Properties of four human embryonic stem cell lines maintained in a feeder‐free culture system

Carpenter, Melissa K.; Rosler, Elen S.; Fisk, Gregory J.; Brandenberger, Ralph; Ares, Ximena; Miura, Takumi; Lucero, Mary T.; Rao, Mahendra S.

doi:10.1002/dvdy.10431

Cited by 261 publications

(186 citation statements)

References 28 publications

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“…Although a number of feeder-free systems have been described (1,3,7,9,10,12,13,21), all hES lines to date have been established and repeatedly passaged on mouse feeders prior to transfer to a feeder free-system for cell propagation. Thus far, the feeder free protocols which we have tried (3,21), have not been compatible with the DA differentiation of hES cell lines developed in this protocol.…”

Section: Discussionmentioning

confidence: 99%

“…Currently, there are a number of established hES lines available from NIH-approved sources (Wicell, Bresagen) and a number of new lines provided by the Howard Hughes Institute of Harvard University. All of these cell lines were originally propagated on mouse feeder layers, although several have recently been transferred to feeder free support systems (1,3,7,9,12,21). While the differentiation of DA traits in some hES lines has been described previously (4,5,16,17,18,26), with the exception of one study (19) these protocols involve the prolonged use of complex media containing serum or other undefined reagents and/or cell conditioned media (CM) or co-culture with these cells (ie.…”

Section: Introductionmentioning

confidence: 99%

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A protocol for the differentiation of human embryonic stem cells into dopaminergic neurons using only chemically defined human additives: Studies in vitro and in vivo

et al. 2007

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Our ability to use human embryonic stem (hES) cells in cell replacement therapy for Parkinson's disease depends on the discovery of ways to simply and reliably differentiate a dopaminergic (DA) phenotype in these cells. Although several protocols exist for the differentiation of DA traits in hES, they involve the prolonged use of complex media with undefined components, cell conditioned media and/or co-culture with various cells, usually of animal origin. In this study, several well-characterized (H9, BG01) and several new uncharacterized (HUES7, HUES8) hES cell lines were studied for their capacity to differentiate into DA neurons in culture using a novel rapid protocol which uses only chemically-defined human-derived media additives and substrata. Within 3 weeks, cells from all 4 cell lines progressed from the undifferentiated state to β-tubulin III positive cells expressing DA markers in vitro. Moreover, transplantation of these cells into the striata of 6-hydroxydopaminetreated rats at the neuronal progenitor stage resulted in the appearance of differentiated DA traits in vivo 2-3 weeks later.

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Section: Discussionmentioning

confidence: 99%

Section: Introductionmentioning

confidence: 99%

A protocol for the differentiation of human embryonic stem cells into dopaminergic neurons using only chemically defined human additives: Studies in vitro and in vivo

et al. 2007

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“…When transferred to feeder-free conditions, hESCs were reported to grow initially as three-dimensional colonies [15] composed of three distinct cell types [16]. At the core were ICM-like polygonal cells with large nuclei, relatively little cytosol, few desmosomes, and no gap junctions.…”

Section: Introductionmentioning

confidence: 99%

Feeder-Free Monolayer Cultures of Human Embryonic Stem Cells Express an Epithelial Plasma Membrane Protein Profile

et al. 2008

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Human embryonic stem cells (hESCs) are often cocultured on mitotically inactive fibroblast feeder cells to maintain their undifferentiated state. Under these growth conditions, hESCs form multilayered colonies of morphologically heterogeneous cells surrounded by flattened mesenchymal cells. In contrast, hESCs grown in feeder cell-conditioned medium on Matrigel instead tend to grow as monolayers with uniform morphology. Using mass spectrometry and immunofluorescence microscopy, we showed that hESCs under these conditions primarily express proteins belonging to epithelium-related cell-cell adhesion complexes, including adherens junctions, tight junctions, desmosomes, and gap junctions. This indicates that monolayers of hESCs cultured under feeder-free conditions retain a homogeneous epithelial phenotype similar to that of the upper central cell layer of colonies maintained on feeder cells. Notably, feeder-free hESCs also coexpressed vimentin, which is usually associated with mesenchyme, suggesting that these cells may have undergone epithelium-to-mesenchyme transitions, indicating differentiation. However, if grown on a "soft" substrate (Hydrogel), intracellular vimentin levels were substantially reduced. Moreover, when hESCs were transferred back to feeder cells, expression of vimentin was again absent from the epithelial cell population. These results imply that on tissue culture substrates, vimentin expression is most likely a stressinduced response, unrelated to differentiation. STEM

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“…The colonies are very compact with many tightly spaced cells of large nucleus-to-cytoplasm ratios. The surface antigens include the stage-specific pluripotent antigens SSEA-4 and SSEA-3 and the teratocarcinoma recognition antigens TRA-1 −60 and TRA-1−81 [25]. Specific transcription factors expressed that are associated with the expression of particular elements of the pluripotent genome [115] include the POU (pit-octunc)-domain transcription factor Octamer-4 (Oct-4), Nanog, and Sox2.…”

Section: A Human Pluripotent Stem Cellsmentioning

confidence: 99%

“…Many EBs tend to show cell types of only one or two germ layers, but in an unpredictable manner. Thus, with appropriate subculture conditions and physical removal of colonies showing specific morphologies, behaviors, or proteins, it is possible to establish cultures that are enriched for particular cell types or mixtures of cell types [25]. However, this cell behavior is unpredictable and the sorting is not completely effective.…”

Section: A Human Pluripotent Stem Cellsmentioning

confidence: 99%

Differentiation of neural lineage cells from human pluripotent stem cells

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et al. 2008

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Human pluripotent stem cells have the unique properties of being able to proliferate indefinitely in their undifferentiated state and to differentiate into any somatic cell type. These cells are thus posited to be extremely useful for furthering our understanding of both normal and abnormal human development, providing a human cell preparation that can be used to screen for new reagents or therapeutic agents, and generating large numbers of differentiated cells that can be used for transplantation purposes. Critical among the applications for the latter are diseases and injuries of the nervous system, medical approaches to which have been, to date, primarily palliative in nature. Differentiation of human pluripotent stem cells into cells of the neural lineage, therefore, has become a central focus of a number of laboratories. This has resulted in the description in the literature of several dozen methods for neural cell differentiation from human pluripotent stem cells. Among these are methods for the generation of such divergent neural cells as dopaminergic neurons, retinal neurons, ventral motoneurons, and oligodendroglial progenitors. In this review, we attempt to fully describe most of these methods, breaking them down into five basic subdivisions: 1) starting material, 2) induction of loss of pluripotency, 3) neural induction, 4) neural maintenance and expansion, and 5) neuronal/glial differentiation. We also show data supporting the concept that undifferentiated human pluripotent stem cells appear to have an innate neural differentiation potential. In addition, we evaluate data comparing and contrasting neural stem cells differentiated from human pluripotent stem cells with those derived directly from the human brain.

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Properties of four human embryonic stem cell lines maintained in a feeder‐free culture system

Abstract: Several laboratories have begun evaluating human ES (hES

Cited by 261 publications

References 28 publications

A protocol for the differentiation of human embryonic stem cells into dopaminergic neurons using only chemically defined human additives: Studies in vitro and in vivo

A protocol for the differentiation of human embryonic stem cells into dopaminergic neurons using only chemically defined human additives: Studies in vitro and in vivo

Feeder-Free Monolayer Cultures of Human Embryonic Stem Cells Express an Epithelial Plasma Membrane Protein Profile

Differentiation of neural lineage cells from human pluripotent stem cells

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