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ABSTRACT Human pluripotent stem cells (hPSCs) offer an unprecedented opportunity to model diverse cell types and tissues. To enable systematic exploration of the programming landscape

mediated by transcription factors (TFs), we present the Human TFome, a comprehensive library containing 1,564 TF genes and 1,732 TF splice isoforms. By screening the library in three hPSC

lines, we discovered 290 TFs, including 241 that were previously unreported, that induce differentiation in 4 days without alteration of external soluble or biomechanical cues. We used four

of the hits to program hPSCs into neurons, fibroblasts, oligodendrocytes and vascular endothelial-like cells that have molecular and functional similarity to primary cells. Our

cell-autonomous approach enabled parallel programming of hPSCs into multiple cell types simultaneously. We also demonstrated orthogonal programming by including oligodendrocyte-inducible

hPSCs with unmodified hPSCs to generate cerebral organoids, which expedited in situ myelination. Large-scale combinatorial screening of the Human TFome will complement other strategies for

cell engineering based on developmental biology and computational systems biology. Access through your institution Buy or subscribe This is a preview of subscription content, access via your

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subscriptions * Read our FAQs * Contact customer support SIMILAR CONTENT BEING VIEWED BY OTHERS EFFICIENT GENERATION OF FUNCTIONAL NEURONS FROM MOUSE EMBRYONIC STEM CELLS VIA NEUROGENIN-2

EXPRESSION Article 18 August 2023 IDENTIFICATION OF ASCL1 AS A DETERMINANT FOR HUMAN IPSC-DERIVED DOPAMINERGIC NEURONS Article Open access 15 November 2021 ITERATIVE TRANSCRIPTION FACTOR

SCREENING ENABLES RAPID GENERATION OF MICROGLIA-LIKE CELLS FROM HUMAN IPSC Article Open access 10 June 2025 DATA AVAILABILITY. Next-generation sequencing data that support the findings of

the study are available in the Gene Expression Omnibus using accession code GSE159786. CODE AVAILABILITY The code that supports the findings of this study is available from the corresponding

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H. Lee for critical feedback and the Church and Busskamp laboratories for support. We acknowledge technical support from the Harvard Biopolymers Facility, the Harvard Division of Immunology

Flow Cytometry Core Facility, the Beth Israel Deaconess Medical Center Flow Cytometry Core, the Wyss Flow Cytometry and Microscopy Core, M. Ericsson and P. Coughlin at the Harvard Medical

School Electron Microscopy Facility, M. T. Gianatasio at the Dana-Farber/Harvard Cancer Center Specialized Histopathology Core and Rodent Histopathology Core (both supported, in part, by

National Cancer Institute Cancer Center Support grant NIH 5 P30 CA06516) and Harvard Medical School Orchestra Research Computing. We also thank the TU Dresden Center for Molecular and

Cellular Bioengineering Advanced Imaging, Deep Sequencing, Flow Cytometry and Stem Cell Engineering core facilities. We would also like to thank J. Gray’s laboratory for electrophysiology

support, S. Jeanty and J. Lee (Church lab, Harvard Medical School) for the PGP1 Sendai virus hiPSC line, G. Sheynkman and W. Glindmeyer for helpful discussions, A. Jolma, K. Nitta and K.

Said for technical assistance and M. Lemieux and J. McDade for their support in depositing the library to Addgene. A.H.M.N. was supported by an NSERC Postgraduate Fellowship and a Peter and

Carolyn Lynch Foundation Fellowship. J.E.R.A. was supported by the DIGS-BB program. S.L.S. is a Shurl and Kay Curci Foundation Fellow of the Life Sciences Research Foundation. The Ellison

Foundation and Institute Sponsored Research funds from the DFCI Strategic Initiative supported M.V. and D.E.H. The project was supported by the Volkswagen Foundation (Freigeist - A110720),

the European Research Council (ERC-StG-678071 - ProNeurons) and the Deutsche Forschungsgemeinschaft (SPP2127, EXC-2068-390729961 - Cluster of Excellence - Physics of Life at TU Dresden and

EXC-2151-390873048 – Cluster of Excellence – ImmunoSensation2 at the University of Bonn) to V.B. G.M.C. acknowledges funding from National Human Genome Research Institute grants P50 HG005550

‘Center for Casual Variation’, RM1 HG008525 ‘Center for Genomically Engineered Organs’, the Simons Foundation for Autism Research Initiative (368485), the Blavatnik Biomedical Accelerator

at Harvard University, the FunGCAT program from the Office of the Director of National Intelligence Intelligence Advanced Research Projects Activity, via the Army Research Office, under

federal award no. W911NF-17-2-0089 and research funding from R. Merkin and the Merkin Family Foundation. AUTHOR INFORMATION Author notes * Jesus Eduardo Rojo Arias Present address:

Wellcome-MRC Cambridge Stem Cell Institute, Jeffrey Cheah Biomedical Centre, Cambridge Biomedical Campus, University of Cambridge, Cambridge, UK * These authors contributed equally: Alex H.

