Live imaging can provide powerful insights into developmental and cellular processes but availability of multiplexable reporters has been limiting. Here we describe ORACLE, a cell fate reporter class in which fluorescent proteins fused with the nucleoporin POM121 are driven by promoters of transcription factors of interest. ORACLE’s nuclear rim localisation therefore enables multiplexing with conventional nuclear reporters. We applied ORACLE to investigate the dynamics of pluripotency exit at single-cell level, using human pluripotent stem cells (hPSCs) imaged by multi-day time-lapse high-content microscopy. Using an ORACLE-OCT4 pluripotency marker we reveal that G1 phase length and OCT4 level are strongly coupled and that spatial location in a colony impacts the timing of pluripotency exit. Combining ORACLE-OCT4 and an ORACLE-SOX1 early neuronal differentiation marker, we visualize in real-time the dynamics of cell fate transition between pluripotency and early neural fate, and show that pluripotency exit and differentiation onset are likely not tightly coupled in single-cells. Thus ORACLE is a powerful tool to enable quantitative studies of spatiotemporal cell fate control.

Predicting how stem cells become patterned and differentiated into target tissues is key for optimising human tissue design. Here, we established DEEP-MAP - for deep learning-enhanced morphological profiling - an approach that integrates single-cell, multi-day, multi-colour microscopy phenomics with deep learning and allows to robustly map and predict cell fate dynamics in real-time without a need for cell state-specific reporters. Using human pluripotent stem cells (hPSCs) engineered to co-express the histone H2B and two-colour FUCCI cell cycle reporters, we used DEEP-MAP to capture hundreds of morphological- and proliferation-associated features for hundreds of thousands of cells and used this information to map and predict spatiotemporally single-cell fate dynamics across germ layer cell fates. We show that DEEP-MAP predicts fate changes as early or earlier than transcription factor-based fate reporters, reveals the timing and existence of intermediate cell fates invisible to fixed-cell technologies, and identifies proliferative properties predictive of cell fate transitions. DEEP-MAP provides a versatile, universal strategy to map tissue evolution and organisation across many developmental and tissue engineering contexts.

A preclinical evaluation using a regenerative medicine methodology comprising an additively manufactured medical-grade ε-polycaprolactone β-tricalcium phosphate (mPCL-TCP) scaffold with a corticoperiosteal flap was undertaken in eight sheep with a tibial critical-size segmental bone defect (9.5 cm 3 , M size) using the regenerative matching axial vascularization (RMAV) approach. Biomechanical, radiological, histological, and immunohistochemical analysis confirmed functional bone regeneration comparable to a clinical gold standard control (autologous bone graft) and was superior to a scaffold control group (mPCL-TCP only). Affirmative bone regeneration results from a pilot study using an XL size defect volume (19 cm 3 ) subsequently supported clinical translation. A 27-year-old adult male underwent reconstruction of a 36-cm near-total intercalary tibial defect secondary to osteomyelitis using the RMAV approach. Robust bone regeneration led to complete independent weight bearing within 24 months. This article demonstrates the widely advocated and seldomly accomplished concept of “bench-to-bedside” research and has weighty implications for reconstructive surgery and regenerative medicine more generally.