Synthetic genetic circuits to uncover and enforce the OCT4 trajectories of successful reprogramming of human fibroblasts

Katherine Ilia; Nika Shakiba; Trevor Bingham; Ross D Jones; Michael M Kaminski; Eliezer Aravera; Simone Bruno; Sebastian Palacios; Ron Weiss; James J Collins; Domitilla Del Vecchio; Thorsten M Schlaeger

doi:10.1101/2023.01.25.525529

Synthetic genetic circuits to uncover and enforce the OCT4 trajectories of successful reprogramming of human fibroblasts

bioRxiv [Preprint]. 2023 Jan 25:2023.01.25.525529. doi: 10.1101/2023.01.25.525529.

Authors

Katherine Ilia^{1

2

3}, Nika Shakiba^{1

3

4}, Trevor Bingham^{5

6}, Ross D Jones^{1

3

4}, Michael M Kaminski^{1

3

7}, Eliezer Aravera^{1

3

8}, Simone Bruno⁹, Sebastian Palacios^{1

3

10}, Ron Weiss^{1

3

10}, James J Collins^{1

2

3

11

12}, Domitilla Del Vecchio^{3

9}, Thorsten M Schlaeger⁵

Affiliations

¹ Department of Biological Engineering, Massachusetts Institute of Technology (MIT), Cambridge, MA, USA.
² Institute for Medical Engineering and Science, MIT, Cambridge, MA, USA.
³ Synthetic Biology Center, Massachusetts Institute of Technology, Cambridge, MA, 02139, USA.
⁴ School of Biomedical Engineering, University of British Columbia, Vancouver, British Columbia, V6T 1Z3 Canada.
⁵ Boston Children's Hospital Stem Cell Program, Boston Children's Hospital, Boston, MA, 02115, USA.
⁶ Harvard University, Boston, MA, 02115, USA.
⁷ Max Delbrück Center for Molecular Medicine, Berlin, 13125, Germany.
⁸ College of Engineering and Applied Sciences, Stony Brook University, Stony Brook, NY, 11794, USA.
⁹ Department of Mechanical Engineering, Massachusetts Institute of Technology, Cambridge, MA, 02139, USA.
¹⁰ Department of Electrical Engineering and Computer Science, Massachusetts Institute of Technology, Cambridge, MA, 02139, USA.
¹¹ Wyss Institute for Biologically Inspired Engineering, Harvard University, Boston, MA, USA.
¹² Broad Institute of MIT and Harvard, Cambridge, MA, 02139, USA.

Abstract

Reprogramming human fibroblasts to induced pluripotent stem cells (iPSCs) is inefficient, with heterogeneity among transcription factor (TF) trajectories driving divergent cell states. Nevertheless, the impact of TF dynamics on reprogramming efficiency remains uncharted. Here, we identify the successful reprogramming trajectories of the core pluripotency TF, OCT4, and design a genetic controller that enforces such trajectories with high precision. By combining a genetic circuit that generates a wide range of OCT4 trajectories with live-cell imaging, we track OCT4 trajectories with clonal resolution and find that a distinct constant OCT4 trajectory is required for colony formation. We then develop a synthetic genetic circuit that yields a tight OCT4 distribution around the identified trajectory and outperforms in terms of reprogramming efficiency other circuits that less accurately regulate OCT4. Our synthetic biology approach is generalizable for identifying and enforcing TF dynamics for cell fate programming applications.

Publication types

Preprint

Grants and funding

R01 EB024591/EB/NIBIB NIH HHS/United States