• International Journal of Stem Cells
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• v.15 no.1
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• pp.104-111
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• 2022

Many of early findings regarding intestinal stem cells (ISCs) and their niche in the human intestine have relied on colorectal cancer cell lines and labor-intensive and time-consuming mouse models. However, these models cannot accurately recapitulate the physiologically relevant aspects of human ISCs. In this study, we demonstrate a reliable and robust culture method for 3D expanding intestinal spheroids (InS^exp) mainly comprising ISCs and progenitors, which can be derived from 3D human intestinal organoids (HIOs). We did functional chararcterization of InS^exp derived from 3D HIOs, differentiated from human pluripotent stem cells, and optimization culture methods. Our results indicate that InS^exp can be rapidly expanded and easily passaged, and show enhanced growth rates via WNT pathway activation. InS^exp are capable of exponential cell expansion and cryopreservation. Furthermore, in vitro-matured HIO-derived InS^exp proliferate faster than immature HIO-derived InS^exp with preservation of the parental HIO characteristics. These findings may facilitate the development of scalable culture systems for the long-term maintenance of human ISCs and provide an alternative platform for studying ISC biology.

• International Journal of Stem Cells
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• v.16 no.1
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• pp.36-43
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• 2023

Background and Objectives: Lymphoblastoid cell lines (LCLs) deposited from disease-affected individuals could be a valuable donor cell source for generating disease-specific induced pluripotent stem cells (iPSCs). However, generation of iPSCs from the LCLs is still challenging, as yet no effective gene delivery strategy has been developed. Methods and Results: Here, we reveal an effective gene delivery method specifically for LCLs. We found that LCLs appear to be refractory toward retroviral and lentiviral transduction. Consequently, lentiviral and retroviral transduction of OCT4, SOX2, KFL4 and c-MYC into LCLs does not elicit iPSC colony formation. Interestingly, however we found that transfection of oriP/EBNA-1-based episomal vectors by electroporation is an efficient gene delivery system into LCLs, enabling iPSC generation from LCLs. These iPSCs expressed pluripotency makers (OCT4, NANOG, SSEA4, SALL4) and could form embryoid bodies. Conclusions: Our data show that electroporation is an effective gene delivery method with which LCLs can be efficiently reprogrammed into iPSCs.

• Archives of Plastic Surgery
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• v.34 no.5
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• pp.537-542
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• 2007

Purpose: Adipose tissue contains a population of pluripotent stem cells capable of differentiating along multiple mesenchymal cell lineages. It is well known that fat depots from different part of our body shows different nature not only in morphological aspect but also physiologic aspect. The authors compared the adipogenic potentials and osteogenic potentials of adipose stem cells from different anatomical sites of human. Methods: After laparotomy by surgery team, the authors isolated these adipose stem cells successfully from 7 men with an average age of 58, and induced differentiation along adipogenic and osteogenic lineages in vitro. On the 14th day, cells cultured in adipogenic media differentiated into adipocytes in vitro, as evidenced by positive Oil Red O staining of lipid vacuoles. On the 21st day, cells cultured in osteogenic media differentiated into osteoblasts in vitro as demonstrated by Alizarin red staining of a calcified extracellular matrix. Results: After exposure to adipogenic and osteogenic differentiation medium, subcutaneous adipose stem cells were found to possess greater adipogenic and osteogenic potentials than cells isolated from visceral adipose tissues. Conclusion: This study indicates that adipogenic and osteogenic potentials of adipose stem cells vary by their anatomical sites, with subcutaneous adipose stem cells exhibiting higher adipogenic and osteogenic potential than those isolated from visceral fat.

