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JoVE Journal Medicine

Medicine

Electroporation-Based Genetic Modification of Primary Human Pigment Epithelial Cells Using the Sleeping Beauty Transposon System

Published: February 4, 2021 doi: 10.3791/61987
Automatic Translation
1, 2,3, 4,5, 4, 6, 7, 1, 2,3
1Department of Ophthalmology, University Hospital RWTH Aachen, 2Experimental Ophthalmology, University of Geneva, 3Department of Ophthalmology, University Hospitals of Geneva, 4Université de Paris, CNRS, INSERM, UTCBS, Unité des technologies Chimiques et Biologiques pour la Santé, 5Chimie ParisTech, PSL Research University, 6Max Delbrück Center for Molecular Medicine in the Helmholtz Association, 7Division of Medical Biotechnology, Paul-Ehrlich-Institute

Summary

We have developed a protocol to transfect primary human pigment epithelial cells by electroporation with the gene encoding pigment epithelium-derived factor (PEDF) using the Sleeping Beauty (SB) transposon system. Successful transfection was demonstrated by quantitative polymerase chain reaction (qPCR), immunoblotting, and enzyme-linked immunosorbent assay (ELISA).

Abstract

Our increasingly aging society leads to a growing incidence of neurodegenerative diseases. So far, the pathological mechanisms are inadequately understood, thus impeding the establishment of defined treatments. Cell-based additive gene therapies for the increased expression of a protective factor are considered as a promising option to medicate neurodegenerative diseases, such as age-related macular degeneration (AMD). We have developed a method for the stable expression of the gene encoding pigment epithelium-derived factor (PEDF), which is characterized as a neuroprotective and anti-angiogenic protein in the nervous system, into the genome of primary human pigment epithelial (PE) cells using the Sleeping Beauty (SB) transposon system. Primary PE cells were isolated from human donor eyes and maintained in culture. After reaching confluence, 1 x 104 cells were suspended in 11 µL of resuspension buffer and combined with 2 µL of a purified solution containing 30 ng of hyperactive SB (SB100X) transposase plasmid and 470 ng of PEDF transposon plasmid. Genetic modification was carried out with a capillary electroporation system using the following parameters: two pulses with a voltage of 1,100 V and a width of 20 ms. Transfected cells were transferred into culture plates containing medium supplemented with fetal bovine serum; antibiotics and antimycotics were added with the first medium exchange. Successful transfection was demonstrated in independently performed experiments. Quantitative polymerase chain reaction (qPCR) showed the increased expression of the PEDF transgene. PEDF secretion was significantly elevated and remained stable, as evaluated by immunoblotting, and quantified by enzyme-linked immunosorbent assay (ELISA). SB100X-mediated transfer allowed for a stable PEDF gene integration into the genome of PE cells and ensured the continuous secretion of PEDF, which is critical for the development of a cell-based gene addition therapy to treat AMD or other retinal degenerative diseases. Moreover, analysis of the integration profile of the PEDF transposon into human PE cells indicated an almost random genomic distribution.

Introduction

Advanced age is described to be the main risk for neurodegenerative diseases. Age-related macular degeneration (AMD), a polygenic disease leading to severe vision loss in patients older than 60 years of age, belongs to the four most common causes of blindness and vision impairment1 and is expected to increase to 288 million people in 20402. Dysfunctions of the retinal pigment epithelium (RPE), a single layer of tightly packed cells located between the choriocapillaris and the retinal photoreceptors, contribute to the pathogenesis of AMD. The RPE fulfills multiple tasks that are essential for a normal retinal function3 and secretes a variety of growth factors and factors essential to maintain the structural integrity of the retina and the choriocapillaris, thereby supporting photoreceptor survival and providing a basis for the circulation and the supply of nutrients.

In healthy eyes, pigment epithelium-derived factor (PEDF) is responsible for balancing the effects of vascular endothelial growth factor (VEGF) and protects neurons against apoptosis, prevents endothelial cell proliferation, and stabilizes the capillary endothelium. A shifted VEGF-to-PEDF ratio is related to ocular neovascularization, which was observed in animal models4,5 as well as in samples of patients with choroidal neovascularization (CNV) due to AMD and proliferative diabetic retinopathy6,7,8,9,10. The enhanced VEGF concentration is the target for the current standard treatment. The anti-VEGF pharmaceuticals bevacizumab, ranibizumab, aflibercept and, most recently, brolucizumab improve visual acuity in about one third of CNV patients or rather stabilize vision in 90% of cases11,12,13. However, the frequent, often monthly, intravitreal injections bear the risk of adverse events14, impair patient compliance, and represent a significant economic burden to healthcare systems15. Moreover, a certain percentage of patients (2%-20%) do not respond or only poorly react to the anti-VEGF therapy16,17,18,19. These negative concomitants necessitate the development of alternative treatments, e.g., intraocular implants, cell and/or gene therapeutic approaches.

