Feeder-independent culture of human embryonic stem cells

Ludwig, Tenneille E.; Bergendahl, Veit; Levenstein, Mark E.; Yu, Junying; Probasco, Mitchell D.; Thomson, James A.

doi:10.1038/nmeth902

Cited by 609 publications

(471 citation statements)

References 16 publications

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“…Human embryonic stem cells (lines H1, H7, and H9) were maintained in feeder-independent media (TeSR) as described in ref. 42. Cells were treated for 0, 3, 15, 30, 60, and 75 h with 12-O-tetradecanoylphorbol-13-acetate (TPA) (Sigma-Aldrich) at a final concentration of 50 ng/ml.…”

Section: Methodsmentioning

confidence: 99%

Mass spectrometry identifies and quantifies 74 unique histone H4 isoforms in differentiating human embryonic stem cells

Phanstiel

Brumbaugh²,

Berggren³

et al. 2008

Proc. Natl. Acad. Sci. U.S.A.

162

175

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Epigenetic regulation through chromatin is thought to play a critical role in the establishment and maintenance of pluripotency. Traditionally, antibody-based technologies were used to probe for specific posttranslational modifications (PTMs) present on histone tails, but these methods do not generally reveal the presence of multiple modifications on a single-histone tail (combinatorial codes). Here, we describe technology for the discovery and quantification of histone combinatorial codes that is based on chromatography and mass spectrometry. We applied this methodology to decipher 74 discrete combinatorial codes on the tail of histone H4 from human embryonic stem (ES) cells. Finally, we quantified the abundances of these codes as human ES cells undergo differentiation to reveal striking changes in methylation and acetylation patterns. For example, H4R3 methylation was observed only in the presence of H4K20 dimethylation; such context-specific patterning exemplifies the power of this technique.electron transfer dissociation ͉ epigenetics ͉ posttranslational modification ͉ histone code ͉ pluripotency P luripotency-the ability to differentiate into any specialized lineage-is the hallmark of embryonic stem (ES) cells and the basis for their experimental and therapeutic potential. The precise molecular mechanisms that define pluripotency remain elusive; however, a number of recent works suggest a central role for epigenetic regulation through chromatin (1-6). Either by recruiting or shielding certain factors, modifications on histone proteins modulate a gene's local environment and thereby regulate expression (7-12). Concerted changes in histone modification states occur during differentiation (13). For example, high levels of histone H3 and H4 acetylation are characteristic of pluripotent cells in mice and the abundance of these marks decreases during differentiation (14, 15). Methylation of R2, R17, and R26 of histone H3 by CARM1 also correlates with cell fate and potency (6). Cells with higher levels of methylation, at these residues, were enriched in the embryonic part of the blastocyst. Next, specific patterns of histone H3K4me3 and H3K27me3 are observed at promoter regions of genes that are regulated during differentiation (1, 2). Finally, demethylation of H3K27me3 is required for activation of certain HOX genes essential for proper development (16,17). The demethylase responsible interacts directly with MLL 2/3 complexes, which methylate histone H3K4 (18). Taken together these experiments have shed new light on the power of epigenetic regulation within ES cells; however, the precise details and role(s) of such combinatorial PTM patterns remain largely unknown.Technological limitations in our ability to discover and quantify combinatorial histone PTMs has, and continues to, present a major obstacle. Most of our knowledge of epigenetics has been derived by antibody-based approaches. Antibodies require a priori knowledge of individual modifications, are subject to epitope occlusion, and have difficulty distingu...

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Section: Methodsmentioning

confidence: 99%

Mass spectrometry identifies and quantifies 74 unique histone H4 isoforms in differentiating human embryonic stem cells

Phanstiel

Brumbaugh²,

Berggren³

et al. 2008

Proc. Natl. Acad. Sci. U.S.A.

