A whole new generation

Induced pluripotent stem cells promise much for clinical and pure research, but the reliable and robust generation of these cells has proved to be a difficult task. Here we learn of a new method which offers vector free iPSCs The ability to create induced pluripotent stem cells (iPSCs) has created new opportunities in both clinical and research settings. Patient-specific cell replenishment therapies are under development, and iPSCs are enabling construction of human models of complex diseases and revealing important insights that can lead to a more personalised approach to medicine. In addition, researchers are using iPSCs for drug testing, screening potential therapeutics against large numbers of patient-specific cells prior to initiating clinical trials in order to create studies that are more targeted and likely to succeed. iPSCs are also being used in investigative toxicity studies to help identify drug candidates with toxicity concerns earlier in the discovery process, reducing the development costs associated with late-stage clinical trial failures. Despite the tremendous potential of iPSCs, researchers had been held back the by time-consuming and inefficient methods required to generate these cells. Though several key developments have improved iPSC generation techniques, challenges still remain. [caption id="attachment_37339" align="alignright" width="200"] Figure 1. A rapid and reliable assay to validate TAT-Cre transduction and recombination activities. A 293T cell line stably expressing a double fluorescent reporter construct was used to monitor Cre recombination (A). Cells express RFP before Cre-recombination. Cre-mediated recombination induces the expression of the GFP, by deleting the LoxP-flanking RFP gene. Maximal GFP expression was achieved when 4 mM TAT-Cre was used to treat the cells overnight. (B, C). Dose dependent increases of GFP expressing cells were quantified using flow cytometry analysis (C). Percentage of GFP positive cells were plotted against the dosage of TAT-Cre used (D). Consistent lot-to-lot performance was observed.[/caption] In 2007, the first iPSCs were created by converting somatic skin cells through simultaneous co-infection with four separate retroviral expression vectors (Oct-4, Klf4, Sox-2, and c-Myc)¹.Each vector carried one transcription factor, resulting in a high number of genomic integrations. This posed a safety risk and could result in a heterogeneous cell population. Alternative methods, including using plasmids and non-integrating adenovirus vectors to deliver the transcription factors, were far less efficient than using retroviral vectors. A new approach then allowed researchers to generate human and mouse iPSCs using a single polycistronic lentiviral vector. STEMCCA reprogramming kits use a single lentiviral vector that expresses a “stem cell cassette” containing the four transcription factors. Use of a single vector significantly reduces the number of viral integrations required for the derivation of iPSCs; in some cases, iPSC clones possessing only a single viral integrant can be isolated². Reprogramming of somatic cells using viral transduction of defined transcription factors remains a widely used method to obtain iPSCs. However, the presence of viral transgenes in iPSCs is undesirable, as they have been shown to affect differentiation potential and raise the possibility of insertional mutagenesis leading to malignant transformation. Various techniques have attempted to address this issue, including RNA transfection, non-integrating viruses, protein transduction and site-specific recombinases to excise the transgenes after reprogramming. [caption id="attachment_37340" align="alignleft" width="200"] Figure 2. Schematic of cell-permeant TAT-Cre fusion protein. The amino acid sequence of the amino terminus is depicted showing the TAT peptide sequence in red (A). Purification of recombinant TAT-Cre from bacteria, as analysed by Coomassie blue staining of an SDS-PAGE (B). The lanes represent the following: 1 – total lysates; 2 – insoluble fraction; 3 – supernatant; 4 – flow through; 5 – control; 10 – 1 hour IPTG induced culture. Numbers on the left indicate molecular weight (kDA) of marker proteins. Cre protein is approximately 41 kDa[/caption] This article describes the efficient generation of transgene-free mouse and human iPSCs through the use of a Cre-excisable polycistronic lentiviral vector expressing the “stem cell cassette” comprised of all four transcription factors, followed by exposure of the fully reprogrammed iPSC to cell-permeable TAT-Cre recombinant protein. In addition, this article describes a simple and robust PCR strategy that enables fast identification of deleted clones directly from primary iPSC colonies. Materials and Methods A 293T cell line stably expressing a double fluorescent reporter construct was used to validate and monitor TAT-Cre recombination (Figure 1). Cells express RFP before Cre-recombination, and Cre-mediated recombination induces the expression of the GFP by deleting the LoxP-flanking RFP gene. Maximal GFP expression was achieved when 4 mM TAT-Cre was used to treat the cells overnight. Human iPSC excision Human iPSCs were transitioned to feeder-free conditions. Rho-associated protein kinase (ROCK) inhibitor was added one day before passaging, and the cells were dissociated into a single cell suspension. A 12-well plate was used, with 50,000-100,000 cells added to each well. To allow the cells to attach, the wells were incubated overnight; they were then incubated with 2-5 mM of the protein TAT-Cre (Figure 2). Colonies started to re-emerge and could be expanded after 7-9 days. Finally, genomic DNA was extracted for real-time quantitative PCR analysis (Figure 3). [caption id="attachment_37342" align="alignright" width="200"] Figure 3. Human excision: Time course of TAT-Cre treatment (A). Individual colonies were picked at 9-14 days post-treatment and added directly to Lysis Buffer for real time quantitative PCR analysis. The Ct value of WPRE in the excised samples should correlate with the negative controls, untreated hiPSCs and no template control (B).