Establishment of the Embryo-derived Stem (ES) Cell Lines from Mouse Blastocysts: Effects of the Feeder Cell Layer (original) (raw)
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Establishment of an embryonic stem cell line from blastocyst stage mouse embryos
Romanian journal of morphology and embryology = Revue roumaine de morphologie et embryologie, 2011
Embryonic stem cells have the ability to remain undifferentiated and proliferate in vitro while maintaining the potential to differentiate into derivatives of all three embryonic germ layers. The aim of the present study was to establish mouse ES lines from blastocyst stage embryos obtained after CD1/EGFP mice superovulation. We isolated, cultured and determined the characteristics of mouse embryonic stem cells in early passages, which were first described by Evans M and Kaufman M. Therefore, we evaluated the morphological criteria for the approval of ES cells in early expansion stage. Two cell lines were isolated (CDE1 and CDE2) and analyzed. They showed similar characteristics to those reported earlier for blastocyst-derived ES cell lines.
The effect of mouse embryonic fibroblast in direct differentiation of mouse embryonic stem cells
Iranian Journal of Reproductive Medicine
Background: Since embryonic stem (ES) cells have the dual ability to proliferate indefinitely and differentiate into multiple tissue types, ES cells could potentially provide an unlimited cell supply for human transplantation. Objective: In order to study the differentiation of mouse embryonic stem (mES) cells, they were cultured in suspension by using ES media without Leukemia Inhibitory Factor (LIF) to induce spontaneous differentiation. Cellular morphology of differentiated derivatives was then evaluated. Materials and Methods: Undifferentiated mES from our laboratory were cultured in three different settings by using ES media containing 0.1% / 1mM trypsin/EDTA and removing LIF; in the absence of murine embryonic fibroblast (MEF) feeder cells (group 1), in the presence of MEF feeder cells with a density of 0.5×10 5 cells/ml (group 2), and 0.5×10 6 cells/ml (group 3). Five days after the initiation of cell culture, and inducing mES cells to form embryoid bodies (EBs), they were removed from dish by centrifugation, and then they were cultured on collagen coated dishes for 20 days. The dishes were fixed and stained by Wright-Gimsa method at the end of the study period. Results: In group 1, mES cells showed spontaneous differentiation to all derivatives of three germ cells, including: epithelia like, fibroblast like and neron-like cells. In group 2, almost all ES cells were found to be differentiated into granular progenitor cells including hematopoietic cell lineages. In group 3, various morphologies including nerve cell lineages and fibroblast-like cells were detected. Conclusion: Differentiation of mES cells can be a dose response process, depending on the factors that may be released from MEF feeder layer to ES media in a coculture system. Our results indicated that in the presence of low numbers of MEF cells, mES cells can spontaneously differentiate into hematopoeitic cell lineages.
Isolation and differentiation of mouse embryonic stem cells
Iranian Journal of Reproductive Medicine
Background: Recently, embryonic stem (ES) cells have become very important resources in basic medical researches. These cells can differetiate into derivatives of all primary germ layers. Objectives: In order to isolate embryonic stem cells in vitro, the blastocyst were cultured and the morphological aspects, population doubling time, alkalin phosphatse and differentiation properties of the cells were investigated. Materials and Methods: The balstocysts from NMRI mice were cultured for 3 days up to time that inner cell mass (ICM) reach to the outgrowth stage. The cells were disaggregated and trypsinized every 3 days until the appearance of the colonies of ES cells. The colony positive cells were fixed and stained for alkaline phosphatase. The ES cells were cultured in suspension state for 5 days, at the same time Leukaemia Inhibitory Factor (LIF) was removed from media to form embryoid bodies(EBs). The EBs were cultured for 8 -20 days on collagen coated dish to induce the spontaneouse differentiation. Results: During the 6-9 days after the disaggregation of ICM in the expansion stage , the colony of ES cells appeared as a flat monolayer mass with strike boundaries and nondistinguish cytoplasm including a few nuclei. In colony formation stage, the morphology changed from flat monolayer to round multilayer with strike define boundaries. Undifferentiated cells were seen as intensely small cells attached together compactly with high nucleus/cytoplasm (N/C) ratio. The cells of colonies tend to differetiate by separation from each other and became larger and diffused on substrate by attaching to dish. The positive alkaline phosphatase cells were seen in typical morphology of ES colonies. The EBs cells were seen in culture after 5 days in suspension and began to spontaneously differentiate into various types of cells such as nerve and hematopoitic lineages. Conclusion: Despite strike morphology of ES colonies, it is difficult to distinguish the differentiated from undifferentiated cell colonies in the colony formation stage. New ES cells are capable to give rise into EBs and are susceptible of spontaneously differentiation in various type of cells.
