• BMB Reports
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• v.54 no.12
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• pp.626-631
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• 2021

Janus kinase 2 (JAK2), a non-receptor tyrosine kinase, is a critical component of cytokine and growth factor signaling pathways regulating hematopoietic cell proliferation. JAK2 mutations are associated with multiple myeloproliferative neoplasms. Although physiological and pathological functions of JAK2 in hematopoietic tissues are well-known, such functions of JAK2 in the nervous system are not well studied yet. The present study demonstrated that JAK2 could negatively regulate neuronal differentiation of mouse embryonic stem cells (ESCs). Depletion of JAK2 stimulated neuronal differentiation of mouse ESCs and activated glycogen synthase kinase 3β, Fyn, and cyclin-dependent kinase 5. Knockdown of JAK2 resulted in accumulation of GTP-bound Rac1, a Rho GTPase implicated in the regulation of cytoskeletal dynamics. These findings suggest that JAK2 might negatively regulate neuronal differentiation by suppressing the GSK-3β/Fyn/CDK5 signaling pathway responsible for morphological maturation.

• Korean Journal of Fisheries and Aquatic Sciences
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• v.50 no.2
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• pp.160-168
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• 2017

Optimizing the conditions for stem cell culture is an essential prerequisite for the efficient utilization of stem cells. In the culture of fish embryonic stem cells (ESCs) or ESC-like cells, embryo extracts are important for stable growth, but there is no rule for determining the developmental stage of the embryos used to obtain extracts. Therefore, this study investigated the effects of the developmental stage of extract donor embryos on the culture of Oryzias dancena ESC-like cells. O. dancena ESC-like cells were cultured in different media containing each of four types of embryo extract depending on the developmental stage of the extract donor embryos. Growth, morphology, colony-forming ability, alkaline phosphatase (AP) activity, and embryoid body (EB) formation of the cells were investigated. While the developmental stage of the extract donor embryos did not influence the growth, morphology, AP activity, or EB formation of ESC-like cells, colony-forming ability was affected and the pattern of the effects differed completely between the two ESC-like cells investigated. These results suggest that the developmental stage of extract donor embryos should be selected carefully for the culture of ESC-like cells, according to the research purpose and type of cell line.

• Clinical and Experimental Reproductive Medicine
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• v.38 no.4
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• pp.216-221
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• 2011

Objective: To differentiate the human embryonic stem cells (hESCs) into the retinal pigment epithelium (RPE) in the defined culture condition and determine its therapeutic potential for the treatment of retinal degenerative diseases. Methods: The embryoid bodies were formed from hESCs and attached on the matrigel coated culture dishes. The neural structures consisting neural precursors were selected and expanded to form rosette structures. The mechanically isolated neural rosettes were differentiated into pigmented cells in the media comprised of N2 and B27. Expression profiles of markers related to RPE development were analyzed by reverse transcription-polymerase chain reaction and immunostaining. Dissociated putative RPE cells ($10^5$ cells/5 ${\mu}L$) were transplanted into the subretinal space of rat retinal degeneration model induced by intravenous sodium iodate injection. Animals were sacrificed at 1, 2, and 4 weeks after transplantation, and immnohistochemistry study was performed to verify the survival of the transplanted cells. Results: The putative RPE cells derived from hESC showed characteristics of the human RPE cells morphologically and expressed molecular markers and associated with RPE fate. Grafted RPE cells were found to survive in the subretinal space up to 4 weeks after transplantation, and the expression of RPE markers was confirmed with immunohistochemistry. Conclusion: Transplanted RPE cells derived from hESC in the defined culture condition successfully survived and migrated within subretinal space of rat retinal degeneration model. These results support the feasibility of the hESC derived RPE cells for cell-based therapies for retinal degenerative disease.

