Regulation of cell proliferation and apoptosis of HiB5 hippocampal progenitor cells by all-trans- and 9-cis-retinoic acids : 해마 HiB5 세포주에서 retinoic acid 에 의한 세포증식과 세포사의 조절

정진주 서울대학교 대학원 1999 국내박사

Although recent evidence suggests that retinoids play a role in physiological functions of the central nervous system, the effect of retinoids on the hippocampal cells is largely unknown. The present study examined the effects of all-trans- and 9-cis-retinoic acid (9-cis-RA) on the proliferation and apoptotic death of hippocampal progenitor cell line HiB5. Since these cells were immortalized using temperature sensitive allele tsA58 of SV40 large T antigen, these cells grow continuously at 32C, the permissive temperature of T antigen in medium containing 10% fetal bovine serum (FBS) in an undifferentiated state. At 39C, in chemically defined medium (N2), however, they stop proliferation and start to differentiate into heterogeneous cell populations following apoptotic deaths of large fraction of cells. Thus, HiB5 cell line is a good model for the study of hippocampal cell development and differentiation. At 32C, treatment of HiB5 cells with all-traps-RA or 9-cis-RA significantly increased trypan-blue-exclusiveviable cell number in N2, but not in DMEM containing 10% FBS. Both retinoids also attenuated the decrease in cell number up to 4 days after treatment at 39C. Accordingly, DNA synthesis rate was significantly increased by all-trans-RA and 9-cis-RA in N2 medium as revealed by [^(3)H]-thymidine incorporation assay. The protein levels of p53 and SV40 T antigen were increased by all-trans-RA and 9cis-RA in N2 medium both at 32C and 39C. Along with the increases in viable cells, both retinoids increased dead floating cells in N2 both at 32C and 39C under microscopic observation. DNA fragmentation analysis showed that DNA laddering was induced in a time-related manner in these conditions. DAPI-stained morphology of nucleus showed the typical characteristics of apoptosis, supporting again that dying cells are undergoing apoptosis. However, flow cytometric analysis of DNA contents showed that the proportion of apoptotic cells was not affected by retinoids in N2 media while that of the cells in S phase was significantly increased, which parallels with the increase in cell number. In addition, both retinoids increased BcI-2 mRNA level at 32 and 39C, but only 9cis-RA was able to alleviate the increase in Bax mRNA expression at 39C. As a whole, the ratio of BcI-2 to Bax expression was increased by both retinoids indicating that retinoids increased the survival rate of cell, which may explain, in part, the increased number of viable cells. In an attempt to study the molecular mechanisms of retinoid action in HiB5 cells, the expressions of retinoic acid receptors (RARs) and retinoid X receptors (RXRs) were examined. HiB5 cells proliferating under DMEM containing 10% FBS expressed mRNAs for RARα, RARr, RXRα and RXRβ but not RARβ and RXRr. When the cells were transferred to N2 medium, addition of retinoids induced RARr at both 32C and 39C. Of special note is that all-trans-RA had stronger effect on the induction of RARr than 9-cis-RA. Moreover, functional retinoid receptor contents correlated well with changes in these transcriptlevels, as measured by transient transfections with strong retinoic acid response element (RARE) reporter construct. Electromobility shift assays also revealed that the binding activities to synthetic oligonucleotides corresponding to the retinoic acid response elements were increased in retinoid-treated groups at 32C, while not in complete accordance with the results of 39C. Taken together, these data strongly suggest that both retinoids are heavily involved in the survival of HiB5 hippocampal progenitor cells by increasing proliferation, but not apoptosis and that all-trans-RA and 9-cis-RA might act via different mechanism in these processes.

Study on the mitochondrial DNA release of innate-like B1 cells

오소영 세종대학교 대학원 2020 국내석사

Cell-free DNA, both nuclear and mitochondrial DNA (mtDNA), have been known to be a potential biomarker in numerous diseases, such as cancer, autoimmune disease, and viral infection. In most cases, leakage of extracellular mtDNA is mediated in a ROS-dependent manner. It is known to promote sterile inflammation. Recently, one study showed that C type ODN could induce mtDNA release from B-cell chronic lymphocytic leukemia (CLL) in both TLR9- and ROS-independent manner. However, it remains unclear what type of B cells respond to C type ODNs. Thus, we wanted to investigate the effect of GpC-C on B cells in the aspect of mtDNA release. We found that B1a cells, innate-like CD5+ B subset, are the primary source to release mtDNA extracellularly in response to GpC-C included in C type ODN. Besides, mtDNA secretion was more remarkable in IL10+ (regulatory) B1a subpopulation. Furthermore, the unmethylated G/C stem-loop structure of C type ODNs is critical to mtDNA secretion, and ODNs with this structure was designated as G/C rich ODNs in this study. Taken together, our findings demonstrated that G/C rich ODNs could be a useful reagent for modulating the functions of regulatory B1a cells and might apply to various immune diseases such as cancers and autoimmune diseases.

