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

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-cultu...

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Bibliographic Details
Published in:PloS one 2014-04, Vol.9 (4), p.e90454
Main Authors: Lim, Jung Jin, Shim, Myung Sun, Lee, Jeoung Eun, Lee, Dong Ryul
Format: Article
Language:English
Subjects:
Alginic acid
Amino acids
Analysis
Animals
Biology and Life Sciences
Biomarkers - metabolism
Biomedical materials
Bone morphogenetic protein 4
Calcium
Calcium alginate
Cell culture
Cell Culture Techniques - methods
Cell differentiation
Cell Differentiation - drug effects
Cell Lineage - drug effects
Cell proliferation
Cell Proliferation - drug effects
Cells, Cultured
DEAD-box RNA Helicases - metabolism
Differentiation
Embryo cells
Embryoid Bodies - cytology
Embryonic stem cells
Fertility
Fibroblast Growth Factor 2 - pharmacology
Fibroblasts
Gene expression
Gene Expression Regulation - drug effects
Germ cells
Glial Cell Line-Derived Neurotrophic Factor - pharmacology
Growth factors
Human Embryonic Stem Cells - cytology
Human Embryonic Stem Cells - drug effects
Human Embryonic Stem Cells - metabolism
Humans
Leukemia Inhibitory Factor - pharmacology
Male
Media (culture)
Medicine
Methods
Mice
Phosphatase
Physiological aspects
Proteins
Research and Analysis Methods
Rodents
Somatic cells
Sperm
Spermatozoa - cytology
Spermatozoa - drug effects
Stem cells
Testis - cytology
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Summary: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-α1 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.
ISSN:1932-6203
1932-6203
DOI:10.1371/journal.pone.0090454