The excitement and controversy surrounding the potential role of human embryonic stem (ES)1,2 cells in transplantation therapy have often overshadowed their potentially more important use as a basic research tool for understanding the development and function of human tissues. Human ES cells can proliferate without a known limit and can form advanced derivatives of all three embryonic germ layers. What is less widely appreciated is that human ES cells can also form the extra-embryonic tissues that differentiate from the embryo before gastrulation. The use of human ES cells to derive early human trophoblast is particularly valuable, because it is difficult to obtain from other sources and is significantly different from mouse trophoblast. Here we show that bone morphogenetic protein 4 (BMP4), a member of the transforming growth factor-β (TGF-β) superfamily, induces the differentiation of human ES cells to trophoblast. DNA microarray, RT-PCR, and immunoassay analyses demonstrate that the differentiated cells express a range of trophoblast markers and secrete placental hormones. When plated at low density, the BMP4-treated cells form syncytia that express chorionic gonadotrophin (CG). These results underscore fundamental differences between human and mouse ES cells, which differentiate poorly, if at all, to trophoblast3. Human ES cells thus provide a tool for studying the differentiation and function of early human trophoblast and could provide a new understanding of some of the earliest differentiation events of human postimplantation development.
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We thank Leann Crandall, Jessica Antosiewicz, Christine Daigh, and Rachel Lewis for technical support, and Thaddeus G. Golos and staff of the Thomson laboratory for critical reading of the manuscript. This work was supported by the WiCell Research Institute, a non-profit subsidiary of the Wisconsin Alumni Research Foundation. Human ES cells are made available by the WiCell Research Institute to academic/non-profit researchers on a cost-recovery basis under the terms of a Memorandum of Understanding and Simple Letter Agreement. Research at Stanford University was supported by grants from the National Cancer Institute and Howard Hughes Medical Institute (HHMI) to P.O.B. X.C. was a Howard Hughes fellow of the Life Sciences Research Foundation. Patrick Brown of HHMI and Department of Biochemistry, Stanford University School of Medicine, asked to have his name removed from the paper because of his commitment to the Public Library of Science (PLoS) (http://www.publiclibraryofscience.org).
The authors declare no competing financial interests.
Supplementary Fig. 1.
Vesicles formed by BMP4-treated H1 cells. H1 cells (cultured in conditioned medium (CM) with bFGF) were treated with 100 ng/ml BMP4 for 7 days. The cell layer was removed from the culture dish by treatment of 1 mg/ml dispase for 10 min. followed by gentle scraping with a policemen rubber. The cell clumps were transferred to a T25 flask and cultured in unconditioned medium for another 7 days and then photographed. (JPG 11 kb)
Supplementary Fig. 2.
Microinjection of rhodamine-dextran in mononuclear (lower panels) and early syncytial trophoblast (upper panels). The early syncytial cell (five days of differentiation compared to 14 days of differentiation in Fig.1) was injected immediately after the first morphological evidence of fusion (see syncytial cell fusion in time-lapse clip 1). H1 cells (cultured in CM with bFGF) were treated with 100 ng/ml BMP4 for 5 days and injected with rhodamine-dextran (Sigma) through a 0.5 m capillary (eppendorf) at 150 hPa injection pressure for 0.2 sec. The nuclei were stained by Hoechst 33342 (Sigma), and photographed under phase (left panels) and epifluorescent microscopy (right panels). (JPG 48 kb)
Supplementary Fig. 3.
RT-PCR analysis of H1 cells cultured in CM, unconditioned medium, or CM + BMP4 (100 ng/ml) for 7 days, all in the continuous presence of bFGF. Genes known to be expressed in trophoblast (such as CG-β, GCM1, ERR- β, Hash2, MET, HLA-G1, cytokeratin 7, and CD9), pluripotent cell marker genes (such as Oct4 and TERT), and HLA class I genes (such as HLA-A and HLA-B) were examined. β-Actin expression was used as an internal control for equal RNA loading. Reactions processed without reverse transcriptase (-RT) serve as negative controls. (JPG 27 kb)
Supplementary Fig. 4.
Microarray comparison between HeLa cells, BMP4-treated H1 cells and untreated H1 cells. H1 cells (cultured in CM with bFGF) were treated with or without 100 ng/ml BMP4 for 7 days. HeLa cells were cultured in DMEM plus 10% fetal calf serum. RNA from each cell sample was extracted for microarray in reference to a human RNA pool. Microarray is shown for 698 cDNA clones (A) that were selected as described in the Experimental Protocol. Expanded views for genes encoding some typical trophoblast marker genes and tumor antigens are shown in B-D. (JPG 52 kb)
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Xu, RH., Chen, X., Li, D. et al. BMP4 initiates human embryonic stem cell differentiation to trophoblast. Nat Biotechnol 20, 1261–1264 (2002). https://doi.org/10.1038/nbt761
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