Gene therapy has evolved as promising treatment for hereditary and non-hereditary diseases and intends to restore non-functional gene sequences or suppress malfunctioned ones. For polygenic diseases, where identification and replacement of the causative factors is hardly possible, strategies aim for the continuous delivery of a protective factor. In the case of AMD, various additive therapies have been developed, such as the stable expression of endostatin and angiostatin20, the VEGF antagonist soluble fms-like tyrosine kinase-1 (sFLT-1)21,22, the complement regulatory protein cluster of differentiation 59 (CD59)23 or PEDF24,25. The eye, and especially the retina, is an excellent target for a gene-based medication due to the enclosed structure, good accessibility, small size, and immune privilege, thus allowing for a localized delivery of low therapeutic doses and making transplants less susceptible to rejection. Moreover, the eye enables non-invasive monitoring, and the retina can be examined by different imaging techniques.

Viral vectors are, because of their high transduction efficiency, the main vehicle to deliver therapeutic genes into target cells. However, depending on the viral vector used, different adverse reactions have been described, such as immune and inflammatory responses26, mutagenic and oncogenic effects27,28, or dissemination in other tissues29. Practical limitations include a restricted packaging size30 as well as difficulties and costs associated with the production of clinical grade lots31,32. These drawbacks have promoted the further development of non-viral, plasmid-based vectors that are transferred via lipo-/polyplexes, ultrasound or electroporation. However, genomic integration of the transgene into the host genome is usually not promoted with plasmid vectors, thus resulting in a transient expression.

Transposons are naturally occurring DNA fragments that change their position within the genome, a characteristic that has been adopted for gene therapy. Due to an active integration mechanism, transposon-based vector systems allow for a continuous and constant expression of the inserted transgene. The Sleeping Beauty (SB) transposon, reconstituted from an ancient Tc1/mariner-type transposon found in fish33 and further improved by molecular evolution resulting in the hyperactive variant SB100X34, enabled efficient transposition in various primary cells and was used for the phenotypic correction in different disease models35. At present, 13 clinical trials have been initiated using the SB transposon system. The SB100X transposon system consists of two components: the transposon, which comprises the gene of interest flanked by terminal inverted repeats (TIRs), and the transposase, which mobilizes the transposon. Following plasmid DNA delivery to the cells, the transposase binds the TIRs and catalyzes the excision and integration of the transposon into the cell's genome.

We have developed a non-viral cell-based additive therapy for the treatment of neovascular AMD. The approach comprises the electroporation-based insertion of the PEDF gene into primary pigment epithelial (PE) cells by means of the SB100X transposon system36,37,38. The genetic information of the transposase and PEDF are provided on separate plasmids, thereby enabling the adjustment of the ideal SB100X-to-PEDF transposon ratio. Electroporation is performed using a pipette-based capillary transfection system that is characterized by a maximized gap size between the electrodes while minimizing their surface area. The device was shown to achieve excellent transfection rates in a wide range of mammalian cells39,40,41. The small electrode surface area provides a uniform electric field and reduces the various side effects of electrolysis42.

The anti-angiogenic functionality of PEDF secreted by transfected pigment epithelial cells was shown in various in vitro experiments analyzing the sprouting, migration, and apoptosis of human umbilical vein endothelial cells43. In addition, transplantation of PEDF-transfected cells in a rabbit model of corneal neovascularization44 as well as a rat model of CNV43,45,46 showed decline of neovascularization.

Here, we describe a detailed protocol for the stable insertion of the PEDF gene into primary human RPE cells via the SB100X transposon system using a capillary transfection system. The transfected cells were kept in culture for 21 days and subsequently analyzed in terms of PEDF gene expression by quantitative polymerase chain reaction (qPCR) and in terms of PEDF protein secretion by immunoblotting and enzyme-linked immunosorbent assay (ELISA, Figure 1).

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Protocol

Human donor eyes were obtained from the Aachen Cornea Bank of the Department of Ophthalmology (University Hospital RWTH Aachen) after obtaining informed consent in accordance with the Declaration of Helsinki protocols. Procedures for the collection and use of human samples have been approved by the institutional ethics committee.

