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Synthetic gene circuits for cell state detection and protein tuning in human pluripotent stem cells

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posted on 2023-06-23, 14:03 authored by Laura Prochazka, Yale S Michaels, Charles Lau, Ross D Jones, Mona Siu, Ting Yin, Diana Wu, Esther Jang, Mercedes Vázquez‐Cantú, Penney M Gilbert, Himanshu Kaul, Yaakov Benenson, Peter W Zandstra

During development, cell state transitions are coordinated through changes in the identity of molecular regulators in a cell type- and dose-specific manner. The ability to rationally engineer such transitions in human pluripotent stem cells (hPSC) will enable numerous applications in regenerative medicine. Herein, we report the generation of synthetic gene circuits that can detect a desired cell state using AND-like logic integration of endogenous miRNAs (classifiers) and, upon detection, produce fine-tuned levels of output proteins using an miRNA-mediated output fine-tuning technology (miSFITs). Specifically, we created an “hPSC ON” circuit using a model-guided miRNA selection and circuit optimization approach. The circuit demonstrates robust PSC-specific detection and graded output protein production. Next, we used an empirical approach to create an “hPSC-Off” circuit. This circuit was applied to regulate the secretion of endogenous BMP4 in a state-specific and finetuned manner to control the composition of differentiating hPSCs. Our work provides a platform for customized cell state-specific control of desired physiological factors in hPSC, laying the foundation for programming cell compositions in hPSC-derived tissues and beyond. 

Funding

P is supported by a Medicine by Design postdoctoral fellowship from the University of Toronto's Medicine by Design initiative, which received funding from Canada First Research Excellence Fund (CFREF) (MPDF-2017-01). This work is further supported by a CIHR grant (MOP 57885). PWZ is the Canada Research Chair in Stem Cell Bioengineering. PMG is the Canada Research Chair in Endogenous Repair. YB is funded by ERC (StG CellControl), Swiss National Science Foundation, NCCR “Molecular Systems Engineering” (31003A_149802) and ETH Zurich. HK acknowledges funding support by the Royal Academy of Engineering (Grant #RF\201920\19\275) and Michael Smith Foundation for Health Research (Trainee Award #18427). YM is supported by the Michael Smith Foundation for Health Research (Trainee Award #18453) anda CIHR Banting Postdoctoral Fellowship (BPF 170702). RDJ is supported by the Michael Smith Foundation for Health Research (Trainee Award #RT-2021-1946).

History

Citation

Molecular Systems Biology (2022) 18: e10886. https://doi.org/10.15252/msb.202110886

Author affiliation

School of Engineering

Version

  • VoR (Version of Record)

Published in

Molecular Systems Biology

Volume

18

Issue

11

Pagination

e10886

Publisher

EMBO

issn

1744-4292

eissn

1744-4292

Acceptance date

2022-10-17

Copyright date

2022

Available date

2023-06-23

Language

en

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