• Journal of the Korean Association of Oral and Maxillofacial Surgeons
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• v.32 no.4
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• pp.327-333
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• 2006

Future cell-based therapies such as tissue engineering will benefit from a source of autogenous pluripotent stem cells. There are embryonic stem cells (ESC) and autologous adult stem cells, two general types of stem cells potentilally useful for these applications. But practical use of ESC is limited due to potential problems of cell regulation and ethical considerations. To get bone marrow stem cells is relatively burden to patients because of pain, anesthesia requirement. The ideal stem cells are required of such as the following advantages: easy to obtain, minimal patient discomfort and a capability of yielding enough cell numbers. Adipose autologus tissue taken from intraoral fatty pad or abdomen may represent such a source. Our study designed to demonstrate the ability of human adipose tissue-derived stromal cells (hATSC) from human abdominal adipose tissue diffentiating into osteocyte and adipocyte under culture in vitro conditions. As a result of experiment, we identified stromal cell derived adipose tissue has the multilineage potentiality under appropriate culture conditions. And the adipose stromal cells expressed several mesenchymal stem cell related antigen (CD29, CD44) reactions. Secondary, we compared the culture results of a group of hATSC stimulated with TGF-${\beta}$1, bFGF with a hATSC group without growth factors to confirm whether cytokines have a important role of the proliferation in osteogenic differentiation. The role of cytokines such as TGF-${\beta}$1, bFGF increased hATSC's osteogenic differentiation especially when TGF-${\beta}$1 and bFGF were used together. These results suggest that adipose stromal cells with growth factors could be efficiently available for cell-based bone regeneration.

• BMB Reports
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• v.48 no.5
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• pp.256-265
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• 2015

Cardiovascular and neurodegenerative diseases are major health threats in many developed countries. Recently, target tissues derived from human embryonic stem (hES) cells and induced pluripotent stem cells (iPSCs), such as cardiomyocytes (CMs) or neurons, have been actively mobilized for drug screening. Knowledge of drug toxicity and efficacy obtained using stem cell-derived tissues could parallel that obtained from human trials. Furthermore, iPSC disease models could be advantageous in the development of personalized medicine in various parts of disease sectors. To obtain the maximum benefit from iPSCs in disease modeling, researchers are now focusing on aging, maturation, and metabolism to recapitulate the pathological features seen in patients. Compared to pediatric disease modeling, adult-onset disease modeling with iPSCs requires proper maturation for full manifestation of pathological features. Herein, the success of iPSC technology, focusing on patient-specific drug treatment, maturation-based disease modeling, and alternative approaches to compensate for the current limitations of patient iPSC modeling, will be further discussed. [BMB Reports 2015; 48(5): 256-265]

• International Journal of Oral Biology
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• v.37 no.4
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• pp.175-180
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• 2012

Skin-derived precursor cells (SKPs) are multipotent, sphere-forming and embryonic neural crest-related precursor cells that can be isolated from dermis. It is known that the properties of porcine SKPs can be enhanced by leukemia inhibitory factor (LIF) which is an essential factor for the generation of embryonic stem cells in mice. In our present study, to enhance or maintain the properties of murine SKPs, LIF was added to the culture medium. SKPs were treated with 1,000 IU LIF for 72 hours after passage 3. Quantitative real time RT-PCR was then performed to quantify the expression of the pluripotent stem cell specific genes Oct4, Nanog, Klf4 and c-Myc, and the neural crest specific genes Snai2 and Ngfr. The results show that the expression of Oct4 is increased in murine SKPs by LIF treatment whereas the level of Ngfr is decreased under these conditions. Interestingly, LIF treatment reduced Nanog expression which is also important for cell proliferation in adult stem cells and for osteogenic induction in mesenchymal stem cells. These findings implicate LIF in the maintenance of stemness in SKPs through the suppression of lineage differentiation and in part through the control of cell proliferation.