CD8+ T cell responses and antigen presentation during Mycobacterium tuberculosis infection in humans

조장은 Graduate School, Yonsei University 2005 국내박사

Cell-mediated immune responses are a major protective mechanism against Mycobacterium tuberculosis infection. These cells mediated immune responses are composed of T cells and macrophages. In order to understand the immune mechanism for tuberculosis (TB), three aspects of the immune responses regarding the major histocompatibility complex (MHC) class I-restricted CD8+ T cells and the processing of M. tuberculosis antigens were researched. The three aspects included : CD8+ T cell responses to M. tuberculosis-derived peptides, the antigen processing mechanism of M. tuberculosis somatic antigen, and the effect of M. tuberculosis on interferon-gamma (IFN-g)-induced genes.First, ThyA30-38, RpoB127-135, PstA175-83, and 85B15-23 were previously identified as M. tuberculosis-derived peptides specific for HLA-A*0201-restricted CD8+ T cells. This study characterized these peptide-specific CD8+ T cells in individuals that were either actively (TB patients) or latently infected with M. tuberculosis (PPD+). CD8+ T cell responses to these peptides were induced in all study groups (PPD+ healthy subjects, pulmonary TB patients, and TB pleurisy patients), except the PPD- subjects. However, the M. tuberculosis antigen-specific CD8+ T cell immunity appeared to be depressed in patients with advanced stages of TB. These M. tuberculosis-derived peptides also induced CD8+ T cell responses in subjects expressing the subtypes HLA-A*0203, A*0206, and A*0207, suggesting that these epitopes are A2 supertype peptides. Among these four peptides, the immunodominant peptide that induced the highest number of IFN-g secreting CD8+ T cells differed depending on the subjects. Short-term cell lines specific for these peptides proliferated in vitro and secreted IFN-g upon antigenic stimulation in PPD+ subjects. HLA-A*0201 dimer assays indicated that the PstA175-83-specific CD8+ T cell population in PPD+ healthy subjects was functionally heterogeneous, since only one-half or one-fourth of the cells produced IFN-g upon peptide stimulation. In addition, by assaying cytotoxic T lymphocyte (CTL) activities, we observed that CTL responses specific for these M. tuberculosis-derived peptides could be induced in Bacille Calmette-Guerin (BCG)-vaccinated subjects. This result suggests that CD8+ T cells may be involved in controlling TB in BCG-vaccinated or PPD+ healthy people.Secondly, M. tuberculosis resides and replicates inside macrophages. In our previous publication, CD8+ T cell-mediated immune responses specific for the peptide RpoB127-135, which was derived from the RNA polymerase beta-subunit of the M. tuberculosis protein, could be induced in TB patients. In order to demonstrate that CD8+ T cells can recognize RpoB127-135 that was processed by M. tuberculosis-infected macrophages, CD8+ T cell lines specific for the RpoB127-135 peptide were generated from the peripheral blood mononuclear cells (PBMCs) of healthy HLA-A*0201 and A*0206 subjects, using in vitro immunization techniques. These CD8+ T cell lines specifically recognized and destroyed M. tuberculosis infected-macrophages. In addition, the presentation of the M. tuberculosis-derived epitope peptide, RpoB127-135, to CD8+ T cells did not seem to be inhibited by brefeldin-A treatment, which blocks the classical MHC class I-restricted antigen presentation pathway in macrophages. Therefore, the RpoB127-135 peptide may be processed by accessing the alternative MHC class I processing pathway, which was previously suggested as the processing pathway for the cytoplasmic proteins of M. tuberculosis. Since the RpoB gene of M. tuberculosis was reported to be actively expressed inside macrophages, the RpoB protein or derived peptides may be useful for the development of TB vaccines. This study also suggests that not only secreted but also somatic proteins of M. tuberculosis need to be screened for TB vaccines and therapeutic agents.Lastly, the effect of M. tuberculosis infection on the expression of IFN-g induced genes involved in MHC class I-restricted antigen processing pathway of the host cells was investigated. IFN-g is a principal mediator of the bactericidal activation of macrophages. M. tuberculosis is either resistant to the IFN-g responsive microbicidal mechanisms of macrophages or alternatively may block the macrophage response to IFN-g. This study demonstrates that the M. tuberculosis infection selectively affected the transcription of IFN-g responsive genes. While the transcription of CD64 decreased, IFN-g responsive genes involved in the MHC class I Ag processing pathway were either unaffected or induced by M. tuberculosis. Further studies are needed to elucidate the underlying mechanisms whereby M. tuberculosis inhibits cellular responses to IFN-g. 결핵에 대한 보호면역반응은 세포매개면역반응으로 T세포와 대식세 포가 중요한 역할을 한다. 결핵에 대한 면역기전을 이해하기 위해 MHC class I에 제한적인CD8+ T세포와 결핵항원의 처리과정에 대한 면역반 응을 세가지 측면, 즉 결핵균에서 유래된 펩티드에 대한 CD8+ T세포반응, 결핵균 균체 (somatic) 항원의 처리과정, 결핵균이 IFN-g에 의해 유도되는 유전자에 미치는 영향에 대해 연구하였다. 첫번째로 ThyA30-38, RpoB127-135, PstA175-83, 85B15-23이 HLA-A*0201에 제한적인 CD8+ T 세포에 특 정한 결핵균에서 유도된 펩티드임이 이미 보고된바있다. 활동성 결핵환 자와 PPD+인 사람에서 이 펩티드에 특정한CD8+ T 세포의 특징을 알 아보고자 하였다. 위의 펩티드는 HLA-A*0203, A*206, A*0207 type을 가진 사람에서 CD8+ T 세포반응을 유도하였고, 따라서 항원결정기가 A2 supertype 펩티드임을 제시하였다. 펩티드에 대한 CD8+ T 세포 면역반응 은 PPD+ 건강인, 폐결핵환자, 흉막성 결핵환자에서 유도되었다. 그러나 결핵 항원에 특정한CD8+ T세포 면역반응은 결핵이 심하게 진전된 환자 에서 감소되었다. 이러한 4개의 펩티드중에서 많은 양의 IFN-g의 분비를 유도하는 면역우세 (immunodominant) 펩티드는 사람마다 달랐다. 펩티드 에 특정한 IFN-g를 분비하는 단기세포주 (short term cell line)는 in vitro 에서 증식하였고, 항원자극 후PPD+인 사람에서 IFN-g를 분비하였다. 또 한HLA-A*0201 dimer assay를 수행한 결과, PPD+ 건강인에서 PstA175-83에 특정한 CD8+ T세포 집단의 12에서 14만이 IFN-g를 생성함을 확인하였고, PPD+ 건강인에서 IFN-g를 생성하는 PstA175-83에 특정한 CD8+ T 세포집단이 다양함을 알수있었다. 또한 BCG백신을 맞은 사람에서 결핵균 유래펩티드에 특정한 기억독성면역 (recall CTL) 반응을 알아보 았다. 이 결과는 CD8+ T세포가 BCG 백신을 맞은 사람 들에서 결핵에 대한 보호면역반응을 유도할 수 있음을 제시한다.두번째로 결핵균은 대식세포에서 살고 증식한다. 결핵균 단백의 RNA polymerase로부터 유래한 RpoB127-135 펩티드에 특정한 면역반응을 매개 하는CD8+ T세포는 결핵환자에서 유도되었다. 이는 CD8+ T세포가 결 핵균이 감염된 대식세포에서 처리된RpoB127-135를 인지하는지 알아보기 위해 RpoB127-135 펩티드에 특정한 CD8+ T세포를 in vitro immunization 방법으로 정상인 HLA-A*0201와A*0206 type을 가진 정상인의 말초혈 액을 사용하여 만들었다. 이렇게 만들어진 CD8+ T 세포는 결핵균이 감염 된 대식세포를 특이적으로 인지하여 파괴하였다. 또한 결핵균에서 유도된 RpoB127-135 펩티드를 CD8+ T세포에 제시하는 기전은 brefeldin A 처리 후에 억제되지 않았다. 따라서 결핵균에서 유도된 RpoB127-135 펩티드는 결 핵균의 세포질단백의 처리방법으로 제시된 alternative MHC class I 경로 로 처리되어 면역세포에 전달되고 인식된다고 사료된다. 결핵균의 RpoB 유전자가 대식세포안에서 잘표현되었고, 따라서 RpoB단백질이나 RpoB 단백질에서 유도된 펩티드는 결핵백신개발에 유용할 것으로 기대된다.마지막으로 IFN-g는 대식세포를 활성화시키는 주된 매개자이고, 결핵 균은 대식세포에서IFN-g에 의한 세균억제기전에 저항하거나 IFN-g에 의한 대식세포의 활성화를 막을 수 있을 것이다. 이번 연구는 결핵균으로 감 염된 대식세포와 수상돌기세포에서 IFN-g에 반응하는 MHC class I 처리와 제시에 관여하는 유전자의 전사에 대한 억제와 유도가 유전자마다 다 른것을 증명하였다. 따라서 다음 단계에서는 결핵균이 IFN-g의 세포반응 을 억제하는 기전을 이해하는 것이 필요하고, 이런 연구는 결핵에 대한 백 신과 치료제 개발에 기여하리라 사료된다.

