Toward Precision Bioengineering of Human-Induced Pluripotent Stem Cells: A Modular Platform for Transcriptional Reprogramming and Targeted Genomic Integration

Abstract

Cellular reprogramming marks a new era of regenerative medicine and immunotherapy, enabling stem cell researchers to develop new models, therapeutics, and technologies with human-induced pluripotent stem cells (hiPSCs) at their core. However, hiPSC translational research is often limited by slow differentiation kinetics, heterogeneity within differentiated populations, transgene silencing during lineage commitment and unpredictable genomic profiles. Current methods for bioengineering hiPSCs lack the precision and modularity required to develop regenerative medicine-based therapeutics and complex modelling applications. This thesis describes the development of a modular bioengineering platform that demonstrates accelerated transcriptional reprogramming and precise, stable targeted genomic integration in human hiPSCs. By characterizing the feasibility of these two approaches, a framework and toolkit for downstream precise stem cell-based bioengineering were established.

Here, we designed a multiplexed CRISPR activation system for the directed differentiation of hiPSCs by targeting coordinated transcriptional programs and genes. This modular approach generated human iPSC-derived brain endothelial-like cells (iBECs), shortening conventional differentiation timelines by 61% while maintaining and expanding key phenotypic markers. Moreover, to achieve stable and safe transgene expression, we describe a site-specific engineering platform for hiPSCs across four genomic safe harbours (GSH): AAVS1, CCR5, hRosa26 and the novel hRogi1 locus. We evaluated the performance of modular genetic cassettes, benchmarking the stability of traditional (CAG, EFS) and unique (CBX3.1) promoters to mitigate gene silencing during lineage differentiation into germ layers. Finally, by using recombinase-mediated cassette exchange (RMCE), we demonstrate a proof-of-principle “plug-and-play” pipeline that enables the swapping of synthetic cargoes, providing a modular platform for downstream translational applications. Collectively, this research delivers a comprehensively optimized bioengineering toolkit and blueprint for hiPSCs that improves differentiation fidelity and the reliability of genomic modifications. The modularity of this toolkit positions this research uniquely and lays the basis for the future advancement of next-generation cell therapies and complex models.