• 한국임상수의학회지
• /
• 제28권4호
• /
• pp.394-398
• /
• 2011

Mesenchymal stem cells (MSC) are pluripotent cells that can be found in umbilical cord blood from new borne babies as well as placenta, bone marrow, adipose tissue, amniotic fluid, muscle, et al. MSC are capable of renewing themselves without differentiation in long-term culture, also can be differentiated into various tissues under specific condition. Formulating a cryopreservation protocol for the MSC is required because these cells cannot survive for long periods under in vitro culture conditions and a new formulation of harmless cryoprotectant is needed for the direct injection of MSC into patients. The undifferentiated MSC were frozen with a vitrification solution of 40% ethylene glycol, 20% Ficoll-70 and 0.3M sucrose. The survival rate after thawing and their proliferation rate were examined and compared with slow rate cooling methods using dimethylsulfoxide (DMSO). The vitrification method showed high survival rate after thawing and proliferation capacity comparable to DMSO. It can be suggested that ultra-rapid cooling method by vitrification is reliable methods for long term preservation of MSC and the vitrification solution with ethylene glycol, Ficoll-70 and sucrose will be more beneficially used for direct transplantation of MSC into patients than DMSO solution.

• 한국임상수의학회지
• /
• 제28권1호
• /
• pp.57-62
• /
• 2011

The shortage of transplantable kidneys has many efforts to regenerate bioartificial kidneys using transgenic animals and diverse kinds of scaffolds which are important tools for cell seeding. However, there are many limitations for clinical applications so far. Recently, decellularized bioscaffolds using animal organs come into spotlight because of its many superior advantages. In current study, we produced decellularized kidney bioscaffolds of pig which is an attractive animal as a clinical model for human. We decellularized pig kidneys with 1% SDS detergent solution using peristaltic pump systems for 12h. After decellularization process, the kidney bioscaffolds preserved intact 3D morphology including glomerular structure and almost DNA from pig was entirely removed. In addition, this process could preserve micro vascular network which is necessary for cell survival. Although, additional studies for recellularization and transplantation should be required, the decellular vascularized kidney bioscaffolds might have many potentials for kidney regeneration.

• Molecules and Cells
• /
• 제42권11호
• /
• pp.739-746
• /
• 2019

Significant knowledge about the pathophysiology of Alzheimer's disease (AD) has been gained in the last century; however, the understanding of its causes of onset remains limited. Late-onset AD is observed in about 95% of patients, and APOE4-encoding apolipoprotein E4 (ApoE4) is strongly associated with these cases. As an apolipoprotein, the function of ApoE in brain cholesterol transport has been extensively studied and widely appreciated. Development of new technologies such as human-induced pluripotent stem cells (hiPSCs) and CRISPR-Cas9 genome editing tools have enabled us to develop human brain model systems in vitro and readily manipulate genomic information. In the context of these advances, recent studies provide strong evidence that abnormal cholesterol metabolism by ApoE4 could be linked to AD-associated pathology. In this review, we discuss novel discoveries in brain cholesterol dysregulation by ApoE4. We further elaborate cell type-specific roles in cholesterol regulation of four major brain cell types, neurons, astrocytes, microglia, and oligodendrocytes, and how its dysregulation can be linked to AD pathology.

• Reproductive and Developmental Biology
• /
• 제38권2호
• /
• pp.79-84
• /
• 2014

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.

• Clinical and Experimental Reproductive Medicine
• /
• 제29권1호
• /
• pp.1-12
• /
• 2002

Objective: In order to acquire the technique for the establishment of human embryonic stem cells (ESe) derived from the human frozen-thawed embryos produced in IVF-ET program, this study was performed to establish mouse ESC derived from the in vitro fertilized embryos. Materials and Methods: After Fl hybrid (C57BL female $\times$ CBA mael) female mice were superovulated with PMSG and hCG treatment, their oocytes were retrieved and inseminated, and the fertilized embryos were cultured for 96-120 hours until the expected stages of blastocysts were obtained. To isolate the inner cell mass (ICM), either the blastocysts were treated with immunosurgery, or the whole embryos were cultured for 4 days. Isolated ICMs were then cultured onto STO feeder cell layer, and the resultant ICM colonies were subcultured with trypsin-EDTA treatment. During the subculture process, ESC-like cell colonies were observed with phase contrast microscopy. To identify ESC in the subcultured ESC-like cell colonies, alkaline phosphatase activity and Oct-4 (octamer-binding transcription factor-4) expression were examined by immunohistochemistry and RT-PCR, respectively. To examine the spontaneous differentiation, ESC-like cell colonies were cultured without STO feeder cell layer and leukemia inhibitory factor (LIF). Results: Seven ESC-like cell lines were established from ICMs isolated from the in vitro fertilized embryos. According to the developmental stage, the growth of ICMs isolated from the expanded blastocysts was significantly better than that of ICMs isolated from the hatched blastocysts (80.3% vs. 58.7%, p<0.05). ESC-like cell colonies were only obtained from ICMs of expanded blastocysts. However, the ICMs isolated from the embryos treated with immunosurgery were poorly grown and frequently differentiated during the culture process. The established ESC-like cell colonies were positively stained with alkaline phosphatase and expressed Oct-4, and their morphology resembled that observed in the previously reported mouse ESC. In addition, following the extended in vitro culture process, they maintained their expression of cell surface markers characteristic of the pluripotent stem cells such as alkaline phosphatase and Oct-4. When cultured without STO feeder cell layer and LIF, they were spontaneously differentiated into the various types of cells. Conclusion: The findings of this study suggest that the establishment of mouse ESC can be successfully derived from the in vitro fertilized embryos. The established ESC-like cells expressed the cell surface markers characteristic of the pluripotent stem cells and spontaneously differentiated into the various types of cells.

