Isolation and differentiation of mouse embryonic stem cells (original) (raw)
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Biotechnology and Bioengineering, 2002
Embryonic stem (ES) cells have tremendous potential as a cell source for cell-based therapies. Realization of that potential will depend on our ability to understand and manipulate the factors that in¯uence cell fate decisions and to develop scalable methods of cell production. We compared four standard ES cell differentiation culture systems by measuring aspects of embryoid body (EB) formation ef®ciency and cell proliferation, and by tracking development of a speci®c differentiated tissue typeÐbloodÐusing functional (colony-forming cell) and phenotypic (Flk-1 and CD34 expression) assays. We report that individual murine ES cells form EBs with an ef®ciency of 42 9%, but this value is rarely obtained because of EB aggregationÐa process whereby two or more individual ES cells or EBs fuse to form a single, larger cell aggregate. Regardless of whether EBs were generated from a single ES cell in methylcellulose or liquid suspension culture, or aggregates of ES cells in hanging drop culture, they grew to a similar maximum cell number of 28,000 9,000 cells per EB. Among the three methods for EB generation in suspension culture there were no differences in the kinetics or frequency of hematopoietic development. Thus, initiating EBs with a single ES cell and preventing EB aggregation should allow for maximum yield of differentiated cells in the EB system. EB differentiation cultures were also compared to attached differentiation culture using the same outputs. Attached colonies were not similarly limited in cell number; however, hematopoietic development in attached culture was impaired. The percentage of early Flk-1 and CD34 expressing cells was dramatically lower than in EBs cultured in suspension, whereas hematopoietic colony formation was almost completely inhibited. These results provide a foundation for development of ef®cient, scalable bioprocesses for ES cell differentiation, and inform novel methods for the production of hematopoietic tissues.
Development of Defined Medium for Mouse, Monkey and Human ES Cell Culture
wwwsoc.nii.ac.jp
Embryonic stem (ES) cells have great potential for tissue engineering application. However, major limitations in the use of the human ES cells for a variety of biotechnological applications are the current requirement for the use of feeder cells, and relatively poor understanding of their responses to growth factors to manipulate cell differentiation. Studies using mouse or monkey ES cells are useful for understanding of ES cell characteristic. Generally, mouse ES cells are maintained on mouse embryonic fibroblast (MEF) cells in a medium supplemented with serum, and human ES cells are also maintained on MEF in a medium supplemented with knock-out serum-replacement or on matrigel with an MEF-conditioned medium (CM). These culture conditions hamper the cell biological analysis of ES cells due to the presence of various unknown products. We have established a defined serum-free ESF7 medium for mouse ES cells without feeders or BMP4. In this medium, mouse ES cells express Oct-3/4, Nanog and Sox2. Building on this experience, we have developed a defined serum-free medium for the culture of human ES cells. We are now testing this culture medium for monkey ES cells. These culture conditions may be useful for understanding of ES cell responses to differentiation factors.
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.
Blood island formation in attached cultures of murine embryonic stem cells
Developmental Dynamics, 1996
Differentiation of murine embryonic stem cells in suspension culture results in the formation of cystic embryoid bodies that develop blood islands. In this study pre-cystic embryoid bodies were attached to a substratum, and the program of differentiation was monitored. The attached ES cell cultures formed blood islands on a cell layer that migrated out from the center of attachment and beneath a mesothelial-like cell layer. Morphological and in situ marker analysis showed benzidine-positive hematopoietic cells surrounded by vascular endothelial cells that expressed PECAM and took up DiI-Ac-LDL. Waves of morphological differentiation were evident, suggesting a graded response to differentiation signals. Electron microscopy of the blood islands showed that they were similar to blood islands of cystic embryoid bodies and mouse yolk sacs, and cell-cell junctions were evident among the blood island cells. RNA expression analysis was consistent with the presence of hematopoietic precursor cells of several lineages and a primitive vascular endothelium in the cultures. Thus a program of vascular and hematopoietic development can be elaborated in attached ES cell cultures, and these blood islands are accessible to experimental manipulation. 0 1996 Wiley-Liss, Inc.
Development, Growth and Differentiation, 1987
The ES cell lines are embryo-derived stem cell lines directly isolated from the inner cell mass of mouse blastocysts using feeder cell layer. We have established a number of ES cell lines from 129 or C57BL/6 strain mice by using the feeder layer of the STO cells (from ATCC) or the primary embryonic fibroblasts, which was obtained by trypsinizing the 16-day-old BALB/c mouse fetus. The ES cell lines established on the STO feeder layer showed differentiation into various tissues in solid tumors when injected into syngenic mice. The ES cell lines established on the primary fibroblasts exhibited differentiation into larger variety of tissues in solid tumors. Karyotype was almost diploid and majority of the cells kept normal set of chromosomes in G-banding. We conclude that the primary fibroblasts are better feeder layer than the STO cells for establishment and maintenance of the ES cell lines. Karyotype was, however, nearly tetraploid.
Stem Cells, 2008
Human embryonic stem cells (hESCs) can be maintained in vitro as immortal pluripotent cells but remain responsive to many differentiation-inducing signals. Investigation of the initial critical events involved in differentiation induction would be greatly facilitated if a specific, robust, and quantitative assay for pluripotent hESCs with self-renewal potential were available. Here we describe the results of a series of experiments to determine whether the formation of adherent alkaline phosphatase-positive (AP+) colonies under conditions optimized for propagating undifferentiated hESCs would meet this need. The findings can be summarized as follows. (a) Most colonies obtained under these conditions consist of ≥30 AP+ cells that coexpress OCT4, NANOG, SSEA3, SSEA4, TRA-1-60, and TRA-1-81. (b) Most such colonies are derived from SSEA3+ cells. (c) Primary colonies contain cells that produce secondary colonies of the same composition, including cells that initiate multilineage differen...
Journal of Veterinary Science
Background: Conditioned medium is the medium obtained from certain cultured cells and contained secretome from the cells. The secretome, which can be in the form of growth factors, cytokines, exosomes, or other proteins secreted by the cells, can induce the differentiation of cells that still have pluripotent or multipotent properties. Objectives: This study examined the effects of conditioned medium derived from E17 rat brain cells on cells with pluripotent properties. Methods: The conditioned medium used in this study originated from E17 rat brain cells. The CM was used to induce the differentiation of primary colonies of mice blastocysts. Primary colonies were stained with alkaline phosphatase to analyze the pluripotency. The morphological changes in the colonies were examined, and the colonies were stained with GFAP and Neu-N markers on days two and seven after adding the conditioned medium. Results: The conditioned medium could differentiate the primary colony, beginning with the formation of embryoid-body-like structure; round GFAP positive cells were identified. Finally, neuron-like cells testing positive for Neu-N were observed on the seventh day after adding the conditioned medium. Conclusions: Conditioned medium from different species, in this case, E17 rat brain cells, induced and promoted the differentiation of the primary colony from mice blastocysts into neuron-like cells. The addition of CM mediated neurite growth in the differentiation process.