Preparation of Autogenic Human Feeder Cells for Growth of Human Embryonic Stem Cells

Gonzalez, Rodolfo; Snyder, Evan Y.

doi:10.1002/9780470151808.sc01c05s4

Cited by 7 publications

(5 citation statements)

References 12 publications

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“…Since the initially high activin A production decreased much more slowly and was maintained on a higher level compared to the other bioreactors, the results from gene expression analysis and from histological studies suggest that differentiation in this reactor was suppressed by a ‘feeder cell‐like’ activity. This could be due to differentiation of some hESCs into fibroblasts or fibroblast‐like cells with the ability to secrete activin A, and that are able to support the self‐renewal of undifferentiated hESCs, as has been described in 2D cultures in the literature (Xu et al ., ; Stojkovic et al ., ; Yoo et al ., ; Gonzalez et al ., ).…”

Section: Resultsmentioning

confidence: 97%

“…A possible explanation for the origin of this ‘feeder cell‐like’ activity is that, under the influence of the culture medium used, some of the hESCs differentiated into cells exhibiting self‐renewal supportive activity. This is in accordance with studies by other groups that showed that autologous hESC‐derived feeder cells are able to support the self‐renewal of undifferentiated hESCs (Xu et al ., ; Stojkovic et al ., ; Yoo et al ., ; Gonzalez et al ., ).…”

Section: Discussionmentioning

confidence: 97%

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Teratoma formation of human embryonic stem cells in three-dimensional perfusion culture bioreactors

Stachelscheid

Wulf-Goldenberg

Eckert

et al. 2012

J Tissue Eng Regen Med

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Teratoma formation in mice is today the most stringent test for pluripotency that is available for human pluripotent cells, as chimera formation and tetraploid complementation cannot be performed with human cells. The teratoma assay could also be applied for assessing the safety of human pluripotent cell-derived cell populations intended for therapeutic applications. In our study we examined the spontaneous differentiation behaviour of human embryonic stem cells (hESCs) in a perfused 3D multi-compartment bioreactor system and compared it with differentiation of hESCs and human induced pluripotent cells (hiPSCs) cultured in vitro as embryoid bodies and in vivo in an experimental mouse model of teratoma formation. Results from biochemical, histological/immunohistological and ultrastuctural analyses revealed that hESCs cultured in bioreactors formed tissue-like structures containing derivatives of all three germ layers. Comparison with embryoid bodies and the teratomas revealed a high degree of similarity of the tissues formed in the bioreactor to these in the teratomas at the histological as well as transcriptional level, as detected by comparative whole-genome RNA expression profiling. The 3D culture system represents a novel in vitro model that permits stable long-term cultivation, spontaneous multi-lineage differentiation and tissue formation of pluripotent cells that is comparable to in vivo differentiation. Such a model is of interest, e.g. for the development of novel cell differentiation strategies. In addition, the 3D in vitro model could be used for teratoma studies and pluripotency assays in a fully defined, controlled environment, alternatively to in vivo mouse models.

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Section: Resultsmentioning

confidence: 97%

Section: Discussionmentioning

confidence: 97%

Teratoma formation of human embryonic stem cells in three-dimensional perfusion culture bioreactors

Stachelscheid

Wulf-Goldenberg

Eckert

et al. 2012

J Tissue Eng Regen Med

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“…hESC-derived fibroblasts were generated as described [14] for use in global gene expression comparisons. These fibroblasts were grown in DMEM+10% FBS at 37°C/5% CO 2 and fed on alternate days and passaged weekly with 0.05% trypsin (Gibco).…”

Section: Methodsmentioning

confidence: 99%

Propagation of human embryonic and induced pluripotent stem cells in an indirect co-culture system

Abraham

Sheridan

Laurent

et al. 2010

Biochemical and Biophysical Research Communications

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We have developed and validated a microporous poly(ethylene terephthalate) membrane-based indirect co-culture system for human pluripotent stem cell (hPSC) propagation, which allows realtime conditioning of the culture medium with human fibroblasts while maintaining the complete separation of the two cell types. The propagation and pluripotent characteristics of a human embryonic stem cell (hESC) line and a human induced pluripotent stem cell (hiPSC) line were studied in prolonged culture in this system. We report that hPSCs cultured on membranes by indirect coculture with fibroblasts were indistinguishable by multiple criteria from hPSCs cultured directly on a fibroblast feeder layer. Thus this co-culture system is a significant advance in hPSC culture methods, providing a facile stem cell expansion system with continuous medium conditioning while preventing mixing of hPSCs and feeder cells. This membrane culture method will enable testing of novel feeder cells and differentiation studies using co-culture with other cell types, and will simplify stepwise changes in culture conditions for staged differentiation protocols.

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“…They were harvested at passage 41. hESC-derived fibroblasts (hESC-Fibro) were derived as a stable proliferating population from spontaneously differentiating passage 40 WA09 hESC cells (Gonzalez et al 2008); after their differentiation they were expanded in two batches, to passage 11 and passage 13 before harvesting. The neonatal fibroblast line (Fibro) cell line was obtained from GlobalStem, Inc (newborn human foreskin fibroblasts, untreated) and was harvested for analysis at passage 13.…”

Section: Cell Culture and Dna Preparationmentioning

confidence: 99%

Dynamic changes in the human methylome during differentiation

Laurent¹,

Wong²,

Li³

et al. 2010

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SingaporeDNA methylation is a critical epigenetic regulator in mammalian development. Here, we present a whole-genome comparative view of DNA methylation using bisulfite sequencing of three cultured cell types representing progressive stages of differentiation: human embryonic stem cells (hESCs), a fibroblastic differentiated derivative of the hESCs, and neonatal fibroblasts. As a reference, we compared our maps with a methylome map of a fully differentiated adult cell type, mature peripheral blood mononuclear cells (monocytes). We observed many notable common and cell-type-specific features among all cell types. Promoter hypomethylation (both CG and CA) and higher levels of gene body methylation were positively correlated with transcription in all cell types. Exons were more highly methylated than introns, and sharp transitions of methylation occurred at exon-intron boundaries, suggesting a role for differential methylation in transcript splicing. Developmental stage was reflected in both the level of global methylation and extent of non-CpG methylation, with hESC highest, fibroblasts intermediate, and monocytes lowest. Differentiation-associated differential methylation profiles were observed for developmentally regulated genes, including the HOX clusters, other homeobox transcription factors, and pluripotence-associated genes such as POU5F1, TCF3, and KLF4. Our results highlight the value of high-resolution methylation maps, in conjunction with other systems-level analyses, for investigation of previously undetectable developmental regulatory mechanisms.

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Preparation of Autogenic Human Feeder Cells for Growth of Human Embryonic Stem Cells

Cited by 7 publications

References 12 publications

Teratoma formation of human embryonic stem cells in three-dimensional perfusion culture bioreactors

Teratoma formation of human embryonic stem cells in three-dimensional perfusion culture bioreactors

Propagation of human embryonic and induced pluripotent stem cells in an indirect co-culture system

Dynamic changes in the human methylome during differentiation

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