1. Isolation of primary human RPE cells

  1. Lay out sterile protective clothing and gloves. Place a sterile drape under a laminar flow.
  2. Place sterile preparation instruments and other necessary sterile equipment under the laminar flow.
  3. Record the receipt of the eye globes, the beginning of the preparation as well as possible abnormalities. Register donor age, gender, cause of death, and the period between time of death and eye removal.
  4. Place the eye globe in a sterile gauze compress and hold it in one hand.
  5. Separate the anterior segment from the posterior segment by a circumferential cut approximately 3.5 mm posterior to the limbus using a scalpel and an extra fine pointed eye scissor.
  6. After tilting the posterior segment downwards, carefully remove the vitreous and the retina using colibri forceps.
  7. Discreetly rearrange the collapsed RPE/choroidea complex using straight iris forceps and subsequently hold it in position with curved iris forceps.
  8. Fill the posterior eye cup with 1 mL of Dulbecco's Modified Eagle's Medium/Ham's F-12 Nutrient Mixture (DMEM/F12) supplemented with 10% fetal bovine serum (FBS), 80 U/mL penicillin and 80 µg/mL streptomycin (Pen/Strep), and 2.5 µg/mL amphotericin B (AmphoB).
    ​NOTE: Contrary to the isolation of primary RPE cells from pig or bovine eyes36,37, the posterior eye cup doesn't have to be filled and incubated with trypsin.
  9. Harvest the RPE cells by gently brushing the retinal pigment epithelium from the optic nerve toward the limbus with a fire-polished curved glass Pasteur pipette, while fixating the RPE/choroidea complex at the limbus using colibri forceps. Transfer the cell suspension into a Petri dish.
  10. Repeat the steps 1.8 and 1.9 and collect all cells in the Petri dish. Resuspend the cell suspension carefully by pipetting up and down using a single channel pipette (100-1,000 µL).
  11. Seed the RPE cell suspension of each eye globe into three wells of a 24-well cell culture plate and fill them up to 1 mL with DMEM/F12 supplemented with FBS, Pen/Strep, and AmphoB.
  12. Maintain the RPE cell cultures at 37 °C in a humidified atmosphere of 95% air and 5% CO2 until confluence is reached. Change the cell culture medium twice a week.

2. Electroporation of primary human RPE cells

  1. Preparation of the SB100X transposase/PEDF transposon plasmid mixture
    1. Purify the SB100X transposase and PEDF transposon plasmid DNA using customary plasmid purification kits, which are identified to be suitable for the use in applications such as transfection, according to the protocol of the manufacturer.
    2. Quantify the plasmid DNA contents using a microvolume spectrophotometer and adjust them to a concentration of 250 ng/µL with 10 mM Tris-HCl (pH 8.5).
    3. Mix one portion of 250 ng/µL SB100X transposase plasmid DNA (e.g., 2.5 µL) with 16 portions of 250 ng/µL PEDF transposon plasmid DNA (e.g., 40 µL). Residual plasmid mixture can be stored at -20 °C.
      ​NOTE: Multiple repeated freezing and thawing cycles of the plasmid mixture should be avoided.
    4. Place 2 µL of the SB100X transposase/PEDF transposon plasmid mixture, containing 29.4 ng of SB100X transposase plasmid and 470.6 ng of PEDF transposon plasmid, in a sterile 1.5 mL safe-lock microcentrifuge tube. Keep on ice until mixed with cells.
  2. Set-up of the capillary transfection system
    NOTE: Electroporation of primary human RPE cells has been performed via a capillary transfection system using the 10 µL kit according to the manufacturer's protocol. The system comprises the transfection device, the transfection pipette, and the pipette station. The kit contains 10 µL transfection tips, buffer tubes, electrolytic buffer E (buffer E), and resuspension buffer R (buffer R).
    1. Connect the pipette station to the transfection device.
    2. Fill a buffer tube with 3 mL of buffer E and insert it into the pipette station until a click sound is heard.
      NOTE: Ensure that the side electrode of the buffer tube is connected to the side ball plunger of the pipette station.
    3. For the electroporation of primary human RPE cells, set the following pulse conditions on the transfection device: 1,100 V (pulse voltage), 20 ms (pulse width), two pulses (pulse number).
  3. Preparation of the cultivated primary human RPE cells
    1. Document shape and morphology of the primary RPE cell cultures via phase contrast microscopy. Take low magnification micrographs to demonstrate the growth of the RPE cells to a confluent and integrated monolayer as well as higher magnification micrographs to point out the typical cobblestone morphology of the RPE cells.
    2. Aspirate the cell culture medium and wash the cells twice with 1 mL buffered phosphate saline (PBS, pH 7.4).
    3. Trypsinize primary human RPE cells with 0.5 mL 0.05% trypsin-EDTA at 37 °C in a humidified atmosphere of 95% air and 5% CO2 for 7-15 min (maximum). Check the cell detachment microscopically.
    4. Stop trypsinization with 1 mL of DMEM/F12 supplemented with FBS. Take a 20 µL aliquot for cell counting using a hemocytometer. Centrifuge the RPE cell suspension at 106 x g for 10 min.
    5. Mix the 20 µL aliquot with 20 µL trypan blue solution. Pipette 10 µL in each half of the hemocytometer and count the cells under a phase contrast microscope.
    6. After centrifugation, resuspend the RPE cell pellet in 1 mL of PBS.
    7. Take 10,000-100,000 RPE cells per transfection reaction and centrifuge them at 106 x g for 10 min.
      ​NOTE: Beside the transfection reactions with electric field application and addition of plasmid DNA, each approach also includes two different control cultures: (1) without electric field application and without plasmid DNA; (2) with electric field application, but without plasmid DNA.
    8. Resuspend the cell pellet in 11 µL of buffer R and combine it with 2 µL of the SB100X transposase/PEDF transposon plasmid mixture (see step 2.1.4).
      ​NOTE: After resuspension in buffer R, the cells must be processed within 15 min to avoid a reduction in cell viability and transfection efficiency.
    9. Insert the head of the transfection pipette into the 10 µL transfection tip until the clamp fully picks up the mounting stem of the piston. Draw up the cell/plasmid solution into the transfection tip.
      NOTE: Avoid generation of air bubbles and their aspiration into the transfection tip.
    10. Provide 1 mL of DMEM/F12 supplemented with FBS, but without Pen/Strep and without AmphoB, into the required number of wells of a 24-well cell culture plate.
  4. Electroporation process
    1. Insert the transfection pipette into the buffer tube placed in the pipette station (see step 2.2.2) until a click sound is heard.
      ​NOTE: Ensure the metal head of the transfection pipette is connected to the ball plunger inside of the pipette station and to the buffer tube.
    2. Press Start on the touchscreen of the transfection device. Before application of the electric pulse, the device automatically checks whether the buffer tube and the transfection pipette are properly inserted. After delivery of the electric pulses, Complete is displayed on the touchscreen.
    3. Carefully remove the transfection pipette from the pipette station and immediately release the electroporated cells from the 10 µL transfection tip by pipetting the cell/plasmid solution into the prepared wells of the cell culture plate (see step 2.3.10).
    4. Maintain transfected RPE cell cultures at 37 °C in a humidified atmosphere of 95% air and 5% CO2. Add Pen/Strep and AmphoB with the first medium exchange 3 days after electroporation.

