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Stable Fluorescent Labelling of human iPSCs aid Cell Tracking after Differentiation and Transplantation: Strategies and Pitfalls!

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Review of “Stable Enhanced Green Fluorescent Protein Expression After Differentiation and Transplantation of Reporter Human Induced Pluripotent Stem Cells Generated by AAVS1 Transcription Activator-Like Effector Nucleases” from Stem Cells TM by Stuart P. Atkinson.

The generation of a method to track pluripotent cell-derived cell types post-transplantation would aid the efficient monitoring of cell survival, migration, and incorporation into the recipient organs. However, silencing of transgene expression used to label cells usually accompanies differentiation. Targeted genome editing through the use of an engineered nuclease may aid the generation of pluripotent cells which express a fluorescent transgene such as eGFP from a site which is ubiquitously expressed and not silenced during differentiation. TALENs comprise a non-specific FokI nuclease domain fused to a customized Transcription Activator-Like Effector (TALE) DNA binding domain [1] and previous studies have utilised them to target human embryonic stem cells (hESCs) and human induced pluripotent stem cells (hiPSCs) [2]. With these studies and requirements in mind, the group of Jizhong Zou at the National Institute of Arthritis and Musculoskeletal and Skin Diseases, Bethesda, USA have now designed and optimized an eGFP constructs and targeting strategy to generate human induced pluripotent stem cell (hiPSC) reporter lines [3].

The non-pathogenic adeno-associated virus 2 (AAV2) can integrate at the adeno-associated virus integration site 1 (AAVS1) locus (part of PPP1R12C gene loci) without any deleterious effects and so if considered a “safe harbour”, and deemed a useful site for the generation of hiPSC reporter lines. The researchers generated specific AAVS1-targeting TALENs, as shown in the adjoined figure, which displayed higher functionality with higher cutting activity than commercially available ZFNs, assessed using an optimized EGFP rescue assay which measures the success of TALEN-mediated homology-directed repair occurs [4]. Additionally, the researchers also used a non-homologous end joining (NHEJ) assay to assess mutations generated from TALEN-caused DNA double-break repair, and this demonstrated that pZT-AAVS1 TALENs were highly effective and specific.

The authors then assessed the integration of two fluorescent proteins within a donor plasmid (amilRFP, driven by the CMV7 promoter, and copGFP, driven by the EF1a-short promoter, alongside a Puromycin resistance cassette) into an hiPSC line (NCRM5) using the using the pZTAAVS1 TALENs. Following nucleofection and 12 days of antibiotic selection, no amilRFP-positive cells could be found, although copGFP-positive clones were visible. In depth analyses of the causes of the weakness/silencing of the promoter constructs suggested that the CMV7 and EF1a short promoters were not optimal and required the consideration of different promoters. Furthermore, off-target integration also led to some errant GFP expression, underlining the necessity for thorough analysis of reporter constructs. Similar in depth studies utilising an alternate promoter - CAG, which has higher activity than CMV and EF1a [5], found an ability for high capacity EGFP expression at the AAVS1 site. Embryoid-body mediated spontaneous differentiation of pAAVS1-CAG-EGFP hiPSCs also demonstrated that EGFP expression was persistent during in vitro differentiation, with more than 90% of cells being EGFP+ at 10–14 days, with EGFP present in mesodermal, ectodermal, and endodermal cell types. Teratoma formation assessment in mice found well-encapsulated cystic tumors within 6–7 weeks composed of cells of all three primary embryonic germ layers with more than 98% of the cells expressing EGFP, altogether suggesting that cells retained their ability to differentiate into three germ lines while continuing to express EGFP. This was further examined in differentiated cells after in vivo engraftment using cardiomyocytes as differentiated cells. Before xeno-engraftment into infarcted mouse heart hearts, cells expressed EGFP at a high level, and at 3 weeks and 7 weeks after transplantation EGFP-positive cells existed on the border zone of the infarcted area.

The study goes into great depth to assess, construct, validate, and test the targeting of a reporter into a “safe harbour” site in hiPSCs, and may be of great use in long term transplant studies to explore their use in translational medicine. This includes the facilitation in vivo imaging to study the survival, localization, and safety of transplanted cells in preclinical studies, and has the possibility of use in the delivery of gene products for therapy. This highly detailed and useful study also comes with an added bonus extra; the authors have deposited the AAVS1-TALEN vectors and AAVS1-CAG-EGFP hiPSC reporter lines in non-profit plasmid and cell repositories, respectively, which will be available to non-profit organizations and for research use on request.

References

  1. Boch J and Bonas U Xanthomonas AvrBs3 family-type III effectors: discovery and function. Annu Rev Phytopathol 2010;48:419-436.
  2. Hockemeyer D, Wang H, Kiani S, et al. Genetic engineering of human pluripotent cells using TALE nucleases. Nat Biotechnol 2011;29:731-734.
  3. Luo Y, Liu C, Cerbini T, et al. Stable Enhanced Green Fluorescent Protein Expression After Differentiation and Transplantation of Reporter Human Induced Pluripotent Stem Cells Generated by AAVS1 Transcription Activator-Like Effector Nucleases. Stem Cells Transl Med 2014;
  4. Zou J, Maeder ML, Mali P, et al. Gene targeting of a disease-related gene in human induced pluripotent stem and embryonic stem cells. Cell Stem Cell 2009;5:97-110.
  5. Chen CM, Krohn J, Bhattacharya S, et al. A comparison of exogenous promoter activity at the ROSA26 locus using a PhiiC31 integrase mediated cassette exchange approach in mouse ES cells. PLoS One 2011;6:e23376.