Lentiviral Vector Design and Imaging Approaches to Visualize the Early Stages of Cellular Reprogramming

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.

Reticular dysgenesis–associated AK2 protects hematopoietic stem and progenitor cell development from oxidative stress

“…Western blot analysis from patient fibroblasts and CD3 + T cells demonstrated reduced protein expression compared with control (Henderson et al, 2013), and a PROVEAN score of 3.871 predicted this mutation to be deleterious. The cultured fibroblasts were reprogrammed to pluripotency by using an excisable, self-inactivating, codon-optimized lentiviral vector carrying OCT4, SOX2, KLF4, cMYC, and the reporter dTomato in a single cassette (Warlich et al, 2011). Immunofluorescence imaging and quantitative realtime PCR analysis confirmed the stemness of the generated iPSC line by expression of the characteristic pluripotency markers TRA-1-81, TRA-1-60, NANOG, OCT-4, SSEA-3, and SSEA-4 (Fig.…”

BAF Complex Enhances Reprogramming of Adult Human Fibroblasts

“…Since it has been shown recently that, contrary to the BAF complex in mouse stem cells, the BAF complex in human embryonic stem cells also contains Baf170 [29], and there might be differences in its functional conservation between mouse and human. To investigate the role of Brg1 and Baf155 in human reprogramming, we utilized a reprogramming cassette consisting of optimized human Oct4, Sox2, Klf4 and c-Myc along with a tomato reporter gene (SF hOSKM-idTomato) [30]. 100,000 F134 cells were infected with SF hOSKM-idTomato.…”

“…Human adult fibroblasts F134 [32] were reprogrammed with a lentiviral vector encoding the human cDNAs of OCT4, SOX2, KLF4 and c-MYC under the control of the SFFV promoter [30]. Retroviral vectors encoding Brg1 and Baf155 [2] and the lentiviral vector were…”

Letter to the Editor: Production of Mature Healthy Hematopoietic Cells from Induced Pluripotent Stem Cells Derived from an AML Diagnostic Sample Containing the t(8;21) Translocation

“…Transduction of primary AML patient cells (0.35 3 10 6 ) was accomplished on a Retronectin (Takara Bio, Shiga, Otsu, Japan) coated 24-well plate with vesicular stomatitis virus glycoprotein-pseudotyped lentiviral vector particles encoding for the reprogramming cassette following standard protocols [2]. Three days after transduction, AML patient cells were transferred to a six-well plate containing irradiated murine embryonic fibroblasts and iPSC growth conditions according to published protocols [3].…”

“…We used a polycistronic 4-in-1 lentiviral vector to transfer the four Yamanaka reprogramming factors to generate iPSCs [2] from a pretherapy diagnostic bone marrow sample from a 29-year-old male patient with FAB M2 AML following informed consent according to the Declaration of Helsinki. Routine genetic analyses revealed that the AML sample had wild-type NPM1 and FLT3, contained no inv16/ t(16;16) or t(15;17) aberrations, but did harbor the t(8;21).…”