The spatial self-organization within pluripotent stem cell colonies is continued in detaching aggregates (bulk RNA-Seq)

Colonies of induced pluripotent stem cells (iPSCs) reveal aspects of self-organization even under culture conditions that maintain pluripotent state. To investigate the dynamics of this process under spatial confinement we used either PDMS pillars or micro-contact printing of vitronectin. OCT4, E-cadherin, and NANOG were progressively upregulated within about 70 µm from the outer rim of iPSC colonies. Single-cell RNA-sequencing demonstrated that OCT4high subsets have pronounced up-regulation of the TGF-ß pathway, particularly of NODAL and its inhibitor LEFTY, at the rim of the colonies. Furthermore, calcium-dependent cell-cell interaction seemed to be relevant for the self-organization. After 5 to 7 days, the iPSC colonies detached spontaneously from micro-contact printed substrates to form 3D aggregates. This new method allowed generation of embryoid bodies (EBs) of controlled size, without any enzymatic or mechanical treatment. Within the early 3D aggregates, the radial organization and differential gene expression continued in analogy to the changes observed during self-organization of flat iPSC colonies. Our results provide further insight into the gradual self-organization within iPSC colonies and at their transition into EBs. Overall design: In this study, we analyzed the heterogeneity of gene expression within self-organized iPSC colonies and early iPSC aggregates. We used micro-contact printing of vitronectin to confine cell growth to circles of 600 µm in diameter on tissue culture plastic. We seeded iPSCs (two replicas with different cell lines) on the substrates. We carried out single-cell RNA-sequencing (sc-RNA-seq) using the Chromium platform (10x Geneomics) at day 5 after cell seeding. At this stage the cells reached confluent state and self-organized with up-regulation of pluripotency markers at the rim of the colony. Furthermore, we analyzed early aggregates of iPSCs that formed by self-detachment from the substrates at about day 6. The non-adherent aggregates were then analyzed by sc-RNA-seq at day 8 after the initial seeding on the substrates.