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Three-step method for proliferation and differentiation of human embryonic stem cell (hESC)-derived male germ cells.

ABSTRACT: The low efficiency of differentiation into male germ cell (GC)-like cells and haploid germ cells from human embryonic stem cells (hESCs) reflects the culture method employed in the two-dimensional (2D)-microenvironment. In this study, we applied a three-step media and calcium alginate-based 3D-culture system for enhancing the differentiation of hESCs into male germ stem cell (GSC)-like cells and haploid germ cells. In the first step, embryoid bodies (EBs) were derived from hESCs cultured in EB medium for 3 days and re-cultured for 4 additional days in EB medium with BMP4 and RA to specify GSC-like cells. In the second step, the resultant cells were cultured in GC-proliferation medium for 7 days. The GSC-like cells were then propagated after selection using GFR-?1 and were further cultured in GC-proliferation medium for 3 weeks. In the final step, a 3D-co-culture system using calcium alginate encapsulation and testicular somatic cells was applied to induce differentiation into haploid germ cells, and a culture containing approximately 3% male haploid germ cells was obtained after 2 weeks of culture. These results demonstrated that this culture system could be used to efficiently induce GSC-like cells in an EB population and to promote the differentiation of ESCs into haploid male germ cells.

SUBMITTER: Lim JJ

PROVIDER: S-EPMC3972183 | biostudies-literature | 2014

REPOSITORIES: biostudies-literature

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Three-step method for proliferation and differentiation of human embryonic stem cell (hESC)-derived male germ cells.

Lim Jung Jin JJ Shim Myung Sun MS Lee Jeoung Eun JE Lee Dong Ryul DR

PloS one 20140401 4

The low efficiency of differentiation into male germ cell (GC)-like cells and haploid germ cells from human embryonic stem cells (hESCs) reflects the culture method employed in the two-dimensional (2D)-microenvironment. In this study, we applied a three-step media and calcium alginate-based 3D-culture system for enhancing the differentiation of hESCs into male germ stem cell (GSC)-like cells and haploid germ cells. In the first step, embryoid bodies (EBs) were derived from hESCs cultured in EB m ...[more]

PMID: 24690677

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Project description:The mechanisms by which human embryonic stem cells (hESC) differentiate to endodermal lineage have not been extensively studied. Mathematical models can aid in the identification of mechanistic information. In this work we use a population-based modeling approach to understand the mechanism of endoderm induction in hESC, performed experimentally with exposure to Activin A and Activin A supplemented with growth factors (basic fibroblast growth factor (FGF2) and bone morphogenetic protein 4 (BMP4)). The differentiating cell population is analyzed daily for cellular growth, cell death, and expression of the endoderm proteins Sox17 and CXCR4. The stochastic model starts with a population of undifferentiated cells, wherefrom it evolves in time by assigning each cell a propensity to proliferate, die and differentiate using certain user defined rules. Twelve alternate mechanisms which might describe the observed dynamics were simulated, and an ensemble parameter estimation was performed on each mechanism. A comparison of the quality of agreement of experimental data with simulations for several competing mechanisms led to the identification of one which adequately describes the observed dynamics under both induction conditions. The results indicate that hESC commitment to endoderm occurs through an intermediate mesendoderm germ layer which further differentiates into mesoderm and endoderm, and that during induction proliferation of the endoderm germ layer is promoted. Furthermore, our model suggests that CXCR4 is expressed in mesendoderm and endoderm, but is not expressed in mesoderm. Comparison between the two induction conditions indicates that supplementing FGF2 and BMP4 to Activin A enhances the kinetics of differentiation than Activin A alone. This mechanistic information can aid in the derivation of functional, mature cells from their progenitors. While applied to initial endoderm commitment of hESC, the model is general enough to be applicable either to a system of adult stem cells or later stages of ESC differentiation.

Dataset Information

Three-step method for proliferation and differentiation of human embryonic stem cell (hESC)-derived male germ cells.

Publications

Three-step method for proliferation and differentiation of human embryonic stem cell (hESC)-derived male germ cells.

Similar Datasets

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