WO2023233176A1 - Novel human induced pluripotent stem cell line stably expressing a reporter - Google Patents

Novel human induced pluripotent stem cell line stably expressing a reporter Download PDF

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WO2023233176A1
WO2023233176A1 PCT/HU2023/050031 HU2023050031W WO2023233176A1 WO 2023233176 A1 WO2023233176 A1 WO 2023233176A1 HU 2023050031 W HU2023050031 W HU 2023050031W WO 2023233176 A1 WO2023233176 A1 WO 2023233176A1
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reporter
cell
sequence
promoter
sequence encoding
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Anita FEHÉR
Andrea SCHNÚR
András DINNYÉS
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Biotalentum Tudásfejlesztő Kft.
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    • C12N15/90—Stable introduction of foreign DNA into chromosome
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    • C12N15/63—Introduction of foreign genetic material using vectors; Vectors; Use of hosts therefor; Regulation of expression
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    • C12N2830/40—Vector systems having a special element relevant for transcription being an insulator

Definitions

  • the invention relates to human induced pluripotent stem cell (hiPSC) expressing a reporter, in particular iRFP720.
  • hiPSC human induced pluripotent stem cell
  • iRFP720 a reporter
  • the hiPSC and cells differentiated thereof are useful for real-time imaging and tracking of transplanted cells in preclinical studies, testing novel cellular products in regenerative therapies as well as for applications in disease modelling, drug development and toxicological studies.
  • hESC Human embryonic stem cells
  • hiPSC induced pluripotent stem cells
  • Pluripotent stem cells have the potential to address the shortage of cell source, and in addition to their renewable capacity, when patient-derived iPSCs are used the allogeneic immune response can also be avoided 8 .
  • a bioimaging method should be the least invasive, non-toxic for the patient, and provide an exclusive visualization of the viable grafted cells.
  • reporter genes such as green, red or blue fluorescent proteins (eGFP, DsRed, mCherry, BFP) could provide an attractive option to trace transplanted cells 10-12
  • the expression of these reporter genes generates easily measurable signal suitable for cell monitoring and changes in signal intensity can indicate cell death or proliferation.
  • the in vivo use of this type of reporters is limited due to the low penetration depths of visible light and fading of the signal in the body due to absorption by haemoglobin and myoglobin 13 14 .
  • the conventional reporters eGFP, DsRed, mCherry, BFP
  • NIRPs near-infrared fluorescent proteins
  • DrBphP bacterial phytochrome of De- inococcus radiodurans have very good spectral characteristics (high extinction coefficient, low fluorescence quantum yield), their fluorescence from deep tissues exceeds by at least one order of magnitude the fluorescence of other reporter proteins.
  • Tissue autofluorescence is generally much lower in the 700-1000 nm spectrum 16 leading to even more significant improvement in sensitivity and resolution.
  • NIRFPs can be triggered by infrared laser light and as a result they change in shape generating an ultrasound-signal, that can be sensitively detected by optoacoustic detectors and noninvasively imaged in live animals with very good signal to background ratio, making possible the monitoring of cells deeper in the body without killing the subject animal.
  • iRFP720 has been shown to be easily detected in vivo due to its minimal absorption in mammalian tissues, thus providing a new perspective in the application for protein labelling, live cell imaging and in vivo tracking 17 . This imaging approach offers the potential for earlier detection of rejection or dysfunction of the transplanted cells, tissues, or organs 18 19 .
  • iRFP7 0 labelled cells have been efficiently used to mark ovarian cancer, lung cancer, and breast cancer as well as mesenchymal stem cell grafts in the brain 20-23 and were able to track cell engraftment in heart 24-26 .
  • AAVS1 The Adeno-Associated Virus Site 1 (AAVS1), located in the 1st intron of PPP1R12C (protein phosphatase 1 regulatory subunit 12C) on human chromosome 19, considered favourable and well-characterized candidate in human iPSC transgenesis 46-50 . Insertion of exogenous DNA into the AAVS1 locus promises predictable and strong transgene expression without noticeable functional and phenotypic alteration in the modified cell lines 46,51 . Based on these characteristics, AAVS1 is commonly referred and used as a “safe-harbour” site. The expression of the transgene inserted into the AAVS1 locus is robust and persistent, which on one hand explained by the maintenance of an open -chromatin configuration in this locus 52 .
  • the invention provides a cell, preferably a recombinant cell, preferably a human induced pluripotent stem cell (hiPSC) expressing a reporter, wherein the DNA-sequence encoding the reporter is integrated into the Adeno- Associated Virus Site 1 (AAVS 1) by a targeting vector comprising
  • LHA Left Homology Arm
  • RHA Right Homology Arm
  • a targeting vector is provided to transform a cell, preferably human induced pluripotent cell to express a reporter, comprising:
  • LHA Left Homology Arm
  • RHA Right Homology Arm
  • the reporter is a protein, preferably a fluorescent protein, highly preferably the near-infrared fluorescent protein iRFP720.
  • the promoter driving the expression of the reporter is selected from a EFl -alpha promoter, a PGK promoter, a CMV promoter and a CAG promoter, preferably the promoter driving the expression of the reporter is a CAG promoter.
  • the selection marker is a resistance marker, preferably selected from a neomycin resistance gene, a hygromycin resistance gene and a puromycin resistance gene, highly preferably the resistance marker is a puromycin resistance gene.
  • the expression of the selection marker is driven by the endogenous promoter of the PPP1R12C gene.
  • sequence comprising a transcription termination and polyadenylation signal sequence is selected from BGH, SV40 and the rabbit beta globin poly(A), preferably the rabbit beta globin poly(A).
  • the sequence encoding the reporter-cassette is flanked by insulator sequences, preferably wherein the insulator sequences are cHS4 insulator sequences.
  • the targeting vector comprises or has the sequence according to SEQ ID No. 1.
  • sequence encoding the reporter protein is integrated into AAV S 1 by the CRISPR/Cas9 method.
  • sequence encoding the reporter is present in the genome of the cell in a single copy or 2 copies.
  • the selection marker is expressed by the cell.
  • the DNA-sequence encoding the reporter is stably integrated into the genome of the cell.
  • the sequence encoding the selection marker is stably integrated into the genome of the cell.
  • the invention relates to a differentiated human cell expressing a reporter, wherein the differentiated human cell is produced by differentiating the hiPSC.
  • the use of a differentiated human cell for real-time imaging and/or tracking of transplanted cells is provided, wherein the differentiated human cell is produced by differentiating the hiPSC.
  • hiPSC Use of a hiPSC as described above is provided for producing differentiated human cells expressing a reporter.
  • a method for producing a cell expressing a reporter comprising the insertion of the sequence coding for the reporter into a human cell using the targeting vector described above.
  • the introduction of the sequence coding for the reporter is performed by the CRISPR/Cas9 method.
  • the cell is hiPSC.
  • Donor vector used to target the locus is depicted above.
  • LHA/RHA Left and Right Homology Arms
  • 2A- puro 2A self-cleaving peptide sequence and the puromycin resistance gene
  • cHS4 chicken hypersensitive site-4 insulator sequence
  • CAG CMV early enhancer/chicken 0 actin promoter
  • iRFP+rGbPAS iRFP720 near-infrared fluorescent protein coding sequence terminated with rabbit beta globin polyadenylation signal sequence.
  • the first 2 exons of PPP1R12C gene in the AAVS1 locus are shown with black boxes.
  • CRISPR/Cas9 mediated DSB (double-strand break) and the restriction cut sites are indicated by arrows. The probes used for Southern blot analysis are depicted accordingly.
  • FIG. 1 Genetic screening. a Junction PCRs were performed using locus-specific primers that bind to genomic sequences outside of the homology regions in combination with vector-specific primers. Expected fragment sizes for positive samples: 1196 bp for the left region and 1789 bp for the right region. Three clones tested positive in the screening that contained correctly integrated donor DNA at both ends. C+: positive control hiPSC line containing genome integrated eGFP sequence in the AAVS1 locus, C-: negative control SBAD2 hiPSC line, NTC: no template control. The original uncropped gels are presented in Figure 9. b Southern blot analysis of the candidate clones (A5-04, A7-09, A7-10) and negative control SBAD2 hiPSC line.
  • gDNA samples were digested with Ncol or Apal restriction enzymes and tested with AAVS1-LHA or AAVS1-RHA specific probes, respectively.
  • WT negative control SBAD2 hiPSC line
  • M DIG-labeled DNA Molecular Weight Marker VII (Roche), the ladder scale specified by the manufacturer is shown on the right side of the blots. The original uncropped blots are presented in Figure 10.
  • FIG. 3 Live cell fluorescent imaging and FACS-analysis of the SBAD2-iRFP720 reporter hiPSCs.
  • Bar charts on iRFP720 images show average fluorescence intensities (arbitrary intensity values) measured from 10 different 50 x 50 pm area. Scale bar: 150 pm.
  • b Flow cytometry analysis of the iRFP720 expressing SBAD2 hiPSC clones. Excitation of 638 nm was used, and emission window was set to 720/30 nm. The clones were proved to be homogeneous for the reporter expression.
  • iRFP720 expressing hiPSCs show normal stem cell characteristics and karyotype.
  • FIG. 5 Pluripotency tests. a Representative immunofluorescent micrographs of undifferentiated SBAD2-iRFP720 reporter hiPSCs, that were positively stained for stem cell markers OCT4, NANOG and TRA1 -81 (in green), nuclei were labeled with DAPI (in blue), b SBAD2-iRFP720 hiPSCs were spontaneously differentiated and analyzed by immunocytochemistry. Multilineage differentiation potential was confirmed by immuno staining for endodermal (GATA4), mesodermal (BRACHYURY) and ectodermal (TUBB3, NESTIN) germ layers (in green), nuclei were labeled with DAPI (in blue). Scale bar: 50 pm.
