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Isolation and characterization of node/notochord-like cells from mouse embryonic stem cells.

ABSTRACT: The homeobox gene Noto is expressed in the node and its derivative the notochord. Here we use a targeted Noto-GFP reporter to isolate and characterize node/notochord-like cells derived from mouse embryonic stem cells. We find very few Noto-expressing cells after spontaneous differentiation. However, the number of Noto-expressing cells was increased when using Activin A to induce a Foxa2- and Brachyury-expressing progenitor population, whose further differentiation into Noto-expressing cells was improved by simultaneous inhibition of BMP, Wnt, and retinoic acid signaling. Noto-GFP(+) cells expressed the node/notochord markers Noto, Foxa2, Shh, Noggin, Chordin, Foxj1, and Brachyury; showed a vacuolarization characteristic of notochord cells; and can integrate into midline structures when grafted into Hensen's node of gastrulating chicken embryos. The ability to generate node/notochord-like cells in vitro will aid the biochemical characterization of these developmentally important structures.

SUBMITTER: Winzi MK

PROVIDER: S-EPMC3928718 | biostudies-literature | 2011 Nov

REPOSITORIES: biostudies-literature

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Isolation and characterization of node/notochord-like cells from mouse embryonic stem cells.

Winzi Maria K MK Hyttel Poul P Dale Jacqueline Kim JK Serup Palle P

Stem cells and development 20110406 11

The homeobox gene Noto is expressed in the node and its derivative the notochord. Here we use a targeted Noto-GFP reporter to isolate and characterize node/notochord-like cells derived from mouse embryonic stem cells. We find very few Noto-expressing cells after spontaneous differentiation. However, the number of Noto-expressing cells was increased when using Activin A to induce a Foxa2- and Brachyury-expressing progenitor population, whose further differentiation into Noto-expressing cells was ...[more]

PMID: 21351873

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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.

Dataset Information

Isolation and characterization of node/notochord-like cells from mouse embryonic stem cells.

Publications

Isolation and characterization of node/notochord-like cells from mouse embryonic stem cells.

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