Single-cell RNA sequencing reveals metallothionein heterogeneity during hESC differentiation to definitive endoderm.
Junjie Lu, Anna Baccei, Edroaldo Lummertz da Rocha, Christelle Guillermier, Sean McManus, Lydia A Finney, Cheng Zhang, Matthew L Steinhauser, Hu Li, Paul H Lerou
Author Information
Junjie Lu: Department of Pediatrics, Division of Neonatology and Newborn Medicine, Massachusetts General Hospital for Children, Harvard Medical School, Boston, MA 02114, USA.
Anna Baccei: Department of Pediatrics, Division of Neonatology and Newborn Medicine, Massachusetts General Hospital for Children, Harvard Medical School, Boston, MA 02114, USA.
Edroaldo Lummertz da Rocha: Center for Individualized Medicine, Department of Molecular Pharmacology & Experimental Therapeutics, Mayo Clinic, Rochester, MN 55905, USA.
Christelle Guillermier: Department of Medicine, Division of Genetics, Center for NanoImaging, Brigham and Women's Hospital, Harvard Medical School, Boston, MA 02115, USA.
Sean McManus: Department of Pediatrics, Division of Neonatology and Newborn Medicine, Massachusetts General Hospital for Children, Harvard Medical School, Boston, MA 02114, USA.
Lydia A Finney: Leadership Institute, Argonne National Laboratory, 9700 S Cass Ave, Lemont, IL 60439, USA.
Cheng Zhang: Center for Individualized Medicine, Department of Molecular Pharmacology & Experimental Therapeutics, Mayo Clinic, Rochester, MN 55905, USA.
Matthew L Steinhauser: Department of Medicine, Division of Genetics, Center for NanoImaging, Brigham and Women's Hospital, Harvard Medical School, Boston, MA 02115, USA.
Hu Li: Center for Individualized Medicine, Department of Molecular Pharmacology & Experimental Therapeutics, Mayo Clinic, Rochester, MN 55905, USA.
Paul H Lerou: Department of Pediatrics, Division of Neonatology and Newborn Medicine, Massachusetts General Hospital for Children, Harvard Medical School, Boston, MA 02114, USA. Electronic address: plerou@partners.org.
Differentiation of human pluripotent stem cells towards definitive endoderm (DE) is the critical first step for generating cells comprising organs such as the gut, liver, pancreas and lung. This in-vitro differentiation process generates a heterogeneous population with a proportion of cells failing to differentiate properly and maintaining expression of pluripotency factors such as Oct4. RNA sequencing of single cells collected at four time points during a 4-day DE differentiation identified high expression of metallothionein genes in the residual Oct4-positive cells that failed to differentiate to DE. Using X-ray fluorescence microscopy and multi-isotope mass spectrometry, we discovered that high intracellular zinc level corresponds with persistent Oct4 expression and failure to differentiate. This study improves our understanding of the cellular heterogeneity during in-vitro directed differentiation and provides a valuable resource to improve DE differentiation efficiency.
