A Novel Culture Technique for Human Embryonic Stem Cells Using Porous Membranes

Kim, Sinae; Ahn, Seong Eun; Lee, Jae Ho; Lim, Do-Seon; Kim, Kwangsoo; Chung, Hyung-Min; Lee, Soo−Hong

doi:10.1634/stemcells.2006-0814

Cited by 36 publications

(34 citation statements)

References 20 publications

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“…Co-culture-iPSCs (CiPSCs) have the same characteristics, differentiation, expression of pluripotent markers, and morphology as embryonic stem cells (ESCs) grown in colonies in vitro. A previous study found that human ESCs and iPSCs can be propagated using this method (Kim et al, 2007;Abraham et al, 2010). In our study, we provide evidence that this culture system is convenient and simple to use in research concerning the reprogramming mechanism of stem cells in an in vitro environment.…”

Section: Introductionsupporting

confidence: 62%

“…Furthermore, C-iPSCs had the ability to form teratomas after injection into nude mice, and three germ layers formed within 30 days. This method conveniently allows for the easy separation of feeder cells without enzyme treatment from iPSCs midway through the process of induction (Kim et al, 2007). In addition, MEF cells can be used for feeder cells directly.…”

Section: Discussionmentioning

confidence: 99%

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Generation of induced pluripotent mouse stem cells in an indirect co-culture system

Dong¹,

Kaleri²,

Lu³

et al. 2012

Genet. Mol. Res.

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ABSTRACT. Typically, production of induced pluripotent stem cells requires direct contact with feeder cells. However, once the stem cells have reached the appropriate maturation point, it is difficult to separate them from feeder cells, which must be irradiated with γ-rays or treated with the antibiotic mitomycin-C. We used a microporous poly-membrane-based indirect contact co-culture system with mouse embryonic fibroblasts to induce mouse pluripotent stem cells without radiation or antibiotics. We found that induced pluripotent stem cells induced by this co-culture method had a reprogramming efficiency and time similar to those induced using traditional methods. Furthermore, strongly expressed pluripotent markers showed a normal karyotype and formation and contained all three germ layers in a teratoma.

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Section: Introductionsupporting

confidence: 62%

Section: Discussionmentioning

confidence: 99%

Generation of induced pluripotent mouse stem cells in an indirect co-culture system

Dong¹,

Kaleri²,

Lu³

et al. 2012

Genet. Mol. Res.

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“…The PET membrane with 1 mm pore size was selected because it has been reported that only the 1 mm pore can effectively minimize the migration of mouse embryonic fibroblast feeder cells to zero during culture, while the 3 and 8 mm pores cannot. 18 This method of 3D culture is called the ''sandwich method'' in the rest of the article. Limbal epithelial cells in single-cell suspension were cultured for 14-21 days and collected before confluence.…”

Section: Standard and Sandwich Culture Methodsmentioning

confidence: 99%

A Three-Dimensional Culture Method to Expand Limbal Stem/Progenitor Cells

Mei

González

Nakatsu

et al. 2014

Tissue Engineering Part C: Methods

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The current standard method to culture human limbal stem/progenitor cells (LSCs) in vitro is to culture limbal epithelial cells directly on a layer of murine 3T3 feeder cells (standard method). The direct contact between human cells and murine feeder cells poses the potential risk of incomplete removal of feeder cells after culture and cross-contamination in clinical applications. We present here a novel three-dimensional (3D) sandwich method in which LSCs and feeder cells were separately cultured on opposite sides of a porous membrane. Limbal epithelial cells in the form of single-cell suspensions, cell clusters, and tissue explants were subjected to standard culture or to a 3D sandwich culture method. The 3D sandwich method consistently yielded LSCs derived from cell clusters and tissue explants. The expanded LSCs exhibited a small, compact, cuboidal stem-cell morphology and stem cell phenotypes comparable to those of LSCs derived from the standard culture method. Limbal epithelial cell clusters cultured with the sandwich method had a significantly higher proliferation rate than did those cultured with the standard method. The 3D sandwich method did not favor the propagation of single LSCs. In summary, the 3D sandwich method permits complete separation between cultured cells and feeder cells, while providing an even and maximal proximity between them. This alternative method permits culturing of LSCs without the risk of feeder cell contamination.

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“…Three different hESC lines were used in this study: SNUhES3 (Institute of Reproductive Medicine and Population, Medical Research Center, Seoul National University, Korea), 11 CHA3-hESC (Stem Cell Research Laboratory, CHA Stem Cell Institute, Pochon CHA University, Korea), 12 and H9 (Wisconsin Regional Primate Research Center, University of Wisconsin). Mitomycin-C-treated (Sigma, St. Louis, MO) SIM mouse embryo-derived thioguanine and ouabain resistant (STO) feeder system and mitomycin-C-treated mouse embryonic fibroblast feeder system were used for SNUhES3, CHA3-hESC, and H9, respectively, in 0.1% gelatin (Sigma)-coated tissue culture dishes at 378C and 5% CO 2 in an air atmosphere.…”

Section: Culture Of Hescsmentioning

confidence: 99%

Novel Embryoid Body–Based Method to Derive Mesenchymal Stem Cells from Human Embryonic Stem Cells

Lee

Kang

et al. 2010

Tissue Engineering Part A

Self Cite

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Application of human embryonic stem cells (hESCs) to stem-cell therapy is not feasible because of the risk of tumorigenicity and rejection. In contrast, human mesenchymal stem cells (hMSCs) are free from the risk of tumorigenicity and also have immune privilege. However, hMSCs obtained from adults have infinite variety in terms of the biological characteristics and functionality. We report here a new derivation method of hMSCs from hESCs. The derivation of hMSCs from three different hESC lines (SNUhES3, CHA3-hESC, and H9) was performed by embryoid bodies formation and subsequent culture with stage-different media without using inductive xenogenic feeder and mechanical selection procedure. The derived cells were morphologically similar to the unique fingerprint-like pattern of hMSCs and grew stably for at least 35 passages in vitro. These cells had hMSCs-like immunophenotypes: negative for CD34 and CD45; positive for CD29, CD44, CD73, CD90, and CD105. They could be differentiated into multiple lineages including osteocytes, chondrocytes, adipocytes, and myocytes. They maintained normal karyotype during the long-term cultivation and did not show tumorigenicity when transplanted into the immunodeficient mice. In conclusion, the new embryoid body-based derivation method of hMSCs from hESCs is simple, safe, and reproducible in three different hESC lines. We expect that this method will provide a more effective and powerful tool to derive hMSCs from various hESC lines.

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A Novel Culture Technique for Human Embryonic Stem Cells Using Porous Membranes

Cited by 36 publications

References 20 publications

Generation of induced pluripotent mouse stem cells in an indirect co-culture system

Generation of induced pluripotent mouse stem cells in an indirect co-culture system

A Three-Dimensional Culture Method to Expand Limbal Stem/Progenitor Cells

Novel Embryoid Body–Based Method to Derive Mesenchymal Stem Cells from Human Embryonic Stem Cells

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