  • GATA4 endodermal
  • BRACHYURY mesodermal
  • TUBB3, NESTIN ectodermal
  • FIG. 1 Immunocytochemical analysis of stage specific marker expression during pancreatic progenitor differentiation.
  • SBAD2-iRFP720 reporter hiPSCs were differentiated in 3D culture system. Spheroids were fixed, cryosectioned, and the highest diameter middle sections were immunostained after 4 and 13 days of differentiation. Top panels represent the overview of the cryosectioned cultures, while the rest of the panels show higher magnifications on day 4 (left) or day 13 (right). On day 4, most of the cells express definitive endoderm markers F0XA2 (green) and SOX17 (red). Note the high expression of the key transcription factors in pancreatic cell development on day 13 (PDX1 in green, NKX6.1 in red). Nuclei were counterstained with DAPI (blue). Scale bars: 200 pm and 50 pm.
  • Figure 9 Original uncropped gel pictures of the junction PCRs.
  • junction PCRs were performed using locus-specific primers that bind to genomic sequences outside of the homology regions in combination with vector-specific primers. Expected fragment sizes for positive samples: 1196 bp for the left region and 1789 bp for the right region. Three clones tested positive in the screening that contained correctly integrated donor DNA at both ends. C+: positive control hiPSC line containing genome integrated eGFP sequence in the AAVS1 locus, C-: negative control SBAD2 hiPSC line, NTC: no template control.
  • Figure 10 Original uncropped Southern blot images.
  • the newly developed iRFP720-reporter human iPSC line will become a valuable tool in a variety of biological applications: for real-time imaging and tracking of transplanted cells in preclinical studies (it allows for continuous longitudinal non-invasive monitoring of transplanted stem cells and their derivatives with high sensitivity) to test novel cellular products in regenerative therapies as well as for applications in disease modelling, drug development and toxicological studies.
  • SBAD2 hiPSC line was used to generate the reporter cells by inserting a CAG-promoter driven iRFP720 cassette into the AAVS1 safe harbor locus.
  • CAG promoter was chosen after careful consideration of multiple promoter options. It enables strong and stable expression of transgenes/reporters with little risk of silencing and helps to detect the transplanted cells continuously, independently from their differentiation status and activation of tissue-specific promoters 30-32 .
  • the CRISPR/Cas9 technology was used for targeted integration of the reporter cassette into the AAVS1 locus to prevent transgene silencing and position effect related expression variations that can affect the proper endogenous gene expression pattern.
  • a puromycin resistance gene was inserted into the AAVS1 locus, driven by the endogenous PPP1R12C promoter, along with the CAG-iRFP720 reporter cassette, which was flanked by cHS4 insulator elements to block the potential interactions between the transgene and the target cell genome (Fig. 1).
  • SBAD2 hiPSCs were nucleofected with the donor vector and CRISPR/Cas9 components (applied as RNP complexes), then the cells were selected by puromycin. Targeting efficiency was relatively low, with 0.001% of cells surviving the puromycin treatment. After selection, individual drug-resistant colonies were isolated, propagated, and screened by locus-specific junction PCRs (Fig. 2a, Fig. 9). The junction PCR results showed that several clones contained only partially integrated donor sequences. Three correctly modified PCR-positive clones were found and then analyzed by Southern blot (A5-04, A7-09 and A7- 10). The Southern blot experiments (Fig. 2b, Fig.
  • iRFP720 expressing hiPSCs show normal stem cell characteristics. Two iRFP720 expressing hiPSC clones (A7-09 and A7-10) were further tested and characterized. Expression of the iRFP720 reporter gene was both detected with fluorescence live cell imaging (Fig.
  • iRFP720 fluorescence signal was 500-900-fold higher than the basal autofluorescence level and the cell populations were found to be homogeneous for the reporter expression.
  • A7-09 cells displayed 1.8x more red emission as compared to A7-10 cells.
  • Both clones showed proper hiPSC-morphology, the colonies were tightly packed, round-shaped, and the cells had a high nuclear/cytoplasm ratio, which was indistinguishable from that of the parental SBAD2 hiPSCs (Fig. 4).
  • the iRFP720 reporter cell lines were also checked for chromosomeintegrity and showed normal diploid 46, XY karyotype (Fig. 4).
  • An important consideration when manipulating hiPSCs is the maintenance of pluripotency. Gene targeting is stressful, and it may have negative effect on cell survival and proliferative capacity.
  • EBs embryoid bodies
  • the differentiated cultures were characterized for the expression of the three germ layer markers. We found that the undifferentiated reporter iPSCs were positively stained for the major pluripotency markers (OCT3/4, NANOG, TRA-1-81, Fig.
  • A7-10 iRFP720 expressing iPSCs are able to efficiently differentiate into pancreatic precursor cells. As summarized above, both clones were found to show well-detectable, homogeneous iRFP720 expression. Taking into account the potential induction of endoplasmic reticulum stress in the cells due to overexpression of the reporter protein, the heterozygous A7-10 clone was selected for further studies and to generate pancreatic progenitor cells expressing the iRFP720 reporter gene.
  • the heterozygous cell line containing only one copy of the reporter was preferred based on theoretical considerations to prevent or at least minimize the possibility of ER machinery overload and subsequent ER stress that may be caused by long-term and high-level overexpression of foreign, exogenous proteins in the cells.
  • 3D three-dimensional
  • the spheroids showed an appropriate marker expression profile at each stage, as genes and transcription factors related to pancreatic development were selectively upregulated at the specific stages: SOX17 and F0XA2 in definitive endoderm stage, PDX1 and NKX6.1 in pancreatic progenitors.
  • pluripotency marker expression NAN0G1, OCT4 downregulated upon differentiation (Fig. 6b).
  • Immunohistochemical analysis also confirmed the proper expression of the stage-specific differentiation markers (Fig. 7).
  • iRFP720 expression is stable during pancreatic differentiation.
  • epigenetic silencing by DNA methylation and/or loss of the transgene can contribute to reductions in the transgene expression 34,35 .
  • SBAD202-01 human iPSC line http://stem-schulc.org, 65
  • SBAD2 Normal Adult Human Dermal Fibroblast cells
  • hiPSCs were cultured at 37°C in a humidified atmosphere containing 5% C02 in a feeder-free system on Matrigel (BD Biosci- ences)-coated tissue culture plates.
  • mTeSR 1 culture medium StemCell Technologies
  • EDTA 0.02%, Versene, Lonza
  • Expression cassette of iRFP720 under the control of a CAG promoter was obtained from pCAG-iRFP720 plasmid (Addgene_89687) and inserted into a modified AAVS1 targeting vector backbone (constructed in house from Addgene_22212) through pCR-II-Blunt-TOPO cloning (Thermo Fisher Scientific).
  • cHS4 insulator sequences, flanking the expression cassette and obtained from pLNHX_cHS4_650 plasmid, were also incorporated into the donor vector to protect reporter expression from silencing 66 .
  • junction PCRs were performed using locus-specific genomic primers that bind outside of the AAVS1 homology regions, in combination with donor vector-specific primers (Table 1). The fragments were amplified by Phusion Hot Start II High-Fidelity DNA Polymerase (Thermo Fisher Scientific).
  • Phusion Hot Start II High-Fidelity DNA Polymerase Thermo Fisher Scientific.
  • NEB Ncol restriction enzyme
  • Hybond N+ nylon membrane Amersham.
  • DIG-labeled DNA probes were prepared by random primed labeling for the AAVS1 left and right homology regions (581 and 622 bp, respectively).
  • Probe labeling, hybridization and detection were performed using DIG-High Prime DNA Labeling and Detection Starter Kit II (Roche), following instmctions of the manufacturer.
  • Kodak Gel Logic 1500 Imaging System was used to document the gels and blots.
  • the iRFP720 reporter hiPSCs were treated with Demecolcine solution (10 pg/mL in Hanks' Balanced Salt solution (HBSS)) and processed with standard methods. Giemsa-banded karyotype analysis was performed for a minimum of 20 metaphase cells and the chromosomes were classified according to the International System of Human Cytogenetic Nomenclature (ISCN).
  • ISCN International System of Human Cytogenetic Nomenclature
  • iPSC cultures and differentiated spheroids were dispersed into single-cell suspension by Accutase and Trypsin, respectively, then the cells were suspended in PBS containing 10 mM HEPES and analyzed using an Attune NxT flow cytometer (Thermo Fisher Scientific).
  • iRFP720 fluorescence was measured with 638 nm excitation and emission at 720/30 nm. Analysis of the results was performed using Attune NxT 3.1.2 software.
  • Live cell iRFP imaging Live cells were washed once with PBS and incubated at room temperature for least 5 minutes in 5 pg/ L fluorescein diacetate (FDA) working solution which was prepared freshly in PBS from a thawed stock solution of 5 mg/ml FDA in DMSO.
  • FDA fluorescein diacetate
  • Olympus FV1000 confocal laser scanning microscope was used for imaging of cells with 488 nm excitation and 500-530 nm emission ("fluorescein” dye combination) for FDA, or with 633 nm excitation and LP650 nm emission ("Cy5" dye configuration) for iRFP720.
  • the sections were permeabilized with 0.1% Triton X-100 in PBS and blocked for 1 hour at 24°C with 3% BSA in PBS. The sections were then incubated with primary antibodies (overnight, 4°C; Table 2). On the next day, the sections were washed 3 times in PBS, the isotype specific secondary antibodies were diluted in blocking buffer and applied for 1 hour at RT. The sections were then washed again 3 times with PBS and covered using Vectashield® mounting medium containing DAPI (1.5 pg/mL; Vector Laboratories) (1 hour, RT). Negative controls for the secondary antibodies were prepared in the same way by omitting the primary antibodies.
  • Dominguez-Bendala J., Inverardi, L. & Ricordi, C. Stem cell-derived islet cells for transplantation. Curr Opin Organ Transplant 16, 76-82, doi:10.1097/MOT.0b013e32834252b5 (2011).