3. Analyses of transfected primary human RPE cells

  1. Sample preparation
    1. After a cultivation time of 3 weeks, ultimately incubate the RPE cell cultures in a defined volume of 1.0 mL DMEM/F12 supplemented with FBS, Pen/Strep, and AmphoB for a defined time of 24 h.
    2. Take the cell culture supernatants and store them at -20 °C until further near-time processing or at -80 °C for thermally unstable samples and long-term storage.
    3. Trypsinize transfected RPE cell cultures with 0.5 mL of 0.05% trypsin-EDTA at 37 °C in a humidified atmosphere of 95% air and 5% CO2 for 10 min.
    4. Stop trypsinization with 1 mL of DMEM/F12 supplemented with FBS. Take small aliquots for cell counting using a hemocytometer (see step 2.3.5). Centrifuge the RPE cell suspensions at 106 x g for 10 min.
    5. Store the RPE cell pellets at -80 °C until further processing.
  2. Evaluation of PEDF secretion by western blotting
    NOTE: For a qualitative assessment, cell culture supernatants (see step 3.1.2) are analyzed via sodium dodecyl sulfate polyacrylamide gel electrophoresis (SDS-PAGE) and subsequent western blotting. Depending on the PEDF transposon plasmid DNA used, the supernatants have to be processed differently.
    1. Purification of His-tagged PEDF fusion proteins from cell culture supernatants
      1. Take 30 µL nickel-nitrilotriacetic acid (Ni-NTA) slurry per sample using a bevel-cut tip and pellet the Ni-NTA resin by centrifugation (2,660 x g for 30 s).
      2. Carefully resuspend the Ni-NTA resin with 200 µL of 1x incubation buffer (50 mM NaH2PO4, 300 mM NaCl, 10 mM imidazole, pH 8.0) and pellet it by centrifugation (2,660 x g for 30 s).
      3. Repeat the previous step.
      4. Carefully resuspend the Ni-NTA resin with 40 µL 4x incubation buffer (200 mM NaH2PO4, 1.2 M NaCl, 40 mM imidazole, pH 8.0) per sample.
      5. Mix 900 µL of cell culture supernatant with 260 µL of 4x incubation buffer and 55 µL of pretreated Ni-NTA slurry (see step 3.2.1.4).
      6. Incubate the mixture on a rocking shaker at room temperature for 60 min.
      7. Pellet the Ni-NTA resin by centrifugation (2660 x g for 60 s).
      8. Carefully resuspend the Ni-NTA resin with 175 µL of 1x incubation buffer and pellet resin by centrifugation (2,660 x g for 60 s).
      9. Repeat the previous step.
      10. Carefully resuspend the Ni-NTA resin with 30 µL of elution buffer (50 mM NaH2PO4, 300 mM NaCl, 250 mM imidazole, pH 8.0) and incubate the mixture on a rocking shaker at room temperature for 20 min.
      11. Pellet the Ni-NTA resin by centrifugation (2,660 x g for 30 s).
      12. Carefully take the supernatant and mix it with 2x SDS sample buffer47.
      13. Heat the mixture at 95 °C for 5 min and separate the Ni-NTA purified proteins on a 10% SDS-polyacrylamide gel.
      14. Perform western blotting using anti-Penta-His antibodies (mouse monoclonal, 1:500) and horseradish peroxidase (HRP)-conjugated anti-mouse antibodies (rabbit polyclonal, 1:1,000) as previously described36,37.
    2. Direct analysis of non-tagged PEDF proteins
      1. Take 15 µL of cell culture supernatant and mix it with 2x SDS sample buffer47.
      2. Heat the mixture at 95 °C for 5 min and separate the proteins on a 10% SDS-polyacrylamide gel.
      3. Perform western blotting using anti-human PEDF antibodies (rabbit polyclonal, 1:4,000) and HRP-conjugated anti-rabbit antibodies (goat polyclonal, 1:2,000) as previously described38.
    3. Quantification of PEDF secretion by ELISA
      1. Analyze cell culture supernatants (see step 3.1.2) using a human PEDF ELISA kit according to the manufacturer's protocol.
      2. Relate the amount of secreted PEDF to the time and the cell number determined for each transfection reaction (see step 3.1.4).
    4. Analysis of PEDF gene expression in transfected RPE cells
      1. Isolate total RNA from the RPE cell pellets (see step 3.1.5) using a commercially available kit according to the manufacturer's protocol.
      2. Quantify RNA contents using a microvolume spectrophotometer.
      3. Carry out reverse transcription on 0.1 µg RNA using a reverse transcription system according to the manufacturer's protocol.
      4. Perform real-time qPCR as previously described38.
    5. Analysis of transgene insertion sites in transfected RPE cells
      1. Isolate genomic DNA from the RPE cell pellets (see step 3.1.5) using a commercially available kit according to the manufacturer's protocol.
      2. Quantify DNA contents using a microvolume spectrophotometer.
      3. Generate insertion site libraries using a computation-assisted hemi-specific PCR scheme as previously described38.
      4. Perform computational analysis as previously described38.

