• 한국동물생명공학회지
• /
• 제38권4호
• /
• pp.199-208
• /
• 2023

Background: Canine induced pluripotent stem cells (iPSCs) are an attractive source for veterinary regenerative medicine, disease modeling, and drug development. Here we used vitamin C (Vc) to improve the reprogramming efficiency of canine iPSCs, and its functions in the reprogramming process were elucidated. Methods: Retroviral transduction of Oct4, Sox2, Klf4, c-Myc (OSKM), and GFP was employed to induce reprogramming in canine fetal fibroblasts. Following transduction, the culture medium was subsequently replaced with ESC medium containing Vc to determine the effect on reprogramming activity. Results: The number of AP-positive iPSC colonies dramatically increased in culture conditions supplemented with Vc. Vc enhanced the efficacy of retrovirus transduction, which appears to be correlated with enhanced cell proliferation capacity. To confirm the characteristics of the Vc-treated iPSCs, the cells were cultured to passage 5, and pluripotency markers including Oct4, Sox2, Nanog, and Tra-1-60 were observed by immunocytochemistry. The expression of endogenous pluripotent genes (Oct4, Nanog, Rex1, and telomerase) were also verified by PCR. The complete silencing of exogenously transduced human OSKM factors was observed exclusively in canine iPSCs treated with Vc. Canine iPSCs treated with Vc are capable of forming embryoid bodies in vitro and have spontaneously differentiated into three germ layers. Conclusions: Our findings emphasize a straightforward method for enhancing the efficiency of canine iPSC generation and provide insight into the Vc effect on the reprogramming process.

• BMB Reports
• /
• 제52권5호
• /
• pp.295-303
• /
• 2019

Breakthroughs in stem cell technology have contributed to disease modeling and drug screening via organoid technology. Organoid are defined as three-dimensional cellular aggregations derived from adult tissues or stem cells. They recapitulate the intricate pattern and functionality of the original tissue. Insulin is secreted mainly by the pancreatic ${\beta}$ cells. Large-scale production of insulin-secreting ${\beta}$ cells is crucial for diabetes therapy. Here, we provide a brief overview of organoids and focus on recent advances in protocols for the generation of pancreatic islet organoids from pancreatic tissue or pluripotent stem cells for insulin secretion. The feasibility and limitations of organoid cultures derived from stem cells for insulin production will be described. As the pancreas and gut share the same embryological origin and produce insulin, we will also discuss the possible application of gut organoids for diabetes therapy. Better understanding of the challenges associated with the current protocols for organoid culture facilitates development of scalable organoid cultures for applications in biomedicine.

• 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.

• BMB Reports
• /
• 제45권1호
• /
• pp.20-25
• /
• 2012

The present study aimed to investigate the function of translationally controlled tumor protein (TCTP) in the regulation of Oct4 in mouse embryonic carcinoma P19 cells and mouse J1 embryonic stem (ES) cells. The mRNA level of endogenous TCTP in somatic cells was 2-4 folds higher than that in pluripotent P19 and J1 ES cells. Overexpression of TCTP in mouse pluripotent cells not only reduced the level of Oct4 transcription, but also decreased the pluripotency of stem cells. The N-terminal end of TCTP (amino acids 1-60) played an important role in suppressing the Oct4 promoter. Moreover, overexpression of TCTP in P19 cells suppressed the Oct4 promoter activity in a dose- and a time-dependent manner. In addition, knockdown of TCTP by small interfering RNA increased the expression of Oct4. Our study indicates that TCTP downregulates the Oct4 expression by binding the Sf1 site of Oct4 promoter in mouse pluripotent cells.

• International Journal of Stem Cells
• /
• 제17권2호
• /
• pp.194-203
• /
• 2024

Evaluating cell metabolism is crucial during pluripotent stem cell (PSC) differentiation and somatic cell reprogramming as it affects cell fate. As cultured stem cells are heterogeneous, a comparative analysis of relative metabolism using existing metabolic analysis methods is difficult, resulting in inaccuracies. In this study, we measured human PSC basal metabolic levels using a Seahorse analyzer. We used fibroblasts, human induced PSCs, and human embryonic stem cells to monitor changes in basal metabolic levels according to cell number and determine the number of cells suitable for analysis. We evaluated normalization methods using glucose and selected the most suitable for the metabolic analysis of heterogeneous PSCs during the reprogramming stage. The response of fibroblasts to glucose increased with starvation time, with oxygen consumption rate and extracellular acidification rate responding most effectively to glucose 4 hours after starvation and declining after 5 hours of starvation. Fibroblasts and PSCs achieved appropriate responses to glucose without damaging their metabolism 2~4 and 2~3 hours after starvation, respectively. We developed a novel method for comparing basal metabolic rates of fibroblasts and PSCs, focusing on quantitative analysis of glycolysis and oxidative phosphorylation using glucose without enzyme inhibitors. This protocol enables efficient comparison of energy metabolism among cell types, including undifferentiated PSCs, differentiated cells, and cells undergoing cellular reprogramming, and addresses critical issues, such as differences in basal metabolic levels and sensitivity to normalization, providing valuable insights into cellular energetics.