Cell cycle regulation by ankyrin repeat-rich membrane spanning scaffold protein and size-dependent fractionation of human mesenchymal stem cells using microfluidic chip filtration

정희경 서울대학교 대학원 2017 국내박사

As one of the adaptor proteins, ankyrin rich membrane spanning protein (ARMS/Kidins220) is highly expressed in the nervous system, such as the hippocampus, olfactory burb, and motor neurons in the spiral cord. Generally, ARMS/Kidins220 interacts with the neurotrophin, ephrin, vascular endothelial growth factor (VEGF) and glutamate receptors, which have many essential roles in the nervous system such as neuronal survival, neuronal differentiation, dendrites and synapse developments. Recent studies have reported that this protein yields sustained signal of the mitogen-activated protein kinase (MAPK) via the CrkL-C3G complex and Rap1, which are both highly expressed in melanoma. This suggests the ARMS is a potential oncogene and is involved in controlling cell cycle. Cell proliferation is tightly controlled by cyclins and cyclin-dependent kinase (CDKs) in G1, S, M, and G2 phases. First, we observed that knockdown of ARMS/Kidins220 inhibited mouse neuroblastoma cell proliferation and resulted in a slowdown of cell cycle during G1 phase by reductions of cyclin D1 and CDK4. In addition, these decrease in cyclin D1 and CDK4 protein levels cause a subsequent reduction of pRb hyperphosphorylation which occurs at the G1/S phase transition. Moreover, we observed an upregulation of p21, a CDK4 inhibitor, with downregulation of ARMS/Kidins220. Taken together, these data suggest that p21 inhibits the kinase activity of cyclin D1-CDK4 and prevents the hyperphosphorylation of pRb, resulting in down regulation of cell cycle progression. Therefore, we report that the low ARMS/Kidins220 has a role as a signaling mediator to regulate neuroblastoma proliferation and could be worked as a potential oncogene. Human mesenchymal stem cells (hMSCs) can self-renew and differentiate into multiple cell types, which make them suitable for use in tissue engineering and cell therapy. However, their clinical application is hindered by the need for in vitro culture with growth factors in order for their expansion to reach therapeutically useful levels. Moreover, hMSCs consist of a heterogeneous population that exhibits variable morphology, a limited capacity for self-renewal, and inefficient differentiation. Thus, obtaining purified hMSCs with high potential is an important step toward increasing the efficiency of stem cell therapy. For the purpose of flow-based identification of hMSC subpopulations, optimally designed microfluidic chips were developed based on the hydrodynamic filtration (HDF) principle. In these chips, hydrodynamic effects for passive separation are combined with fluid interaction along a series of channels. Microfluidic chip design parameters resulted from complete analysis of laminar flow for flow fraction and complicated networks of main and multi-branched channels, which were validated for microfluidic cell sorting by HDF to fractionate hMSCs into three size-dependent subpopulations: small rapidly self-renewing (RS, < 25 µm); medium spindle-shaped (SS, 25–40 µm); and large flattened (FL, > 40 µm) cells. In our HDF chip, a virtual cut-off width (WC) boundary causes hMSCs to migrate to sidewalls, whereby they enter branch channels in response to specific ratios between main and side flows. Our results showed continuous and rapid sorting of hMSCs into three subpopulations with highly efficient recovery (>86%) and complete purity rates, and without damage to cells. Analysis of surface marker expression revealed that RS and SS cells showed high levels of CD73, CD90, and CD105, whereas FL cells did not express CD73. Subsequently, to compare the multipotency of sorted cells, each subpopulation was induced to undergo adipogenic, osteogenic, and Schwann cell (SC) differentiation. Phenotypic and gene marker expression analyses of differentiation indicated that sorted RS and SS subpopulations showed higher potential for adipogenic, osteogenic, and Schwann cell differentiation than the FL subpopulation. Moreover, RS and SS cells displayed significantly increased expression of SC markers and growth factors such as hepatocyte growth factor (HGF) and vascular endothelial growth factor (VEGF). Therefore, our results indicate that the microfluidic chip successfully sorted hMSCs based on size and target cells with high multilineage potentials were obtained from hMSCs.

