Trophoblast lineage cells derived from human induced pluripotent stem cells

Abstract

Background: During implantation, the blastocyst trophectoderm attaches to the endometrial epithelium and continues to differentiate into all trophoblast subtypes, which are the major components of a placenta. Aberrant trophoblast proliferation and differentiation are associated with placental diseases. However, due to ethical and practical issues, there is almost no available cell or tissue source to study the molecular mechanism of human trophoblast differentiation, which further becomes a barrier to the study of the pathogenesis of trophoblast-associated diseases of pregnancy. In this study, our goal was to generate a proof-of-concept model for deriving trophoblast lineage cells from induced pluripotency stem (iPS) cells from human fibroblasts. In future studies the generation of trophoblast lineage cells from iPS cells established from patient's placenta will be extremely useful for studying the pathogenesis of individual trophoblast-associated diseases and for drug testing.

Methods and results: Combining iPS cell technology with BMP4 induction, we derived trophoblast lineage cells from human iPS cells. The gene expression profile of these trophoblast lineage cells was distinct from fibroblasts and iPS cells. These cells expressed markers of human trophoblasts. Furthermore, when these cells were differentiated they exhibited invasive capacity and placental hormone secretive capacity, suggesting extravillous trophoblasts and syncytiotrophoblasts.

Conclusion: Trophoblast lineage cells can be successfully derived from human iPS cells, which provide a proof-of-concept tool to recapitulate pathogenesis of patient placental trophoblasts in vitro.

Keywords: Human indcued pluripotent stem cells; Trophoblast lineage cells.

Fig. 1

Induced pluripotent stem cells (iPS cells) established from human fibroblast. (A) Human fetal fibroblast (IMR-90). (B) iPS cells generated from IMR-90. (C) Alkaline phosphatase (ALP) was expressed in iPS cells. (D, E and F) NANOG/TRA-1-81, NANOG/SSEA-4 and NANOG/TRA-1-60 were expressed in iPS cells. (Scale bars, D are 100 μm and the remaining is 200 μm.) (G). Endogenous 4 pluripotent factors POU5F1, SOX2, NANOG and LIN28 as well as FOXD3 and TERT were used to measure their respective expression levels. Expression was detected from all 6 loci in the iPS cells, compared to donor fibroblasts (P<0.05). The expression level relative to fibroblast=1.

Fig. 2

iPS cells (iPSC) generated from fibroblasts were differentiated into trophoblast lineage cells (TC) with BMP4. (A) TC derived from iPSC. (B–D) TC identity is confirmed with CDX2, EOMES and CK-7 protein expression in cultures. Scale bar, 200μM. (E) TC express pluripotent associated transcripts: FOXD3, TERT and POU5F1, as well as NANOG, SOX2 and SALL4 (P<0.05); and trophoblast specific transcripts: CDX2, HAND1, GATA3 and GATA4. (F) DNA methylation status of iPSC, TC and fibroblasts on POU5F1, NANOG, H19-IGF2.

Fig. 3

Transcriptome analysis iPS cells (iPSC), trophoblast lineage cells (TC), and fibroblasts. (A) Clustering analysis indicates that iPSC and TC form a sub-cluster separately from fibroblasts. (B–D) The scatter plots reveal that the correlation between iPSC and TC (coefficient r = 0.967) is much higher than the correlation between iPSC and fibroblast (r = 0.838). (E) There were 3238 features common between the significantly altered 5120 features in iPSC and 7714 features in TC. These observations indicate greater similarity between iPSC and TC expression profiles compared to fibroblasts. (F) From PCA, the projection on two principal components (PCs) covering 78% of the total variance reveals that both iPSC and TC are differentially expressed from fibroblasts along PC #1 (57% variance). The differences between iPSC and TC are smaller as depicted along PC #2 that covers only 21% of the total variance.

Fig. 4

Trophoblast lineage cells (TC) derived from iPS cells (iPSC) differentiated into trophoblasts with 100ng/ml BMP4 for 7-day. (A) Multinuclear trophoblasts formed, nuclei marked by red. (B) CK-7 (green) positive cells. (C) Estradiol, hCG and Progesterone detected from D-7 culture medium. (n=3). (D) TC showed significant invasive capability compare to donor fibroblast in 20~22 hours invasion assay (n=3). Scale bar, 200μM.