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Representative Results

Cultivation and electroporation of primary human RPE cells
We have shown that seeding of a sufficient number of primary RPE cells of animal origin allows for the cultivation and growth to an integrated monolayer of pigmented, hexagonally shaped cells36,37,48. Their capability to form tight junctions, to exhibit phagocytic activity, and to express specific marker genes in vitro48 reflects substantial tasks of the retinal pigment epithelium in vivo. Cultivated primary RPE cells isolated from human donor eyes also showed the typical cobblestone morphology, regardless of the donor's age (65.3 ± 9.94 a, min: 49 a, max: 83 a, n = 12), the post-mortem time of isolation (37.3 ± 17.0 h, min: 16 h, max: 68 h, n = 12), and the cultivation time (27.6 ± 14.1 d, min: 13 d, max: 61 d, n = 12) (Figure 2, left panel). The application of short-term electrical pulses to primary human RPE cells using the capillary transfection system did not adversely affect the epithelial morphology (Figure 2, right panel). Testing of various electric parameters revealed that two pulses with a pulse voltage of 1,200 V and a pulse width of 20 ms yielded good and stable transfection efficiencies and the highest number of viable RPE cells (unpublished data).

SB100X-mediated insertion of the PEDF gene into cultivated primary human RPE cells
We have tested plasmid DNA ratios of SB100X-to-PEDF transposon ranging from 250 ng (0.08 pmol) SB100X transposase plus 250 ng (0.052 pmol) PEDF transposon to 12.2 ng (0.0039 pmol) SB100X transposase plus 487.8 ng (0.1 pmol) PEDF transposon. This approach identified the two combinations 29.4 ng (0.0094 pmol) SB100X transposase plus 470.6 ng (0.098 pmol) PEDF transposon and 23.8 ng (0.0076 pmol) plus 476.2 ng (0.099 pmol) as the ones that obtained the best transposition efficiencies37. In cultivated primary human RPE cells, delivery of the PEDF transgene using the SB100X transposon system allowed for a continuously increased PEDF gene expression and PEDF protein secretion. For His-tagged recombinant PEDF, western blot analysis of cell culture media from transfected primary human RPE cells demonstrated PEDF secretion at constant levels without transgene silencing for more than 500 days (Figure 3A). For non-tagged PEDF, secretion in transfected primary human RPE cells was also shown to be significantly increased compared to non-transfected cells38. A representative western blot analysis of cell culture media from serially performed transfections clearly indicated the universally higher PEDF secretion rate at 21 days after transfection (Figure 3B), and in a pursued culture, long-term elevated PEDF secretion was proven for at least 165 days (Figure 3C). ELISA-based quantification demonstrated a 20-fold increase of total PEDF secretion in transfected primary human RPE cells compared to respective non-transfected control cells (Figure 3D). This increment was also affirmed on gene expression level, where the total PEDF expression was raised more than 30-fold (Figure 3E).