Myc Choreographs the Crosstalk Between Tumor Cells and Macrophages to Drive Immune Evasion in Cancer

Dhanasekaran, Renumathy Stanford University ProQuest Dissertations & These 2022 해외박사(DDOD)

소속기관이 구독 중이 아닌 경우 오후 4시부터 익일 오전 9시까지 원문보기가 가능합니다.

The MYC oncogene is one of the most commonly activated oncogenes in human cancer [1]. Hepatocellular carcinoma (HCC), an aggressive malignancy with prognosis, shows MYC activation in up to 70% of patients [1]. The coordinated effects of MYC on a multitude of cellular processes results in enforcement of the hallmarks of cancer. While the role of MYC in regulating cancer cell autonomous functions like cell growth, proliferation and metabolic dysfunction are well characterized [2-4], the mechanisms by which MYC modulates the host immune response are not fully understood. Macrophages are immune cells part of the mononuclear phagocyte innate immune system which play a crucial role in tumor progression and metastasis [5,6]. They are one of the largest immune populations infiltrating tumors and have a strong prognostic and therapeutic role [7]. This dissertation focuses on the mechanistic and therapeutic role of the MYC oncogene in regulating the crosstalk between liver cancer cells and macrophages.Chapter 1 of this dissertation provides a broad overview of the role of the MYC oncogene in enabling the cancer cells to evade host immune surveillance. I describe the major MYC genetic aberrations and alternate mechanisms by which the MYC-related pathways are activated in most cancers. I summarize recent studies which demonstrate how MYC signaling enables tumor cells to dysregulate their microenvironment and reprogram the host immune response. I discuss how MYC pathways not only dictate cancer cell pathophysiology but also suppress the host immune response against that cancer by promoting exclusion of surveilling lymphocytes, regulating the expression of immune checkpoints and reprogramming innate immune cells.Currently, there are very limited therapeutic options for the management of liver cancer [8]. While MYC is a prominent therapeutic target in HCC, it is generally considered undruggable. In chapter 2 I present data which establishes the therapeutic role of MYC in liver cancer. Antisense oligonucleotides (ASOs) were used to target and reduce the expression of MYC to impede tumor progression and phenotypically elicits oncogene addiction in the transgenic mouse models of MYC-driven primary hepatocellular carcinoma (HCC). I show that the ASOs effectively depleted MYC mRNA and protein expression without significant toxicity. Treatment with MYC ASO in vivo, but not with a control ASO, decreased proliferation, induced apoptosis, increased senescence, and remodeled the tumor microenvironment by recruitment of CD4+ T cells. I further demonstrate that MYC ASO inhibits the growth of human liver cancer xenografts in vivo. Thus, our results illustrate the feasibility of therapeutically targeting MYC in liver cancer.Metastasis is a major cause of mortality in cancer. In chapter 3 I demonstrate how MYC cooperates with the transcription factor Twist1 to promote metastasis in liver cancer. We generated an autochthonous transgenic mouse model whereby conditional expression of MYC and Twist1 enables hepatocellular carcinoma (HCC) to metastasize in >90% of mice. We show that MYC and Twist1 cooperate to elicit a transcriptional program associated with the expression of a cytokinome that leads to the recruitment and polarization of tumorassociated macrophages (TAM). Systemic treatment with CCL2 and IL13 induced MYCHCCs to metastasize; whereas, blockade of CCL2 and IL13 abrogated MYC/Twist1-HCC metastasis. Further, I show that MYC and TWIST1 in 33 human cancers (n=9502) predicts poor survival, CCL2/IL13 expression and TAM infiltration. Finally, I conducted a prospective clinical study to confirm that plasma levels of CCL2 and IL13 predicted invasive liver tumors. We conclude that MYC and TWIST1 cooperate to elicit crosstalk between cancer cells and macrophages to drive metastasis.The role of MYC and macrophages in determining response to immune checkpoint therapy is not fully appreciated. In chapter 4, I show MYC oncogene induces a Th2-like immune profile, with reduced CD8 T cell infiltration, activates immune checkpoint expression, and predicts responsiveness to immune checkpoint inhibition in human HCC. I confirm experimentally that MYC-driven tumors suppress pro-inflammatory antigen-presenting macrophages with increased CD40 and MHCII expression, which in turn impedes T cell response. Further, I demonstrate that the MYC-driven suppression of macrophages can be reversed by combined but not individual blockades of PDL1 and CTLA4. Moreover, depletion of macrophages abrogated the anti-neoplastic effects of PDL1 and CTLA4 blockade. Hence, I establish that MYC is a predictor of responsiveness to immunotherapy through suppression of pro-inflammatory macrophages, and this immune evasion can be overcome by combined PDL1 and CTLA4 blockade.MYC inactivation leads to reversible tumor dormancy in liver cancer [9]. The immunogenic mechanisms governing MYC-induced tumor dormancy are illustrated in chapter 5. Using transgenic mouse models of MYC driven liver cancer and human HCC therapy-resistant dormancy, I establish that the TGFβ pathway is activated in quiescent cancer stem cell-like dormant tumor cells. Single cell RNA sequencing and multiplex immunofluorescence demonstrate that immunosuppressive PDL1 expressing macrophages engulf the dormant tumor cells in the perivascular niche and secrete TGFβ1 thus enabling their survival. Lastly, I confirm that inhibition of both TGFβ and PDL1 pathways is required to eliminate dormant tumor cells and prevent cancer recurrence. I show the translational relevance of these findings using human samples therapy-resistant HCC. Thus, I have identified a novel, therapeutically vulnerable, mechanism governing macrophage-mediated dormancy in MYC-driven cancers.

