ArticleMolecular therapy : the journal of the American Society of Gene Therapy2011
Lentiviral vector design and imaging approaches to visualize the early stages of cellular reprogramming.
Article in Molecular therapy : the journal of the American Society of Gene Therapy, 2011. The graph could read no effect estimate from its abstract, so it casts no vote on the map. Cited by 159 papers.
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159 citing papers in PubMed.
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- Germ line LCP1 mutations cause immunodeficiency with neutropenia, monocytopenia, lymphopenia, and defective cytokinesis.Blood advances · 2026Article
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- The nuclear matrix stabilizes primed-specific genes in human pluripotent stem cells.Nature cell biology · 2025Article
- ARID1A-BAF coordinates ZIC2 genomic occupancy for epithelial-to-mesenchymal transition in cranial neural crest specification.American journal of human genetics · 2024Article
- Long Terminal Repeats of Gammaretroviruses Retain Stable Expression after Integration Retargeting.Viruses · 2024Article
- A Simple Nonviral Method to Generate Human Induced Pluripotent Stem Cells Using SMAR DNA Vectors.Genes · 2024Article
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- Graft-derived neurons and bystander effects are maintained for six months after human iPSC-derived NESC transplantation in mice's cerebella.Scientific reports · 2024Article
- Interactions of the Immune System with Human Kidney Organoids.Transplant international : official journal of the European Society for Organ Transplantation · 2024Article
99 more citing papers are in PubMed but not listed here.
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13 authors.
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Abstract
Induced pluripotent stem cells (iPSCs) can be derived from somatic cells by gene transfer of reprogramming transcription factors. Expression levels of these factors strongly influence the overall efficacy to form iPSC colonies, but additional contribution of stochastic cell-intrinsic factors has been proposed. Here, we present engineered color-coded lentiviral vectors in which codon-optimized reprogramming factors are co-expressed by a strong retroviral promoter that is rapidly silenced in iPSC, and imaged the conversion of fibroblasts to iPSC. We combined fluorescence microscopy with long-term single cell tracking, and used live-cell imaging to analyze the emergence and composition of early iPSC clusters. Applying our engineered lentiviral vectors, we demonstrate that vector silencing typically occurs prior to or simultaneously with the induction of an Oct4-EGFP pluripotency marker. Around 7 days post-transduction (pt), a subfraction of cells in clonal colonies expressed Oct4-EGFP and rapidly expanded. Cell tracking of single cell-derived iPSC colonies supported the concept that stochastic epigenetic changes are necessary for reprogramming. We also found that iPSC colonies may emerge as a genetic mosaic originating from different clusters. Improved vector design with continuous cell tracking thus creates a powerful system to explore the subtle dynamics of biological processes such as early reprogramming events.
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