• Clinical and Experimental Pediatrics
• /
• 제53권8호
• /
• pp.786-789
• /
• 2010

Induced pluripotent stem (iPS) cells are generated by epigenetic reprogramming of somatic cells through the exogenous expression of transcription factors. Recently, the generation of iPS cells from patients with a variety of genetic diseases was found to likely have a major impact on regenerative medicine, because these cells self-renew indefinitely in culture while retaining the capacity to differentiate into any cell type in the body, thereby enabling disease investigation and drug development. This review focuses on the current state of iPS cell technology and discusses the potential applications of these cells for disease modeling; drug discovery; and eventually, cell replacement therapy.

• Molecules and Cells
• /
• 제45권12호
• /
• pp.923-934
• /
• 2022

Human pluripotent stem cell-derived cardiomyocytes (hPSC-CMs) have great potential in applications such as regenerative medicine, cardiac disease modeling, and in vitro drug evaluation. However, hPSC-CMs are immature, which limits their applications. During development, the maturation of CMs is accompanied by a decline in their proliferative capacity. This phenomenon suggests that regulating the cell cycle may facilitate the maturation of hPSC-CMs. Aurora kinases are essential kinases that regulate the cell cycle, the role of which is not well studied in hPSC-CM maturation. Here, we demonstrate that CYC116, an inhibitor of Aurora kinases, significantly promotes the maturation of CMs derived from both human embryonic stem cells (H1 and H9) and iPSCs (induced PSCs) (UC013), resulting in increased expression of genes related to cardiomyocyte function, better organization of the sarcomere, increased sarcomere length, increased number of mitochondria, and enhanced physiological function of the cells. In addition, a number of other Aurora kinase inhibitors have also been found to promote the maturation of hPSC-CMs. Our data suggest that blocking aurora kinase activity and regulating cell cycle progression may promote the maturation of hPSC-CMs.

• Asian-Australasian Journal of Animal Sciences
• /
• 제16권6호
• /
• pp.910-927
• /
• 2003

Transgenesis is a very powerful tool not only to help understanding the basics of life science but also to improve the efficiency of animal production. Since the first transgenic mouse was born in 1980, rapid development and wide application of this technique have been made in laboratory animals as well as in domestic animals. Although pronuclear injection is the most widely used method and nuclear transfer using somatic cells broadens the choice of making transgenic domestic animals, the demand for precise manipulation of the genome leads to the utilization of gene targeting. To make this technique possible, a pluripotent embryonic cell line such as embryonic stem (ES) cell is required to carry genetic mutation to further generations. However, ES cell, well established in mice, is not available in domestic animals even though many attempt to establish the cell line. An alternate source of pluripotent cells is embryonic germ (EG) cells derived from primordial germ cells (PGCs). To make gene targeting feasible in this cell line, a better culture system would help to minimize the unnecessary loss of cells in vitro. In this review, general methods to produce transgenic domestic animals will be mentioned. Also, it will focus on germ cell engineering and methods to improve the establishment of pluripotent embryonic cell lines in domestic animals.

• Reproductive and Developmental Biology
• /
• 제35권1호
• /
• pp.1-8
• /
• 2011

Techniques to evaluate gene expression profiling, such as sufficiently sensitive cDNA microarrays or real-time quantitative PCR, are efficient methods for monitoring human pluripotent stem cell (hESC/iPSC) cultures. However, most of these high-throughput tests have a limited use due to high cost, extended turn-around time, and the involvement of highly specialized technical expertise. Hence, there is an urgency of rapid, cost-effective, robust, yet sensitive method development for routine screening of hESCs/hiPSCs. A critical requirement in hESC/hiPSC cultures is to maintain a uniform undifferentiated state and to determine their differentiation capacity by showing the expression of gene markers representing all three germ layers, including ectoderm, mesoderm, and endoderm. To quantify the modulation of gene expression in hESCs/hiPSC during their propagation, expansion, and differentiation via embryoid body (EB) formation, we developed a simple, rapid, inexpensive, and definitive multimarker, semiquantitative multiplex RT-PCR platform technology. Among the 9 gene primers tested, 5 were pluripotent markers comprising set 1, and 3 lineage-specific markers were combined as set 2, respectively. We found that these 2 sets were not only effective in determining the relative differentiation in hESCs/hiPSCs, but were easily reproducible. In this study, we used the hES/hiPS cell lines to standardize the technique. This multiplex RT-PCR assay is flexible and, by selecting appropriate reporter genes, can be designed for characterization of different hESC/hiPSC lines during routine maintenance and directed differentiation.

• Molecules and Cells
• /
• 제41권11호
• /
• pp.971-978
• /
• 2018

The stem cell factor (SCF)/c-KIT axis plays an important role in the hematopoietic differentiation of human pluripotent stem cells (hPSCs), but its regulatory mechanisms involving microRNAs (miRs) are not fully elucidated. Here, we demonstrated that supplementation with SCF increases the hematopoietic differentiation of hPSCs via the interaction with its receptor tyrosine kinase c-KIT, which is modulated by miR-221 and miR-222. c-KIT is comparably expressed in undifferentiated human embryonic and induced pluripotent stem cells. The inhibition of SCF signaling via treatment with a c-KIT antagonist (imatinib) during hPSC-derived hematopoiesis resulted in reductions in the yield and multi-lineage potential of hematopoietic progenitors. We found that the transcript levels of miR-221 and miR-222 targeting c-KIT were significantly lower in the pluripotent state than they were in terminally differentiated somatic cells. Furthermore, suppression of miR-221 and miR-222 in undifferentiated hPSC cultures induced more hematopoiesis by increasing c-KIT expression. Collectively, our data implied that the modulation of c-KIT by miRs may provide further potential strategies to expedite the generation of functional blood cells for therapeutic approaches and the study of the cellular machinery related to hematologic malignant diseases such as leukemia.

