Reporter gene-engineering of human induced pluripotent stem cells during differentiation renders in vivo traceable hepatocyte-like cells accessible

Abstract

HighlightsiPSC-derived hepatocyte-like cells (HLCs) rendered traceable in vivo.Reproducible lentivirus-based gene transfer during the differentiation process.Protocol and reporter expression did not negatively impact on HLC maturation.Proof-of-principle shown for whole-body SPECT/CT-afforded HLC in vivo tracking.

Spatiotemporal in vivo tracking of polyclonal human regulatory T cells (Tregs) reveals a role for innate immune cells in Treg transplant recruitment

“…The fluorescent co-reporter GFP in our fusion reporter NIS-GFP helped streamline generation of traceable cells for preclinical experimentation. 27 , 28 , 36 Notably, NIS-fluorescent protein fusion reporters have been shown previously (1) to be correctly localized to the plasma membrane and function in several different cell types (rodent and human cancer cells, 20 , 27 , 28 , 29 , 36 human CAR T cells, 49 and hepatocyte-like cells 29 ) and (2) to mediate uptake of various substrates similarly to unmodified NIS (for similar relative substrate uptake of NIS substrates, compare Figure S9 A, Diocou et al. 36 , and Khoshnevisan et al.…”

“… 16 , 22 One of the most promising host reporter genes is the human sodium iodide symporter (NIS), which has been shown to be well tolerated in different cell types, including cancer cells, cardiomyocytes, stem cells, and T cells. 27 , 28 , 29 , 30 In a proof-of-principle study employing retroviral transduction methodology, we demonstrated ex vivo engineering of murine Tregs to express NIS and detected the cells in vivo 24 h after administration by SPECT imaging. 31 Importantly, it has also been shown recently that the radioactivity levels that can be expected to be taken up into T cells through NIS use during imaging did not cause lasting DNA damage in T cells.…”

Non-invasive Reporter Gene Imaging of Cell Therapies, including T Cells and Stem Cells

“…Section 3.1). Mammalian NIS has been used in a variety of cell tracking applications in animal models spanning a wide range of different cell types [45,[84][85][86][87][88][89][90][91][92]. This is a testament to both its excellent contrast in many applications, as NIS is only endogenously expressed in the thyroid and a few extra-thyroidal tissues (salivary glands, mammary glands, stomach and small intestine, testes; [22]), and its small anionic radiotracers being readily available for both PET and SPECT imaging (Tab.1).…”

“…Whilst PSC-derived therapies are costly to produce, they have already entered early clinical trials [124], and as their use for treating a greater range of injuries and diseases comes ever closer to clinical reality, reporter gene studies become increasingly important. Most early PSC therapy tracking studies incorporated the reporter cassette via lentiviral transduction and utilised the firefly luciferase Radionuclide reporter gene imaging could help overcome this limitation, but it has been employed only by a few studies using human PSCs to date [92,123,125,126]. Additionally, while studies using lentiviral-mediated reporter expression have mostly demonstrated stable reporter expression following both differentiation and PSC expansion, the risk of epigenetic silencing and viral integration at unwanted genomic sites remains.…”

“…Preclinical studies utilising reporter gene tracking of stem cell therapies. Human MSCs Monitoring of MSC homing and therapeutic potential in breast cancer hNIS SPECT Adenovirus [228] Human MSCs Tracking of long-term fate and trafficking of MSCs Triple fusion protein: fLuc-mRFP-HSV1-sr39tk BLI and PET/CT Lentivirus [229] Human MSCs Understanding MSC fate in tissue repair Mutant of dopamine type 2 receptor (D2R80A) PET Lentivirus [230] Human MSCs Evaluating myocardial tracking potential with a PET reporter in small (rat) and large animal studies (swine) HSV1-sr39tk PET Adenovirus [125] hESCs and their differentiated progeny [236] hESCs/H9 line Safety study: analysis number of contaminating undifferentiated hESCs required to yield a teratoma Fusion protein: fLuc-eGFP BLI Lentivirus [122] hESC-derived MSCs Studied the distribution of human MSCs in a rat hind limb ischemic injury model immediately after transplantation and also analysed the recipient tissue response to transplanted cells fLuc BLI Lentivirus [237] hESCs TGL fusion protein: HSV1-tk-eGFP-fLuc BLI Lentivirus [239] hESC-derived skeletal myoblasts/H1 and H9 lines Assessment of long-term myoblast engraftment and survival with monitoring for teratoma formation TGL fusion protein: HSV1-tk-eGFP-fLuc BLI Lentivirus [240] hESCs/H1 and H9 Monitoring stem cell engraftment and teratoma formation Bicistronic fLuc and GFP and fusion of HSV1-tk-GFP BLI and SPECT/CT Lentivirus [241] hiPSCs and their differentiated progeny [242] hiPSC-derived HLCs Potential for tracking transplanted HLC populations in vivo hNIS-eGFP fusion SPECT/CT Lentivirus [243] hiPSC-derived neural stem/progenitor cells Determining the feasibility of tumour ablation following hiPSC-NS/PC spinal cord transplantation utilising immunoregulation Fusion protein Venus-fLuc BLI Lentivirus [244] hiPSC-derived endothelial cells Analysis of potential of iPSC-derived ECs to promote perfusion of ischaemic tissue in model of peripheral arterial disease Fusion protein: fLuc-eGFP BLI Lentivirus [245] hiPSCs Evaluation of transplanted hiPSC survival, engraftment, and distribution of in a pig model of myocardial infarction Bicistronic ratNIS and Venus SPECT/CT Plasmid transfection [246] 55 hiPSCs evaluating systems to purge residual hiPSCs before graft without compromising hematopoietic repopulation capability fLuc BLI Lentivirus [247] hiPSC-derived cardiomyocytes Assessment of relationship between transplanted cell number and engraftment rate in myocardial injury Bicistronic fLuc and GFP BLI Lentivirus [248] Abbreviations…”