Human Embryonic Stem Cells as Models for Trophoblast Differentiation

Phase contrast images showing the morphology of H1 hESC colonies cultured in the absence (a) or presence of 10ng/ml BMP4 for 3 (b, c) days. Cells were cultured in low (a, b) or atmospheric (c) oxygen, and in the presence (a) or absence (b, c) of FGF2. An area of morphologically differentiated cells, characterized by a cobblestone appearance, becomes visible at the periphery of the colony, and progresses inwards in cultures in which BMP4 was added (b, c). Central areas in b and c contain small, presumably undifferentiated cells, and appear quite similar to cells in a. BMP4-driven differentiation was slower under low oxygen. The scale bar shown in panel c represents 1mm.

Differentiation in BMP4 treated H9 hESC colonies proceeds from the periphery inward. (A) On day 5, cells at the periphery express syncytiotrophoblast marker hCGα (green) and hCGβ (red). Nuclei are counterstained with TO-PRO 3 (blue). (B) At day 6, cells in the core stain positively for pluripotency marker OCT4 (red), whereas the rest of the colony expresses the trophoblast marker cytokeratin 7 (green). Scale bars represent 500μm.

Changes in microarray expression (with values normalized to the median intensity of the array) for four genes associated with a pluripotent phenotype (POU5F1, NANOG, SOX2, and LEFTY2) and four genes that are regularly used as trophoblast markers (CGA, CGB5, KRT7, and KRT8). H1 hESC were exposed to BMP4 (10ng/ml) in either 4% (gray bar) or 20% (black bar) oxygen and collected after 0, 3, 12, 24, 72 and 120h. These data were obtained on Whole Human Genome Oligo microarrays (Agilent). Genes that were changing over time within each oxygen condition were identified by ANOVA. The genes shown here were selected from 33,814 genes of which 5194 changed expression more than 2-fold at P<0.01 in at least one time point. Changes in gene expression have been verified by real-time PCR with cDNA from H1 and H9 cells for POU5F1, NANOG, SOX2, LEFTY2, CGA, and CGB.