Single-cell transcriptomics reveal differences between chorionic and basal plate cytotrophoblasts and trophoblast...

Area of Science:

• Human placental development
• Trophoblast biology
• Stem cell research

Background:

• Cytotrophoblast (CTB) cells are bipotent progenitors in early human placenta, differentiating into invasive extravillous trophoblast (EVT) and syncytiotrophoblast (STB).
• The cell-of-origin for trophoblast stem cells (TSC) remains unidentified, despite their bipotentiality.

Purpose of the Study:

• To investigate the transcriptional diversity of early and late first trimester villous CTB (vCTB).
• To compare vCTB transcriptional profiles with trophoblast stem cells (TSC).
• To identify distinct CTB states and their relationship to TSC and differentiation potential.

Main Methods:

• Single-cell RNA sequencing (scRNA-seq) of placental villous tissue from early (6-8 weeks) and late (12-14 weeks) first trimester.
• scRNA-seq and GeoMx spatial transcriptomics of CTB from basal and chorionic regions of early placenta.
• scRNA-seq of TSC lines and CTB during TSC derivation.
• Flow cytometry analysis of TSC lines for specific markers.

Main Results:

• CTB clusters showed distinctions based on gestational age and location (basal vs. chorionic plate).
• Identified trophoblast states including 'initial state' vCTB, CTB subtypes, precursor STB, and precursor/mature EVT.
• CTB progenitors were enriched in early basal plate, with basal CTB biased toward EVT and chorionic CTB toward STB precursors.
• TSC clustered most closely with EVT precursor cells and lost specific in vivo markers (e.g., PAGE4) during in vitro culture.
• ITGA5+ CTB could form EVT in 2D culture but not organoids, while ITGA5- CTB formed organoids but not EVT in 2D.

Conclusions:

• Distinct CTB states exist regionally in the placenta as early as six weeks gestation.
• Current TSC lines are transcriptionally similar to ITGA5+ CTB, biased toward the EVT lineage.
• Cell surface markers like ITGA5 differentiate CTB with distinct self-renewal and differentiation potentials.