  • TALEN Transcription activator-like effector nuclease-mediated CLYBL targeting enables enhanced transgene expression and one-step generation of dual reporter human induced pluripotent stem cell (iPSC) and neural stem cell (NSC) lines.
  • iPSC human induced pluripotent stem cell
  • NSC neural stem cell

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Abstract

The invention relates to human induced pluripotent stem cell (hiPSC) expressing a reporter, in particular 1RFP720. The hiPSC and cells differentiated thereof are useful for real-time imaging and tracking of transplanted cells in preclinical studies, testing novel cellular products in regenerative therapies as well as for applications in disease modelling, drug development and toxicological studies.

Description

NOVEL HUMAN INDUCED PLURIPOTENT STEM CELL LINE STABLY EXPRESSING A REPORTER
FIELD OF THE INVENTION
The invention relates to human induced pluripotent stem cell (hiPSC) expressing a reporter, in particular iRFP720. The hiPSC and cells differentiated thereof are useful for real-time imaging and tracking of transplanted cells in preclinical studies, testing novel cellular products in regenerative therapies as well as for applications in disease modelling, drug development and toxicological studies.
BACKGROUND OF THE INVENTION
Over the past decade, cellular therapies have emerged as the new frontier for the treatment of various chronic diseases, including diabetes1. Because various types of diabetes are linked to a loss of beta cells, cell therapy research focuses on beta-cell replenishment strategies to compensate for insulin deficiency2-4. Human embryonic stem cells (hESC) and induced pluripotent stem cells (hiPSC) are considered as very attractive sources of surrogate beta cells, because of their ability to differentiate into all major somatic cell lineages, including endoderm, where the beta cells originate5-7. Pluripotent stem cells have the potential to address the shortage of cell source, and in addition to their renewable capacity, when patient-derived iPSCs are used the allogeneic immune response can also be avoided8.
Monitoring of cell homing and the fate of the delivered cellular products, including death, survival, proliferation, migration and differentiation, is fundamental for clarification of the regenerative process and its safety. Advanced biosafety visualization of the grafted cells and tracking their fate in the host has a great importance in preclinical assessment of novel cell-based therapies. This can be achieved by reporter gene expression or physical labelling using nanoparticles9. A reliable in vivo imaging method, in addition to monitoring cell viability, would also provide information on the in vivo migration of the transplanted cells, thus allowing a systematic investigation of cell therapy, which is crucial for proper scientific interpretation. Ideally, a bioimaging method should be the least invasive, non-toxic for the patient, and provide an exclusive visualization of the viable grafted cells.
Cell labelling with reporter genes such as green, red or blue fluorescent proteins (eGFP, DsRed, mCherry, BFP) could provide an attractive option to trace transplanted cells 10-12 The expression of these reporter genes generates easily measurable signal suitable for cell monitoring and changes in signal intensity can indicate cell death or proliferation. However, the in vivo use of this type of reporters is limited due to the low penetration depths of visible light and fading of the signal in the body due to absorption by haemoglobin and myoglobin13 14. The conventional reporters (eGFP, DsRed, mCherry, BFP) can be detected in the visible spectral range, where autofluorescence is relatively high, thereby significantly increasing the background and interfering with imaging15. In contrast, near-infrared fluorescent proteins (NIRFPs), developed from the DrBphP bacterial phytochrome of De- inococcus radiodurans have very good spectral characteristics (high extinction coefficient, low fluorescence quantum yield), their fluorescence from deep tissues exceeds by at least one order of magnitude the fluorescence of other reporter proteins. Tissue autofluorescence is generally much lower in the 700-1000 nm spectrum16 leading to even more significant improvement in sensitivity and resolution. NIRFPs can be triggered by infrared laser light and as a result they change in shape generating an ultrasound-signal, that can be sensitively detected by optoacoustic detectors and noninvasively imaged in live animals with very good signal to background ratio, making possible the monitoring of cells deeper in the body without killing the subject animal. iRFP720 has been shown to be easily detected in vivo due to its minimal absorption in mammalian tissues, thus providing a new perspective in the application for protein labelling, live cell imaging and in vivo tracking17. This imaging approach offers the potential for earlier detection of rejection or dysfunction of the transplanted cells, tissues, or organs18 19. iRFP7 0 labelled cells have been efficiently used to mark ovarian cancer, lung cancer, and breast cancer as well as mesenchymal stem cell grafts in the brain20-23 and were able to track cell engraftment in heart24-26.
Conventional random integration of reporter genes is not favourable due to a number of limitations69. Location of the insertion site can affect the reporter expression leading to inadequate epigenetic modifications and altered regulation70-71, and multiple integrations of the transgene may result in overexpression artefacts or inadequate expression patterns72. To overcome these obstacles, several reporter cell lines have already been generated by targeted insertion of different promoter-driven reporter genes into the genome, e.g. into landing pads40, into the CLYBL locus on human chromosome 1341-43, or into the AAVS1 locus30,31’44’45. The Adeno-Associated Virus Site 1 (AAVS1), located in the 1st intron of PPP1R12C (protein phosphatase 1 regulatory subunit 12C) on human chromosome 19, considered favourable and well-characterized candidate in human iPSC transgenesis46-50. Insertion of exogenous DNA into the AAVS1 locus promises predictable and strong transgene expression without noticeable functional and phenotypic alteration in the modified cell lines 46,51. Based on these characteristics, AAVS1 is commonly referred and used as a “safe-harbour” site. The expression of the transgene inserted into the AAVS1 locus is robust and persistent, which on one hand explained by the maintenance of an open -chromatin configuration in this locus52. On the other hand, the presence of an insulator site has been found to prevent the spread of heterochromatin, thus ensuring stable transgene expression 51. However, promoter silencing and clonedependent variations in transgene expression has been observed in different cell types during directed differentiation when AAVS 1 site was used for transgene insertion, drawing attention to the need for careful clone-screening before and throughout the differentiation53-56.
SUMMARY OF THE INVENTION
The invention provides a cell, preferably a recombinant cell, preferably a human induced pluripotent stem cell (hiPSC) expressing a reporter, wherein the DNA-sequence encoding the reporter is integrated into the Adeno- Associated Virus Site 1 (AAVS 1) by a targeting vector comprising
- an AAVS1 Left Homology Arm (LHA) comprising a sequence homologous with the endogenous genomic sequence uptream of the genomic DNA cleavage site in the 1st intron of the PPP1R12C gene,
- a sequence encoding a selection marker,
- a reporter-cassette comprising
- a promoter driving the expression of the reporter,
- a sequence encoding the reporter,
- a sequence comprising a transcription termination and polyadenylation signal sequence,
- insulator sequences flanking the reporter-cassette,
- an AAVS1 Right Homology Arm (RHA) comprising a sequence homologous with the endogenous genomic sequence downstream of the genomic DNA cleavage site in the 1st intron of the PPP1R12C gene.
A targeting vector is provided to transform a cell, preferably human induced pluripotent cell to express a reporter, comprising:
- an AAVS1 Left Homology Arm (LHA) comprising a sequence homologous with the endogenous genomic sequence uptream of the genomic DNA cleavage site in the 1st intron of the PPP1R12C gene, - a sequence encoding a selection marker,
- a reporter-cassette comprising
- a promoter driving the expression of the reporter,
- a sequence encoding the reporter,
- a sequence comprising a transcription termination and polyadenylation signal sequence,
- insulator sequences flanking the reporter-cassette,
- an AAVS1 Right Homology Arm (RHA) comprising a sequence homologous with the endogenous genomic sequence downstream of the genomic DNA cleavage site in the 1st intron of the PPP1R12C gene.
Preferably the reporter is a protein, preferably a fluorescent protein, highly preferably the near-infrared fluorescent protein iRFP720.
Preferably the promoter driving the expression of the reporter is selected from a EFl -alpha promoter, a PGK promoter, a CMV promoter and a CAG promoter, preferably the promoter driving the expression of the reporter is a CAG promoter.
Preferably the selection marker is a resistance marker, preferably selected from a neomycin resistance gene, a hygromycin resistance gene and a puromycin resistance gene, highly preferably the resistance marker is a puromycin resistance gene.
Preferably the expression of the selection marker is driven by the endogenous promoter of the PPP1R12C gene.
Preferably the sequence comprising a transcription termination and polyadenylation signal sequence is selected from BGH, SV40 and the rabbit beta globin poly(A), preferably the rabbit beta globin poly(A).
Preferably the sequence encoding the reporter-cassette is flanked by insulator sequences, preferably wherein the insulator sequences are cHS4 insulator sequences.
Preferably the targeting vector comprises or has the sequence according to SEQ ID No. 1.
Preferably the sequence encoding the reporter protein is integrated into AAV S 1 by the CRISPR/Cas9 method.
Preferably the sequence encoding the reporter is present in the genome of the cell in a single copy or 2 copies.
Preferably the selection marker is expressed by the cell.
Preferably the DNA-sequence encoding the reporter is stably integrated into the genome of the cell. Preferably the sequence encoding the selection marker is stably integrated into the genome of the cell.
The invention relates to a differentiated human cell expressing a reporter, wherein the differentiated human cell is produced by differentiating the hiPSC. The use of a differentiated human cell for real-time imaging and/or tracking of transplanted cells is provided, wherein the differentiated human cell is produced by differentiating the hiPSC.
Use of a hiPSC as described above is provided for producing differentiated human cells expressing a reporter.
A method for producing a cell expressing a reporter, comprising the insertion of the sequence coding for the reporter into a human cell using the targeting vector described above. Preferably the introduction of the sequence coding for the reporter is performed by the CRISPR/Cas9 method. Preferably only clones having a single copy or two copies of the sequence coding for the reporter in their genome are selected. Preferably the cell is hiPSC. BRIEF DESCRIPTION OF THE DRAWINGS
Figure 1. Targeting of the iRFP720 reporter construct into the AAVS1 locus of SBAD2 hiPSCs.
