You are hereMarch 8, 2011 | Pluripotent Stem Cells
Engineered colour coded lentiviral vectors to visualise iPSC generation
From the laboratory of Axel Schambach in Germany comes a report describing a system which can be utilized to study the subtle dynamics of early reprogramming. Published in Molecular Therapy, Warlich et al.1 demonstrate the use of specially engineered colour-coded lentiviral vectors containing reprogramming factors (Oct4, Klf4, Sox2 and c-Myc) under the control of a retroviral promoter which induce rapid high level expression of reprogramming factors followed by rapid silencing. Using mouse embryonic fibroblasts containing an Oct4-EGFP reporter, the authors were able to observe cell-intrinsic stochastic processes following the induction of pluripotency, enabling the visualisation of the conversion of fibroblasts to induced pluripotent stem cells (iPSC). In their system, the expression of red fluorescent protein in transduced cells indicates reprogramming factor expression whilst green fluorescence indicates the emergence of iPSC. Using fluorescence microscopy, long term single cell tracking and live cell imaging, they demonstrate that vector silencing occurs prior to or at the onset of the expression of the pluripotency marker Oct4. They reveal that stochastic epigenetic changes are required for reprogramming and that early reprogrammed colonies can emerge as a genetic mosaic formed on the basis of epigenetic variability, particularly under conditions that increase reprogramming efficiency (e.g. the addition of valproic acid during induction). They observed heterogeneity of EGFP expression in iPSC colonies and report that not all cells within an iPSC colony expressed EGFP - some cells within the colony maintained the expression of exogenous reprogramming factors and failed to reprogramme. The observed heterogeneity within clonal colonies of genetically identical cells undergoing reprogramming in this study is particularly interesting and supports Yamanaka’s hypothesis of a stochastic model for induced pluripotency in somatic cells2. Studies such as this help to discern the mechanisms underlying and variables guiding the reprogramming process by gene transfer of reprogramming transcription factors into somatic cells.
1 Warlich E. Et al., ‘Lentiviral Vector Design and Imaging Approaches to Visualize the Early Stages of Cellular Reprogramming’ Molecular Therapy. 1 February 2011 doi:10.1038/mt.2010.314
2 Yamanaka S. ‘Elite and stochastic models for induced pluripotent stem cell generation’ Nature. 2009 Jul 2;460(7251):49-52.