal and mesodermal differentiation of induced pluripotent stem cells using graphene. Graphene has not only enhanced the adhesion and proliferation of mesenchymal stem cells, but also led to the faster differentiation even without any other exogenous signals. Nonetheless, graphene has some cytotoxicities in its amount-response manner, which is critical to regenerative medicine. The cytotoxicities of graphene were compared with those of grapheneoxide and carbon nanotubes.N

수소화된 비정질 규소의 비평형 정상상태 전하 수송에 관한 연구

학위논문(박사) - 한국과학기술원 : 물리학과, 1989.2, [ iii, 81 p. ]Steady-state photoconductivity ($\sigma_{th}$) in hydrogenated amorphous silicon(a-Si:H) has been studied theorically and experimentally in order to understand the recombination processes of carriers and to obtain the information on the localized states in the mobility gap. Although the experimental data-published by many groups show somewhat different features, it is commonly observed that the $\sigma_{th}$ shows activated behavior in the temperature range between about 200 and 100 K. The activation energy depends on the doping concentration and incident light intensity, but it lies between 0.1 and 0.2 eV. The exponent $\gamma$ obtained from the intensity dependence of $\sigma_{ph}(\sigma_{ph}\,\propto\,I^\gamma$, here I is incident light intensity) is 0.5 for n-type a-Si:H films. In order to explain the temperature and intensity dependence of $\sigma_{ph}$,we developed a numerical model in which the recombination of free carriers through the exponentially distributed band-tail states and through the Gaussian-distributed dangling bonds were calculated in terms of Shockley-Read theory and the occupation statistics of correlated dangling bonds. From the comparison between calculated $\sigma_{ph}$ and observed $\sigma_{ph}$, it is concluded that the recombination of free carriers in band edge through the dangling bonds are dominant at room temperature. In the temperature range between 200 and 100 K, the recombination of carriers in band-tail states through the dangling bonds explain the observed experimental results of activated from and bimolecular behavior in $\sigma$ph.한국과학기술원 : 물리학과

질의 능력 기반의 멀티데이터베이스 질의 처리기 설계

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