Journal
BIOMATERIALS
Volume 282, Issue -, Pages -Publisher
ELSEVIER SCI LTD
DOI: 10.1016/j.biomaterials.2022.121389
Keywords
Heterogeneity; Pluripotent stem cells; Self-organization; Micro-contact printing; EB formation; Single-cell RNA-Sequencing
Funding
- German Research Foundation (DFG) [WA 1706/8-1, WA 1706/11-1, WA 1706/12-1/CRU344, 363055819/GRK2415]
- Interdisciplinary Center for Clinical Research (IZKF) within the faculty of Medicine at the RWTH Aachen University [O3-3]
- Ministry for Innovation, Science and Research of German Federal State of North Rhine-Westphalia, Duesseldorf, Germany
- CAPES-Alexander von Humboldt postdoctoral fellowship
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Colonies of induced pluripotent stem cells (iPSCs) exhibit self-organization and transition into 3D aggregates. This transition is characterized by gradual upregulation of specific genes and the formation of embryoid bodies (EBs). The use of polydimethylsiloxane (PDMS) pillars or micro-contact printing of vitronectin plays a role in spatial confinement and the formation of controlled size EBs without enzymatic or mechanical treatment.
Colonies of induced pluripotent stem cells (iPSCs) reveal aspects of self-organization even under culture conditions that maintain pluripotency. To investigate the dynamics of this process under spatial confinement, we used either polydimethylsiloxane (PDMS) pillars or micro-contact printing of vitronectin. There was a progressive upregulation of OCT4, E-cadherin, and NANOG within 70 mu m from the outer rim of iPSC colonies. Single-cell RNA-sequencing and spatial reconstruction of gene expression demonstrated that OCT4(high) subsets, residing at the edge of the colony, have pronounced up-regulation of the TGF-beta pathway, particularly of NODAL and its inhibitor LEFTY. Interestingly, after 5-7 days, 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 enzymatic or mechanical treatment. Within the early 3D aggregates, radial organization and differential gene expression continued in analogy to the changes observed during self-organization of iPSC colonies. Early self-detached aggregates revealed up-regulated germline-specific gene expression patterns as compared to conventional EBs. However, there were no marked differences after further directed differentiation toward hematopoietic, mesenchymal, and neuronal lineages. Our results provide further insight into the gradual self organization within iPSC colonies and at their transition into EBs.
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