Change of parthenogenetic DNA methylation pattern following cell fusion induced reprogramming

장현식 건국대학교 대학원 2015 국내석사

Pluripotent stem cells can self-renew and differentiate into all three germ layers in vitro and in vivo. Differentiated somatic cells can be reprogrammed into pluripotent state by cell-cell fusion. Fusion-induced reprogramming also entails epigenetic modification of somatic cell genome, such as DNA demethylation, X chromosome reactivation and histone modification. Here we investigated whether embryonic stem cells (ESCs) reprogram genomic imprinting pattern of somatic cells through cell fusion. To facilitate detection of imprinting change, we used parthenogenetic cells as a somatic and a pluripotent fusion partner cells, which were fused with biparental partner cells; ESCs were fused with parthenogenetic neural stem cells (pNSCs) and parthenogenetic ESCs (pESCs) were fused with biparental NSCs. Oct4-GFP-positive ES-pNSC and pES-NSC hybrid cells expressed pluripotency markers, Oct4 and Nanog. DNA methylation patterns of imprinted genes in ES-pNSC and pES-NSCs were similar to pluripotent fusion partners, ESCs and pESCs. However, differentially methylated pattern of Peg3 in NSCs was not changed by fusion with pESCs, which showed completely methylated patterns of Peg3. This finding indicates that imprinting pattern of somatic cells, if not all imprinted genes, may not be changed into that of pluripotent stem cells by cell-fusion induced reprogramming. 만능 줄기세포는 자가 재생산 능력을 가지고, 체내의 다양한 세포로 분화가 가능하다. 이미 분화된 체세포는 세포융합을 통해 만능성 상태로 리프로그램이 될 수 있다는 보고가 있었다. 체세포가 만능성 상태로 리프로그램이 되면 후생유전학적인 변화가 함께 일어나게 되는데 DNA 메틸화, 히스톤의 변형, X 염색체의 활성 그리고 각인 유전자의 변화 등이 이에 속한다. 본 연구에서는 배아줄기세포가 세포융합을 통해 배아 줄기세포가 체세포의 각인 유전자 양상을 리프로그램 시키는지 조사해보았다. 실험에 사용된 처녀생식 세포는 각인 유전자의 변화를 확인하기 위해서 사용하였다. 처녀생식 배아 줄기세포는 일반적인 신경 줄기세포와, 처녀생식 신경 줄기세포는 일반적인 배아 줄기세포와 세포융합을 진행하였다. 세포융합이 된 세포는 Oct4-GFP 형광을 통해 확인 할 수 있었고, Oct4와 Nanog 같은 만능성 관련 유전자들이 발현하였다. 융합된 세포에서 각인 유전자의 DNA 메틸화 양상은 융합에 사용된 만능성 세포와 유사함을 확인 할 수 있었다. 하지만 Peg3의 DNA 메틸화 양상은 만능성 세포와 유사성이 다소 떨어진다. 따라서 모든 각인 유전자에서 DNA 메틸화 양상은 세포 융합으로 리프로그래밍 되었을 때 만능성 세포와 유사하게 변하지 않음을 보여준다.