• International Journal of Stem Cells
• /
• 제17권2호
• /
• pp.141-146
• /
• 2024

Recent advancements in organoid technology have led to a vigorous movement towards utilizing it as a substitute for animal experiments. Organoid technology offers versatile applications, particularly in toxicity testing of pharmaceuticals or chemical substances. However, for the practical use in toxicity testing, minimal guidance is required to ensure reliability and relevance. This paper aims to provide minimal guidelines for practical uses of kidney organoids derived from human pluripotent stem cells as a toxicity evaluation model in vitro.

• Molecules and Cells
• /
• 제37권10호
• /
• pp.705-712
• /
• 2014

The early steps of neural development in the vertebrate embryo are regulated by sets of transcription factors that control the induction of proliferative, pluripotent neural precursors, the expansion of neural plate stem cells, and their transition to differentiating neural progenitors. These early events are critical for producing a pool of multipotent cells capable of giving rise to the multitude of neurons and glia that form the central nervous system. In this review we summarize findings from gain- and loss-of-function studies in embryos that detail the gene regulatory network responsible for these early events. We discuss whether this information is likely to be similar in mammalian embryonic and induced pluripotent stem cells that are cultured according to protocols designed to produce neurons. The similarities and differences between the embryo and stem cells may provide important guidance to stem cell protocols designed to create immature neural cells for therapeutic uses.

• International Journal of Stem Cells
• /
• 제10권1호
• /
• pp.1-11
• /
• 2017

Human cardiomyocytes (CMs) cease to proliferate and remain terminally differentiated thereafter, when humans reach the mid-20s. Thus, any damages sustained by myocardium tissue are irreversible, and they require medical interventions to regain functionality. To date, new surgical procedures and drugs have been developed, albeit with limited success, to treat various heart diseases including myocardial infarction. Hence, there is a pressing need to develop more effective treatment methods to address the increasing mortality rate of the heart diseases. Functional CMs are not only an important in vitro cellular tool to model various types of heart diseases for drug development, but they are also a promising therapeutic agent for cell therapy. However, the limited proliferative capacity entails difficulties in acquiring functional CMs in the scale that is required for pathological studies and cell therapy development. Stem cells, human pluripotent stem cells (hPSCs) in particular, have been considered as an unlimited cellular source for providing functional CMs for various applications. Notable progress has already been made: the first clinical trials of hPSCs derived CMs (hPSC-CMs) for treating myocardial infarction was approved in 2015, and their potential use in disease modeling and drug discovery is being fully explored. This concise review gives an account of current development of differentiation, purification and maturation techniques for hPSC-CMs, and their application in cell therapy development and pharmaceutical industries will be discussed with the latest experimental evidence.

• The Korean Journal of Pain
• /
• 제32권4호
• /
• pp.245-255
• /
• 2019

Stem cells are attracting attention as a key element in future medicine, satisfying the desire to live a healthier life with the possibility that they can regenerate tissue damaged or degenerated by disease or aging. Stem cells are defined as undifferentiated cells that have the ability to replicate and differentiate themselves into various tissues cells. Stem cells, commonly encountered in clinical or preclinical stages, are largely classified into embryonic, adult, and induced pluripotent stem cells. Recently, stem cell transplantation has been frequently applied to the treatment of pain as an alternative or promising approach for the treatment of severe osteoarthritis, neuropathic pain, and intractable musculoskeletal pain which do not respond to conventional medicine. The main idea of applying stem cells to neuropathic pain is based on the ability of stem cells to release neurotrophic factors, along with providing a cellular source for replacing the injured neural cells, making them ideal candidates for modulating and possibly reversing intractable neuropathic pain. Even though various differentiation capacities of stem cells are reported, there is not enough knowledge and technique to control the differentiation into desired tissues in vivo. Even though the use of stem cells is still in the very early stages of clinical use and raises complicated ethical problems, the future of stem cells therapies is very bright with the help of accumulating evidence and technology.

• 산업기술연구
• /
• 제42권1호
• /
• pp.29-36
• /
• 2022

The CRISPR system has revolutionized gene editing field. Cas9-mediated gene editing such as Indel induction or HDR enable targeted gene disruption or precise correction of mutation. Moreover, CRISPR-based new editing tools have been developed such as base editors. In this review, we focus on gene editing in human pluripotent stem cells, which is principal technique for gene correction therapy and disease modeling. Pluripotent stem cell-specific drug YM155 enabled selection of target gene-edited pluripotent stem cells. Also, we discussed base editing for treatment of congenital retina disease. Adenine base editor delivery as RNP form provide an approach for genetic disease treatment with safe and precise in vivo gene correction.