Theriogenology, 2010
The domestic cat is a focal mammalian species that is used as a model for developing assisted reproductive technologies for preserving endangered cats and for studying human diseases. The generation of stable characterized cat embryonic stem cells (ESC) lines to use as donor nuclei may help to improve the efficiency of interspecies somatic cell nuclear transfer for preserving endangered cats and allow the creation of knockout cell lines to generate knockout cats for studying function of specific genes related to human diseases. It will also enable the possibility of producing gametes in vitro from ESC of endangered cats. In the present study, we report the generation of cat embryonic stem-like (cESL) cells from blastocysts derived entirely in vitro. We generated 32 cESL cell lines from 331 in vitro derived blastocysts from which inner cell masses were isolated by immunosurgery or by a mechanical method. Inhibition of cat dermal fibroblast (CDF) proliferation after exposure to mitomycin-C was both dose and time dependent, where doses of 30 to 40 g/mL for 5 h were most efficient. These dosages were higher than that required to inhibit cell proliferation of mouse fetal fibroblasts (MFF; 10 g/mL for 2.5 h). Mitomycin-C did not significantly increase necrosis of cells from either species, and had an anti-proliferative effect at concentrations below cytotoxicity. A clear species-specific relationship between feeder layers and derivation of cESL cell lines was observed, where higher numbers of cESL cell lines were generated on homologous cat feeder layers (n ϭ 26) than from those derived on heterologous mouse feeder layers (n ϭ 6). Three cESL cell lines generated from immunosurgery and cultured on CDF maintained self-renewal and were morphologically undifferentiated for nine and twelve passages (69 -102 days). These lines showed a tightly packed dome shaped morphology, exhibited alkaline phosphatase activity and immuno-expression of the pluripotent marker OCT-4 and surface marker SSEA-1. Primary colonies at P0 to P3 and cat blastocysts expressed transcription factors OCT-4, NANOG and SOX-2 and the proto-oncogene C-MYC. However, expression was at levels significantly lower than in vitro produced blastocysts. During culture, cESL colonies spontaneously differentiated into fibroblasts, cardiomyocytes, and embryoid bodies. Development of techniques to prevent differentiation of cESL cells will be essential for maintaining defined cell lines.
Derivation of Stem Cell Lines from Mouse Preimplantation Embryos
Journal of Visualized Experiments
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.
Biology of Reproduction, 2009
Recently, F0 embryonic stem (ES) cell mice have been produced by injection of ES cells into eight-cell embryos using either laser-or piezo-assisted injection systems. To simplify the injection procedure, we have optimized the conventional blastocyst injection method, free of laser-or piezo-assisted micromanipulation systems, to produce F0 ES cell pups. To increase the efficiency of producing mice from ES cell injection into eight-cell and blastocyst stage embryos, we have tested: 1) the effect of activating ES cell before injection, 2) the effect of in vitro culture in medium optimized for the survival of both ES cells and embryos, and 3) the effect of transferring the micromanipulated embryos into the oviduct versus into the uterus of CD1 foster mice. Two B6D2 hybrid ES cell lines were used for injection in a multifactorial analysis to evaluate the efficiency of producing live chimeric and F0 ES cell mice. Our results demonstrate that the activation of ES cells and the appropriate culture conditions are crucial parameters influencing the generation of F0 ES cell offspring. Transfer of blastocysts injected with ES cells into the oviduct of 0.5-day postcoitum pseudopregnant females increased the number of live animals with higher chimera proportion. Under these conditions, injections into eight-cell embryos produce a high number of F0 ES mice, and the conventional blastocyst injection method produces a lower number of F0 ES cell pups; however, the efficiency of production of chimeric mice with germline transmission was high. We have developed an economical and efficient technique for producing fully ES cell-derived F0 mice with full germline transmission that can be applied in many laboratories without the use of piezo or laser instruments. cell activation, culture media, embryo transfer, embryonic stem cell, mouse