Figure 1
Figure 1: Workflow for the electroporation-based insertion of the PEDF gene into primary human RPE cells by means of the SB100X transposon system. The scheme describes the chronological course of (A) the isolation of primary human RPE cells and their subsequent cultivation to a confluent monolayer, (B) the single steps of the electroporation process, including the purification of the SB100X transposase and the PEDF transposon plasmid DNA, the preparation of the SB100X transposase/PEDF transposon plasmid mixture, the set-up of the capillary transfection system, the preparation of the cultivated primary human RPE cells, the electroporation, the seeding, and the cultivation of the transfected RPE cells, as well as (C) the analyses of the transfected primary human RPE cells, comprising the preparation of the cell culture supernatants and transfected cell samples, the evaluation and quantification of PEDF secretion, and the analysis of PEDF gene expression. Please click here to view a larger version of this figure.

Figure 2
Figure 2: Phase contrast micrographs of RPE cells isolated from different donor eyes. Cultures of primary human RPE cells, differing in donor age, post-mortem time of isolation, and cultivation time prior to their application for electroporation (left panel), as well as cultures of transfected primary human RPE cells, exposed to the electric parameters 1,100 V (pulse voltage), 20 ms (pulse width), and 2 pulses (number of pulses) using the capillary transfection system (right panel), exhibited a confluent integrated monolayer of pigmented cells in a cobblestone-like pattern (scale bars: 500 µm). Please click here to view a larger version of this figure.

Figure 3
Figure 3: PEDF secretion and PEDF gene expression after SB100X-mediated transfection of primary human RPE cells. (A-C) Immunoblot-based analyses of PEDF secretion stability in cultivated primary human RPE cells transfected with a mixture of 29.4 ng (0.0094 pmol) SB100X transposase plasmid plus 470.6 ng (0.098 pmol) PEDF transposon plasmid using the capillary transfection system. (A) RPE cells from a 26-year-old donor (female, post-mortem time of isolation: 26 h, cultivation time before electroporation: 14 days) were transfected and maintained in culture for more than 500 days. His-tagged recombinant PEDF (~48 kDa) was purified from cell culture media at different time points and evaluated by western blotting using anti-Penta-His antibodies. (B) For non-tagged recombinant PEDF, RPE cells from a 53-year-old donor (female, post-mortem time of isolation: 28 h, cultivation time before transfection: 19 days) were electroporated without plasmid DNA (control cultures #1 and #2) or with the addition of plasmid DNA (PEDF-transfected cell cultures #3 to #7) and maintained in culture for 21 days. Cell culture supernatants were not purified, but directly analyzed by immunoblotting using anti-PEDF antibodies. Western blot analysis of cell lysates showed the level of intracellular PEDF in controls (cultures #1 and #2) and transfected cells (cultures #3 and #4). Loading of similar protein amounts was indicated by the equal density of GAPDH protein bands (~36 kDa). (C) For the analysis of long-term PEDF secretion, RPE cells from a 63-year-old donor (male, post-mortem-time of isolation: 26 h, cultivation time before transfection: 15 days) were electroporated without plasmid DNA (control cultures #1 and #2) or with plasmid DNA (PEDF-transfected cell culture #3) and maintained in culture for at least 165 days. Cell culture media were not purified, but directly analyzed by immunoblotting using anti-PEDF antibodies. (D) ELISA-based quantification of total PEDF secretion in transfected primary human RPE cells (two samples) and non-transfected control cells (four samples). Secretion of the transfected cells was compared to the secretion of the non-transfected cells (*p = 0.0264, unpaired t test with Welch's correction). (E) Endogenous and total (endogenous plus recombinant) PEDF gene expression in transfected primary human RPE cells were related to the expression in non-transfected control cells, whose expression was set to 1 (dashed line). Data is presented as a box-and-whisker plot (whiskers: min to max). Total PEDF gene expression was compared to endogenous PEDF gene expression (not significant, unpaired t test with Welch's correction). The results for the non-tagged recombinant PEDF (B-E) represent two units of the entire data set of primary RPE cells from up to 27 donors transfected with a SB100X-to-PEDF transposon ratio of 29.4 ng (0.0094 pmol) plus 470.6 ng (0.098 pmol), which was described by Thumann et al. 201738. Please click here to view a larger version of this figure.

