Conditions and Techniques for Mouse Embryonic Stem Cell Derivation and Culture

Lee, Kun-Hsiung

doi:10.5772/55105

Cited by 5 publications

(7 citation statements)

References 142 publications

(274 reference statements)

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“…According to their ability to yield mESC, mouse strains can be classified into permissive strains, such as 129S2 and C57BL, and non-permissive strains, like CBA, NOD, and FVB [19,20]. Despite the wide use of hybrid strains like B6D2F1 and B6CBAF1 in procedures such as somatic cell nuclear transfer [21][22][23], these strains have seldom been used for mESC derivation [24][25][26] and their permissiveness for mESC derivation is yet unclear.…”

Section: Introductionmentioning

confidence: 99%

Single blastomeres as a source of mouse embryonic stem cells: effect of genetic background, medium supplements, and signaling modulators on derivation efficiency

Vila-Cejudo

Massafret

Santaló

et al. 2018

J Assist Reprod Genet

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Purpose To assess the role of the genetic background, the culture medium supplements, and the presence of modulators of signaling pathways on mouse embryonic stem cell derivation from single blastomeres from 8-cell embryos. Methods Mice from permissive and non-permissive genetic backgrounds, different culture media supplements, knockout serum replacement (KSR) and N2B27, and the presence or absence of 2i treatment were used to derive mouse embryonic stem cells (mESC) from single blastomeres isolated from 8-cell embryos and from control embryos at the blastocyst stage. After the sixth passage, the putative mESC were analyzed by immunofluorescence to assess their pluripotency and, after in vitro differentiation induction, their ability to differentiate into derivatives of the three primary germ layers. Selected mESC lines derived from single blastomeres in the most efficient culture conditions were further characterized to validate their stemness. Results In control embryos, high mESC derivation efficiencies (70-96.9%) were obtained from permissive backgrounds or when embryos were cultured in medium complemented with 2i regardless of their genetic background. By contrast, only blastomeres isolated from embryos from permissive background cultured in KSR-containing medium complemented with 2i were moderately successful in the derivation of mESC lines (22.9-24.5%). Moreover, we report for the first time that B6CBAF2 embryos behave as permissive in terms of mESC derivation. Conclusions Single blastomeres have higher requirements than whole blastocysts for pluripotency maintenance and mESC derivation. The need for 2i suggests that modulation of signaling pathways to recreate a commitment towards inner cell mass could be essential to efficiently derive mESC from single blastomeres.

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Section: Introductionmentioning

confidence: 99%

Single blastomeres as a source of mouse embryonic stem cells: effect of genetic background, medium supplements, and signaling modulators on derivation efficiency

Vila-Cejudo

Massafret

Santaló

et al. 2018

J Assist Reprod Genet

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“…Another pivotal factor is the source of feeder cells and their culture. Several studies have demonstrated that HFF, Mouse Embryonic Fibroblasts (MEF) and mouse STO fibroblasts are equally able to support ESC derivation and culture 15 . However, HFF are up to two times more durable than MEF and STO in culture 16 , allowing more time for the mESC to grow.…”

Section: Disclosuresmentioning

confidence: 99%

Derivation of Stem Cell Lines from Mouse Preimplantation Embryos

Vila-Cejudo

Ibáñez

Santaló

2017

JoVE

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Mouse embryonic stem cell (mESC) derivation is the process by which pluripotent cell lines are established from preimplantation embryos. These lines retain the ability to either self-renew or differentiate under specific conditions. Due to these properties, mESC are a useful tool in regenerative medicine, disease modeling, and tissue engineering studies. This article describes a simple protocol to obtain mESC lines with high derivation efficiencies (60-80%) by culturing blastocysts from permissive mouse strains on feeder cells in defined medium supplemented with leukemia inhibitory factor. The protocol can also be applied to efficiently derive mESC lines from non-permissive mouse strains, by the simple addition of a cocktail of two small-molecule inhibitors to the derivation medium (2i medium). Detailed procedures on the preparation and culture of feeder cells, collection and culture of mouse embryos, and derivation and culture of mESC lines are provided. This protocol does not require specialized equipment and can be carried out in any laboratory with basic mammalian cell culture expertise.

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“…Without external factors in the medium, neurosphere behaviour and fate are mediated by the properties of the adherent substrate 89 . FBS is currently the most universal component in the culture of numerous primary and immortalized cell types 90,91 . In cultures of hESCs, it promotes apoptosis of undifferentiated cells and spontaneous differentiation into cardiomyocytes 92 .…”

Section: 3a -Fbsmentioning

confidence: 99%

“…It increases proliferation in a concentration dependent manner, downregulating the pro-apoptic tumour suppressor gene p53 91 . Its constituents are not fully defined, but it is known to contain growth factors, hormones, iron transporters, vitamins, amino acids, and carbohydrates 93 , as well as components inducing stem cell differentiation and replication, as well as those affecting plating efficiency 88,90 . EGF, PDGF, IGF and insulin are FBS components likely involved in functional proliferation, iPSC reprogramming, and promotion of differentiation 91 .…”

Section: 3a -Fbsmentioning

confidence: 99%

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Electrophysiological Profile of Differentiating Human Central Canal Ependymal Stem-Progenitor Cells

Malone¹

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Following spinal cord injury (SCI), current treatments look to halt the spread of tissue damage to minimize pain, with limited effectiveness. Unfortunately, there is no widely used approach for promoting re-growth across the lesion site, a necessity for functional recovery. To that end, stem cell transplant and niche manipulation therapies are a promising tool for repairing damage from SCI and other neurodegenerative conditions. As most stem cell studies are based exclusively on animal models, we aimed to address several gaps in understanding that are fundamental for potential translation to humans. Since immunocytochemistry alone cannot adequately determine whether stem cells have differentiated into mature neurons, we used patchclamp electrophysiology to assess the passive and active electrical properties of stem cells from the central canal of the spinal cord throughout the in vitro differentiation process. Rat ependymal-stem progenitor cells (epSPCs) differentiate towards a majority astrocytic fate under intrinsic differentiation conditions in vitro. Human epSPCs, on the other hand, differentiate towards a majority neuronal fate. Electrophysiological recordings of these cells in intrinsic FBScontaining differentiation media and under BDNF-, GDNF-and RA-guided differentiation show no action potential firing from two to ten weeks in vitro. Passive membrane properties fail to reach that of typical mature neurons within this time frame. Surprisingly, the vast majority of cells showed voltage-dependent spontaneous synaptic currents with reversal near 0 mV, outward rectification, and decay kinetics that are consistent with excitatory glutamatergic responses. Further studies investigating the necessary timeline and the most effective differentiation media required for development of active membrane properties are needed, as is identification of the receptor subtypes responsible for the observed synaptic currents. This will help inform future transplant and stem cell niche manipulation strategies for the treatment of human neurological disorders.iii

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Conditions and Techniques for Mouse Embryonic Stem Cell Derivation and Culture

Cited by 5 publications

References 142 publications

Single blastomeres as a source of mouse embryonic stem cells: effect of genetic background, medium supplements, and signaling modulators on derivation efficiency

Single blastomeres as a source of mouse embryonic stem cells: effect of genetic background, medium supplements, and signaling modulators on derivation efficiency

Derivation of Stem Cell Lines from Mouse Preimplantation Embryos

Electrophysiological Profile of Differentiating Human Central Canal Ependymal Stem-Progenitor Cells

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