• Development and Reproduction
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• v.24 no.2
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• pp.135-147
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• 2020

Polyvinylidene fluoride (PVDF) is a stable and biocompatible material that has been broadly used in biomedical applications. Due to its piezoelectric property, the electrospun nanofiber of PVDF has been used to culture electroactive cells, such as osteocytes and cardiomyocytes. Here, taking advantage of the piezoelectric property of PVDF, we have fabricated a PVDF nanofiber scaffolds using an electrospinning technique for differentiating human embryonic stem cells (hESCs) into neural precursors (NPs). Surface coating with a peptide derived from vitronectin enables hESCs to firmly adhere onto the nanofiber scaffolds and differentiate into NPs under dual-SMAD inhibition. Our nanofiber scaffolds supported the differentiation of hESCs into SOX1-positive NPs more significantly than Matrigel. The NPs generated on the nanofiber scaffolds could give rise to neurons, astrocytes, and oligodendrocyte precursors. Furthermore, comparative transcriptome analysis revealed the variable expressions of 27 genes in the nanofiber scaffold groups, several of which are highly related to the biological processes required for neural differentiation. These results suggest that a PVDF nanofiber scaffold coated with a vitronectin peptide can serve as a highly efficient and defined culture platform for the neural differentiation of hESCs.

• BMB Reports
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• v.55 no.3
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• pp.142-147
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• 2022

Human pluripotent stem cells (PSCs) have been utilized as a promising source in regenerative medicine. However, the risk of teratoma formation that comes with residual undifferentiated PSCs in differentiated cell populations is most concerning in the clinical use of PSC derivatives. Here, we report that a monoclonal antibody (mAb) targeting PSCs could distinguish undifferentiated PSCs, with potential teratoma-forming activity, from differentiated PSC progeny. A panel of hybridomas generated from mouse immunization with H9 human embryonic stem cells (hESCs) was screened for ESC-specific binding using flow cytometry. A novel mAb, K312, was selected considering its high stem cell-binding activity, and this mAb could bind to several human induced pluripotent stem cells and PSC lines. Cell-binding activity of K312 was markedly decreased as hESCs were differentiated into embryoid bodies or by retinoic acid treatment. In addition, a cell population negatively isolated from undifferentiated or differentiated H9 hESCs via K312 targeting showed a significantly reduced expression of pluripotency markers, including Oct4 and Nanog. Furthermore, K312-based depletion of pluripotent cells from differentiated PSC progeny completely prevented teratoma formation. Therefore, our findings suggest that K312 is utilizable in improving stem cell transplantation safety by specifically distinguishing residual undifferentiated PSCs.

• Journal of Life Science
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• v.33 no.1
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• pp.64-72
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• 2023

Despite several advances in identification of cardiac transcription factors, there are still needs to find new bioactive molecules that promote cardiomyogenesis from stem cells to highly efficient myocardial differentiation. We analyzed Illumina expression microarray data of mouse embryonic stem cells (mESCs)-derived cardiomyocytes. 276 genes were upregulated (≥ 4fold) in mESCs-derived cardiomyocytes compared undifferentiated ESCs. Secreted phosphoprotein 2 (Spp2) is one of candidates and is known to inhibit bone morphogenetic protein 2 (BMP2) signal transduction as a pseudoreceptor for BMP2. However, its function in cardiomyogenesis is unknown. We confirmed that Spp2 expression increased during the differentiation into functional cardiomyocytes using mESCs, TC-1/Kh2 and E14. Interestingly, Spp2 secretion transiently increased 3 days after formation of embryoid bodies (EBs), indicating that the extracellular secretion of Spp2 is involved in the differentiation of ESCs into cardiomyocytes. To characterize Spp2, we performed experiments using the C2C12 mouse myoblast cell line, which has the property of shifting the differentiation pathway from myoblastic to osteoblastic by treatment with BMP2. Similar to the differentiation of ESCs, transcription of Spp2 increased as C2C12 myoblasts differentiated into myotubes. In particular, Spp2 secretion increased dramatically in the early stage of differentiation. Furthermore, treatment with Spp2-Flag recombinant protein promoted the differentiation of C2C12 myoblasts into myotubes. Taken together, we suggest a novel bioactive protein Spp2 that differentiates ESCs into cardiomyocytes. This may be useful for understanding the molecular pathways of cardiomyogenesis and for experimental or clinical promotion of stem cell therapy for ischemic heart diseases.

