• Asian-Australasian Journal of Animal Sciences
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• v.26 no.4
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• pp.588-595
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• 2013

Mesenchymal stem cells (MSCs) are often known to have a therapeutic potential in the cell-mediated repair for fatal or incurable diseases. In this study, canine umbilical cord MSCs (cUC-MSCs) were isolated from umbilical cord matrix (n = 3) and subjected to proliferative culture for 5 consecutive passages. The cells at each passage were characterized for multipotent MSC properties such as proliferation kinetics, expression patterns of MSC surface markers and self-renewal associated markers, and chondrogenic differentiation. In results, the proliferation of the cells as determined by the cumulative population doubling level was observed at its peak on passage 3 and stopped after passage 5, whereas cell doubling time dramatically increased after passage 4. Expression of MSC surface markers (CD44, CD54, CD61, CD80, CD90 and Flk-1), molecule (HMGA2) and pluripotent markers (sox2, nanog) associated with self-renewal was negatively correlated with the number of passages. However, MSC surface marker (CD105) and pluripotent marker (Oct3/4) decreased with increasing the number of subpassage. cUC-MSCs at passage 1 to 5 underwent chondrogenesis under specific culture conditions, but percentage of chondrogenic differentiation decreased with increasing the number of subpassage. Collectively, the present study suggested that sequential subpassage could affect multipotent properties of cUC-MSCs and needs to be addressed before clinical applications.

• BMB Reports
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• v.44 no.12
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• pp.793-798
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• 2011

Recently, pluripotency induction or cellular reprogramming by introducing critical transcription factors has been extensively studied, but has been demonstrated only in vitro. Based on reports that Oct4 is critically involved in transforming neural stem cells into pluripotent cells, we used the lentiviral vector to introduce the Oct4 gene into the hippocampal dentate gyrus (DG) of adult mice. We examined whether this manipulation led to cellular or behavioral changes, possibly through processes involving the transformation of NS cells into pluripotent cells. The Oct4 lentivirus-infused group and the green fluorescent protein lentivirus-infused group showed a similar thickness of the DG and a comparable level of synaptophysin expression in the DG. Furthermore, our behavioral analyses did not show any differences between the groups concerning exploratory activity, anxiety, or memory abilities. This first trial for pluripotency induction in vivo, despite negative results, provides implications and information for future studies on in vivo cellular reprogramming.

• Development and Reproduction
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• v.17 no.1
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• pp.9-16
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• 2013

Coactivator-associated arginine methyltransferase 1 (CARM1) is included in the protein arginine methyltransferase (PRMT) family, which methylates histone arginine residues through posttranslational modification. It has been proposed that CARM1 may up-regulate the expression of pluripotency-related genes through the alteration of the chromatin structure. Mouse embryonic stem cells (mESCs) are pluripotent and have the ability to self-renew. The cells are mainly used to study the genetic function of novel genes, because the cells facilitate the transmission of the manipulated genes into target mice. Since the up-regulated methylation levels of histone arginine residue lead to the maintenance of pluripotency in embryos and stem cells, it may be suggested that CARM1 overexpressing mESCs elevate the expression of pluripotency-related genes in reconstituted embryos for transgenic mice and may resist the differentiation into trophectoderm (TE). We constructed a fusion protein by connecting CARM1 and 7X-arginine (R7). As a cell-penetrating peptide (CPP), can translocate CARM1 protein into mESCs. CPP-CARM1 protein was detected in the nuclei of the mESCs after a treatment of 24 hours. Accordingly, the expression of pluripotency-related genes was up-regulated in CPP-CARM1-treated mESCs. In addition, CPP-CARM1-treated mESC-derived embryoid bodies (EBs) showed an elevated expression of pluripotency-related genes and delayed spontaneous differentiation. This result suggests that the treatment of recombinant CPP-CARM1 protein elevates the expression of pluripotency-related genes of mESCs by epigenetic modification, and this protein-delivery system could be used to modify embryonic fate in reconstituted embryos with mESCs.