Donor vector used to target the locus is depicted above. LHA/RHA: Left and Right Homology Arms, 2A- puro: 2A self-cleaving peptide sequence and the puromycin resistance gene, cHS4: chicken hypersensitive site-4 insulator sequence, CAG: CMV early enhancer/chicken 0 actin promoter, iRFP+rGbPAS: iRFP720 near-infrared fluorescent protein coding sequence terminated with rabbit beta globin polyadenylation signal sequence. The first 2 exons of PPP1R12C gene in the AAVS1 locus are shown with black boxes. CRISPR/Cas9 mediated DSB (double-strand break) and the restriction cut sites are indicated by arrows. The probes used for Southern blot analysis are depicted accordingly.
Figure 2. Genetic screening. a Junction PCRs were performed using locus-specific primers that bind to genomic sequences outside of the homology regions in combination with vector-specific primers. Expected fragment sizes for positive samples: 1196 bp for the left region and 1789 bp for the right region. Three clones tested positive in the screening that contained correctly integrated donor DNA at both ends. C+: positive control hiPSC line containing genome integrated eGFP sequence in the AAVS1 locus, C-: negative control SBAD2 hiPSC line, NTC: no template control. The original uncropped gels are presented in Figure 9. b Southern blot analysis of the candidate clones (A5-04, A7-09, A7-10) and negative control SBAD2 hiPSC line. gDNA samples were digested with Ncol or Apal restriction enzymes and tested with AAVS1-LHA or AAVS1-RHA specific probes, respectively. WT: negative control SBAD2 hiPSC line, M: DIG-labeled DNA Molecular Weight Marker VII (Roche), the ladder scale specified by the manufacturer is shown on the right side of the blots. The original uncropped blots are presented in Figure 10.
Figure 3. Live cell fluorescent imaging and FACS-analysis of the SBAD2-iRFP720 reporter hiPSCs. a iRFP720 expressing (red, excitation/detection: 633/LP650) and control cells were stained with viability indicator dye fluorescein diacetate (FDA, green, excitation/detection: 488/500-530). Bar charts on iRFP720 images show average fluorescence intensities (arbitrary intensity values) measured from 10 different 50 x 50 pm area. Scale bar: 150 pm. b Flow cytometry analysis of the iRFP720 expressing SBAD2 hiPSC clones. Excitation of 638 nm was used, and emission window was set to 720/30 nm. The clones were proved to be homogeneous for the reporter expression.
Figure 4. iRFP720 expressing hiPSCs show normal stem cell characteristics and karyotype.
Brightfield images depicting the proper iPSC-morphology of the SBAD2 and SBAD2-iRFP720 reporter hiPSCs. Scale bars: 200 pm (left panels) and 100 pm (middle panels). Karyogram of the cell lines showed normal 46 chromosomes (XY).
Figure 5. Pluripotency tests. a Representative immunofluorescent micrographs of undifferentiated SBAD2-iRFP720 reporter hiPSCs, that were positively stained for stem cell markers OCT4, NANOG and TRA1 -81 (in green), nuclei were labeled with DAPI (in blue), b SBAD2-iRFP720 hiPSCs were spontaneously differentiated and analyzed by immunocytochemistry. Multilineage differentiation potential was confirmed by immuno staining for endodermal (GATA4), mesodermal (BRACHYURY) and ectodermal (TUBB3, NESTIN) germ layers (in green), nuclei were labeled with DAPI (in blue). Scale bar: 50 pm.
Figure 6. Pancreatic differentiation of SBAD2-iRFP720 reporter hiPSCs in 3D culture system. a Schematic for the generation of pancreatic progenitor cells, b mRNA expression of pluripotency markers (0CT4 and NANOG), definitive endoderm (F0XA2, SOX17) and pancreatic progenitor (NKX6.1, PDX1) markers at the indicated differentiation stages determined by RT-qPCR measurements. Data are presented as mean±SEM, n=3.
Figure 7. Immunocytochemical analysis of stage specific marker expression during pancreatic progenitor differentiation.
SBAD2-iRFP720 reporter hiPSCs were differentiated in 3D culture system. Spheroids were fixed, cryosectioned, and the highest diameter middle sections were immunostained after 4 and 13 days of differentiation. Top panels represent the overview of the cryosectioned cultures, while the rest of the panels show higher magnifications on day 4 (left) or day 13 (right). On day 4, most of the cells express definitive endoderm markers F0XA2 (green) and SOX17 (red). Note the high expression of the key transcription factors in pancreatic cell development on day 13 (PDX1 in green, NKX6.1 in red). Nuclei were counterstained with DAPI (blue). Scale bars: 200 pm and 50 pm.
Figure 8. iRFP720 expression in SBAD2-iRFP720 reporter hiPSC-derived spheroids during pancreatic differentiation. Cells were differentiated for 13 days and analyzed. a iRFP720-mRNA expression at the indicated differentiation stages was determined by RT-qPCR measurements. Data are presented as mean±SEM, n=3. b iRFP720 fluorescence of control and reporter hiPSC-derived differentiated samples was measured by flow cytometry. Excitation of 638 nm was used, and emission window was set to 720/30 nm. Samples from left to right: Control SBAD2 iPSC, iRFP720 reporter iPSC (Dayl), iRFP720 reporter P. Progenitor (Dayl3). c Confocal microscopic analysis of control (top panels) and iRFP720-expressing spheroids on day 4 and day 13 of pancreatic differentiation (middle and bottom panels). Right Left panels show representative merged images of the nuclear DAPI stain (blue) with bright field pictures of the cryosectioned spheroids, left right panels demonstrate the iRFP720 expression (in red, excitation/detection: 633/LP650). Scale bar: 50 pm.
Figure 9. Original uncropped gel pictures of the junction PCRs.
Junction PCRs were performed using locus-specific primers that bind to genomic sequences outside of the homology regions in combination with vector-specific primers. Expected fragment sizes for positive samples: 1196 bp for the left region and 1789 bp for the right region. Three clones tested positive in the screening that contained correctly integrated donor DNA at both ends. C+: positive control hiPSC line containing genome integrated eGFP sequence in the AAVS1 locus, C-: negative control SBAD2 hiPSC line, NTC: no template control.
Figure 10. Original uncropped Southern blot images.
Southern blot analysis of the candidate clones (A5-04, A7-09, A7-10) and negative control SBAD2 hiPSC line. gDNA samples were digested with Ncol or Apal restriction enzymes and tested with AAVS1 -LHA or AAVS1-RHA specific probes, respectively. WT: negative control SBAD2 hiPSC line, M: DIG-labeled DNA Molecular Weight Marker VII (Roche).
DETAILED DESCRIPTION OF THE INVENTION
In this study, we established and characterized a novel human iPSC line that stably expresses the near-infrared fluorescent protein, by inserting the iRFP720 coding sequence into the AAVS1 safe harbor locus of human iPSCs. To generate the genetically modified reporter iPSCs, the CRISPR/Cas9 technology was applied which is more efficient than the conventional methods and offers powerful tool for accurate genome editing to generate improved cellular models27-29. The newly established iRFP720 reporter hiPSC line might become an ideal tool for real-time imaging, enabling robust readout that helps both cell therapy development and drug screening applications.
We have demonstrated the retained genomic stability, pluripotency and multilineage differentiation ability of the newly generated iPSC lines. Moreover, we have found that the reporter iPSCs were able to efficiently differentiate into pancreatic progenitor cells with maintained, uniform, and high level iRFP720 expression during the differentiation. Although a slight decrease was observed in the fluorescence intensity, the iRFP720 mRNA expression remained stable, indicating that there was no downregulation or transgene silencing, and this observation is more likely due to the metabolic shift which is a hallmark of differentiated cells62-64.
The newly developed iRFP720-reporter human iPSC line will become a valuable tool in a variety of biological applications: for real-time imaging and tracking of transplanted cells in preclinical studies (it allows for continuous longitudinal non-invasive monitoring of transplanted stem cells and their derivatives with high sensitivity) to test novel cellular products in regenerative therapies as well as for applications in disease modelling, drug development and toxicological studies.
Targeting strategy. SBAD2 hiPSC line was used to generate the reporter cells by inserting a CAG-promoter driven iRFP720 cassette into the AAVS1 safe harbor locus. CAG promoter was chosen after careful consideration of multiple promoter options. It enables strong and stable expression of transgenes/reporters with little risk of silencing and helps to detect the transplanted cells continuously, independently from their differentiation status and activation of tissue-specific promoters 30-32. Instead of random insertion, the CRISPR/Cas9 technology was used for targeted integration of the reporter cassette into the AAVS1 locus to prevent transgene silencing and position effect related expression variations that can affect the proper endogenous gene expression pattern. To generate the transgenic cell line, a puromycin resistance gene was inserted into the AAVS1 locus, driven by the endogenous PPP1R12C promoter, along with the CAG-iRFP720 reporter cassette, which was flanked by cHS4 insulator elements to block the potential interactions between the transgene and the target cell genome (Fig. 1).