• BMB Reports
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• v.52 no.8
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• pp.482-489
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• 2019

Reproductive biotechnology has developed rapidly and is now able to overcome many birth difficulties due to infertility or the transmission of genetic diseases. Here we introduce the next generation of assisted reproductive technologies (ART), such as mitochondrial replacement technique (MRT) or genetic correction in eggs with micromanipulation. Further, we suggest that the transmission of genetic information from somatic cells to subsequent generations without gametes should be useful for people who suffer from infertility or genetic diseases. Pluripotent stem cells (PSCs) can be converted into germ cells such as sperm or oocytes in the laboratory. Notably, germ cells derived from nuclear transfer embryonic stem cells (NT-ESCs) or induced pluripotent stem cells (iPSCs) inherit the full parental genome. The most important issue in this technique is the generation of a haploid chromosome from diploid somatic cells. We hereby examine current science and limitations underpinning these important developments and provide recommendations for moving forward.

• Journal of Microbiology and Biotechnology
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• v.32 no.1
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• pp.126-140
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• 2022

Stem cells can be applied usefully in basic research and clinical field due to their differentiation and self-renewal capacity. The aim of this study was to establish an effective novel therapeutic cellular source and create its molecular expression profile map to elucidate the possible therapeutic mechanism and signaling pathway. We successfully obtained a mesenchymal stem cell population from human embryonic stem cells (hESCs) cultured on chemically defined feeder-free conditions and treated with connective tissue growth factor (CTGF) and performed the expressive proteomic approach to elucidate the molecular basis. We further selected 12 differentially expressed proteins in CTGF-induced hESC-derived mesenchymal stem cells (C-hESC-MSCs), which were found to be involved in the metabolic process, immune response, cell signaling, and cell proliferation, as compared to bone marrow derived-MSCs(BM-MSCs). Moreover, these up-regulated proteins were potentially related to the Wnt/β-catenin pathway. These results suggest that C-hESC-MSCs are a highly proliferative cell population, which can interact with the Wnt/β-catenin signaling pathway; thus, due to the upregulated cell survival ability or downregulated apoptosis effects of C-hESC-MSCs, these can be used as an unlimited cellular source in the cell therapy field for a higher therapeutic potential. Overall, the study provided valuable insights into the molecular functioning of hESC derivatives as a valuable cellular source.

• Asian-Australasian Journal of Animal Sciences
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• v.28 no.11
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• pp.1565-1572
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• 2015

Porcine embryonic stem cells (pESCs) have become an advantageous experimental tool for developing therapeutic applications and producing transgenic animals. However, despite numerous reports of putative pESC lines, deriving validated pESC lines from embryos produced in vitro remains difficult. Here, we report that embryo aggregation was useful for deriving pESCs from in vitro-produced embryos. Blastocysts derived from embryo aggregation formed a larger number of colonies and maintained cell culture stability. Our derived cell lines demonstrated expression of pluripotent markers (alkaline phosphatase, Oct4, Sox2, and Nanog), an ability to form embryoid bodies, and the capacity to differentiate into the three germ layers. A cytogenetic analysis of these cells revealed that all lines derived from aggregated blastocysts had normal female and male karyotypes. These results demonstrate that embryo aggregation could be a useful technique to improve the efficiency of deriving ESCs from in vitro-fertilized pig embryos, studying early development, and deriving pluripotent ESCs in vitro in other mammals.

• BMB Reports
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• v.44 no.12
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• pp.799-804
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• 2011

Gangliosides play an important role in neuronal differentiation processes. The regulation of ganglioside levels is related to the induction of neuronal cell differentiation. In this study, the ST8Sia5 gene was transfected into mESCs and then differentiated into neuronal cells. Interestingly, ST8Sia5 gene transfected mESCs expressed GQ1b by HPTLC and immunofluorescence analysis. To investigate the effects of GQ1b over-expression in neurogenesis, neuronal cells were differentiated from GQ1b expressing mESCs in the presence of retinoic acid. In GQ1b expressing mESCs, increased EBs formation was observed. After 4 days, EBs were co-localized with GQ1b and nestin, and GFAP. Moreover, GQ1b co-localized with MAP-2 expressing cells in GQ1b expressing mESCs in 7-day-old EBs. Furthermore, GQ1b expressing mESCs increased the ERK1/2 MAP kinase pathway. These results suggest that the ST8Sia5 gene increases ganglioside GQ1b and improves neuronal differentiation via the ERK1/2 MAP kinase pathway.