Development of Versatile Drug Delivery Systems for Cell Therapy

Shrestha Prakash 영남대학교 대학원 2022 국내박사

다양한 식품 유도 물질의 독성 평가를 위해 인간의 소화 시스템을 모방하는 조직칩

노비안절린 중앙대학교 대학원 2020 국내석사

전세계적으로 암이 29%의 사망률을 기록했기 때문에, 더 나은 형태의 항암제를 개발하려는 엄청난 시도는 치료 중에 건강한 세포와 악성 세포를 향한 독성으로 인해 방해 받았습니다. 쿠르쿠마 롱가 뿌리에서 추출한 폴리페놀인 커큐민은 정상 세포를 향한 유해한 효과 없이 임상 치료의 유망한 항암제로 나타났습니다. 여기서는 전기화학적 탐지를 통해 인간 위암세포(MKN-28)와 인간 간암세포(HepG2), 인간 뇌암세포(SH-SY5Y)와 지엽종(U-87MG)의 독성 측정에 매우 민감한 몇 가지 변형 나노구조 플랫폼을 제안했습니다. 인간의 위암 세포에서 섬유질과 콜라겐과 같은 두 가지 다른 종류의 세포외 매트릭스(ECM) 단백질이 나노 구조 기질 표면에서 변형되어 시차펄스전압측정기(DPV)에 노출되었습니다. 파이브로넥틴을 코팅한 플랫폼 중 하나는 MKN-28에 대한 커큐민 및 커큐민 나노캐리어의 항암효과 선별에 생체적합성과 전기적 신호 강도 면에서 최고의 성능을 보였습니다. 그 결과, 10-100 μM 범위 내에서 커큐민-NLC의 독성이 높은 것으로 확인되었으며, 이는 전기화학 및 기존 색도측정법(CCK-8 분석)에 의해 확인되었습니다. 사람 위암 세포의 독성 평가뿐 아니라 간암 세포에 대한 커큐민의 간독성도 평가됐습니다. ECM을 코팅 하지 않은 나노 구조 플랫폼이 최적임을 확인 하였으며, 전기화학 신호 탐지를 이용하여 커큐민의 간독성 평가에 계속 사용하였습니다. 커큐민의 반값 최대 억제 농도(IC50)의 전기화학적 결과는 23.63 μM으로 기존 CCK-8 결과(IC50 = 87.88 μM)보다 3.71배 높은 민감도를 보였습니다. 한편 뇌종양 세포에서는 신경성 뇌종양과 지엽종 공동 배양 결과 세포의 3차원 스피드로이드 모델을 만들어냈습니다. 이 지엽종 세포는 신경성 블라스토마 세포의 연결과 결합에 도움을 주었고, 이로 인해 스피드로이드가 커지고 전기화학 신호가 높아졌습니다. DPV와 CCK-8 분석을 이용한 전기화학적 검출 시, 70 μM에서 커큐민이 스페로이드에 미치는 신경 독성 영향을 검출하기 위한 민감도가 각각 66.7%, 23%씩 감소하는 것으로 나타났습니다. 세포 분류 분석 데이터 또한 전기화학적 결과를 뒷받침했습니다. 이것은 이 플랫폼을 이용한 스페로이드 형성의 공배양 방법, 그리고 전기화학적 검출은 스테로이드 형성의 비표지 기반 고속 약물 스크리닝 및 세포 활성 평가를 위한 매우 유망한 조합입니다. As cancer recorded for 29% of the human death worldwide, tremendous attempts for developing better types of anticancer drugs were hindered with the toxicity toward both healthy and malignant cells during treatment. Curcumin, a polyphenol extracted from Curcuma longa root, has been showed as a promising anticancer drug for clinical therapy without harmful manner toward normal cells. Here, we proposed several modified nanostructured platforms that are highly sensitive in measuring the toxicity of curcumin on human stomach cancer cells (MKN-28), human liver cancer cells (HepG2), and human brain cancer cells (co-culture of neuroblastoma (SH-SY5Y) and glioblastoma (U-87MG)) through electrochemical detection. In human stomach cancer cells, two different types of extracellular matrix (ECM) proteins, such as fibronectin and collagen, were modified on the surface of nanostructured substrates and were exposed to differential pulse voltammetry (DPV). One of the platforms, which was coated with fibronectin, showed the best performance in terms of both biocompatibility and the electrical signal intensity, was chosen for screening of anticancer effects of curcumin and curcumin nanocarrier on MKN-28. As a result, we found that the curcumin-NLC is highly toxic within the range of 10-100 µM, which was confirmed by both the electrochemical and conventional colorimetric methods (CCK-8 analysis). As well as the toxicity assessment of human stomach cancer cells, hepatoxicity of curcumin on liver cancer cells was assessed. The nanostructured platform with and without modification of ECM were tested and revealed to have unmodified nanostructure platform as the highest electrocatalytic towards liver cancer cells and continued to use for the hepatoxicity assessment of curcumin. The electrochemical results of the half maximal inhibitory concentration (IC50) of curcumin was 23.63 µM, which is 3.71 fold higher sensitivity than conventional CCK-8 results (IC50 = 87.88 µM). Meanwhile in the brain cancer cells, the co-culture of neuroblastoma and glioblastoma turned out to produce a three-dimensional spheroid model of cells. The glioblastoma cells helped the connection and binding of neuroblastoma cells, which helped spheroid became bigger and has higher electrochemical signal. When electrochemical detection using DPV and CCK-8 analysis were employed, the sensitivity for detecting the neurotoxicity effects of curcumin towards spheroid were revealed in 70 µM with decreasing of 66.7% and 23% of live cells, respectively. Cell sorting analysis data supported the electrochemical result as well, which made the harmony of this platform, co-culture method in spheroid formation, and electrochemical detection are very promising combination for a label-free high-throughput screening of spheroid formation, cells viabilities and toxicity assessment.