162

175

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“…induced pluripotent stem cells (hiPSCs) have the potential to grow indefinitely in culture and to make any cell in the adult human body, they represent important resources for regenerative medicine (1) and for disease modeling efforts that seek to recreate a patient's disease pathogenesis in the laboratory (2,3). hESCs and hiPSCs are typically grown on a "feeder" cell layer of mitotically inactivated mouse embryonic fibroblasts (mEFs) or on "feeder-free" culture systems composed of a variety of ECM/ serum proteins coated onto tissue culture dishes (4)(5)(6)(7)(8)(9) or synthetic materials (10)(11)(12)(13)(14). Nearly all these systems have been reported to promote hESC/hiPSC self-renewal when the cells are passaged in multicellular clumps and seeded at a suitably high cell density.…”

mentioning

confidence: 99%

“…We found that a 300-μm spot diameter could support most applications of routine cell culture with hESCs/ hiPSCs (Figs. [4][5][6], and similarly sized colonies were routinely observed on standard feeder substrates before they needed to be passaged (Fig. S10C).…”

mentioning

confidence: 99%

Surface-engineered substrates for improved human pluripotent stem cell culture under fully defined conditions

Saha

Mei²,

Reisterer³

et al. 2011

Proc. Natl. Acad. Sci. U.S.A.

134

109

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The current gold standard for the culture of human pluripotent stem cells requires the use of a feeder layer of cells. Here, we develop a spatially defined culture system based on UV/ozone radiation modification of typical cell culture plastics to define a favorable surface environment for human pluripotent stem cell culture. Chemical and geometrical optimization of the surfaces enables control of early cell aggregation from fully dissociated cells, as predicted from a numerical model of cell migration, and results in significant increases in cell growth of undifferentiated cells. These chemically defined xeno-free substrates generate more than three times the number of cells than feeder-containing substrates per surface area. Further, reprogramming and typical gene-targeting protocols can be readily performed on these engineered surfaces. These substrates provide an attractive cell culture platform for the production of clinically relevant factor-free reprogrammed cells from patient tissue samples and facilitate the definition of standardized scale-up friendly methods for disease modeling and cell therapeutic applications.

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“…(A.) Activin A [43,61] and the other members of the TGF␤ ligand family (TGF␤, Nodal) [62] have been shown to support the maintenance of pluripotent state under feeder-cell free conditions. The TGF␤ family members, through the activation of TGFb receptor and subsequently Smad 2, 3, initiate the transcriptional activation of NANOG, and generate a well characterized gene product for pluripotency.…”

Section: Q7mentioning

confidence: 99%

Calcium signaling in human pluripotent stem cells

2016

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a b s t r a c tHuman pluripotent stem cells provide new tools for developmental and pharmacological studies as well as for regenerative medicine applications. Calcium homeostasis and ligand-dependent calcium signaling are key components of major cellular responses, including cell proliferation, differentiation or apoptosis. Interestingly, these phenomena have not been characterized in detail as yet in pluripotent human cell sates. Here we review the methods applicable for studying both short-and long-term calcium responses, focusing on the expression of fluorescent calcium indicator proteins and imaging methods as applied in pluripotent human stem cells. We discuss the potential regulatory pathways involving calcium responses in hPS cells and compare these to the implicated pathways in mouse PS cells. A recent development in the stem cell field is the recognition of so called "naïve" states, resembling the earliest potential forms of stem cells during development, as well as the "fuzzy" stem cells, which may be alternative forms of pluripotent cell types, therefore we also discuss the potential role of calcium homeostasis in these PS cell types.

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Feeder-independent culture of human embryonic stem cells

Cited by 609 publications

References 16 publications

Mass spectrometry identifies and quantifies 74 unique histone H4 isoforms in differentiating human embryonic stem cells

Mass spectrometry identifies and quantifies 74 unique histone H4 isoforms in differentiating human embryonic stem cells

Surface-engineered substrates for improved human pluripotent stem cell culture under fully defined conditions

Calcium signaling in human pluripotent stem cells

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