Gene targeting, genetic screening, clone testing. SBAD2 hiPSCs were nucleofected with the donor vector and CRISPR/Cas9 components (applied as RNP complexes), then the cells were selected by puromycin. Targeting efficiency was relatively low, with 0.001% of cells surviving the puromycin treatment. After selection, individual drug-resistant colonies were isolated, propagated, and screened by locus-specific junction PCRs (Fig. 2a, Fig. 9). The junction PCR results showed that several clones contained only partially integrated donor sequences. Three correctly modified PCR-positive clones were found and then analyzed by Southern blot (A5-04, A7-09 and A7- 10). The Southern blot experiments (Fig. 2b, Fig. 10) showed proper integration of the reporter cassette into the AAVS1 locus and confirmed the heterozygous targeting event in the A7-10 clone, demonstrating a single copy integration into the targeted genomic locus, while in case of A7-09 cells both AAVS1 alleles were modified (bial- lelic targeting). An additional fragment was detected in A5-04 sample, suggesting extra vector-integration into another genomic location in this clone. Thus, the overall efficiency of precise genetic modification was found to be quite low (0.0002%), with a homozygote to heterozygote ratio of 1 :2. DNA sequencing of the targeted genomic region in the A7-09 and A7-10 clones verified the accurate genome editing, and potential CRISPR/Cas9 mediated off-target cleavages were assessed by sequence analysis of the four most likely predicted off-target sites. The results showed that there was no CRISPR/Cas9 mediated nonspecific cleavage at any of these off-target sites. iRFP720 expressing hiPSCs show normal stem cell characteristics. Two iRFP720 expressing hiPSC clones (A7-09 and A7-10) were further tested and characterized. Expression of the iRFP720 reporter gene was both detected with fluorescence live cell imaging (Fig. 3a) as well as by flow cytometry, confirming high-level of iRFP720 expression in both clones (Fig. 3b). iRFP720 fluorescence signal was 500-900-fold higher than the basal autofluorescence level and the cell populations were found to be homogeneous for the reporter expression. Based on microscopic fluorescence intensity quantification and flow cytometric measurements, A7-09 cells displayed 1.8x more red emission as compared to A7-10 cells. Both clones showed proper hiPSC-morphology, the colonies were tightly packed, round-shaped, and the cells had a high nuclear/cytoplasm ratio, which was indistinguishable from that of the parental SBAD2 hiPSCs (Fig. 4). The iRFP720 reporter cell lines were also checked for chromosomeintegrity and showed normal diploid 46, XY karyotype (Fig. 4). An important consideration when manipulating hiPSCs is the maintenance of pluripotency. Gene targeting is stressful, and it may have negative effect on cell survival and proliferative capacity. To confirm the pluripotency and multilineage differentiation ability of the reporter cells, embryoid bodies (EBs) were formed and cultured for 14 days in differentiation medium, then the differentiated cultures were characterized for the expression of the three germ layer markers. We found that the undifferentiated reporter iPSCs were positively stained for the major pluripotency markers (OCT3/4, NANOG, TRA-1-81, Fig. 5a) and they were able to differentiate into endodermal (GATA4), mesodermal (BRACHYURY) and ectodermal (TUBB3, NESTIN) lineages (Fig. 5b), confirming retained pluripotent stem cell properties of the iRFP720-expressing reporter cells.
A7-10 iRFP720 expressing iPSCs are able to efficiently differentiate into pancreatic precursor cells. As summarized above, both clones were found to show well-detectable, homogeneous iRFP720 expression. Taking into account the potential induction of endoplasmic reticulum stress in the cells due to overexpression of the reporter protein, the heterozygous A7-10 clone was selected for further studies and to generate pancreatic progenitor cells expressing the iRFP720 reporter gene. The heterozygous cell line containing only one copy of the reporter was preferred based on theoretical considerations to prevent or at least minimize the possibility of ER machinery overload and subsequent ER stress that may be caused by long-term and high-level overexpression of foreign, exogenous proteins in the cells. We used three-dimensional (3D) culture system, as it has been reported that the induction rate of PDX1+/NKX6.1+ pancreatic cells is markedly improved with cell aggregation, and this positive effect on pancreatic differentiation has been reproduced with multiple hESC/iPSC lines that were able to differentiate efficiently in 3D cultures33. Further advantages of these 3D aggregation cultures are that large-scale production is more easily achievable and the produced spheroids are readily accessible for transplantation in comparison to two-dimensional (2D) adherent cultures. Therefore, we tested pancreatic progenitor differentiation in a 3D culture system and found that the iRFP720 reporter cell line can be efficiently differentiated towards pancreatic lineage using well-established published protocols23. Cells were differentiated over a 13-days period by sequential media changes in a four-stage differentiation scheme (Fig. 6a). Samples were collected at definitive endodermal and pancreatic progenitor stages for assessment of stage specific differentiation markers by RT-qPCR and immunohistochemistry. The spheroids showed an appropriate marker expression profile at each stage, as genes and transcription factors related to pancreatic development were selectively upregulated at the specific stages: SOX17 and F0XA2 in definitive endoderm stage, PDX1 and NKX6.1 in pancreatic progenitors. In parallel, pluripotency marker expression (NAN0G1, OCT4) downregulated upon differentiation (Fig. 6b). Immunohistochemical analysis also confirmed the proper expression of the stage-specific differentiation markers (Fig. 7). iRFP720 expression is stable during pancreatic differentiation. During hiPSC differentiation, epigenetic silencing by DNA methylation and/or loss of the transgene can contribute to reductions in the transgene expression 34,35. Together, they present a major challenge in maintaining predictable and high yields of reporter protein expression. Therefore, we tested the stability of iRFP720 reporter expression during pancreatic differentiation. A7-10 reporter hiPSCs were differentiated into pancreatic progenitor cells with a 13 -days differentiation protocol, and the cells were analyzed by RT-qPCR measurements, examined by flow cytometry, and imaged at various stages. We found no significant differences in iRFP720 mRNA expression during differentiation compared to the iPSC control (Fig. 8a). Although flow cytometry data and confocal microscopic analysis indicated slightly decreased fluorescent signal-intensity in pancreatic progenitor cells, we were still able to detect a strong iRFP720 transgene expression and fluorescence uniformly distributed in the differentiated spheroids (Figs. 8b-c).
EXAMPLES
Cell lines and in vitro cell culture conditions. In this study, SBAD202-01 human iPSC line (http://stem- bancc.org, 65), referred to as SBAD2 was used, which has been established from Normal Adult Human Dermal Fibroblast cells (Lonza, CC-2511) by reprogramming with non-integrative Sendai virus transduction. hiPSCs were cultured at 37°C in a humidified atmosphere containing 5% C02 in a feeder-free system on Matrigel (BD Biosci- ences)-coated tissue culture plates. Cells were maintained in mTeSR 1 culture medium (StemCell Technologies) which was changed daily, and the cells were passaged every 5-7 days using EDTA (0.02%, Versene, Lonza), according to the manufacturer's instructions. hiPSCs underwent routine mycoplasma screening and karyotyping.
Donor vector construction. Expression cassette of iRFP720 under the control of a CAG promoter was obtained from pCAG-iRFP720 plasmid (Addgene_89687) and inserted into a modified AAVS1 targeting vector backbone (constructed in house from Addgene_22212) through pCR-II-Blunt-TOPO cloning (Thermo Fisher Scientific). cHS4 insulator sequences, flanking the expression cassette and obtained from pLNHX_cHS4_650 plasmid, were also incorporated into the donor vector to protect reporter expression from silencing66.
Transfection of donor vector and CRISPR/Cas9 elements. SBAD2 hiPSC culture at 70-80% confluency was incubated with Accutase (Sigma-Aldrich) at 37°C for 9 min to prepare single-cell suspension for gene targeting. 8x105 cells were nucleofected (in duplicates) with 22.5 pg CRISPR/Cas9 ribonucleoprotein (RNP) complex composed of GeneArt™ Platinum™ Cas9 protein (Thermo Fisher Scientific) and guide RNA (Table 1), along with 3.5 pg donor vector, using Human Stem Cell Nucleofector Kit 1 (Lonza) and program B -016 in AMAXA Nucleo- fector™ 2b Device (Lonza). After nucleofection, the transfected cells were spread in 6 well plate and IX Revi- taCell Supplement (Thermo Fisher Scientific) was added into the mTeSR- 1 culture medium to increase cell recovery. Puromycin selection started 2 days later with 800 ng/mL puromycin (Thermo Fisher Scientific) for 24 hours then continued with 200 ng/mL concentration for a week. After selection, puromycin-resistant colonies were isolated, propagated and harvested for cryobanking and genetic analysis. Table 1. The guide RNA and PCR primer sequences
Figure imgf000010_0001
Genetic screening. Junction PCRs were performed using locus-specific genomic primers that bind outside of the AAVS1 homology regions, in combination with donor vector-specific primers (Table 1). The fragments were amplified by Phusion Hot Start II High-Fidelity DNA Polymerase (Thermo Fisher Scientific). For Southern blot analysis, 5 pg genomic DNA was digested overnight with Apal or Ncol restriction enzyme (NEB) and separated on agarose gel, then transferred onto Hybond N+ nylon membrane (Amersham). DIG-labeled DNA probes were prepared by random primed labeling for the AAVS1 left and right homology regions (581 and 622 bp, respectively). Probe labeling, hybridization and detection were performed using DIG-High Prime DNA Labeling and Detection Starter Kit II (Roche), following instmctions of the manufacturer. Kodak Gel Logic 1500 Imaging System was used to document the gels and blots.
Off-target analysis. The most likely off-target sites were predicted by the CRISPR design tool (http://CRISPR.mit.edu/) and the corresponding genomic regions were PCR-amplified using Phusion Hot Start II High-Fidelity DNA Polymerase. The PCR products were purified with GenElute PCR cleanup kit (Sigma-Aldrich) and sequenced directly using an ABI Prism 3130x1 Genetic Analyzer and BigDye Terminator Cycle Sequencing v3.1 Kit (Applied Biosystems).
Karyotyping. The iRFP720 reporter hiPSCs were treated with Demecolcine solution (10 pg/mL in Hanks' Balanced Salt solution (HBSS)) and processed with standard methods. Giemsa-banded karyotype analysis was performed for a minimum of 20 metaphase cells and the chromosomes were classified according to the International System of Human Cytogenetic Nomenclature (ISCN).
Pluripotency tests. Cell clumps were cultured in suspension for five days in mTeSR- 1. The formed embryoid bodies (EBs) were plated on 0.1% gelatin (Merck) coated surface in differentiation medium (DMEM, 20% FBS, 1% MEM Non-Essential Amino Acid Solution (lOOx), 0.1 mM 0-mercaptoethanol, 1% Pen/Strep). On day 14 of differentiation, the cells were fixed with 4% formaldehyde solution and evaluated for the 3 germ layer markers by immunocytochemistry (Table 2). Cells were analysed under a fluorescence microscope equipped with a 3D imaging module (Axio Imager system with ApoTome; Zeiss) controlled using AxioVision 4.8.1 software (Zeiss).
