Retrospective identification of cell-intrinsic factors that mark pluripotency potential in rare somatic cells

Naveen Jain, Yogesh Goyal, Margaret C. Dunagin, Christopher J. Cote, Ian A. Mellis, Benjamin Emert, Connie L. Jiang, Ian P. Dardani, Sam Reffsin, Miles Arnett, Wenli Yang, Arjun Raj*

*Corresponding author for this work

Research output: Contribution to journalArticlepeer-review

3 Scopus citations

Abstract

Pluripotency can be induced in somatic cells by the expression of OCT4, KLF4, SOX2, and MYC. Usually only a rare subset of cells reprogram, and the molecular characteristics of this subset remain unknown. We apply retrospective clone tracing to identify and characterize the rare human fibroblasts primed for reprogramming. These fibroblasts showed markers of increased cell cycle speed and decreased fibroblast activation. Knockdown of a fibroblast activation factor identified by our analysis increased the reprogramming efficiency. We provide evidence for a unified model in which cells can move into and out of the primed state over time, explaining how reprogramming appears deterministic at short timescales and stochastic at long timescales. Furthermore, inhibiting the activity of LSD1 enlarged the pool of cells that were primed for reprogramming. Thus, even homogeneous cell populations can exhibit heritable molecular variability that can dictate whether individual rare cells will reprogram or not.

Original languageEnglish (US)
Pages (from-to)109-133.e10
JournalCell Systems
Volume15
Issue number2
DOIs
StatePublished - Feb 21 2024

Funding

We thank members of the Arjun Raj Lab, particularly Lauren Beck, Phil Burnham, Lee Richman, Yael Heyman, Karun Kiani, Eric Sanford, Allison Cote, and Cat Triandafillou for insightful discussions related to this work. We thank Blake Caldwell of the Marisa Bartolomei lab, Jingchao Zhang of the Ken Zaret lab, Chris Lengner, as well as Wenli Yang and Rachel Truitt, of the Penn iPSC core for advice on iPSC handling and reprogramming. We thank Daniel Xu of the Shelley Berger lab for assistance with immunoblotting protocols and Felicia Peng of the John Murray lab for assistance with copy editing. We thank the Genomics Facility at the Wistar Institute, especially Sonali Majumdar and Sandy Widura for assistance with single-cell partitioning and addition of 10x cell identifiers. We thank the Flow Cytometry Core Laboratory at the Children’s Hospital of Philadelphia Research Institute for assistance with flow cytometry and fluorescence-activated sorting. Finally, N.J. thanks his beloved cat Brioche for keeping him company on her favorite chair or in her favorite box during hours of writing and proofreading (and for some unanticipated edits) as well as Felicia Peng for her unwavering and invaluable support. N.J. acknowledges support from the Michael Brown Fellowship , NIH T32 GM007170 , and NIH F30 HD103378 . Y.G. acknowledges support from the Burroughs Welcome Fund Career Awards at the Scientific Interface, a grant from Research Catalyst Program from the McCormick School of Engineering at Northwestern University, and Northwestern University’s startup funds. I.A.M. acknowledges support from NIH F30 NS100595 . B.E. acknowledges support from NIH F30 CA236129 , NIH T32 GM007170 , and NIH T32 HG000046 . C.L.J. acknowledges support from NIH F30 HG010822 , NIH T32 DK007780 , and NIH T32 GM007170 . A.R. acknowledges support from TR01 GM137425 , NIH R01 CA238237 , NIH R01 CA232256 , NIH 4DN U01 DK127405 , and NSF EFRI EFMA19-33400 . We thank members of the Arjun Raj Lab, particularly Lauren Beck, Phil Burnham, Lee Richman, Yael Heyman, Karun Kiani, Eric Sanford, Allison Cote, and Cat Triandafillou for insightful discussions related to this work. We thank Blake Caldwell of the Marisa Bartolomei lab, Jingchao Zhang of the Ken Zaret lab, Chris Lengner, as well as Wenli Yang and Rachel Truitt, of the Penn iPSC core for advice on iPSC handling and reprogramming. We thank Daniel Xu of the Shelley Berger lab for assistance with immunoblotting protocols and Felicia Peng of the John Murray lab for assistance with copy editing. We thank the Genomics Facility at the Wistar Institute, especially Sonali Majumdar and Sandy Widura for assistance with single-cell partitioning and addition of 10x cell identifiers. We thank the Flow Cytometry Core Laboratory at the Children's Hospital of Philadelphia Research Institute for assistance with flow cytometry and fluorescence-activated sorting. Finally, N.J. thanks his beloved cat Brioche for keeping him company on her favorite chair or in her favorite box during hours of writing and proofreading (and for some unanticipated edits) as well as Felicia Peng for her unwavering and invaluable support. N.J. acknowledges support from the Michael Brown Fellowship, NIH T32 GM007170, and NIH F30 HD103378. Y.G. acknowledges support from the Burroughs Welcome Fund Career Awards at the Scientific Interface, a grant from Research Catalyst Program from the McCormick School of Engineering at Northwestern University, and Northwestern University's startup funds. I.A.M. acknowledges support from NIH F30 NS100595. B.E. acknowledges support from NIH F30 CA236129, NIH T32 GM007170, and NIH T32 HG000046. C.L.J. acknowledges support from NIH F30 HG010822, NIH T32 DK007780, and NIH T32 GM007170. A.R. acknowledges support from TR01 GM137425, NIH R01 CA238237, NIH R01 CA232256, NIH 4DN U01 DK127405, and NSF EFRI EFMA19-33400. N.J. and A.R. conceived and designed the project with valuable input from Y.G. I.A.M. and B.E. N.J. designed, performed, and analyzed all experiments, supervised by A.R. I.A.M. and W.Y. assisted N.J. with optimizing reprogramming conditions in hiF-T cells. Y.G. I.P.D. S.R. C.L.J. B.E. and M.A. helped with some hiF-T cell maintenance and reprogramming experiments. I.P.D. assisted N.J. with automated RNA FISH and DAPI scans. M.C.D. and C.J.C. assisted N.J. with generating RNA FISH probes and single-blind, manual quantification of RNA FISH counts and iPSC colony counts. S.R. assisted N.J. with generating CRISPR knockdown constructs. The protocols for barcode library generation and the subsequent initial pipeline for retrieval of barcodes from genomic DNA sequencing were developed by B.E. The initial pipeline for retrieval of barcodes from copy DNA and matching single-cell sequencing data was developed by Y.G. The analyses and scripts for measuring similarity in single-cell RNA gene expression profiles were conceived and developed by Y.G. and C.L.J. N.J. and A.R. wrote the manuscript. The authors read and approved the final manuscript. A.R. receives royalties related to Stellaris RNA FISH probes.

Keywords

  • cellular barcoding
  • iPSC reprogramming
  • lineage tracing
  • systems biology

ASJC Scopus subject areas

  • Pathology and Forensic Medicine
  • Histology
  • Cell Biology

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