• Reproductive and Developmental Biology
• /
• 제35권4호
• /
• pp.449-455
• /
• 2011

Induced pluripotent stem (iPS) cells have been generated from mouse and human somatic cells by etopic expression of transcription factors. iPS cells are indistinguishable from ES cells in terms of morphology and stem cell marker expression. Moreover, mouse iPS cells give rise to chimeric mice that are competent for germline transmission. However, mice derived from iPS cells often develop tumors. Furthermore, the low efficiency of iPS cell generation is a big disadvantage for mechanistic studies. Nonviral plasmid.based vectors are free of many of the drawbacks that constrain viral vectors. The histone deacetylase inhibitor valproic acid (VPA) has been shown to improve the efficiency of mouse and human iPS cell generation, and vitamin C (Vc) accelerates gene expression changes and establishment of the fully reprogrammed state. The MEK inhibitor PD0325901 (Stemgent) has been shown to increase the efficiency of the reprogramming of human primary fibroblasts into iPS cells. In this report, we described the generation of mouse iPS cells devoid of exogenous DNA by the simple transient transfection of a nonviral vector carrying 2A-peptide-linked reprogramming factors. We used VPA, Vc, and the MEK inhibitor PD0325901 to increase the reprogramming efficiency. The reprogrammed somatic cells expressed pluripotency markers and formed EBs.

• Molecules and Cells
• /
• 제37권1호
• /
• pp.31-35
• /
• 2014

Induced pluripotent stem cells (iPSCs) are capable of unlimited self-renewal and can give rise to all three germ layers, thereby providing a new platform with which to study mammalian development and epigenetic reprogramming. However, iPSC generation may result in subtle epigenetic variations, such as the aberrant methylation of the Dlk1-Dio3 locus, among the clones, and this heterogeneity constitutes a major drawback to harnessing the full potential of iPSCs. Vitamin C has recently emerged as a safeguard to ensure the normal imprinting of the Dlk1-Dio3 locus during reprogramming. Here, we show that vitamin C exerts its effect in a manner that is independent of the reprogramming kinetics. Moreover, we demonstrate that reprogramming cells under 2i conditions leads to the early upregulation of Prdm14, which in turn results in a highly homogeneous population of authentic pluripotent colonies and prevents the abnormal silencing of the Dlk1-Dio3 locus.

• 한국수정란이식학회지
• /
• 제28권1호
• /
• pp.1-6
• /
• 2013

A lot of works have been dedicated to clarify the reasons why the establishment of embryonic stem cells (ESCs) from pig is more difficult than that from mouse and human. Several concomitant factors such as culture condition including feeder layer, sensitivity of cell to cell contact, definitive markers of pluripotency for evaluation of the validity and optimal timing of derivation have been suggested as the disturbing factors in the establishment of porcine ESCs Traditionally, attempts to derive stem cells from porcine embryos have depend on protocols established for mouse ESCs using inner cell mass (ICM) for the isolation and culture. And more recently, protocols used for primate ESCs were also applied. However, there is no report for the establishment of porcine ESCs. Indeed, ungulate species including pigs have crucial developmental differences unlike rodents and primates. Here we will review recent studies about issues for establishment of porcine ESCs and discuss the promise and strategies focusing on the timing for derivation and pluripotent state of porcine ESCs.

• Molecules and Cells
• /
• 제45권2호
• /
• pp.53-64
• /
• 2022

Three-dimensional cultures of human neural tissue/organlike structures in vitro can be achieved by mimicking the developmental processes occurring in vivo. Rapid progress in the field of neural organoids has fueled the hope (and hype) for improved understanding of brain development and functions, modeling of neural diseases, discovery of new drugs, and supply of surrogate sources of transplantation. In this short review, we summarize the state-of-the-art applications of this fascinating tool in various research fields and discuss the reality of the technique hoping that the current limitations will soon be overcome by the efforts of ingenious researchers.

• BMB Reports
• /
• 제51권10호
• /
• pp.500-507
• /
• 2018

Cell reprogramming has been considered a powerful technique in the regenerative medicine field. In addition to diverse its strengths, cell reprogramming technology also has several drawbacks generated during the process of reprogramming. Telomere shortening caused by the cell reprogramming process impedes the efficiency of cell reprogramming. Transcription factors used for reprogramming alter genomic contents and result in genetic mutations. Additionally, defective mitochondria functioning such as excessive mitochondrial fission leads to the limitation of pluripotency and ultimately reduces the efficiency of reprogramming. These problems including genomic instability and impaired mitochondrial dynamics should be resolved to apply cell reprograming in clinical research and to address efficiency and safety concerns. Sirtuin (NAD+-dependent histone deacetylase) has been known to control the chromatin state of the telomere and influence mitochondria function in cells. Recently, several studies reported that Sirtuins could control for genomic instability in cell reprogramming. Here, we review recent findings regarding the role of Sirtuins in cell reprogramming. And we propose that the manipulation of Sirtuins may improve defects that result from the steps of cell reprogramming.