Defined, Feeder‐Independent Medium for Human Embryonic Stem Cell Culture

Ludwig, Tenneille E.; Thomson, James A.

doi:10.1002/9780470151808.sc01c02s2

Cited by 68 publications

(65 citation statements)

References 21 publications

(15 reference statements)

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“…H1 (WA01) cells, H9 (WA09) cells, and iPSC cells were routinely cultured on a Matrigel substratum on mTeSR1 medium (STEMCELL Technologies) (32). To initiate differentiation toward trophoblast, colonies were subcultured; on the following day, the culture medium was changed from the defined mTeSR1 medium to the MEFconditioned DMEM/F12/KOSR medium (MEF-CM), which contains lower concentrations of recombinant FGF2 (4 ng/mL) than mTeSR1 medium and no supplementary TGF-β (Fig.…”

Section: Resultsmentioning

confidence: 99%

Heightened potency of human pluripotent stem cell lines created by transient BMP4 exposure

Yang

Adachi

Sheridan

et al. 2015

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

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Human pluripotent stem cells (PSCs) show epiblast-type pluripotency that is maintained with ACTIVIN/FGF2 signaling. Here, we report the acquisition of a unique stem cell phenotype by both human ES cells (hESCs) and induced pluripotent stem cells (iPSCs) in response to transient (24-36 h) exposure to bone morphogenetic protein 4 (BMP4) plus inhibitors of ACTIVIN signaling (A83-01) and FGF2 (PD173074), followed by trypsin dissociation and recovery of colonies capable of growing on a gelatin substratum in standard medium for human PSCs at low but not high FGF2 concentrations. The self-renewing cell lines stain weakly for CDX2 and strongly for NANOG, can be propagated clonally on either Matrigel or gelatin, and are morphologically distinct from human PSC progenitors on either substratum but still meet standard in vitro criteria for pluripotency. They form well-differentiated teratomas in immune-compromised mice that secrete human chorionic gonadotropin (hCG) into the host mouse and include small areas of trophoblast-like cells. The cells have a distinct transcriptome profile from the human PSCs from which they were derived (including higher expression of NANOG, LEFTY1, and LEFTY2). In nonconditioned medium lacking FGF2, the colonies spontaneously differentiated along multiple lineages, including trophoblast. They responded to PD173074 in the absence of both FGF2 and BMP4 by conversion to trophoblast, and especially syncytiotrophoblast, whereas an A83-01/PD173074 combination favored increased expression of HLA-G, a marker of extravillous trophoblast. Together, these data suggest that the cell lines exhibit totipotent potential and that BMP4 can prime human PSCs to a self-renewing alternative state permissive for trophoblast development. The results may have implications for regulation of lineage decisions in the early embryo.biological sciences | developmental biology | pluripotent stem cells | totipotent | trophoblast

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Section: Resultsmentioning

confidence: 99%

Heightened potency of human pluripotent stem cell lines created by transient BMP4 exposure

Yang

Adachi

Sheridan

et al. 2015

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

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“…To develop a feeder cell-free method to produce MSCs from ESCs or iPSCs, we first used a widely used commercially available defined medium, mTeSR1 (StemCell Technologies), which, in combination with the cell attachment matrix Matrigel (BD Biosciences, San Diego, http://www.bdbiosciences.com), maintains pluripotency of ESCs/iPSCs without the need for feeder cells or additional bFGF [27,30].…”

Section: Sb431542 Inhibitor Differentiation Methodsmentioning

confidence: 99%

Small Molecule Mesengenic Induction of Human Induced Pluripotent Stem Cells to Generate Mesenchymal Stem/Stromal Cells