• Journal of Embryo Transfer
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• v.18 no.3
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• pp.243-248
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• 2003

Sperrnatogenesis, the process by which the male germ-line stem cells(GSCs; type A spermatogonia) divide and differentiate to produce the mature spermatozoa, occurs in the seminiferous tubules of the testis. The GSCs proliferate actively to produce two types of cells: other GSCs and differentiating spermatogonia. GSCs have unipotentcy, devoted solely to the generation of sperm. The function of GSCs has broad implications for development, disease, and evolution. Spermatogenesis is fundamental for propagation of species and the defects of this system can result in infertility or disease. The ability to identify, isolate, culture, and alter GSCs will allow powerful new approaches in animal transgenesis and human gene therapy relating to infertility. Until recently, research on stem cells in the testis has been limited because of technical difficulties in isolating and identifying these cell populations. Here, we were trying to find out optimal conditions for in vitro culture of GSCs for identifying and isolating GSCs. We collected mouse GSCs from 3-days old mouse by two-step enzyme digestion method. GSCs were plated and grown on mouse embryonic fibroblasts in Dulbecco's modified Eagle's medium (DMEM) containing 15％ fatal bovine serum, 10 mM 2-mercaptoethanol, 1％ non-essential amino acids, 1 ng/$m\ell$ bFGF, 10 $\mu$M forskolin, 1500 U/$m\ell$ human recombinant leukemia inhibitory factor (LIF). Over a period 3∼5 days, GSCs gave rise to large multicellular colonies resembling those of mouse pluripotent stem cells. After 5th passages, cells within the colonies continued to be alkaline phosphatase and Oct-4 positive and tested positive against a panel of two immunological markers(Integrin $\alpha$ 6 and Integrin $\beta$ 1) that have been recognized generally to characterize GSCs. SSEA-1, SSEA-3, and SSEA-4 also showed positive signals. Based on our data, these GSCs-derived cultures meet the criteria for GSCs itself and even other pluripotent stem cells. We reported here the establishment of in vitro cultures from mouse male GSCs.

• 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.

• Molecules and Cells
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• v.19 no.1
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• pp.46-53
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• 2005

Human embryonic stem (hES) cells, unlike most cells derived from adult or fetal human tissues, represent a potentially unlimited source of various cell types for basic clinical research. To meet the increased demand for characterized hES cell lines, we established and characterized nine new lines obtained from frozen-thawed pronucleus-stage embryos. In addition, we improved the derivation efficiency from inner cell masses (to 47.4%) and optimized culture conditions for undifferentiated hES cells. After these cell lines had been maintained for over a year in vitro, they were characterized comprehensively for expression of markers of undifferentiated hES cells, karyotype, and in vitro/in vivo differentiation capacity. All of the cell lines were pluripotent, and one cell line was trisomic for chromosome 3. Improved culture techniques for hES cells should make them a good source for diverse applications in regenerative medicine, but further investigation is needed of their basic biology.

• Reproductive and Developmental Biology
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• v.32 no.4
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• pp.223-227
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• 2008

Human embryonic stem (ES) cells retain the capacity for self-renewal, are pluripotent and differentiate into the three embryonic germ layer cells. The regulatory transcription factors Oct4, Nanog and Sox2 play an important role in maintaining the pluripotency of human ES cells. The aim of this research was to identify unknown genes upregulated in human ES cells along with Oct4, Nanog, and Sox2. This study characterizes an unknown gene, named chromosome 1 open reading frame 31 (C1orf31) mapping to chromosome 1q42.2. The product of C1orf31 is the hypothetical protein LOC388753 having a cytochrome c oxidase subunit VIb (COX6b) motif. In order to compare expression levels of C1orf31 in human ES cells, human embryoid body cells, vascular angiogenic progenitor cells (VAPCs), cord-blood endothelial progenitor cells (CB-EPCs) and somatic cell lines, we performed RT-PCR analysis. Interestingly, C1orf31 was highly expressed in human ES cells, cancer cell lines and SV40-immortalized cells. It has a similar expression pattern to the Oct4 gene in human ES cells and cancer cells. Also, the expression level of C1orf31 was shown to be upregulated in the S phase and early G2 phase of synchronized HeLa cells, leading us to purpose that it may be involved in the S/G2 transition process. For these reasons, we assume that C1orf31 may play a role in on differentiation of human ES cells and carcinogenesis.