Spatial heterogeneity analysis of seeding of human induced pluripotent stem cells for neuroectodermal differentiation

Abstract

Introduction

Preparing a uniform cell population in high–density seeding of adherent human induced pluripotent stem cells (hiPSC) requires stable culture conditions and consistent culture operation. In this study, we evaluated cell distribution patterns by changing cell seeding operations and their impact on differentiation toward the neuroectodermal lineage.

Methods

The hiPSC line 201B7 was seeded at 1.23 × 10⁵ cells/cm² following a conventional operation, prolongated time of cell seeding suspension or vessel tilting during cell seeding operation. Fluorescent imaging of cell nuclei was performed 24 h following cell seeding and used for spatial heterogeneity analysis. Flow cytometric analysis was also performed seven days after cell differentiation induction toward neuroectodermal lineage.

Results

Indices for spatial heterogeneity following high–density cell seeding were proposed to assess cell distribution patterns. Global heterogeneity (H_G) was shown to be mostly affected by vessel tilting during cell seeding operation, while local heterogeneity (H_L) was affected by prolongated time of cell seeding suspension. Changes in both spatial heterogeneities in the hiPSC population resulted in a lower yield of target neuroectodermal cells compared with the control operation.

Conclusion

High–density hiPSC seeding is critical for achieving a higher yield of target cells of neuroectodermal lineage. Understanding the spatial heterogeneity in early stages detects errors in cell culture motion and predicts cell fate in later stages of cell culture.