Table 2. List of antibodies used
Figure imgf000011_0001
Human induced pluripotent stem cell-derived pancreatic differentiation. For initiation of the differentiation process and to form three-dimensional spheroids, SBAD2-A7-10 iPSCs were seeded in 6-well low attachment plates (Costar, 3471) at 9 x 105 cells/mL density in mTeSR-1 media supplemented with RevitaCell. The cells were then placed on an orbital shaker (MaxQ 2000 CO2) for overnight, set at rotation rate 95 rpm in a 37°C incubator, 5% CO2, and 100% humidity. The differentiation was started next day by changing mTeSR-1 to stage specific differentiation medium. During the procedure, media changes were performed according to previously published protocol67. Flow cytometry. iPSC cultures and differentiated spheroids were dispersed into single-cell suspension by Accutase and Trypsin, respectively, then the cells were suspended in PBS containing 10 mM HEPES and analyzed using an Attune NxT flow cytometer (Thermo Fisher Scientific). iRFP720 fluorescence was measured with 638 nm excitation and emission at 720/30 nm. Analysis of the results was performed using Attune NxT 3.1.2 software.
Live cell iRFP imaging. Live cells were washed once with PBS and incubated at room temperature for least 5 minutes in 5 pg/ L fluorescein diacetate (FDA) working solution which was prepared freshly in PBS from a thawed stock solution of 5 mg/ml FDA in DMSO. Olympus FV1000 confocal laser scanning microscope was used for imaging of cells with 488 nm excitation and 500-530 nm emission ("fluorescein" dye combination) for FDA, or with 633 nm excitation and LP650 nm emission ("Cy5" dye configuration) for iRFP720.
Immunocytochemistry of three-dimensional cultures. Cryosectioning and immunocytochemistry were performed based on our previously described methods68. 3D spheroid samples were fixed with 4% formaldehyde in 0.1 mol/L PBS for 1 hour at room temperature (RT) and washed 3 times with PBS. The fixed cultures were then cryoprotected in 30% sucrose in PBS containing 0.01% sodium-azide at 4°C until embedding in Shandon Cryomatrix gel (Thermo Fischer Scientific). 16-pm parallel sections were prepared using cryostat (Leica CM 1850 Cryostat, Leica GmbH), mounted to Superfrost™ Ultra Plus Adhesion Slides (Thermo Fisher Scientific) and stored at -20°C until use. After 10 min air-drying, the sections were permeabilized with 0.1% Triton X-100 in PBS and blocked for 1 hour at 24°C with 3% BSA in PBS. The sections were then incubated with primary antibodies (overnight, 4°C; Table 2). On the next day, the sections were washed 3 times in PBS, the isotype specific secondary antibodies were diluted in blocking buffer and applied for 1 hour at RT. The sections were then washed again 3 times with PBS and covered using Vectashield® mounting medium containing DAPI (1.5 pg/mL; Vector Laboratories) (1 hour, RT). Negative controls for the secondary antibodies were prepared in the same way by omitting the primary antibodies. Highest diameter middle sections with immunoreactivity were analyzed using a BX-41 epifluorescent microscope (Olympus) equipped with a DP-74 digital camera and its CellSens software (VI.18; Olympus,) or using an Olympus FV - 1 Oi- W compact confocal microscope system (Olympus) with Fluoview FV 1 Oi software (V2.1; Olympus). For iRFP720-imaging of cryosectioned and DAPI stained spheroids (Fig. 8c), Olympus FV1000 laser scanning confocal microscope was used with "DAPI" and "Cy5" (iRFP720) dye settings. All images were further processed using the GNU Image Manipulation Program (GIMP 2.10.0) and NIH ImageJ analysis software (imagej.nih.gov/ij).
RT-qPCR. Total RNA was isolated from 25-35 spheroids per sample using RNAqueous Micro Total RNA isolation Kit (Thermo Fisher Scientific) according to the manufacturer's instructions. RNA was transcribed by Maxima First Strand cDNA synthesis kit with DNase (Thermo Fisher Scientific). The amplification reactions were carried out in a total volume of 15 pL with SYBR Green JumpStart Taq ReadyMix (Sigma-Aldrich). Human 18S rRNA was used as a reference. The data were analyzed by REST software (2009 V2.0.13), and the values are expressed as mean ± SEM. Oligonucleotide primers used in this study are listed in Table 1.
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SEQ ID No 1 attattaacg cttacaattt cctgatgcgg tattttctcc ttacgcatct gtgcggtatt 60 tcacaccgca tcaggtggca cttttcgggg aaatgtgcgc ggaaccccta tttgtttatt 120 tttctaaata cattcaaata tgtatccgct catgagatta tcaaaaagga tcttcaccta 180 gatcctttta aattaaaaat gaagttttaa atcaatctaa agtatatatg agtaaacttg 240 gtctgacagt taccaatgct taatcagtga ggcacctatc tcagcgatct gtctatttcg 300 ttcatccata gttgcctgac tccccgtcgt gtagataact acgatacggg agggcttacc 360 atctggcccc agtgctgcaa tgataccgcg agacccacgc tcaccggctc cagatttatc 420 agcaataaac cagccagccg gaagggccga gcgcagaagt ggtcctgcaa ctttatccgc 480 ctccatccag tctattaatt gttgccggga agctagagta agtagttcgc cagttaatag 540 tttgcgcaac gttgttgcca ttgctacagg catcgtggtg tcacgctcgt cgtttggtat 600 ggcttcattc agctccggtt cccaacgatc aaggcgagtt acatgatccc ccatgttgtg 660 caaaaaagcg gttagctcct tcggtcctcc gatcgttgtc agaagtaagt tggccgcagt 720 gttatcactc atggttatgg cagcactgca taattctctt actgtcatgc catccgtaag 780 atgcttttct gtgactggtg agtactcaac caagtcattc tgagaatagt gtatgcggcg 840 accgagttgc tcttgcccgg cgtcaatacg ggataatacc gcgccacata gcagaacttt 900 aaaagtgctc atcattggaa aacgttcttc ggggcgaaaa ctctcaagga tcttaccgct 960 gttgagatcc agttcgatgt aacccactcg tgcacccaac tgatcttcag catcttttac 1020 tttcaccagc gtttctgggt gagcaaaaac aggaaggcaa aatgccgcaa aaaagggaat 1080 aagggcgaca cggaaatgtt gaatactcat actcttcctt tttcaatatt attgaagcat 1140 ttatcagggt tattgtctca tgaccaaaat cccttaacgt gagttttcgt tccactgagc 1200 gtcagacccc gtagaaaaga tcaaaggatc ttcttgagat cctttttttc tgcgcgtaat 1260 ctgctgcttg caaacaaaaa aaccaccgct accagcggtg gtttgtttgc cggatcaaga 1320 gctaccaact ctttttccga aggtaactgg cttcagcaga gcgcagatac caaatactgt 1380 tcttctagtg tagccgtagt taggccacca cttcaagaac tctgtagcac cgcctacata 1440 cctcgctctg ctaatcctgt taccagtggc tgctgccagt ggcgataagt cgtgtcttac 1500 cgggttggac tcaagacgat agttaccgga taaggcgcag cggtcgggct gaacgggggg 1560 ttcgtgcaca cagcccagct tggagcgaac gacctacacc gaactgagat acctacagcg 1620 tgagctatga gaaagcgcca cgcttcccga agggagaaag gcggacaggt atccggtaag 1680 cggcagggtc ggaacaggag agcgcacgag ggagcttcca gggggaaacg cctggtatct 1740 ttatagtcct gtcgggtttc gccacctctg acttgagcgt cgatttttgt gatgctcgtc 1800 a g g g g g g egg agcctatgga aaaacgccag caacgcggcc tttttacggt tcctggcctt 1860 ttgctggcct tttgctcaca tgttctttcc tgcgttatcc cctgattctg tggataaeeg 1920 tattaccgcc tttgagtgag ctgataccgc tcgccgcagc cgaacgaccg agcgcagcga 1980 gteagtgage gaggaagegg aagagcgccc aatacgcaaa ccgcctctcc ccgcgcgttg 2040 gccgattcat taatgeaget ggcacgacag gtttcccgac tggaaagegg gcagtgagcg 2100 caacgcaatt aatgtgagtt agctcactca ttaggcaccc caggctttac aetttatget 2160 tccggctcgt atgttgtgtg gaattgtgag cggataacaa tttcacacag gaaacagcta 2220 tgaccatgat tacgccaagc tcagaattaa ccctcactaa agggactagt cctgcaggtt 2280 taaacgaatt cgccctttgc tttctctgac cagcattctc tcccctgggc ctgtgccgct 2340 ttctgtctgc agcttgtggc ctgggtcacc tctacggctg gcccagatcc ttccctgccg 2400 cctccttcag gttccgtctt cctccactcc ctcttcccct tgctctctgc tgtgttgctg 2460 cccaaggatg ctctttccgg agcacttcct tctcggcgct gcaccacgtg atgtcctctg 2520 agcggatcct ccccgtgtct gggtcctctc cgggcatctc tcctccctca cccaacccca 2580 tgccgtcttc actcgctggg ttcccttttc cttctccttc tggggcctgt gccatctctc 2640 gtttcttagg atggccttct ccgacggatg tctcccttgc gtcccgcctc cccttcttgt 2700 aggcctgcat catcaccgtt tttctggaca accccaaagt accccgtctc cctggcttta 2760 gccacctctc catcctcttg ctttctttgc ctggacaccc cgttctcctg tggattcggg 2820 tcacctctca ctcctttcat ttgggcagct cccctacccc ccttacctct ctagtctgtg 2880 ctagctcttc cagccccctg tcatggcatc ttccaggggt ccgagagctc agctagtctt 2940 cttcctccaa cccgggcccc tatgtccact tcaggacagc atgtttgctg cctccaggga 3000 tcctgtgtcc ccgagctggg accaccttat attcccaggg ccggttaatg tggctctggt 3060 tctgggtact tttatctgtc ccctccaccc cacagtgggg caagcttctg acctcttctc 