Modulation of adhesion microenvironment using mesh substrates triggers self-organization and primordial germ cell-like differentiation in mouse ES cells.

Yuta Ando,Taiji Adachi,Kennedy Omondi Okeyo

doi:10.1063/1.5072761

Abstract

The cell adhesion microenvironment plays contributory roles in the induction of self-organized tissue formation and differentiation of pluripotent stem cells (PSCs). However, physical factors emanating from the adhesion microenvironment have been less investigated largely in part due to overreliance on biochemical approaches utilizing cytokines to drive in vitro developmental processes. Here, we report that a mesh culture technique can potentially induce mouse embryonic stem cells (mESCs) to self-organize and differentiate into cells expressing key signatures of primordial germ cells (PGCs) even with pluripotency maintained in the culture medium. Intriguingly, mESCs cultured on mesh substrates consisting of thin (5 μm-wide) strands and considerably large (200 μm-wide) openings which were set suspended in order to minimize the cell-substrate adhesion area, self-organized into cell sheets relying solely on cell-cell interactions to fill the large mesh openings by Day 2, and further into dome-shaped features around Day 6. Characterization using microarray analysis and immunofluorescence microscopy revealed that sheet-forming cells exhibited differential gene expressions related to PGCs as early as Day 2, but not other lineages such as epiblast, primitive endoderm, and trophectoderm, implying that the initial interaction with the mesh microenvironment and subsequent self-organization into cells sheets might have triggered PGC-like differentiation to occur differently from the previously reported pathway via epiblast-like differentiation. Overall, considering that the observed differentiation occurred without addition of known biochemical inducers, this study highlights that bioengineering techniques for modulating the adhesion microenvironment alone can be harnessed to coax PSCs to self-organize and differentiate, in this case, to a PGC-like state.

Highlights

Pluripotent stem cells (PSCs) can differentiate to most cell lineages and are capable of self-organization to generate multicellular tissues, for instance organoids.1 To induce pluripotent stem cells (PSCs) to self-organize, recent research studies have been focusing on replicating in vivo conditions using three-dimensional culture systems in combination with biochemical factors, such as cytokines, to induce specific differentiation and tissue formation.2–4 a number of studies have reported that mechanical and geometrical factors on fabricated culture substrates, such as substrate stiffness, surface topography or micropattern, could trigger self-organization and differentiation through cell adhesion and cell-cell interaction.5–7 These studies show that the emergence of ordered germ layers and/or self-organized structures from a population of PSCs is governed by mechanical and geometrical factors as well as biochemical factors in the extracellular microenvironment
We report that a mesh culture technique can potentially induce mouse embryonic stem cells to self-organize and differentiate into cells expressing key signatures of primordial germ cells (PGCs) even with pluripotency maintained in the culture medium
To modulate a cell adhesion microenvironment, we fabricated microstructured mesh sheets with narrow mesh strands (5 lm in width) and large mesh openings, which were setup suspended on a culture dish and used as substrates for mouse embryonic stem cells (mESCs) culture (Fig. 1)

Summary

Introduction

Pluripotent stem cells (PSCs) can differentiate to most cell lineages and are capable of self-organization to generate multicellular tissues, for instance organoids. To induce PSCs to self-organize, recent research studies have been focusing on replicating in vivo conditions using three-dimensional culture systems in combination with biochemical factors, such as cytokines, to induce specific differentiation and tissue formation. a number of studies have reported that mechanical and geometrical factors on fabricated culture substrates, such as substrate stiffness, surface topography or micropattern, could trigger self-organization and differentiation through cell adhesion and cell-cell interaction. These studies show that the emergence of ordered germ layers and/or self-organized structures from a population of PSCs is governed by mechanical and geometrical factors as well as biochemical factors in the extracellular microenvironment. To induce PSCs to self-organize, recent research studies have been focusing on replicating in vivo conditions using three-dimensional culture systems in combination with biochemical factors, such as cytokines, to induce specific differentiation and tissue formation.. A number of studies have reported that mechanical and geometrical factors on fabricated culture substrates, such as substrate stiffness, surface topography or micropattern, could trigger self-organization and differentiation through cell adhesion and cell-cell interaction.. A number of studies have reported that mechanical and geometrical factors on fabricated culture substrates, such as substrate stiffness, surface topography or micropattern, could trigger self-organization and differentiation through cell adhesion and cell-cell interaction.5–7 These studies show that the emergence of ordered germ layers and/or self-organized structures from a population of PSCs is governed by mechanical and geometrical factors as well as biochemical factors in the extracellular microenvironment. Bioengineering techniques for designing the physical microenvironment will provide a powerful approach to drive the intrinsic self-organization property of cells.

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Conclusion