• Proceedings of the Korea Society of Poultry Science Conference
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• 2001.11a
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• pp.40-46
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• 2001

The use of pluripotent stem cells has tremendous advantages for various purposes but these cell lines with proven germ-line transmission have been completely established only in the mouse. Embryonic germ (EG) cell lines are also pluripotent and undifferentiated stem cells established from primordial germ cells (PGCs). This study was conducted to establish and characterize the chicken EG cells derived from gonadal primordial germ cells. We isolated gonadal PGCs from 5.5-day-old (stage 28) White leghorn (WL) embryos and established chicken EG cells lines with EG culture medium supplemented with human stem cell factor (hSCF), murine leukemia inhibitory factor (mLIF), bovine basic fibroblast growth factor (bFGF), human interleukin-11 (hIL-11), and human insulin-like growth factor-I (hIGF-I). These cells grew continuously for 4 months (10 passages) on a feeder layer of mitotically active chicken embryonic fibroblasts. These cells were characterized by screening with the Periodic acid-Shiff＇s reaction, anti-SSEA-1 antibody, and a proliferation assay after several passages. As the results, the chicken EG cells maintained characteristics of undifferentiated stem cells as well as that of gonadal PGCs. When cultured in suspension, the chicken EG cells successfully formed an embryoid body and differentiated into a variety of cell types when re-seeded onto culture dish. The chicken EG cells were injected into blastodermal layer at stage X and dorsal aorta of recipient embryo at stage 14 (incubation of 53hrs) and produced chimeric chickens with various differentiated tissues derived from the EG cells. The germline chimeras were also successfully induced by using EG cells. Thus, Chicken EG cells will be useful for the production of transgenic chickena and for studies of germ cell differentiation and genomic imprinting.

• Journal of Embryo Transfer
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• v.28 no.1
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• pp.1-6
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• 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
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• v.25 no.3
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• pp.358-367
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• 2008

The embryonic germ cell (EGCs) of mice is a kind of pluripotent stem cell that can be generated from pre- and post-migratory primordial germ cells (PGCs). Most previous studies on DNA methylation of EGCs were restricted to 12.5 days post coitum (dpc). This study was designed to establish and characterize murine EGC lines from migrated PGCs as late as 13.5 dpc and to estimate the degrees of methylation of their imprinted genes as well as of the non-imprinted locus, Oct4, using an accurate and quantitative method of measurement. We established five independent EGC lines from post migratory PGCs of 11.5-13.5 dpc from C57BL/6 ${\times}$ DBA/2 F1 hybrid mouse fetuses. All the EGCs exhibited the typical features of pluripotent cells including hypomethylation of the Oct4 regulatory region. We examined the methylation status of three imprinted genes; Igf2, Igf2r and H19 in the five EGC lines using bisulfite genomic sequencing analysis. Igf2r was almost unmethylated in all the EGC lines irrespective of the their sex and stage of isolation; Igf2 and H19 were more methylated than Igf2r, especially in male EGCs. Moreover, EGCs derived at 13.5 dpc exhibited higher levels of DNA methylation than those from earlier stages. These results suggest that in vitro derived EGCs acquire different epigenotypes from their parental in vivo migratory PGCs, and that sex-specific de novo methylation occurs in the Igf2 and H19 genes of EGCs.