3120 ttcctcccac agggcctcga gagatctggc agcggagagg gcagaggaag tcttctaaca 3180 tgcggtgacg tggaggagaa tcccggccct aggctcgaga tgaccgagta caagcccacg 3240 gtgcgcctcg ccacccgcga cgacgtcccc agggccgtac gcaccctcgc cgccgcgttc 3300 gccgactacc ccgccacgcg ccacaccgtc gatccggacc gccacatcga gcgggtcacc 3360 gagctgcaag aactcttcct cacgcgcgtc gggctcgaca tcggcaaggt gtgggtcgcg 3420 gacgacggcg ccgcggtggc ggtctggacc acgccggaga gcgtcgaagc gggggcggtg 3480 ttcgccgaga tcggcccgcg catggccgag ttgagcggtt cccggctggc cgcgcagcaa 3540 cagatggaag gcctcctggc gccgcaccgg cccaaggagc ccgcgtggtt cctggccacc 3600 gtcggcgtct cgcccgacca ccagggcaag ggtctgggca gcgccgtcgt gctccccgga 3660 gtggaggcgg ccgagcgcgc cggggtgccc gccttcctgg agacctccgc gccccgcaac 3720 ctccccttct acgagcggct cggcttcacc gtcaccgccg acgtcgaggt gcccgaagga 3780 ccgcgcacct ggtgcatgac ccgcaagccc ggtgcctgat ctagagggcc cgtttaaacc 3840 cgctgatcag cctcgactgt gccttctagt tgccagccat ctgttgtttg cccctccccc 3900 gtgccttcct tgaccctgga aggtgccact cccactgtcc tttcctaata aaatgaggaa 3960 attgcatcgc attgtctgag taggtgtcat tctattctgg ggggtggggt ggggcaggac 4020 agcaaggggg aggattcaat tgacggggac agcccccccc caaagccccc agggatgtaa 4080 ttacgtccct cccccgctag ggggcagcag cgagccgccc ggggctccgc tccggtccgg 4140 cgctcccccc gcatccccga gccggcagcg tgcggggaca gcccgggcac ggggaaggtg 4200 gcacgggatc gctttcctct gaacgcttct cgctgctctt tgagcctgca gacacctggg 4260 gggatacggg gaaaaagctt taggcttgtg tctgagcctg catgtttgat ggtgtctgga 4320 tgcaagcaga aggggtggaa gagcttgcct ggagagatac agctgggtca gtaggactgg 4380 gacaggcagc tggagaattg ccatgtagat gttcatacaa tcgtcaaatc atgaaggctg 4440 gaaaagccct ccaagatccc caagaccaac cccaacccac ccaccgtgcc cactggccat 4500 gtccctcagt gccacatccc cacagttctt catcacctcc agggacggtg acccccccac 4560 ctccgtgggc agctgtgcca ctgcagcacc gctctttgga gaaggtaaat cttgctaaat 4620 ccagcccgac cctcccctgg cacaacgtaa ggccattatc tctcatccaa ctccaggacg 4680 gagtcagtga ggataagggc gaattccagc acactggcgg ccgttactag tggatccgag 4740 ctcggtaccg agctcggatc cactagtaac ggccgccagt gtgctggaat tcgcccttga 4800 cattgattat tgactagtta ttaatagtaa tcaattacgg ggtcattagt tcatagccca 4860 tatatggagt tccgcgttac ataacttacg gtaaatggcc cgcctggctg accgcccaac 4920 gacccccgcc cattgacgtc aataatgacg tatgttccca tagtaacgcc aatagggact 4980 ttccattgac gtcaatgggt ggagtattta cggtaaactg cccacttggc agtacatcaa 5040 gtgtatcata tgccaagtac gccccctatt gacgtcaatg acggtaaatg gcccgcctgg 5100 cattatgccc agtacatgac cttatgggac tttcctactt ggcagtacat ctacgtatta 5160 gtcatcgcta ttaccatggt cgaggtgagc cccacgttct gcttcactct ccccatctcc 5220 cccccctccc cacccccaat tttgtattta tttatttttt aattattttg tgcagcgatg 5280 ggggcggggg gggggggggc gcgcgccagg cggggcgggg cggggcgagg ggcggggcgg 5340 ggcgaggcgg aaaggtgcgg cggcagccaa tcagagcggc gcgctccgaa agtttccttt 5400 tatggcgagg cggcggcggc ggcggcccta taaaaagcga agcgcgcggc gggcgggagt 5460 cgctgcgttg ccttcgcccc gtgccccgct ccgcgccgcc tcgcgccgcc cgccccggct 5520 ctgactgacc gcgttactcc cacaggtgag cgggcgggac ggcccttctc ctccgggctg 5580 taattagcgc ttggtttaat gacggctcgt ttcttttctg tggctgcgtg aaagccttaa 5640 agggctccgg gagggccctt tgtgcggggg ggagcggctc ggggggtgcg tgcgtgtgtg 5700 tgtgcgtggg gagcgccgcg tgcggctccg cgctgcccgg cggctgtgag cgctgcgggc 5760 gcggcgcggg gctttgtgcg ctccgcagtg tgcgcgaggg gagcgcggcc gggggcggtg 5820 ccccgcggtg cggggggggc tgcgagggga acaaaggctg cgtgcggggt gtgtgcgtgg 5880 gggggtgagc agggggtgtg ggcgcggcgg tcgggctgta acccccccct gcacccccct 5940 ccccgagttg ctgagcacgg cccggcttcg ggtgcggggc tccgtacggg gcgtggcgcg 6000 gggctcgccg tgccgggcgg ggggtggcgg caggtggggg tgccgggcgg ggcggggccg 6060 cctcgggccg gggagggctc gggggagggg cgcggcggcc cccggagcgc cggcggctgt 6120 cgaggcgcgg cgagccgcag ccattgcctt ttatggtaat cgtgcgagag ggcgcaggga 6180 cttcctttgt cccaaatctg tgcggagccg aaatctggga ggcgccgccg caccccctct 6240 agcgggcgcg gggcgaagcg gtgcggcgcc ggcaggaagg aaatgggcgg ggagggcctt 6300 cgtgcgtcgc cgcgccgccg tccccttctc cctctccagc ctcggggctg tccgcggggg 6360 gacggctgcc ttcggggggg acggggcagg gcggggttcg gcttctggcg tgtgaccggc 6420 ggctctagag cctctgctaa ccatgttcat gccttcttct ttttcctaca gctcctgggc 6480 aacgtgctgg ttattgtgct gtctcatcat tttggcaaag aattgattaa ttcgagcgaa 6540 cgcgtaccgg tgccgccacc atggcggaag gatccgtcgc caggcagcct gacctcttga 6600 cctgcgacga tgagccgatc catatccccg gtgccatcca accgcatgga ctgctgctcg 6660 ccctcgccgc cgacatgacg atcgttgccg gcagcgacaa ccttcccgaa ctcaccggac 6720 tggcgatcgg cgccctgatc ggccgctctg cggccgatgt cttcgactcg gagacgcaca 6780 accgtctgac gatcgccttg gccgagcccg gggcggccgt cggagcaccg atcactgtcg 6840 gcttcacgat gcgaaaggac gcaggcttca tcggctcctg gcatcgccat gatcagctca 6900 tcttcctcga gctcgagcct ccccagcggg acgtcgccga gccgcaggcg ttcttccgcc 6960 gcaccaacag cgccatccgc cgcctgcagg ccgccgaaac cttggaaagc gcctgcgccg 7020 ccgcggcgca agaggtgcgg aagattaccg gcttcgatcg ggtgatgatc tatcgcttcg 7080 cctccgactt cagcgggtcc gtgatcgcag aggatcggtg cgccgaggtc gagtcaaaac 7140 taggcctgca ctatcctgcc tcattcatcc cggcgcaggc ccgtcggctc tataccatca 7200 acccggtacg gatcattccc gatatcaatt atcggccggt gccggtcacc ccagacctca 7260 atccggtcac cgggcggccg attgatctta gcttcgccat cctgcgcagc gtctcgccca 7320 accatctgga gttcatgcgc aacataggca tgcacggcac gatgtcgatc tcgattttgc 7380 gcggcgagcg actgtgggga ttgatcgttt gccatcaccg aacgccgtac tacgtcgatc 7440 tcgatggccg ccaagcctgc gagctagtcg cccaggttct ggcctggcag atcggcgtga 7500 tggaagagtg agcggccgca ctcctcaggt gcaggctgcc tatcagaagg tggtggctgg 7560 tgtggccaat gccctggctc acaaatacca ctgagatctt tttccctctg ccaaaaatta 7620 tggggacatc atgaagcccc ttgagcatct gacttctggc taataaagga aatttatttt 7680 cattgcaata gtgtgttgga attttttgtg tctctcactc ggaaggacat atgggagggc 7740 aaatcattta aaacatcaga atgagtattt ggtttagagt ttggcaacat atgcccatat 7800 gctggctgcc atgaacaaag gttggctata aagaggtcat cagtatatga aacagccccc 7860 tgctgtccat tccttattcc atagaaaagc cttgacttga ggttagattt tttttatatt 7920 ttgttttgtg ttattttttt ctttaacatc cctaaaattt tccttacatg ttttactagc 7980 cagatttttc ctcctctcct gactactccc agtcatagct gtccctcttc tcttatggag 8040 atccctcgac ctgcagccca agctaagggc gaattctgca gatatccatc acactggcgg 8100 ccgctcgagc atgcatgctc gagcggccgc cagtgtgatg gatatctgca gaattcgccc 8160 tttcgacctc actgactccg tcctggagtt ggatgagaga taatggcctt acgttgtgcc 8220 aggggagggt cgggctggat ttagcaagat ttaccttctc caaagagcgg tgctgcagtg 8280 gcacagctgc ccacggaggt gggggggtca ccgtccctgg aggtgatgaa gaactgtggg 8340 gatgtggcac tgagggacat ggccagtggg cacggtgggt gggttggggt tggtcttggg 8400 gatcttggag ggcttttcca gccttcatga tttgacgatt gtatgaacat ctacatggca 8460 attctccagc tgcctgtccc agtcctactg acccagctgt atctctccag gcaagctctt 8520 ccaccccttc tgcttgcatc cagacaccat caaacatgca ggctcagaca caagcctaaa 8580 gctttttccc cgtatccccc caggtgtctg caggctcaaa gagcagcgag aagcgttcag 8640 aggaaagcga tcccgtgcca ccttccccgt gcccgggctg tccccgcacg ctgccggctc 8700 ggggatgcgg ggggagcgcc ggaccggagc ggagccccgg gcggctcgct gctgccccct 8760 agcgggggag ggacgtaatt acatccctgg gggctttggg ggggggctgt ccccgtaccg 8820 gttgacagaa aagccccatc cttaggcctc ctccttccta gtctcctgat attgggtcta 8880 acccccacct cctgttaggc agattcctta tctggtgaca cacccccatt tcctggagcc 8940 atctctctcc ttgccagaac ctctaaggtt tgcttacgat ggagccagag aggatcctgg 9000 gagggagagc ttggcagggg gtgggaggga agggggggat gcgtgacctg cccggttctc 9060 agtggccacc ctgcgctacc ctctcccaga acctgagctg ctctgacgcg gccgtctggt 9120 gcgtttcact gatcctggtg ctgcagcttc cttacacttc ccaagaggag aagcagtttg 9180 gaaaaacaaa atcagaataa gttggtcctg agttctaact ttggctcttc acctttctag 9240 tccccaattt atattgttcc tccgtgcgtc agttttacct gtgagataag gccagtagcc 9300 agccccgtcc tggcagggct gtggtgagga ggggggtgtc cgtgtggaaa actccctttg 9360 tgagaatggt gcgtcctagg tgttcaccag gtcgtggccg cctctactcc ctttctcttt 9420 ctccatcctt ctttccttaa agagtcccca gtgctatctg ggacatattc ctccgcccag 9480 agcagggtcc cgcttcccta aggccctgct ctgggcttct gggtttgagt ccttggcaag 9540 cccaggagag gcgctcaggc ttccctgtcc cccttcctcg tccaccatct catgcccctg 9600 gctctcctgc cccttcccta caggggttcc tggctctgct ctaagggcaa gggcgaattc 9660 gcggccgcta aattcaattc gccctatagt gagtcgtatt acaattcact ggccgtcgtt 9720 ttaca 9725

Claims

1. A cell expressing a reporter, comprising stably integrated in its genome a DNA-sequence encoding the reporter, wherein the DNA-sequence encoding the reporter is integrated into the Adeno-Associated Virus Site 1 (AAVS1) by a targeting vector comprising
