Wnt signaling associated small molecules improve the viability of pPSCs in a PI3K/Akt pathway dependent way

Area of Science:

• Stem Cell Biology
• Developmental Biology
• Molecular Biology

Background:

• Porcine pluripotent stem cells (pPSCs) derived from blastocysts typically exhibit poor viability and low cloning efficiency after dissociation.
• Previous methods struggled with single-cell passaging and maintaining pluripotency in pPSCs.

Purpose of the Study:

• To derive novel porcine pluripotent stem cells (pPSCs) with enhanced viability and pluripotency.
• To investigate the molecular mechanisms underlying improved pPSC characteristics using specific Wnt signaling regulators.

Main Methods:

• Treatment of porcine blastocyst-derived cells with Wnt signaling pathway regulators CHIR99021 and XAV939.
• Assessment of cell morphology, viability, cloning efficiency, and pluripotency gene expression.
• In vitro and in vivo differentiation assays (embryoid body formation, teratoma formation).
• Analysis of key signaling pathways including WNT/TCF and PI3K/Akt.

Main Results:

• The novel pPSCs exhibited small-domed morphology, significantly improved biological viability, and high cloning efficiency (80-90%).
• CH/XAV-treated cells showed elevated pluripotent gene expression and formed embryoid bodies and teratomas with three germ layers.
• CHIR99021 and XAV939 maintained β-catenin in the cytoplasm, not translocating to nuclei, while E-cadherin localized to the cell membrane, activating the PI3K/Akt pathway.

Conclusions:

• The combinative use of CHIR99021 and XAV939 successfully generated new pPSCs closer to the naïve pluripotent state.
• These improved pPSCs possess enhanced pluripotency, facilitating transgenic manipulation and regenerative medicine applications.
• The study provides insights into porcine embryonic stem cell derivation and understanding pPSC biology.