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

bioRxiv (Cold Spring Harbor Laboratory)

Abstract

Summary 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 µm from the outer rim of iPSC colonies. Single- cell RNA-sequencing demonstrated that OCT4 high subsets have pronounced up-regulation of the TGF-β pathway, particularly of NODAL and its inhibitor LEFTY, at the rim of the colonies. Furthermore, calcium-dependent cell-cell interactions were found to be relevant for the self-organization. Interestingly, 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 iPSC colonies. Our results provide further insight into the gradual self-organization within iPSC colonies and at their transition into EBs.

Authors 14

1. Mohamed H. Elsafi Mabrouk corresponding Aachen Institute for Biomedical Engineering–Cell Biology

   RWTH Aachen University

   Affiliation as printed

   Helmholtz-Institute for Biomedical Engineering, Stem Cell Biology and Cellular Engineering, RWTH Aachen University Medical School, Aachen, 52074, Germany

   Institute for Biomedical Engineering – Cell Biology, RWTH Aachen University Medical School, Aachen, 52074, Germany

2. Roman Goetzke Aachen Institute for Biomedical Engineering–Cell Biology

   RWTH Aachen University

   Affiliation as printed

   Helmholtz-Institute for Biomedical Engineering, Stem Cell Biology and Cellular Engineering, RWTH Aachen University Medical School, Aachen, 52074, Germany

   Institute for Biomedical Engineering – Cell Biology, RWTH Aachen University Medical School, Aachen, 52074, Germany

3. Giulio Abagnale

   St. Anna Children's Cancer Research Institute

   Affiliation as printed

   St. Anna Children’s Cancer Research Institute (CCRI), Zimmermannplatz 10, 1090 Vienna, Austria

   St. Anna Children's Cancer Research Institute (CCRI), Zimmermannplatz 10, 1090 Vienna, Austria

4. Burcu Yesilyurt Aachen Institute of Pathology

   RWTH Aachen University

   Affiliation as printed

   Institute of Pathology, RWTH Aachen University Medical School, Pauwelsstraße 30, 52074, Aachen, Germany

   Institute of Pathology, RWTH Aachen University Medical School, Pauwelsstraße 30, 5207, Aachen, Germany

5. Lucia Salz Aachen Institute for Biomedical Engineering–Cell Biology

   RWTH Aachen University

   Affiliation as printed

   Helmholtz-Institute for Biomedical Engineering, Stem Cell Biology and Cellular Engineering, RWTH Aachen University Medical School, Aachen, 52074, Germany

   Institute for Biomedical Engineering – Cell Biology, RWTH Aachen University Medical School, Aachen, 52074, Germany

6. Kira Zeevaert Aachen Institute for Biomedical Engineering–Cell Biology

   RWTH Aachen University

   Affiliation as printed

   Helmholtz-Institute for Biomedical Engineering, Stem Cell Biology and Cellular Engineering, RWTH Aachen University Medical School, Aachen, 52074, Germany

   Institute for Biomedical Engineering – Cell Biology, RWTH Aachen University Medical School, Aachen, 52074, Germany

7. Zhiyao Ma Aachen Institute for Biomedical Engineering–Cell Biology

   RWTH Aachen University

   Affiliation as printed

   Institute for Biomedical Engineering – Cell Biology, RWTH Aachen University Medical School, Aachen, 52074, Germany

   Institute for Biomedical Engineering – Cell Biology, RWTH Aachen University Medical School, Aachen, 5074, Germany

8. Marcelo A. S. Toledo Aachen Department of Hematology, Oncology Faculty of Medicine

   RWTH Aachen University

   Affiliation as printed

   Department of Hematology, Oncology, Hemostaseology and Stem Cell Transplantation, Faculty of Medicine, RWTH Aachen University, Aachen, Germany

9. Ronghui Li Aachen Institute for Computational Genomics

   RWTH Aachen University

   Affiliation as printed

   Institute for Computational Genomics, RWTH Aachen University Medical School, Pauwelsstraße 19, 52074, Aachen, Germany

10. Ivan G. Costa Aachen Institute for Computational Genomics

    RWTH Aachen University

    Affiliation as printed

    Institute for Computational Genomics, RWTH Aachen University Medical School, Pauwelsstraße 19, 52074, Aachen, Germany

11. Vivek Pachauri Aachen Institute of Materials in Electrical Engineering 1 Chair of Micro- and Nanosystems

    RWTH Aachen University

    Affiliation as printed

    Institute of Materials in Electrical Engineering 1 and Chair of Micro-and Nanosystems, RWTH Aachen University, Aachen, Germany

12. Uwe Schnakenberg Aachen Institute of Materials in Electrical Engineering 1 Chair of Micro- and Nanosystems

    RWTH Aachen University

    Affiliation as printed

    Institute of Materials in Electrical Engineering 1 and Chair of Micro-and Nanosystems, RWTH Aachen University, Aachen, Germany

13. Martin Zenke Aachen Institute for Biomedical Engineering–Cell Biology

    RWTH Aachen University

    Affiliation as printed

    Helmholtz-Institute for Biomedical Engineering, Stem Cell Biology and Cellular Engineering, RWTH Aachen University Medical School, Aachen, 52074, Germany

    Institute for Biomedical Engineering – Cell Biology, RWTH Aachen University Medical School, Aachen, 52074, Germany

14. Wolfgang Wagner corresponding Aachen Institute for Biomedical Engineering–Cell Biology

    RWTH Aachen University

    Affiliation as printed

    Helmholtz-Institute for Biomedical Engineering, Stem Cell Biology and Cellular Engineering, RWTH Aachen University Medical School, Aachen, 52074, Germany

    Institute for Biomedical Engineering – Cell Biology, RWTH Aachen University Medical School, Aachen, 52074, Germany