- an AAVS1 Left Homology Arm (LHA) comprising a sequence homologous with the endogenous genomic sequence upstream of the genomic DNA cleavage site in the 1st intron of the PPP1R12C gene,
- a sequence encoding a selection marker,
- a reporter-cassette comprising
- a promoter driving the expression of the reporter,
- a sequence encoding the reporter,
- a sequence comprising a transcription termination and polyadenylation signal sequence,
- insulator sequences flanking the reporter-cassette,
- an AAVS1 Right Homology Arm (RHA) comprising a sequence homologous with the endogenous genomic sequence downstream of the genomic DNA cleavage site in the 1st intron of the PPP1R12C gene.
2. A targeting vector comprising
- an AAVS1 Left Homology Arm (LHA) comprising a sequence homologous with an endogenous genomic sequence uptream of the genomic DNA cleavage site in the 1st intron of the PPP1R12C gene,
- a sequence encoding a selection marker,
- a reporter-cassette comprising
- a promoter driving the expression of the reporter,
- a sequence encoding the reporter,
- a sequence comprising a transcription termination and polyadenylation signal sequence,
- insulator sequences flanking the reporter-cassette,
- an AAVS1 Right Homology Arm (RHA) comprising a sequence homologous with an endogenous genomic sequence downstream of the genomic DNA cleavage site in the 1st intron of the PPP1R12C gene.
3. Use of the targeting vector according to claim 2 in the production of a cell expressing the reporter, wherein the cell comprises stably integrated in its genome the DNA-sequence encoding the reporter and wherein the DNA- sequence encoding the reporter is integrated into the AAVS1.
4. The cell according to claim 1 or the use according to claim 3, wherein the cell is a human induced pluripotent stem cell (hiPSC).
5. The cell according to claim 1 or 4, the targeting vector according to claim 2 or the use according to claim 3 or 4, wherein the reporter is iRFP720.
6. The cell according to any one of claims 1 and 4-5, the targeting vector according to claim 2 or 5 or the use according to any one of claims 3-5, wherein the promoter driving the expression of the reporter is selected from a EFl -alpha promoter, a PGK promoter, a CMV promoter and a CAG promoter.
7. The cell according to claim 6, the targeting vector according to claim 6 or the use according to claim 6, wherein the promoter driving the expression of the reporter is a CAG promoter.
8. The cell according to any one of claims 1 and 4-7, the targeting vector according to any one of claims 2 and 5- 7 or the use according to any one of claims 3-7, wherein the selection marker is a resistance marker, preferably selected from a neomycin resistance gene, a hygromycin resistance gene and a puromycin resistance gene.
9. The cell according to claim 8, the targeting vector according to claim 8 or the use according to claim 8, wherein the selection marker is a puromycin resistance gene.
10. The cell according to any one of claims 1 and 4-9, the targeting vector according to any one of claims 2 and 5- 9 or the use according to any one of claims 3-9, wherein the sequence comprising a transcription termination and polyadenylation signal sequence is the rabbit beta globin poly(A).
11. The cell according to any one of claims 1 and 4-10, the targeting vector according to any one of claims 2 and 5-10 or the use according to any one of claims 3-10, wherein the insulator sequences are cHS4 insulator sequences.
12. The cell according to any one of claims 1 and 4-11, the targeting vector according to any one of claims 2 and 5-11 or the use according to any one of claims 3-11, wherein the targeting vector comprises or has the sequence according to SEQ ID No. 1.
13. The cell according to any one of claims 1 and 4-12 orthe use according to any one of claims 3-12, wherein the expression of the selection marker is driven by the endogenous promoter of the PPP1R12C gene.
14. The cell according to any one of claims 1 and 4-13 or the use according to any one of claims 3-13, wherein the sequence encoding the reporter protein is integrated into AAVS1 by the CRISPR/Cas9 method.
15. The cell according to any one of claims 1 and 4-14 or the use according to any one of claims 3-14, wherein the sequence encoding the reporter is present in the genome of the cell in two copies or in a single copy.
16. The cell according to claim 15 or the use according to claim 15, wherein the sequence encoding the reporter is present in the genome of the cell in a single copy.
17. A cell expressing a reporter, wherein the cell is obtained by the use according to any one of claims 3-16.
18. A differentiated human cell expressing a reporter, wherein the differentiated human cell is produced by differentiating a cell according to any one of claims 1 and 4-17.
19. The cell according to claim 1, wherein
- the selection marker is a puromycin resistance gene,
- the promoter is a CAG promoter,
- the reporter is iRFP720, the sequence comprising a transcription termination and poly adenylation signal sequence is the rabbit beta globin poly(A),
- the insulator sequences are cHS4 insulator sequences,
- the expression of the selection marker is driven by the endogenous promoter of the PPP1R12C gene,
- the sequence encoding the reporter is present in the genome of the hiPSC in a single copy
- the sequence encoding the reporter protein is integrated into AAVS1 by the CRISPR/Cas9 method or wherein
- the targeting vector comprises or has the sequence according to SEQ ID No 1,
- the sequence encoding the reporter is present in the genome of the hiPSC in a single copy
- the sequence encoding the reporter protein is integrated into AAVS1 by the CRISPR/Cas9 method,
- the cell is a hiPSC.
20. A differentiated human cell expressing a reporter, wherein the differentiated human cell is produced by differentiating a cell according to claim 19.
21. The targeting vector according to claim 2, wherein
- the selection marker is a puromycin resistance gene,
- the promoter is a CAG promoter,
- the reporter is iRFP720, the sequence comprising a transcription termination and polyadenylation signal sequence is the rabbit beta globin poly(A),
- the insulator sequences are cHS4 insulator sequences, or the targeting vector comprises or has the sequence according to SEQ ID No 1.
22. A cell expressing a reporter, wherein the cell is obtained by transfecting a cell with the targeting vector according to claim 21.
23. A differentiated human cell expressing a reporter, wherein the differentiated human cell is produced by differentiating a cell according to claim 22, wherein the cell according to claim 22 is a hiPSC.
24. The use according to claim 3, wherein
- the selection marker is a puromycin resistance gene,
- the promoter is a CAG promoter,
- the reporter is iRFP720, the sequence comprising a transcription termination and polyadenylation signal sequence is the rabbit beta globin poly(A),
- the insulator sequences are cHS4 insulator sequences,
- the expression of the selection marker is driven by the endogenous promoter of the PPP1R12C gene,
- the sequence encoding the reporter is present in the genome of the hiPSC in a single copy
- the sequence encoding the reporter protein is integrated into AAVS1 by the CRISPR/Cas9 method or wherein
- the targeting vector comprises or has the sequence according to SEQ ID No 1,
- the sequence encoding the reporter is present in the genome of the hiPSC in a single copy
- the sequence encoding the reporter protein is integrated into AAVS1 by the CRISPR/Cas9 method,
- the cell is hiPSC.
25. A cell expressing a reporter, wherein the cell is obtained by the use according to claim 24.
26. A differentiated human cell expressing a reporter, wherein the differentiated human cell is produced by differentiating a cell according to claim 25.
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