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Genesis of embryonic stem cells.

ABSTRACT: Embryonic stem (ES) cells are permanent pluripotent stem cell lines established from pre-implantation mouse embryos. There is currently great interest in the potential therapeutic applications of analogous cells derived from human embryos. The isolation of ES cells is commonly presented as a straightforward transfer of cells in the early embryo into culture. In reality, however, continuous expansion of pluripotent cells does not occur in vivo, and in vitro is the exception rather than the norm. Both genetic and epigenetic factors influence the ability to derive ES cells. We have tracked the expression of a key marker and determinant of pluripotency, the transcription factor Oct-4, in primary cultures of mouse epiblasts and used this to assay the effect of experimental manipulations on the maintenance of a pluripotent cell compartment. We find that expression of Oct-4 is often lost prior to overt cytodifferentiation of the epiblast. The rate and extent of Oct-4 extinction varies with genetic background. We report that treatment with the MAP kinase/ERK kinase inhibitor PD98059, which suppresses activation of the mitogen-activated protein kinases Erk1 and Erk2, results in increased persistence of Oct-4-expressing cells. Oct-4 expression is also relatively sustained in cultures of diapause embryos and of isolated inner cell masses. Combination of all three conditions allowed the derivation of germline-competent ES cells from the normally refractory CBA mouse strain. These findings suggest that the genesis of an ES cell is a relatively complex process requiring epigenetic modulation of key gene expression over a brief time-window. Procedures that extend this time-window and/or directly regulate the critical genes should increase the efficiency of ES cell derivation.

SUBMITTER: Buehr M

PROVIDER: S-EPMC1693233 | biostudies-other | 2003 Aug

REPOSITORIES: biostudies-other

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Genesis of embryonic stem cells.

Buehr Mia M Smith Austin A

Philosophical transactions of the Royal Society of London. Series B, Biological sciences 20030801 1436

Embryonic stem (ES) cells are permanent pluripotent stem cell lines established from pre-implantation mouse embryos. There is currently great interest in the potential therapeutic applications of analogous cells derived from human embryos. The isolation of ES cells is commonly presented as a straightforward transfer of cells in the early embryo into culture. In reality, however, continuous expansion of pluripotent cells does not occur in vivo, and in vitro is the exception rather than the norm. ...[more]

PMID: 14511487

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Project description:BackgroundEmbryonic Sertoli cells (eSCs) play an important role in sex determination and in male gonad development which makes them a very useful cell type for therapeutic applications. However, the deriving mechanism of Sertoli cells has been unclear and challenging to create a large number of quality eSCs. Therefore, this study aimed to create the eSCs induced from mouse embryonic stem (mES) cells by regulating defined factors and to explore the relevant regulatory mechanism.MethodsSix inducing factors, Sry, Sox9, SF1, WT1, GATA4, and Dmrt1, were respectively transduced into mES cells by lentiviral infection according to the experimental design. The test groups were identified by development stage-specific markers, AMH, Emx2, SF1, and FasL, using flow cytometry. Induced eSCs were determined by FasL and AMH biomarkers under immunofluorescence, immunocytochemistry, and flow cytometry. Moreover, the pluripotency markers, gonad development-related markers, epithelial markers and mesenchymal markers in test groups were transcriptionally determined by qPCR.ResultsIn this study, the co-overexpression of all the six factors effectively produced a large population of eSCs from mES cells in 35 days of culturing. These eSCs were capable of forming tubular-like and ring-like structures with functional performance. The results of flow cytometry indicated that the upregulation of GATA4 and WT1 contributed to the growth of somatic cells in the coelomic epithelium regarded as the main progenitor cells of eSCs. Whereas, SF1 facilitated the development of eSC precursor cells, and Sry and Sox9 promoted the determination of male development. Moreover, the overexpression of Dmrt1 was essential for the maintenance of eSCs and some of their specific surface biomarkers such as FasL. The cellular morphology, biomarker identification, and transcriptomic analysis aided in exploring the regulatory mechanism of deriving eSCs from mES cells.ConclusionConclusively, we have elucidated a differentiation roadmap of eSCs derived from mES cells with a relevant regulatory mechanism. Through co-overexpression of all these six factors, a large population of eSCs was successfully induced occupying 24% of the whole cell population (1 × 105 cells/cm2). By adopting this approach, a mass of embryonic Sertoli cells can be generated for the purpose of co-culture technique, organ transplantation, gonadal developmental and sex determination researches.

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Genesis of embryonic stem cells.

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Genesis of embryonic stem cells.

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