• International Journal of Stem Cells
• /
• 제16권3호
• /
• pp.281-292
• /
• 2023

Background and Objectives: Human induced pluripotent stem cell (hiPSC)-derived cardiomyocyte (CM) hold great promise as a cellular source of CM for cardiac function restoration in ischemic heart disease. However, the use of animal-derived xenogeneic substances during the biomanufacturing of hiPSC-CM can induce inadvertent immune responses or chronic inflammation, followed by tumorigenicity. In this study, we aimed to reveal the effects of xenogeneic substances on the functional properties and potential immunogenicity of hiPSC-CM during differentiation, demonstrating the quality and safety of hiPSC-based cell therapy. Methods and Results: We successfully generated hiPSC-CM in the presence and absence of xenogeneic substances (xeno-containing (XC) and xeno-free (XF) conditions, respectively), and compared their characteristics, including the contractile functions and glycan profiles. Compared to XC-hiPSC-CM, XF-hiPSC-CM showed early onset of myocyte contractile beating and maturation, with a high expression of cardiac lineage-specific genes (ACTC1, TNNT2, and RYR2) by using MEA and RT-qPCR. We quantified N-glycolylneuraminic acid (Neu5Gc), a xenogeneic sialic acid, in hiPSC-CM using an indirect enzyme-linked immunosorbent assay and liquid chromatography-multiple reaction monitoring-mass spectrometry. Neu5Gc was incorporated into the glycans of hiPSC-CM during xeno-containing differentiation, whereas it was barely detected in XF-hiPSC-CM. Conclusions: To the best of our knowledge, this is the first study to show that the electrophysiological function and glycan profiles of hiPSC-CM can be affected by the presence of xenogeneic substances during their differentiation and maturation. To ensure quality control and safety in hiPSC-based cell therapy, xenogeneic substances should be excluded from the biomanufacturing process.

• International Journal of Stem Cells
• /
• 제16권2호
• /
• pp.215-233
• /
• 2023

Background and Objectives: MYC, also known as an oncogenic reprogramming factor, is a multifunctional transcription factor that maintains induced pluripotent stem cells (iPSCs). Although MYC is frequently upregulated in various cancers and is correlated with a poor prognosis, MYC is downregulated and correlated with a good prognosis in lung adenocarcinoma. MYC and two other MYC family genes, MYCN and MYCL, have similar structures and could contribute to tumorigenic conversion both in vitro and in vivo. Methods and Results: We systematically investigated whether MYC family genes act as prognostic factors in various human cancers. We first evaluated alterations in the expression of MYC family genes in various cancers using the Oncomine and The Cancer Genome Atlas (TCGA) database and their mutation and copy number alterations using the TCGA database with cBioPortal. Then, we investigated the association between the expression of MYC family genes and the prognosis of cancer patients using various prognosis databases. Multivariate analysis also confirmed that co-expression of MYC/MYCL/MYCN was significantly associated with the prognosis of lung, gastric, liver, and breast cancers. Conclusions: Taken together, our results demonstrate that the MYC family can function not only as an oncogene but also as a tumor suppressor gene in various cancers, which could be used to develop a novel approach to cancer treatment.

• Reproductive and Developmental Biology
• /
• 제35권3호
• /
• pp.301-306
• /
• 2011

Fibroblasts of large animals are easy to isolate and to maintain in vitro culture. Thus, these cells are extensively applied to donor cell for somatic cell nuclear transfer, and to substrate cells to generate induced pluripotent stem cells after transfection of requited genes to be essentially required for direct reprogramming. However, limited mitotic activity of fibroblasts to differentiate along a terminal lineage becomes restrictive for their versatile application. Recently, commercial culture medium and systems developed for primary cells are provided by manufactures. In this study, we examined whether one of the systems developed for primary fibroblasts of human are effective on porcine ear skin fibroblasts. To this end, we performed proliferation assay after five days culture in vitro of porcine fibroblasts in medium DMEM, which is generally used for fibroblasts culture, and medium M106 for human dermal fibroblasts, supplemented with various concentrations of FBS and LSGS contained mainly growth factors, respectively. Consequence was that presence of 15% FBS and 0.1 ${\times}$ concentrations of LSGS in DMEM showed most active proliferation of porcine fibroblasts.