Cancer cell discrimination of thyroid gland cells by dominant feature parameters method

나철훈 全南大學校 1994 국내박사

본 연구는 인간의 갑상선세포를 대상으로 암세포의 식별을 위하여 새로운 디지탈 영상기술을 적용하여 해석한 것으로 이를 위하여 세포영상해석에 필요한 개선된 처리방법들을 제안하였다. 세포영상으로 갑상선세포를 수술을 통하여 취득한 뒤 각각 정상세포와 암세포(여포성 종양세포, 유두상 종양세포)로 확진된 세포의 현미경 영상을 사용하였다. 세포영상으로부터 세포핵을 구분하기 위하여 기존의 방법을 개선한 방향각을 갖는 Contour Following법을 시도하여 세포핵의 영상을 매우 효과적으로 얻을 수 있음을 입증하였고, 세포핵의 특징추출을 위하여 공간영역에서 기존의 9가지의 특징파라미터를 도입하였고 7개의 특징파라미터들을 새로 제안하였다. 이와같은 16개의 특징 파라미터들중 식별을을 높이기 위하여 우세특징파라미터 5개를 선택하여 식별율 향상을 꾀하였다. 비교대상으로 주파수 영역에서 기존의 방법중 우수한 성능을 가진것으로 알려진 Fourier 변환의 전력밀도분포함수를 파라미터로 사용하여 특징추출실험을 병행하여 각 세포유형별 특징표본군과 비교, 분석하였다. 실험결과 제안된 방향각을 갖는 Contour Following법을 사용함으로서 세포영상으로부터 세포핵만을 매우 효과적으로 분리해 낼수 있음이 확인되었고 우세특징파라미터들을 사용한 암세포식별법을 적용하여 실험한 결과 평균 90%이상의 식별율을 얻음으로서 기존의 방법들 보다 효과적으로 갑상선의 정상세포와 비정상세포를 식별할 수 있음을 증명하였다. A new method of digital image analysis technique for discrimination of cancer cell was presented in this paper. The object image was the Thyroid Gland cell image that provided by Department of Pathology of Chonnam National University that was diagnosed as normal and abnormal(two types of abnormal : follicular neoplastic cell, and papillary neoplastic cell), respectively. The focus of this paper was automatic discrimination of medical cells image into normal or abnormal by dominant feature parameters method. In this paper, techniques for extracting features in medical cells image were described. To segment the nucleus of cell from background, an improved region segmentation algorithm based upon Contour Following method was proposed. By using the proposed region segmentation algorithm, the cells images were sucessfully segmented into nucleus. To calculate the features of each nucleus, the 16 feature parameters were used, 9 of them were gonerally used feature parameters and rest of them were newly dofined in this paper. After constructing a Look-up table of feature parameters, experiment of discrimination was executed with 1822 cells. As a consequence of using dominant feature parameters method proposed in this paper, discrimination rate of 91.11% was obtained for Thyroid Gland cells.