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Discussion

In our project, we aim for the non-viral production of genetically modified primary human RPE cells that continuously overexpress and secrete an effective factor in order to use the transfected cells as long-term therapeutic for the establishment and maintenance of a protective environment. We have established the introduction of the gene encoding PEDF, an ubiquitously expressed multi-functional protein with anti-angiogenic and neuroprotective functions. The protocol described here can be used to stably and reproducibly transfect primary human RPE cells using the SB100X transposon system. DNA delivery and introduction into the RPE cells is performed with a pipette-based capillary transfection system, which uses specific tips as electroporation chamber instead of cuvettes.

The enrichment of primary RPE cultures consisting of morphologically well-differentiated cells to be used for subsequent transfections is influenced by several factors related to the human donor (age and general health status) as well as the receipt of the eyes (post-mortem time of cell isolation). Freshly isolated and seeded primary RPE cells need at least 2 weeks to develop a monolayer of hexagonally shaped cells. As a general rule, cell cultures that needed more time to reach confluence prior to transfection also showed a decelerated adherence and spreading after transfection. Cell adherence and spreading is also impeded by free pigment depositing from RPE cells damaged during cell isolation or the transfection process.

The SB transposon system is a widely used genetic tool that enables the efficient and safe delivery of therapeutically relevant nucleotide sequences into various types of cells to be subsequently applied in gene-based cell therapies. Especially, its enhanced variant SB100X combines the advantages of viral vectors and non-viral plasmid DNA. The integrating mode of action ensures a stable genomic integration of the expression cassette into the host cell's genome and enables a sustained expression of the gene or sequence of interest. In contrast to retroviral vectors, which preferentially integrate into active transcription units with a high risk of potential mutagenesis and oncogenesis27,28, the SB-based integration profile is random. This was demonstrated in a large number of studies using various cultured and primary mammalian cells, including human cells49,50,51,52, as well as for primary rat and human RPE cells38,46. Moreover, application of the SB transposon system as plasmid DNA provides reduced immunogenicity and facilitates the manufacturing process.

Delivery of the genetic information of the SB100X transposase and the PEDF transgene on separate plasmids is an important aspect, as it allows for a precise adjustment of the optimal SB100X-to-PEDF transposon ratio. As described earlier, the SB transposon system is sensitive to an excess of transposase expression, which results in inhibition of the transpositional activity53. We also observed this effect for ARPE-19 cells, a spontaneously evolved cell line purified by selective trypsinization of a primary human RPE culture54, and primary RPE cells. Here, transfection with SB100X-to-PEDF transposon ratios up to 55.6 ng (0.018 pmol) SB100X transposase plus 444.4 ng (0.093 pmol) PEDF transposon resulted in clearly diminished PEDF secretion rates37. The ideal transposase-to-transposon ratio has to be determined for every newly constructed transposon plasmid.

Non-viral plasmid-based vectors can be transferred via lipo-/polyplexes, nanoparticles, laser, ultrasound, or electroporation. We have already shown that lipofection-based transfection of primary PE cells was not efficient and thus switched to the electroporation-based gene transfer36. Electroporation is defined as application of a short-duration electric field to cells, which results in an increase of the membrane's permeability and the formation of aqueous pores, through which plasmid DNA can enter the cell. In general, a mixture of cells, plasmid DNA, and conducting buffer is filled into a specific electroporation chamber between two electrodes, which are connected to the electroporation device generating the electrical pulses. In this so-called bulk electroporation setup, a conventional cuvette electroporation chamber is characterized by two parallel plate-type electrodes, whose distance is variable (0.1-0.4 mm), depending on the cell type and the number of cells used per electroporation reaction. Despite good transfection efficiencies, different side effects may negatively affect the survival of the transfected cells, e.g., pH changes, heat development, bubble generation, and turbulent flow. The capillary transfection system at least attenuates the cuvette-based side effects by possessing electrodes with a minimized surface area and a maximized gap size between them42 as well as by using specific resuspension and electrolytic buffers; however, their compositions are not disclosed.

Despite the enhancements within the bulk electroporation set-up, which led to an increased cell survival, irreversible damage to a certain percentage of cells is unavoidable. Moreover, a precise control of the amount of plasmid integrated into the cells is not possible. The more recent development of miniaturized electroporation systems has allowed for additional improvements by further reducing the limitations associated with bulk electroporation. These new devices are based on microelectrodes, microfluidic, or nanostructures. They offer new application perspectives in the fields of gene therapy, regenerative medicine, and in situ intracellular investigation (reviewed by Chang et al. and Shi et al.)55,56.

