Multiscale microenvironmental perturbation of pluripotent stem cell fate and self-organization

Yoji Tabata,Matthias P Lutolf

doi:10.1038/srep44711

Abstract

The combination of microfluidics with engineered three-dimensional (3D) matrices can bring new insights into the fate regulation of stem cells and their self-organization into organoids. Although there has been progress in 3D stem cell culturing, most existing in vitro methodologies do not allow for mimicking of the spatiotemporal heterogeneity of stimuli that drive morphogenetic processes in vivo. To address this, we present a perfusion-free microchip concept for the in vitro 3D perturbation of stem cell fate. Stem cells are encapsulated in a hydrogel compartment that is flanked by open reservoirs for the diffusion-driven generation of biomolecule gradients. Juxtaposing additional compartments bearing supportive cells enables investigating the influence of long range cell-cell communication. We explore the utility of the microchips in manipulating early fate choices and self-organizing characteristics of 3D-cultured mouse embryonic stem cells (mESCs) under neural differentiation conditions and exposure to gradients of leukemia inhibitory factor (LIF). mESCs respond to LIF gradients in a spatially dependent manner. At higher LIF concentrations, multicellular colonies maintain pluripotency in contrast, at lower concentrations, mESCs develop into apicobasally polarized epithelial cysts. This versatile system can help to systematically explore the role of multifactorial microenvironments in promoting self-patterning of various stem cell types.

Highlights

The combination of microfluidics with engineered three-dimensional (3D) matrices can bring new insights into the fate regulation of stem cells and their self-organization into organoids
The development of in vitro models that can simulate key characteristics of native stem cell niches has become a promising alternative. Such models must take into account both the biophysical and biochemical properties of the extracellular matrix (ECM), the presence of soluble bioactive molecules, and the presence of other cell types that play a role in supporting stem cells through either direct cell–cell communication or long-range, diffusible signals[3]
Since the operation of the microchip does not rely on active perfusion, cells are not exposed to fluid flow, resulting in much higher cell viability due to an accumulation of important autocrine and paracrine factors in the cell culture chamber. We employed this platform for the 3D culture of mouse embryonic stem cells under neural induction conditions, when their differentiation was locally perturbed by exposure to gradients of soluble, cell secreted and gel-immobilized leukemia inhibitory factor (LIF), a self-renewal factor

Summary

Introduction

The combination of microfluidics with engineered three-dimensional (3D) matrices can bring new insights into the fate regulation of stem cells and their self-organization into organoids. We employed this platform for the 3D culture of mouse embryonic stem cells (mESCs) under neural induction conditions, when their differentiation was locally perturbed by exposure to gradients of soluble, cell secreted and gel-immobilized leukemia inhibitory factor (LIF), a self-renewal factor.

Results

Conclusion