Direct Conversion of Somatic Cells into Neural or Hepatic Lineages

곽태환 건국대학교 대학원 2019 국내박사

Cell replacement therapy is a potential treatment option in future medical science. Thus, in vivo tissue-derived neural stem cells (NSCs) and hepatocytes have long been considered as the highly suitable cell source for restoring nerve and liver tissues in various incurable diseases. However, their limited availability as well as the potential risk of immune rejection upon allogeneic transplantation may prohibit their clinical application. In this reason, induced pluripotent stem cells (iPSCs) technology has been regarded as a proper source of transplantable cell types for future clinical study, because the iPSCs are equivalent to embryonic stem cells, which have self-renewal capacity and pluripotency. Although patient-derived iPSCs might be an unlimited source for transplantable cell types in clinic, however, the autologous transplantation of the iPSC-derived cells has been hindered by their tumorigenic potential from residual undifferentiated PSC upon transplantation. Alternatively, recent studies have reported that ectopic expression of cell type-specific genes could directly switch a somatic cell fate into both NSC- and hepatocyte-like states. Because this cellular reprogramming process is not passing through the pluripotent state, direct conversion has been considered as a valuable strategy to generate the patient-specific cells without the risk of tumorigenesis. Han et al. previously reported that the extrinsic expression of NSC-specific transcription factor combination BSKM can reprogram mouse fibroblasts into induced neural stem cells (iNSCs). More recently, Lim et al. described the generation of induced hepatocytes (iHeps) by using liver-specific transcription factor, Hnf1a together with small molecule cocktail. Although iNSCs and iHeps closely resemble in vivo counterparts, the additional studies have been required for future clinical translation. For instance, the extremely low efficiency and prolonged process of reprogramming are major hurdles for applying reprogramming techniques to clinical study. Moreover, cellular identity of reprogrammed cells should be clearly investigated before applying direct reprogramming strategy to therapy. Although many studies have described the generation of iNSCs in human, several technical limitations that block the translation of iNSC technology into clinical application remain. Most of the previous studies show extremely low efficiency of iNSC generation and a prolonged culture period is required for reprogramming and expansion of NSC-like cells due to slow conversion process. To overcome these technical obstacles, the first study has established a robust and efficient method for generating human iNSCs from fibroblasts by introducing the precisely optimized set of reprogramming factors, which shows highly improved reprogramming efficiency and reproducibility. Furthermore, the hiNSCs closely resemble the embryonic stem cell-derived NSCs in terms of molecular characteristics, cellular features, and in vitro differentiation capacity. This approach could bring advances into their therapeutic application. Lim and colleagues previously described that Hnf1a alone could induce somatic cells to adopt a mature hepatic identity. This observation indicate that exogenous Hnf1a is indeed a master hepatic factor that could confer either a mature hepatic state and even hepatic functionalities onto somatic cells. However, the role of this factor following the successful conversion into the hepatic state remains elusive. Thus, the second study attempted to decipher the role of exogenous Hnf1a in the hepatic conversion process through the controllable expression of transgene. As a result, the current data indicated that the reprogrammed state of iHeps generated by Hnf1a alone is metastable and that the continuous expression of exogenous Hnf1a or small molecules is required for stabilizing this metastable state. This result suggested that the reprogramming factors should be precisely optimized for inducing not only a robust cell fate conversion but also a stably reprogrammed cellular identity for further translation of direct reprogramming technology. Collectively, to overcome the technical limitations of previous methods, the first study established novel protocol to generate functional human iNSCs. The new method could dramatically improve the reprogramming efficiency and facilitate reprogramming progress compared to previous reports. And second study suggested that the careful screening of reprogramming factors is essential for successfully converting somatic cells into functional cell types with a stable reprogramming state. These studies provide essential and valuable information to apply the human iNSCs and iHeps into future clinical study. 지난 수세기 동안 현대의학의 급격한 진일보에도 불구하고 아직까지도 알츠하이머 병, 파킨슨씨 병, 만성 간염 등을 포함하는 다양한 난치성 질환들의 완전한 치료는 현실적으로 불가능하다. 장기이식치료만이 질환으로 인해 손상된 기관의 기능적 회복을 위한 유일한 치료법으로 여겨지고 있으나 장기기증자의 부족, 기술적 한계 등으로 인해 이 역시 쉽지않은 실정이다. 한편 교토대학교의 야마나카 교수가 역분화줄기세포 생산기술을 개발한 이래로 줄기세포기반 임상연구의 큰 발전이 이뤄졌다. 실제로 자가재생능 및 분화전능성 등의 독보적인 특성으로 인해 환자에게 이식 가능한 세포를 생산할 수 있는 대체제로서 역분화줄기세포의 가치가 점차 대두되고 있다. 그러나 이러한 무한한 잠재력에도 불구하고 세포이식 후 잔존한 미분화 세포에서 유래할 수 있는 종양형성의 가능성, 고순도 타겟세포 정제기술의 부재 등, 자가유래 역분화줄기세포의 임상적용을 위해서 반드시 해결해야할 문제들이 남아있다. 이러한 한계점들을 극복하기 위해, 최근 체세포에 세포 특이적 유전자를 도입하여 직접 타겟세포를 생산하는 직접교차분화 기술이 개발되어왔다. 직접교차분화의 경우 전능성상태를 거치지 않기 때문에 잔존 미분화 세포로 인한 종양형성 가능성으로부터 비교적 자유로울 수 있으며, 타겟세포를 얻기 위한 별도의 정제 과정이 불필요한 효율적인 기법으로 세포치료를 위한 차세대 대체전략으로 각광받고 있다. 실제로 최근 5년간 신경줄기세포 특이적 유전자 혹은 야마나카 전사인자를 인간 체세포에 도입하여 유도신경줄기세포를 생산한 연구들이 보고되고 있으나, 낮은 생산효율 및 장기간의 생산과정, 야마나카 전사인자의 임상적용 위험성 등으로 인해 실효적인 연구성과를 거두지 못하고 있다. 이러한 문제점들을 해결하기 위해 첫번째 연구에서는 정밀하게 최적화 된 교차분화 인자 조합을 이용하여 보다 효율적인 인간 유도신경줄기세포 생산기술을 개발하였다. 새로운 생산기술은 유도신경줄기세포의 생산효율 향상 및 생산과정 단축 등 기존의 기술적 한계를 높은 수준으로 개선하였으며, 생산된 유도신경줄기세포는 배아줄기세포로부터 분화된 신경줄기세포와 분자생물학적 특성 및 체외 분화능이 매우 유사하였다. 본 연구결과는 임상적용을 가로막고 있던 기존 유도신경줄기세포 생산방법의 기술적 한계점을 개선함으로써 실제 치료에 적용 가능한 유도신경줄기세포 생산기술을 개발했다는 점에서 큰 의미가 있다. 한편, 최근 간 조직 특이적 유전자인 Hnf1a를 단독으로 섬유아세포에 도입하여 성숙한 간세포를 생산할 수 있다는 연구결과가 보고되었다. 이 결과는 Hnf1a가 섬유아세포에 성숙한 간세포의 분자생리학적 상태를 비롯해 간 기능성을 부여할 수 있는 핵심 전사인자라는 것을 의미한다. 그러나 이 단일 유전자가 어떠한 기전을 통해 간세포로의 교차분화를 유도하는지, 그리고 생산된 유도간세포의 상태가 충분히 안정적인지에 대한 심층적인 연구는 아직까지 이뤄지지 않았다. 따라서 두번째 연구에서는 도입된 전사인자의 발현을 조절함으로써 간세포로의 교차분화에서 전사인자들의 역할에 대해 탐구하고자 하였다. 그 결과 단일 전사인자로부터 유도된 유도간세포는 준안정상태이며 안정화를 위해서는 외래유전자 Hnf1a의 지속적인 발현 또는 신호전달 물질의 지속적인 작용이 필요하다는 것을 규명하였다. 이 연구결과는 교차분화기술의 임상적용을 위해서는 교차분화 인자의 선별과정에서 강력하고 효율적인 교차분화 능력 뿐만 아니라 교차분화세포의 안정성 역시 신중하게 검증되어야 한다는 중요한 사실을 시사한다. 본 연구는 유도신경줄기세포와 유도간세포의 생산기술을 개선하여 기존의 기술적 한계들을 극복하는데 일조하였으며 교차분화 기술의 상용화를 앞당기기 위한 기초자료로써 중요한 역할을 할 수 있을 것이라고 사료된다. 뿐만 아니라 효율성, 생산성 향상 등의 기술적 진보에 초점을 맞추고 있는 현재 교차분화 연구분야에 기술의 실용화를 위한 안전성 연구의 중요성을 역설하는 연구결과로 그 의의가 매우 크다고 할 수 있다.