Exogenous supplementation of Activin A enhances germ cell differentiation of human embryonic stem cells

• Published in: Molecular human reproduction 2015-05, Vol.21 (5), p.410-423
• Main Authors: Duggal, Galbha, Heindryckx, Björn, Warrier, Sharat, Taelman, Jasin, Van der Jeught, Margot, Deforce, Dieter, Chuva de Sousa Lopes, Susana, De Sutter, Petra
• Format: Article
• Language: English
• Subjects: Activins - metabolism; Belgium; Biomarkers - metabolism; Bone Morphogenetic Protein 4 - metabolism; Cell Differentiation; Cell Line; Cell Lineage; Cells, Cultured; DEAD-box RNA Helicases - genetics; DEAD-box RNA Helicases - metabolism; Embryoid Bodies - cytology; Embryoid Bodies - metabolism; GATA4 Transcription Factor - genetics; GATA4 Transcription Factor - metabolism; GATA6 Transcription Factor - genetics; GATA6 Transcription Factor - metabolism; Gene Expression Regulation, Developmental; Germ Cells - cytology; Germ Cells - metabolism; Human Embryonic Stem Cells - cytology; Human Embryonic Stem Cells - metabolism; Humans; Proteins - genetics; Proteins - metabolism; Proto-Oncogene Proteins c-kit - genetics; Proto-Oncogene Proteins c-kit - metabolism; RNA-Binding Proteins - genetics; RNA-Binding Proteins - metabolism; Up-Regulation
• ISSN: 1360-9947; 1460-2407
• DOI: 10.1093/molehr/gav004

Human embryonic stem cells (hESCs) derived in the presence of Activin A (ActA) demonstrate an increased differentiation propensity toward the germ cell lineage. In addition, mouse epiblast stem cells and mouse epiblast-like cells are poised toward germ cell differentiation and are derived in the presence of ActA. We therefore investigated whether supplementation with ActA enhances in vitro hESC differentiation toward germ cell lineage. ActA up-regulated early primordial germ cell (PGC) genes STELLA/DPPA3 (developmental pluripotency associated 3) and tyrosine kinase receptor cKIT in both ActA-derived and standard-derived hESCs indicating its role in priming hESCs toward the PGC lineage. Indeed, ActA plus bone morphogenic protein 4 (BMP4) strongly increased germ cell differentiation potential of hESCs based on the high expression of late PGC markers DAZL (deleted in azoospermia-like) and VASA/DDX4 (DEAD-box polypeptide 4) at mRNA and protein level. Hence, the combination of ActA with BMP4 provides an additional boost for hESCs to develop into postmigratory germ cells. Together with increased VASA expression in the presence of ActA and BMP4, we also observed up-regulation of endoderm-specific genes GATA4 (GATA binding protein 4) and GATA6. Finally, we were able to further mature these in vitro-derived PGC-like cells (PGCLCs) by culturing them in in vitro maturation (IVM) medium, resulting in the formation of germ cell-like clusters and induction of meiotic gene expression. In conclusion, we demonstrate for the first time a synergism between ActA and BMP4 in facilitating germ cell-directed differentiation of hESCs, which is enhanced by extended culture in IVM medium, as shown by cytoplasmic VASA-expressing PGCLCs. We propose a novel relationship between the endoderm and germ cell lineage during hESC differentiation.