Synthetic genetic circuits to uncover 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:10.1126/sciadv.adg8495

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

Sci Adv. 2023 Dec;9(48):eadg8495. doi: 10.1126/sciadv.adg8495. Epub 2023 Nov 29.

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

8

9}, Eliezer Aravera^{1

3

10}, Simone Bruno¹¹, Sebastian Palacios^{1

2

3

11

12}, Ron Weiss^{1

3

12}, James J Collins^{1

2

3

13

14}, Domitilla Del Vecchio^{3

11}, Thorsten M Schlaeger⁵

Affiliations

¹ Department of Biological Engineering, Massachusetts Institute of Technology (MIT), Cambridge, MA 02139, USA.
² Institute for Medical Engineering and Science, MIT, Cambridge, MA 02139, 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 Stem Cell Program, Boston Children's Hospital, Boston, MA 02115, USA.
⁶ Harvard University, Boston, MA 02115, USA.
⁷ Berlin Institute for Medical Systems Biology (BIMSB), Max Delbrück Center for Molecular Medicine in the Helmholtz-Association, Berlin 10115, Germany.
⁸ Department of Nephrology and Medical Intensive Care, Charité - Universitätsmedizin Berlin, Medizinische Klinik m.S. Nephrologie und Intensivmedizin, Berlin 10117, Germany.
⁹ Berlin Institute of Health, Berlin 13125, Germany.
¹⁰ Department of Biomedical Informatics, Stony Brook University, Stony Brook, NY 11794, USA.
¹¹ Department of Mechanical Engineering, MIT, Cambridge, MA 02139, USA.
¹² Department of Electrical Engineering and Computer Science, MIT, Cambridge, MA 02139, USA.
¹³ Wyss Institute for Biologically Inspired Engineering, Harvard University, Boston, MA 02215, USA.
¹⁴ 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. We develop a system that accurately reports OCT4 protein levels in live cells and use it to reveal the trajectories of OCT4 in successful reprogramming. Our system comprises a synthetic genetic circuit that leverages noise to generate a wide range of OCT4 trajectories and a microRNA targeting endogenous OCT4 to set total cellular OCT4 protein levels. By fusing OCT4 to a fluorescent protein, we are able to track OCT4 trajectories with clonal resolution via live-cell imaging. We discover that a supraphysiological, stable OCT4 level is required, but not sufficient, for efficient iPSC colony formation. Our synthetic genetic circuit design and high-throughput live-imaging pipeline are generalizable for investigating TF dynamics for other cell fate programming applications.

MeSH terms

Cell Differentiation / genetics
Cells, Cultured
Cellular Reprogramming* / genetics
Fibroblasts / metabolism
Humans
Induced Pluripotent Stem Cells* / metabolism
Transcription Factors / genetics
Transcription Factors / metabolism

Substances

Transcription Factors
POU5F1 protein, human

Grants and funding

R01 EB024591/EB/NIBIB NIH HHS/United States