The spatial self-organization within pluripotent stem cell colonies is continued in detaching aggregates

Area of Science:

• Stem Cell Biology
• Developmental Biology
• Biotechnology

Background:

• Induced pluripotent stem cells (iPSCs) exhibit self-organization properties during culture.
• Understanding iPSC colony dynamics and transition to 3D structures is crucial for developmental biology and regenerative medicine.

Purpose of the Study:

• To investigate the self-organization dynamics of iPSC colonies under spatial confinement.
• To explore a novel method for generating size-controlled embryoid bodies (EBs) from iPSCs.
• To characterize gene expression patterns during iPSC self-organization and EB formation.

Main Methods:

• Utilized polydimethylsiloxane (PDMS) pillars and micro-contact printing of vitronectin for spatial confinement of iPSC colonies.
• Employed single-cell RNA-sequencing and spatial reconstruction to analyze gene expression.
• Observed spontaneous detachment of iPSC colonies to form 3D aggregates (EBs).

Main Results:

• Progressive upregulation of OCT4, E-cadherin, and NANOG observed near the colony rim.
• OCT4-high subsets at the colony edge showed TGF-β pathway activation, including NODAL and LEFTY.
• Spontaneously formed EBs exhibited radial organization and differential gene expression, with early aggregates showing upregulated germline-specific genes compared to conventional EBs.

Conclusions:

• Spatial confinement influences iPSC self-organization and gene expression.
• Spontaneous detachment offers a new method for generating controlled-size EBs without enzymatic or mechanical disruption.
• Early self-detached EBs display distinct germline gene expression profiles, providing insights into early developmental processes.