Chen

Pelekanos

Ellis

et al. 2012

Stem Cells Translational Medicine

179

180

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The translational potential of mesenchymal stem/stromal cells (MSCs) is limited by their rarity in somatic organs, heterogeneity, and need for harvest by invasive procedures. Induced pluripotent stem cells (iPSCs) could be an advantageous source of MSCs, but attempts to derive MSCs from pluripotent cells have required cumbersome or untranslatable techniques, such as coculture, physical manipulation, sorting, or viral transduction. We devised a single-step method to direct mesengenic differentiation of human embryonic stem cells (ESCs) and iPSCs using a small molecule inhibitor. First, epithelial-like monolayer cells were generated by culturing ESCs/iPSCs in serum-free medium containing the transforming growth factor-␤ pathway inhibitor SB431542. After 10 days, iPSCs showed upregulation of mesodermal genes (MSX2, NCAM, HOXA2) and downregulation of pluripotency genes (OCT4, LEFTY1/2). Differentiation was then completed by transferring cells into conventional MSC medium. The resultant development of MSC-like morphology was associated with increased expression of genes, reflecting epithelial-to-mesenchymal transition. Both ESC-and iPSC-derived MSCs exhibited a typical MSC immunophenotype, expressed high levels of vimentin and N-cadherin, and lacked expression of pluripotency markers at the protein level. Robust osteogenic and chondrogenic differentiation was induced in vitro in ES-MSCs and iPS-MSCs, whereas adipogenic differentiation was limited, as reported for primitive fetal MSCs and ES-MSCs derived by other methods. We conclude that treatment with SB431542 in two-dimensional cultures followed by culture-induced epithelial-to-mesenchymal transition leads to rapid and uniform MSC conversion of human pluripotent cells without the need for embryoid body formation or feeder cell coculture, providing a robust, clinically applicable, and efficient system for generating MSCs from human iPSCs. STEM CELLS TRANSLATIONAL MEDICINE 2012;1:83-95

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“…2A) under feeder-free conditions in the defined medium mTeSR (Ludwig and Thomson, 2007;Puelles and Rubenstein, 2003). When hPSCs were dissociated and plated into gfCDM, they died within several days, even in the presence of the Rho kinase (ROCK) inhibitor Y27632 (Kamiya et al, 2011;Watanabe et al, 2007;Wilson and Rubenstein, 2000).…”

Section: Self-patterning Of Hpscs Into Hypothalamic Progenitorsmentioning

confidence: 99%

“…The self-patterning approach permits organ-like tissue development in vitro via the cell-cell and paracrine interactions that pattern tissues in vivo (Ludwig and Thomson, 2007;Sasai et al, 2012). Self-patterning is a rational choice for hypothalamic differentiation as pluripotent stem cells are predisposed to generate anterior neural structures such as the hypothalamus (Puelles and Rubenstein, 2003;Watanabe et al, 2007) by default (Kamiya et al, 2011;Wilson and Rubenstein, 2000) (Fig.…”

Section: Introductionmentioning

confidence: 99%

Generation of neuropeptidergic hypothalamic neurons from human pluripotent stem cells

et al. 2015

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Hypothalamic neurons orchestrate many essential physiological and behavioral processes via secreted neuropeptides, and are relevant to human diseases such as obesity, narcolepsy and infertility. We report the differentiation of human pluripotent stem cells into many of the major types of neuropeptidergic hypothalamic neurons, including those producing pro-opiolemelanocortin, agoutirelated peptide, hypocretin/orexin, melanin-concentrating hormone, oxytocin, arginine vasopressin, corticotropin-releasing hormone (CRH) or thyrotropin-releasing hormone. Hypothalamic neurons can be generated using a 'self-patterning' strategy that yields a broad array of cell types, or via a more reproducible directed differentiation approach. Stem cell-derived human hypothalamic neurons share characteristic morphological properties and gene expression patterns with their counterparts in vivo, and are able to integrate into the mouse brain. These neurons could form the basis of cellular models, chemical screens or cellular therapies to study and treat common human diseases.

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Defined, Feeder‐Independent Medium for Human Embryonic Stem Cell Culture

Cited by 68 publications

References 21 publications

Heightened potency of human pluripotent stem cell lines created by transient BMP4 exposure

Heightened potency of human pluripotent stem cell lines created by transient BMP4 exposure

Small Molecule Mesengenic Induction of Human Induced Pluripotent Stem Cells to Generate Mesenchymal Stem/Stromal Cells

Generation of neuropeptidergic hypothalamic neurons from human pluripotent stem cells

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