Optimization of Single-Cell Seeding Parameters to Improve Scalability of iPSC-Derived Microglia Differentiation

Document Type

Article

Publication Date

2026

JAX Location

NYSCF

Abstract

Induced pluripotent stem cell (iPSC)-derived microglia have become a valuable tool in disease modeling and precision medicine research; however, existing differentiation protocols are often limited by low microglia yield and scalability. The STEMdiffTM Hematopoietic Kit enables the differentiation of iPSCs into hematopoietic progenitor cells (HPCs), but its reliance on manual passaging limits compatibility with automated platforms like the NSYCF Global Array®, which cannot count or seed cells as aggregates. In this study, two automation-compatible differentiation methods were used to compare HPC production – low-density single-cell seeding and embryoid body (EB) formation. While single-cell seeding at 500 cells per well produced healthy colonies, colony counts exceeded the recommended range for HPC differentiation. In contrast, HPC yield was substantially higher in EB-derived cultures, with CD43+ populations being comparable to previous reports. These preliminary findings are a key step in determining optimal culture conditions and seeding densities for automated, high-throughput microglia differentiation.

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