IntroductionThe vertebrate mesoderm gives rise to a diverse variety of tissues, including the heart, vasculature, and blood. It is formed in the primitive streak (PS) which appears first in the posterior epiblast during gastrulation. The nascent PS then elongates to the anterior part, whereas the epiblast cells ingress through the PS, migrate to appropriate location in the embryo, and become either mesoderm or definitive endoderm. 1 Although the process of mesoderm formation is well understood, the underlying molecular mechanisms remain poorly defined.Previous studies have identified several signaling pathways involved in this process. 2 One is the transforming growth factorbeta (TGF-) superfamily member, bone morphogenetic protein-4 (BMP-4). Like other TGF- superfamily members, BMP-4 binds to and brings together the type I and type II receptors on the cell surface, which allows receptor II to phosphorylate the receptor I kinase domain. After this activation, the type I receptor phosphorylates certain members of the Smad proteins (Smad 1, 5, 8; R-Smad). Two phosphorylated R-Smads, in combination with a common mediator Smad (Smad4; Co-Smad), form a heterotrimeric complex, which then translocates into the nucleus and cooperates with other transcription factors to modulate target gene expression. [3][4][5][6] Genetic studies in mice have shown that BMP-4 signaling is required for mesoderm formation. [7][8][9] Mice deficient in the BMP-4 ligand, 7 in the BMP type II receptor, 8 or in a BMP type I receptor Alk3, 9 all fail to develop mesoderm. It has also been demonstrated that BMP-4 plays critical roles in mesoderm induction and mesoderm lineage differentiation from mouse embryonic stem cells (mES cells). [10][11][12][13][14][15][16][17][18][19] The key role of BMP-4 in mouse embryos and mES cells has led us to investigate its function in human embryonic stem cells (hES cells), the differentiation of which can, at least in part, mimic the human embryogenesis. 20,21 Xu et al 22 showed that long-term BMP-4 treatment (up to 7 days) induces hES cell differentiation into trophoblast. Later, Pera et al 23 demonstrated that activation of the same signaling pathway by BMP-2 in hES cells results in the formation of extra-embryonic endoderm. Surprisingly, we found that short-term BMP-4 treatment (24 hours) initiated mesoderm induction at a high efficiency in hES cells. In addition, these mesoderm progenitor cells were able to differentiate into various mesoderm lineages. The differential effects of short-term and long-term BMP-4 treatments on hES cell differentiation suggest that the BMP signaling pathway might play a flexible and time-dependent role in human embryonic development and cell fate determination.
Methods
Cell culture and differentiationThe H1, H7, and H9 hES cell lines were obtained from WiCell Research Institute (Madison, WI). Cells were cultured and passaged on mitomycin C-treated mouse embryonic fibroblast (MEF) feeders in hES cell culture Submitted February 14, 2007; accepted November 20, 2007. Prepublished...
