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Efficient Generation of Transgene-free iPSCs

Wed, 03/19/2014 - 1:52pm
Vi Tuong Chu, Min Lu, Naomi Guyette, Ming Li, Kadari Asifiqbal, Sandra Meyer, and Frank Edenhofer

Induced pluripotent stem cells (iPSCs) offer strong potential for regenerative medicine, as well as disease modeling and drug screening. While researchers are using these cells for a wide range of applications, traditional methods used to generate iPSCs can be inefficient and time-consuming.

The first iPSCs were created in 2007 when researchers infected skin cells with four transcription factors (Oct-4, Klf4, Sox-2, and c-Myc).1 Converting these somatic cells to iPSCs required simultaneous co-infection with four separate retroviral expression vectors. Each vector carried one transcription factor, resulting in a high number of genomic integrations, which could pose a safety risk or lead to a heterogeneous cell population. Alternative methods used plasmids and nonintegrating adenovirus vectors to deliver the transcription factors, but these approaches are far less efficient than using retroviral vectors.

A new development then made it possible to generate human and mouse iPSCs using a single polycistronic lentiviral vector. STEMCCA reprogramming kits (Merck Millipore, Darmstadt, Germany) 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 isolated.2

Reprogramming of somatic cells using viral transduction of defined transcription factors remains a widely used and efficient method to obtain iPSCs. However, the presence of viral transgenes in iPSCs is undesirable, as it raises the possibility of insertional mutagenesis leading to malignant transformation and has also been shown to affect differentiation potential. Various strategies have been employed to address this issue, including non-integrating viruses, RNA transfection, protein transduction and site-specific recombinases to excise the transgenes after reprogramming.

Figure 1. 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 analyzed by Coomassie® blue staining of an SDS-PAGE (B). CL, cleared lysate; SN, supernatant; FL, flow through; W1, W2, wash fractions 1 and 2; E, eluted fraction. Numbers on the left indicate molecular weight (kDa) of marker proteins, Cre protein is approximately 41 kDa. (Figure from F. Edenhofer, Nature Methods, 2006 Jun;3(6):461-7)

In this article, we describe 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” (STEMCCA) comprised of all four transcription factors, followed by exposure of the fully reprogrammed iPSC to cell-permeable TAT-Cre recombinant protein. Additionally, we present a simple and robust PCR strategy that enables fast identification of deleted clones directly from primary iPSC colonies.

 

Materials and Methods

A rapid assay was used 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. 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. Consistent lot-to-lot performance was observed.

Figure 2. Human excision: Time course of TAT-Cre treatment (A). Individual colonies were picked at 9-14 days post-treatment and added to 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).Human iPSC excision with TAT-Cre

Human iPSCs were transitioned to feeder-free conditions. One day before passaging, Rho-associated protein kinase (ROCK) inhibitor was added and the cells were dissociated into a single cell suspension. A 12-well plate was used, and 50,000-100,000 cells were added to each well. The wells were incubated overnight to allow the cells to attach; they were then incubated with 2-5 mM of the protein TAT-Cre (Figure 1). After 7-9 days, colonies started to re-emerge and could be expanded. Genomic DNA was then extracted for real-time quantitative PCR analysis (Figure 2).

Mouse iPSC excision with TAT-Cre

Mouse iPSC excision was performed using two protocols, one with a feeder-based culture and one with a serum-free, feeder-free culture. For the feeder-based culture protocol, mouse iPSCs were grown on pMEF feeder layer in mESC media. The cells (miPSCs and pMEF) were dissociated into a single cell suspension with Accutase solution. Next, 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, and after 5-6 days, colonies started to re-emerge and could be selectively expanded. Genomic DNA was then extracted for real-time quantitative PCR analysis (Figure 3).

For the feeder-free culture protocol, mouse iPSCs were cultured in ESGRO-2i medium for 2-3 passages. The miPSCs were dissociated to a single cell suspension with Accutase solution, and 100,000 cells were plated onto gelatin-coated 6-well plates. The cells were then incubated overnight with 4 mM TAT-Cre in ESGRO-2i medium. After 9-10 days, colonies started to re-emerge and could be selectively expanded. Finally, genomic DNA was extracted for real-time quantitative PCR analysis.

 

Figure 3. Excision efficiency of mouse STEMCCA Cre-excisable polycistronic (OKSM) iPSCs: real-time qPCR analysis of genomic DNA. In the two experiments shown, ΔCt >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 (ESGRO-2i medium, EMD Millipore Cat# SF016, data not shown).Results

Highly efficient excision could be demonstrated following exposure of iPSCs to 4-6 mM TAT-Cre for 1-2 hours: 100% for mouse iPSCs and up to 60% for human iPSCs. The high degree of efficiency obtained with protein transduction is in marked contrast to 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.

Establishment of transgene-free iPSCs required approximately two weeks from the time of addition of the cell-permeant TAT-Cre protein. Factor-free human and mouse iPSCs expressed appropriate morphological and immunochemical staining characteristics of pluripotent cells. Factor-free human iPSCs possessed a normal karyotype and were capable of differentiating into derivatives of all three germ layers in vivo (Figure 4).

The protocol was simple; the straightforward addition of cell-permeant TAT-Cre protein enabled the robust excision and established transgene-free iPSCs. In addition, a quick qPCR screening assay was able to identify any deleted clones.

 

Figure 4. in vitro and in vivo characterization of post-excised human iPSC clones. Post-excised clones (p17) 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 analyzed 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).Conclusion

In summary, we have 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.

Improvements in the generation of iPSCs are critical, as these cells have created new opportunities in both clinical and research settings. Patient-specific cell-replenishment therapies are in development, and researchers are using iPSCs for disease modeling and drug and toxicity screening.  The stem cells are helping to enable construction of human models of complex diseases and reveal important insights that can lead to a more personalized approach to medicine.

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.

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