• 한국발생생물학회지:발생과생식
• /
• 제12권1호
• /
• pp.87-95
• /
• 2008

만능성 인간 배아줄기세포로부터 확립된 신경줄기세포 또는 신경전구세포는 퇴행성 신경질환 세포치료제로 이용될 수 있는 다양한 종류의 신경세포로 분화 유도될 수 있다. 하지만, 인간 배아줄기세포로부터 신경세포를 생산하기 위한 기술은 아직 많은 장애를 가지고 있다. 인간 배아줄기세포 유래 신경전구세포에서 특징적으로 나타나는 신경관 유사로제트에 대한 이해는 인간 배아줄기세포 신경 분화의 효율을 높이는데 유용한 정보를 제공할 것으로 사료된다. 일반적으로 신경로제트(neural rosette)는 분화 중인 배아체를 부착 배양함으로써 유도하지만, 이 방법은 시간이 걸리고 복잡하다는 단점이 있다. 본 연구에서는 신경로제트가 부착배양을 하지 않고 부유배양으로 형성될 수 있는지 조사하였다. 우선적으로, 배아체 형성 및 신경분화에 인간 배아줄기세포 클럼프(clump) 크기가 영향을 주는지를 조사하였고, 사방 $500\;{\mu}m$ 크기의 인간 배아줄기세포 클럼프가 신경 분화 유도에 가장 효과적임을 확인하였다. 로제트 형성을 유도하기 위해, 사방 $500\;{\mu}m$ 크기의 인간 배아줄기세포 클럼프를 1주일 동안 EB 배양배지에 부유 배양함으로써 균일한 크기의 배아체를 얻은 후, NES 배양 배지에서 부가적으로 $1{\sim}2$주 동안 계속 부유 배양한 결과, $7{\sim}10$일 사이에 신경관 유사 로제트가 형성됨을 확인하였다. 로제트 형성 세포의 신경전구세포로서 특성은 RT-PCR과 면역형광염색법을 이용한 신경전구세포 특이적 마커(vimentivi, nestin, MSI1, MSI2, Sox1, Tuj1) 발현을 통해 확인하였다. 또한, 성장인자를 제외한 NES 배양 배지에서 신경로제트를 $2{\sim}6$주 동안 지속적으로 배양하면 성숙 신경세포로의 말단 분화가 유도됨을 확인하였다. 신경세포 특이적 마커(Tuj1, MAP2, GABA)와 신경아교 특이적 마커($S100{\beta}$, GFAP)는 $2{\sim}3$주 또는 4주 후에 각각 발현이 유도됨을 확인하였고, 희소 돌기아교 특이적 마커(O1과 CNPase)는 $5{\sim}6$주 후에 발현이 증가함을 확인하였다. 본 연구결과는 신경로제트가 부유 배양시스템에서 성공적으로 형성됨을 보여주고 있으며, 이는 인간 배아줄기세포의 신경 분화를 이해하고, 신경전구세포 유도 과정을 단순화하는데 효과적으로 이용될 수 있을 것으로 사료된다.

• International Journal of Stem Cells
• /
• 제16권3호
• /
• pp.326-341
• /
• 2023

Background and Objectives: Osteoarthritis (OA) is a degenerative disease that leads to the progressive destruction of articular cartilage. Current clinical therapeutic strategies are moderately effective at relieving OA-associated pain but cannot induce chondrocyte differentiation or achieve cartilage regeneration. We investigated the ability of wedelolactone, a biologically active natural product that occurs in Eclipta alba (false daisy), to promote chondrogenic differentiation. Methods and Results: Real-time reverse transcription-polymerase chain reaction, immunohistochemical staining, and immunofluorescence staining assays were used to evaluate the effects of wedelolactone on the chondrogenic differentiation of mesenchymal stem cells (MSCs). RNA sequencing, microRNA (miRNA) sequencing, and isobaric tags for relative and absolute quantitation analyses were performed to explore the mechanism by which wedelolactone promotes the chondrogenic differentiation of MSCs. We found that wedelolactone facilitates the chondrogenic differentiation of human induced pluripotent stem cell-derived MSCs and rat bone-marrow MSCs. Moreover, the forkhead box O (FOXO) signaling pathway was upregulated by wedelolactone during chondrogenic differentiation, and a FOXO1 inhibitor attenuated the effect of wedelolactone on chondrocyte differentiation. We determined that wedelolactone reduces enhancer of zeste homolog 2 (EZH2)-mediated histone H3 lysine 27 trimethylation of the promoter region of FOXO1 to upregulate its transcription. Additionally, we found that wedelolactone represses miR-1271-5p expression, and that miR-1271-5p post-transcriptionally suppresses the expression of FOXO1 that is dependent on the binding of miR-1271-5p to the FOXO1 3'-untranscribed region. Conclusions: These results indicate that wedelolactone suppresses the activity of EZH2 to facilitate the chondrogenic differentiation of MSCs by activating the FOXO1 signaling pathway. Wedelolactone may therefore improve cartilage regeneration in diseases characterized by inflammatory tissue destruction, such as OA.