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Comparative Study
. 2013 Jul;34(7):536-43.
doi: 10.1016/j.placenta.2013.03.016. Epub 2013 Apr 28.

Comparison of extravillous trophoblast cells derived from human embryonic stem cells and from first trimester human placentas

B P Telugu  1 , K Adachi, J M Schlitt, T Ezashi, D J Schust, R M Roberts, L C Schulz
Affiliations
Comparative Study

Comparison of extravillous trophoblast cells derived from human embryonic stem cells and from first trimester human placentas

B P Telugu et al. Placenta. 2013 Jul.

Abstract

Introduction: Preeclampsia and other placental pathologies are characterized by a lack of spiral artery remodeling associated with insufficient invasion by extravillous trophoblast cells (EVT). Because trophoblast invasion occurs in early pregnancy when access to human placental tissue is limited, there is a need for model systems for the study of trophoblast differentiation and invasion. Human embryonic stem cells (hESC) treated with BMP4- differentiate to trophoblast, and express HLA-G, a marker of EVT. The goals of the present study were to further characterize the HLA-G(+) cells derived from BMP4-treated hESC, and determine their suitability as a model.

Methods: HESC were treated with BMP4 under 4% or 20% oxygen and tested in Matrigel invasion chambers. Both BMP4-treated hESC and primary human placental cells were separated into HLA-G(+) and HLA-G(-)/TACSTD2(+) populations with immunomagnetic beads and expression profiles analyzed by microarray.

Results: There was a 10-fold increase in invasion when hESC were BMP4-treated. There was also an independent, stimulatory effect of oxygen on this process. Invasive cells expressed trophoblast marker KRT7, and the majority were also HLA-G(+). Gene expression profiles revealed that HLA-G(+), BMP4-treated hESC were similar to, but distinct from, HLA-G(+) cells isolated from first trimester placentas. Whereas HLA-G(+) and HLA-G(-) cells from first trimester placentas had highly divergent gene expression profiles, HLA-G(+) and HLA-G(-) cells from BMP4-treated hESC had somewhat similar profiles, and both expressed genes characteristic of early trophoblast development.

Conclusions: We conclude that hESC treated with BMP4 provide a model for studying transition to the EVT lineage.

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Figures

Fig. 1
Fig. 1
H1 hESC were immunostained for POU5F1 (Oct4) (Green B,D) on days 2 (A–B) and 4 (C–D) of BMP4 treatment. No POU5F1 signals were detected on days 6 or 8 (not shown). Nuclei are counterstained with DAPI (Blue A,B). Scale Bar (D): 200 μm. Negative controls are shown in Supplemental Fig. 2.
Fig. 2
Fig. 2
Acquisition of trophoblast lineage markers by hESC over 8 days of culture in BMP4. H1 hESC were immunostained for HLA-G (Green, A,B,C,D), and CGA (Green, E,F,G,H) on d2, 4, 6 and 8 of treatment with BMP4. Nuclei were counterstained with DAPI (blue) Drawings indicate position of image within an hESC colony relative to the colony edge and central core of less differentiated cells. Scale bar (H): 50 μm. Negative controls are shown in Supplemental Fig. 1.
Fig. 3
Fig. 3
H1 and H9 hESC were plated on matrigel invasion chambers and treated with BMP4 for 5 days. (A) Approximately 70% of cells that passed through the matrigel and adhered to the underside of the chamber membrane were positive for extravillous trophoblast marker HLA-G (green). (B) Nearly 100% of these invaded cells were positive for trophoblast marker KRT7 (green). (C) Proliferation marker Ki67 (red). (D) CDH5 (VE-cadherin), green. (E) ITGA1 (integrin alpha 1), green. Nuclei are counterstained with DAPI (blue). Scale bar (E): 50 μm. Positive and negative controls are shown in Supplemental Fig. 2. The number of invaded cells on the membrane was averaged across five randomly selected fields of view in three trials each in H1 (F) and H9 (G) hESC. *p < 0.05, Holm-Sidak pairwise comparisons.
Fig. 4
Fig. 4
Microarray analysis of gene expression profiles in trophoblast derived from first trimester placental tissues and hESC. (A) Clustering of primary and hESC-derived trophoblast expression profiles for genes that are differentially expressed between primary (placental) HLA-G+ and HLA-G+/TACSTD2- cells (4579 probes >2-fold, paired T-test < 0.05 in placental samples). (B) Clustering of primary and hESC-derived trophoblast expression profiles for genes differentially expressed between undifferentiated hESC and all six trophoblast samples from BMP4-hESC or all six trophoblast samples from first trimester placentas (>6 fold in 6/6 comparisons, 3212 probes). The condition trees were generated using values normalized to the 75th percentile intensity of each array. Red/Orange = up-regulated relative to median. Blue/Grey = down-regulated relative to median of 10 samples.
Fig. 5
Fig. 5
Expression of lineage marker genes by microarray analysis. Expression was normalized to the 75th percentile (=1.0) of fluorescence intensity on the array. This normalized value is shown on the y-axis. Error bars represent standard errors. (A) Endoderm and Mesoderm – expressed genes. (B) Genes expressed in mature trophoblast (log scale). (C) Transcription factor genes expressed in early trophoblast development.
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