M. Ng, Parastoo Khoshakhlagh. AUTHORS AND AFFILIATIONS * Department of Genetics, Blavatnik Institute, Harvard Medical School, Boston, MA, USA Alex H. M. Ng, Parastoo Khoshakhlagh, Evan

Appleton, Kiavash Kiaee, Richie E. Kohman, Matthew Dysart, Kathleen Leeper, Wren Saylor, Jeremy Y. Huang, David E. Hill, Marc Vidal & George M. Church * Wyss Institute for Biologically

Inspired Engineering at Harvard University, Boston, MA, USA Alex H. M. Ng, Parastoo Khoshakhlagh, Evan Appleton, Kiavash Kiaee, Richie E. Kohman, Andyna Vernet, Matthew Dysart, Kathleen

Leeper, Wren Saylor, Jeremy Y. Huang, Amanda Graveline & George M. Church * GC Therapeutics, Inc, Cambridge, MA, USA Alex H. M. Ng, Parastoo Khoshakhlagh, Evan Appleton, Kiavash Kiaee 

& George M. Church * Technische Universität Dresden, Center for Molecular and Cellular Bioengineering (CMCB), Center for Regenerative Therapies Dresden (CRTD), Dresden, Germany Jesus

Eduardo Rojo Arias, Giovanni Pasquini, Anka Swiersy & Volker Busskamp * Department of Cardiac Surgery, Boston Children’s Hospital, Boston, MA, USA Kai Wang & Juan M. Melero-Martin *

Department of Surgery, Harvard Medical School, Boston, MA, USA Kai Wang & Juan M. Melero-Martin * Gladstone Institutes and University of California, San Francisco, San Francisco, CA, USA

Seth L. Shipman * Department of Biochemistry, University of Cambridge, Cambridge, UK Jussi Taipale * Department of Medical Biochemistry and Biophysics, Karolinska Institute, Stockholm,

Sweden Jussi Taipale * Applied Tumor Genomics Program, Faculty of Medicine, University of Helsinki, Helsinki, Finland Jussi Taipale * Center for Cancer Systems Biology (CCSB), Dana-Farber

Cancer Institute, Boston, MA, USA David E. Hill & Marc Vidal * Department of Ophthalmology, Medical Faculty, University of Bonn, Bonn, Germany Volker Busskamp Authors * Alex H. M. Ng

View author publications You can also search for this author inPubMed Google Scholar * Parastoo Khoshakhlagh View author publications You can also search for this author inPubMed Google

Scholar * Jesus Eduardo Rojo Arias View author publications You can also search for this author inPubMed Google Scholar * Giovanni Pasquini View author publications You can also search for

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CONTRIBUTIONS A.H.M.N., P.K., V.B. and G.M.C. conceived the idea, led the study and designed all experiments. A.H.M.N. and P.K. performed most of the experiments and analyses, with

significant technical contributions from J.E.R.A, G.P., K.W., A.S., S.L.S., E.A., K.K., R.E.K., A.V., M.D., K.L., W.S., J.Y.H., A.G., J.T., D.E.H., M.V. and J.M.M.-M. V.B. and G.M.C. oversaw

the study. A.H.M.N., P.K. and V.B. wrote the manuscript with input and feedback from all authors. CORRESPONDING AUTHORS Correspondence to Volker Busskamp or George M. Church. ETHICS

DECLARATIONS COMPETING INTERESTS A.H.M.N., P.K., V.B. and G.M.C. are inventors on patents filed by the Presidents and Fellows of Harvard College. Full disclosure for G.M.C. is available at

http://arep.med.harvard.edu/gmc/tech.html. A.H.M.N., P.K. and G.M.C. are co-founders of and have equity in GC Therapeutics, Inc. No reagents or funding from GC Therapeutics were used in this

study. ADDITIONAL INFORMATION PUBLISHER’S NOTE Springer Nature remains neutral with regard to jurisdictional claims in published maps and institutional affiliations. SUPPLEMENTARY

INFORMATION SUPPLEMENTARY INFORMATION Supplementary Figs. 1–7 REPORTING SUMMARY SUPPLEMENTARY TABLE 1 TFs in the Human TFome SUPPLEMENTARY TABLE 2 TFome screen sequencing statistics

SUPPLEMENTARY TABLE 3 TFome screen differentiation scores SUPPLEMENTARY TABLE 4 Novelty and tissue expression of 290 TF hits SUPPLEMENTARY TABLE 5 RNA-seq statistics and expression profiles

SUPPLEMENTARY TABLE 6 TFs involved in oligodendrocyte development SUPPLEMENTARY TABLE 7 Exact _P_ values for statistical tests RIGHTS AND PERMISSIONS Reprints and permissions ABOUT THIS

ARTICLE CITE THIS ARTICLE Ng, A.H.M., Khoshakhlagh, P., Rojo Arias, J.E. _et al._ A comprehensive library of human transcription factors for cell fate engineering. _Nat Biotechnol_ 39,

510–519 (2021). https://doi.org/10.1038/s41587-020-0742-6 Download citation * Received: 03 November 2019 * Accepted: 19 October 2020 * Published: 30 November 2020 * Issue Date: April 2021 *

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