Development of an Imaged-Based Framework for Differentiation Prediction in Reporter iPSCs

Document Type

Article

Publication Date

2026

JAX Location

NYSCF

Abstract

The intermediate and late-stage analyses conducted with lineage-specific markers and gene expression analysis techniques such as qPCRs and flow cytometry fails to capture the continuous nature of an iPSC differentiation. The use of an endogenously tagged iPSC line- a fluorescent reporter gene attached to a select gene- allows for the use of longitudinal live-cell imaging to capture tagged features across cell states as iPSCs differentiate towards varying cell fates. The nucleus and lysosomes are integral to cellular architecture and are functionally critical for cellular processes (genetic regulation and central dogma and metabolic/autophagy state respectively) and are known to morphologically and phenotypically change across cell states in a differentiation and therefore encode enough information to create a differentiation trajectory of an iPSC differentiation to cardiomyocytes. The creation of such a framework allows for the detection of divergence points of specific phenotypes, such as the differentiation of iPSCs to cardiac fibroblasts; this framework also has applications in disease modeling on mutated or disease-associated iPSCs lines by predicting when a diseased/mutated phenotype can first appear. Pilot experiments to assess the best imaging conditions conducive for single-cell tracking and mitigating phototoxicity will be performed in early weeks.

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