Live single-molecule imaging reveals global shifts in mRNA mobility during human stem cell differentiation

Spatiotemporal regulation of mRNA localisation is fundamental to cell identity specification and function, yet tracking transcript dynamics in living differentiating cells remains technically challenging. Here, we establish a robust pipeline for MS2 tagging of endogenous transcripts in human induced pluripotent stem cells (iPSCs), coupled with single-particle tracking and Hidden Markov Modelling to map mRNA mobility landscapes during differentiation and cell state transitions. Applying this approach to different cytoskeleton-encoding transcripts in diverse contexts — neural organoids, directly programmed neurons, and vascular organoids — we reveal a conserved principle: Both, β -actin and β 2b-tubulin particle dynamics progressively shift towards constrained, compartmentalised patterns as cells acquire cell type identity. Perturbation experiments demonstrate that microtubule-dependent tethering is a common, conserved mechanism controlling β -actin mRNA localisation in all cell types studied, whereas translation-dependent anchoring and actin filaments contribute in a context-dependent manner. Analysis of particle dynamics in migrating blood vessel progenitors further showed that β -actin mRNAs accumulating at cell edges are highly diffusive, while those in perinuclear regions show constrained movement. Together, our integrated framework provides a scalable foundation for mechanistic dissection of mRNA targeting in human developmental and disease models.