[/caption] Mouse iPSC excision Two protocols were used for the mouse iPSC excision; one used a feeder-based culture and the other a serum-free, feeder-free culture. For the feeder-based culture protocol, mouse iPSCs were grown on pMEF feeder layer in mESC media. The miPSCs and pMEF cells were dissociated into a single cell suspension with Accutase solution. Then, 10,000 cells were treated with 4 mM TAT-Cre in 200 mL of mESC media for 2-4 hours in a 96-well plate at 37°C. Cells were transferred to a fresh 6-well plate coated with pMEF feeders. Colonies started to re-emerge and could be selectively expanded after 5-6 days. Finally, genomic DNA was extracted for real-time quantitative PCR analysis (Figure 4). For the feeder-free culture protocol, mouse iPSCs were cultured for 2-3 passages in ESGRO-2i medium. The miPSCs were dissociated to a single cell suspension with Accutase solution, and 100,000 cells were plated onto gelatin-coated 6-well plates. Next, the cells were incubated overnight with 4 mM TAT-Cre in ESGRO-2i medium. Colonies started to re-emerge and could be selectively expanded after 9-10 days. Finally, genomic DNA was extracted for real-time quantitative PCR analysis. [caption id="attachment_37344" align="alignleft" width="200"] Figure 4. Excision efficiency of mouse Cre-excisable polycistronic iPSCs: real-time qPCR analysis of genomic DNA. In the two experiments shown, ?Ct >5 was considered a significant difference of DNA expression levels and indicated a successful excision. The Ct value of WPRE in the excised samples should correlate with the negative controls, mESC and no template control. Similar results were obtained when mouse iPSCs were cultured in serum-free, feeder-free condition (data not shown).[/caption] Results Establishment of transgene-free iPSCs required approximately two weeks from the time of addition of the cell-permeant TAT-Cre protein. Mouse and factor-free human and iPSCs expressed appropriate morphological and immunochemical staining characteristics of pluripotent cells. In addition, factor-free human iPSCs possessed a normal karyotype and were capable of differentiating into derivatives of all three germ layers in vivo (Figure 5). Following exposure of iPSCs to 4-6 mM TAT-Cre for 1-2 hours, highly efficient excision could be demonstrated. For mouse iPSCs, efficiency reached 100%; for human iPSCs, the efficiency was up to 60%. This high degree of efficiency is a large improvement over results obtained with electroporation of a plasmid expressing Cre-recombinase (<10%) and also with adenovirus expressing Cre recombinase, which has been shown to be effective for mouse iPSCs but not for human iPSCs. Therefore, the straightforward addition of cell-permeant TAT-Cre protein enabled robust excision and established transgene-free iPSCs. In addition, a quick qPCR screening assay was able to identify any deleted clones. Conclusion To conclude, this article demonstrated a robust system for highly efficient excision of viral vectors from iPSCs using cell-permeant TAT-Cre protein. Efficient delivery of an active recombinant Cre protein to mammalian cells has broad applications for somatic cell reprogramming, and also serves as a powerful tool for rapid genetic manipulation of mammalian genomes. [caption id="attachment_37346" align="alignright" width="200"] Figure 5. In vitro and in vivo characterisation of post-excised human iPSC clones. Post-excised clones expressed the appropriate pluripotent markers (A-D), alkaline phosphatase (data not shown) and possessed normal karyotype (E). Teratoma analysis (F-I). Individual subclones were selected along with pooled subclones and analysed for presence of transgene. All subclones and pooled demonstrate complete excision after 15 passages and similar results could be observed when expansion was conducted in feeder-based culture (J).[/caption] Continued improvements in the production of iPSCs are crucial for accelerating the application of these cells in both research and clinical settings. As iPSCs are incorporated into an increasing number of studies, the technology to create and characterise them continues to advance, helping these cells reach their strong potential. References

1. Takahashi, K, Tanabe, K et al. Induction of pluripotent stem cells from adult human fibroblasts by defined factor. Cell. 2007; 131: 861-72.
2. Sommer, CA et al. iPS cell generation using a single lentiviral stem cell cassette. Stem Cells. 2009; 27(3): 543-9.

Authors Vi Tuong Chu, Min Lu, Naomi Guyette and Ming Li of EMD Millipore and Kadari Asifiqbal, Sandra Meyer, Frank Edenhofer, Institute of Neurobiology, University of Bonn Medical Centre