• International Journal of Oral Biology
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• v.35 no.4
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• pp.209-214
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• 2010

Cells that have endogenous multipotent properties can be used as a starting source for the generation of induced pluripotent cells (iPSC). In addition, small molecules associated with epigenetic reprogramming are also widely used to enhance the multi- or pluripotency of such cells. Skinderived precursor cells (SKPs) are multipotent, sphereforming and embryonic neural crest-related precursor cells. These cells can be isolated from a juvenile or adult mammalian dermis. SKPs are also an efficient starting cell source for reprogramming and the generation of iPSCs because of the high expression levels of Sox2 and Klf4 in these cells as well as their endogenous multipotency. In this study, valproic acid (VPA), a histone deacetylase (HDAC) inhibitor, was tested in the generation of iPSCs as a potential enhancer of the reprogramming potential of SKPs. SKPs were isolated from the back skins of 5-6 week old C57BL/6 X DBA/2 F1 mice. After passage 3, the SKPs was treated with 2 mM of VPA and the quantitative real time RT-PCR was performed to quantify the expression of Oct4 and Klf4 (pluripotency specific genes), and Snai2 and Ngfr (neural crest specific genes). The results show that Oct4 and Klf4 expression was decreased by VPA treatment. However, there were no significant changes in neural crest specific gene expression following VPA treatment. Hence, although VPA is one of the most potent of the HDAC inhibitors, it does not enhance the reprogramming of multipotent skin precursor cells in mice.

• Interdisciplinary Bio Central
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• v.4 no.3
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• pp.6.1-6.12
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• 2012

Parkinson's disease (PD) is a complicated neurodegenerative disorder although it is oftentimes defined by clinical motor symptoms originated from age dependent and progressive loss of dopaminergic neurons in the midbrain. The pathogenesis of PD involves dopaminergic and nondopaminergic neurons in many brain regions and the molecular mechanisms underlying the death of different cell types still remain to be elucidated. There are indications that PD causing disease processes occur in a global scale ranging from DNA to RNA, and proteins. Several PD-associated genes have been reported to play diverse roles in controlling cellular functions in different levels, such as chromatin structure, transcription, processing of mRNA, translational modulation, and posttranslational modification of proteins. The advent of quantitative high throughput screening (HTS) tools makes it possible to monitor systemic changes in DNA, RNA and proteins in PD models. Combined with dopamine neuron isolation or derivation of dopamine neurons from PD patient specific induced pluripotent stem cells (PD iPSCs), HTS techonologies will provide opportunities to draw PD causing sequences of molecular events in pathologically relevant PD samples. Here I discuss previous studies that identified molecular functions in which PD genes are involved, especially those signaling pathways that can be efficiently studied using HTS methodologies. Brief descriptions of quantitative and systemic tools looking at DNA, RNA and proteins will be followed. Finally, I will emphasize the use and potential benefits of PD iPSCs-derived dopaminergic neurons to screen signaling pathways that are initiated by PD linked gene mutations and thus causative for dopaminergic neurodegneration in PD.

• Journal of Veterinary Science
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• v.22 no.6
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• pp.86.1-86.13
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• 2021

Background: Conditioned medium is the medium obtained from certain cultured cells and contained secretome from the cells. The secretome, which can be in the form of growth factors, cytokines, exosomes, or other proteins secreted by the cells, can induce the differentiation of cells that still have pluripotent or multipotent properties. Objectives: This study examined the effects of conditioned medium derived from E17 rat brain cells on cells with pluripotent properties. Methods: The conditioned medium used in this study originated from E17 rat brain cells. The CM was used to induce the differentiation of primary colonies of mice blastocysts. Primary colonies were stained with alkaline phosphatase to analyze the pluripotency. The morphological changes in the colonies were examined, and the colonies were stained with GFAP and Neu-N markers on days two and seven after adding the conditioned medium. Results: The conditioned medium could differentiate the primary colony, beginning with the formation of embryoid-body-like structure; round GFAP positive cells were identified. Finally, neuron-like cells testing positive for Neu-N were observed on the seventh day after adding the conditioned medium. Conclusions: Conditioned medium from different species, in this case, E17 rat brain cells, induced and promoted the differentiation of the primary colony from mice blastocysts into neuron-like cells. The addition of CM mediated neurite growth in the differentiation process.

• The Korean Journal of Pain
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• v.32 no.4
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• pp.245-255
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• 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.