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Derivation of human triploid trophoblast stem cells

  • Genetics
  • Published:
Journal of Assisted Reproduction and Genetics Aims and scope Submit manuscript

Abstract

Purpose

Human trophoblast stem cells (hTSCs) are counterparts of the precursor cells of the placenta and are valuable cell models for the study of placental development and the pathogenesis of placental diseases. The aim of this work was to establish a triploid human TSC (hTSC3PN) derived from the tripronuclear embryos, which are clinically discarded but readily available, for potential applications in basic placental research and disease modeling.

Methods

Eighteen tripronuclear human zygotes from IVF were collected and cultured for 5–6 days. Five high-quality blastocysts were harvested and were individually cultured in hTSC medium. Finally, two hTSC lines were established after 10 days and could be passaged stably.

Results

The karyotyping analysis showed that hTSC3PN contained three sets of chromosomes. And the hTSC3PN exhibited typical features of hTSCs, with the ability to differentiate into two trophoblast lineages: extravillous cytotrophoblasts (EVTs) and syncytiotrophoblasts (STs). In addition, the hTSC3PN can mimic some vital features of trophoblast, including hormone secretion and invasion. Further studies showed that the proliferation and differentiation of hTSC3PN were reduced compared with normal hTSCs, which may be related to the disturbed metabolic signaling in hTSC3PN.

Conclusions

We established the triploid hTSC lines derived from tripronuclear embryos, which provides a potentially useful research model in vitro to study human placental biology and diseases.

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Authors and Affiliations

  1. Department of Histology and Embryology, Center for Stem Cell Biology and Tissue Engineering, Key Laboratory for Stem Cells and Tissue Engineering, Ministry of Education, Zhongshan School of Medicine, Sun Yat-sen University, Guangzhou, China

    Xuhui Kong & Xin Chen

  2. Reproductive Medicine Center, Sun Yat-sen Memorial Hospital, Sun Yat-sen University, Guangzhou, China

    Songbang Ou, Wenjun Wang & Ruiqi Li

  3. Guangdong Provincial Key Laboratory of Malignant Tumor Epigenetics and Gene Regulation, Sun Yat-sen Memorial Hospital, Guangzhou, China

    Songbang Ou, Wenjun Wang & Ruiqi Li

  4. Reproductive Medicine Center, The First People’s Hospital of Kashgar, Kashgar, China

    Ruiqi Li

Authors
  1. Xuhui Kong
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  2. Xin Chen
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  3. Songbang Ou
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  4. Wenjun Wang
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  5. Ruiqi Li
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Corresponding authors

Correspondence to Wenjun Wang or Ruiqi Li.

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Ethics approval and consent to participate

The study protocol and all subjects who participated in this study were approved by the Ethics Committee of Sun Yat-sen Memorial Hospital of Sun Yat-sen University (2020 Reproductive Ethics No. 15), and informed consent was obtained from all patients prior to their participation in accordance with institutional and national guidelines.

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The authors declare no competing interests.

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Supplementary information

Supplement 1

Establishment of normal hTSCs. (A) Immunofluorescence staining of GATA3, GATA2, and ITGA6 in hTSCs (The scale bars indicate 100 μm). (B) Similarities in RNA-seq among hTSCs and the hTSCs of Okae et al.’s [9] study. TSC1 and TSC2 are the cells we established in this study. (C) Immunofluorescence staining of HLA-G in EVT cells induced by hTSCs. (Scale bar, 100 μm.) (D) Immunofluorescence staining of hCG and SDC1 in ST cells induced by hTSCs. (Scale bar, 100 μm.) (E) The expression levels of EVT-specific marker gene (HLA-G) in ST cells induced by hTSCs. Data were presented as mean ± SD (n = 3). (F) The expression levels of ST-specific marker genes (SDC1, INHA, CGA, CGB) in ST cells induced by hTSCs. Data were presented as mean ± SD (n = 3). *p < 0.05, **p<0.01, ***p<0.001, ****p < 0.0001. (PNG 1538 kb)

High Resolution Image (TIF 14749 kb)

Supplement 2

Establishment of the other 3PN hTSCs, hTSC3PN-2. (A) Human tripronuclear zygote (The scale bars indicate 50 μm). (B) P0 of hTSC3PN-2 (The scale bars indicate 100 μm). (C) P3 of hTSC3PN-2 (The scale bars indicate 100 μm). (D–F) Immunofluorescence staining of GATA3, GATA2, and ITGA6 in hTSC3PN-2 cells (The scale bars indicate 100 μm). (PNG 2342 kb)

High Resolution Image (TIF 19485 kb)

Supplement 3.

Comparison of the similarity between HM and hTSC3PN (A) The flow chart of the comparison between the HM and hTSC3PN. The expression matrix of the GSE138250 dattableaset (HM) [16] was downloaded from the GEO database. The edgeR package [17] was used to analyze the differentially expressed genes of the sample counts. p-value < 0.05 with change ≥ twofold were set to filter the expressed genes based on statistically significant differences. (B) Using the DEGs of HM to draw the heat map of hTSCs and hTSC3PN. The DEGs divided the cells into two clusters. (C) Bubble plot of GO enrichment analysis. The size of the circle represents the number of genes enriched in the signal pathway, and the intensity of the color represents statistical significance. (D) The relative expression of PSG1-6, PSG9, PSG11 in HM. logFC: log(Foldchange). p-value<0.05. (E) The Volcano map of DEGs in hTSCs and hTSC3PN. Red dots and letters mark the PSG family genes. The genes on the left of X-axis 0 are the genes with lower expression in hTSC3PN and the ones on the right are the genes with higher expression in hTSC3PN. (F) The RT-QPCR of PSG1-6, PSG9, PSG11 in ST were derived from hTSC and ST derived from hTSC3PN. Data were presented as mean ± SD (n = 3) (*p < 0.05, **p<0.01, ***p<0.001, ****p < 0.0001) (G) TPM of P57 in hTSC3PN1 and hTSC3PN2. TPM: Transcripts Per Kilobase of exon model per Million mapped reads. (PNG 124 kb)

High Resolution Image (TIF 22131 kb)

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Kong, X., Chen, X., Ou, S. et al. Derivation of human triploid trophoblast stem cells. J Assist Reprod Genet 39, 1183–1193 (2022). https://doi.org/10.1007/s10815-022-02436-w

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