Microwell regulation of pluripotent stem cell self-renewal and differentiation

Abstract

The fates of pluripotent stem cells (PSCs), including survival, self-renewal, and differentiation, are regulated by chemical and mechanical cues presented in the three-dimensional (3D) microenvironment. Most PSC studies have been performed on two-dimensional substrates. However, 3D culture systems have demonstrated the importance of intercellular interactions in regulating PSC self-renewal and differentiation. Microwell culture systems have been developed to generate homogenous PSC colonies of defined sizes and shapes and to study how colony morphology affects cell fate. Using microwells, researchers have demonstrated that PSCs remain in a self-renewing undifferentiated state as a result of autocrine and paracrine signaling. Other studies have shown that microwell regulation of embryoid body size affects lineage commitment during differentiation via cell-cell contact and expression of soluble signals. In this review, we discuss recent advances in the design and utilization of 3D microwell platforms for studying intercellular regulation of PSC cell fate decisions and the underlying molecular mechanisms.

Keywords: 3D microenvironment; cell fate; microwells; pluripotent stem cells.

Fig. 1

(a) Phase contrast images of human embryonic stem cells cultured in microwells of different shapes. Approximately 6 days after seeding cells into the wells and expanding cells in MEF conditioned medium, the wells were confluent and the colonies were removed by enzymatic (Dispase) treatment (b) The resulting EBs possessed the shape of the microwell. (c) Confocal fluorescent image of a hESC colony stained with DAPI (blue) and phalloidin (red) in a microwell is shown. (d) Colonies were removed from microwells shown in c and cultured in suspension for one day to form EBs. Cells were stained with DAPI (blue), anti-E-cadherin antibody (red) and anti-β-catenin antibody (green). After 1 day in suspension culture the cuboidal colony became a spherical EB, and spatial gradients in protein expression were detected.

Fig. 2

Summary of methods for generating 3D aggregates of human pluripotent stem cells. References are provided for examples of these methods.