We have demonstrated that electroporation, which was initially performed using a cuvette-based system and subsequently established using a pipette-based capillary system, is a suitable and efficient method to transfect both the pigment epithelial cell line ARPE-19 as well as primary PE cells36,37,38,57,58. Depending on the cell type used, it is important to define appropriate electroporation protocols applying parameters that allow for both good transfection efficiencies and cell survival. Insufficient electric fields prevent the cells from damage but result in low transfection efficiencies. With the increase in electric field strength, enhanced transfection efficiencies are achieved. However, elevated electric parameters also lead to higher percentages of dead cells because of irreversible cell damage. For the ARPE-19 cell line, using the capillary transfection system, electroporation parameters of 1,350 V, 20 ms, and 2 pulses were shown to result in initial transfection efficiencies of 100%37. The application of these parameters for the transfection of primary RPE cells resulted in good efficiencies, too, but also in a lower number of cultivated cells after transfection when compared to electroporation parameters of 1,100 V, 20 ms, and 2 pulses (data not shown). Therefore, the decision was taken to choose the milder electroporation parameters to prevent cell damage and accept less transfected cells.

The overall aim of this approach lies in the clinical application of PEDF-transfected cells for the treatment of patients suffering from neovascular AMD. The therapy comprises the stable introduction of the PEDF transgene into autologous pigment epithelial cells ex vivo based on the SB100X transposon system, followed by transplantation of the transfected cells to the subretinal space of the patient. Thus, it is important to ensure that the PEDF-transfected cells do not transdifferentiate and do not acquire a fibroblastic shape and growth behavior. It is also important to prove that the transfected cells allow for a sustained and consistent secretion of PEDF. Moreover, it is crucial to know the precise amount of PEDF secreted by a particular number of cells for a specific period of time.

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Disclosures

Zoltán Ivics and Zsuzsanna Izsvák are inventors on several patents on SB transposon technology

Acknowledgments

This work was supported by the European Union's Seventh Framework Programme for research, technological development and demonstration, grant agreement no. 305134. Zsuzsanna Izsvák was funded by the European Research Council, ERC Advanced (ERC-2011-ADG 294742). The authors would like to thank Anna Dobias and Antje Schiefer (Department of Ophthalmology, University Hospital RWTH Aachen) for excellent technical support, and the Aachen Cornea Bank (Department of Ophthalmology, University Hospital RWTH Aachen) for providing the human donor eyes.

Materials

Name Company Catalog Number Comments
Isolation of primary human RPE cells
24-Well Cell Culture Plate Eppendorf, Hamburg, Germany 0030722019
Amphotericin B [250 µg/mL] (AmphoB) Merck, Darmstadt, Germany A2942
Colibri Forceps Geuder, Heidelberg, Germany G-18950
Curved Iris Forceps  Geuder, Heidelberg, Germany G-18856
Disposable Scalpel (No. 11) Feather, Osaka, Japan
Dulbecco’s Modified Eagle’s Medium/Ham’s F-12 Nutrient Mixture (DMEM/F12) PAN-Biotech, Aidenbach, Germany P04-41150
Extra Fine Pointed Eye Scissor  Geuder, Heidelberg, Germany G-19405
Fetal Bovine Serum [0.2 µm Sterile Filtered] (FBS) PAN-Biotech, Aidenbach, Germany P40-37500
Glass Pasteur Pipettes Brand, Wertheim, Germany 747715
Penicillin [10,000 units/mL] and Streptomycin [10 mg/mL] (Pen/Strep) Merck, Darmstadt, Germany P0781
Pipette Tips (1000 µL) Starlab, Hamburg, Germany
Single Channel Pipette (100-1000 µL) Eppendorf, Hamburg, Germany
Sterile Drape Lohmann & Rauscher, Rengsdorf, Germany
Sterile Gauze Compress  Fink-Walter, Merchweiler, Germany 321063
Sterile Gloves Sempermed, Wien, Austria
Sterile Petri Dish (Falcon 60 mm x 15 mm) Corning, Corning, NY 351007
Sterile Surgical Gown Halyard Health, Alpharetta, GA
Straight Iris Forceps  Geuder, Heidelberg, Germany G-18855
Electroporation of primary human RPE cells
10 mM Tris-HCl (pH 8.5)
12-Well Cell Culture Plate Thermo Fisher Scientific, Waltham, MA 150628
24-Well Cell Culture Plate Eppendorf, Hamburg, Germany 0030722019
Amphotericin B [250 µg/mL] (AmphoB) Merck, Darmstadt, Germany A2942
Dulbecco’s Modified Eagle’s Medium/Ham’s F-12 Nutrient Mixture (DMEM/F12) PAN-Biotech, Aidenbach, Germany P04-41150
Safe-Lock Microcentrifuge Tubes (1.5 mL) Eppendorf, Hamburg, Germany
Fetal Bovine Serum [0.2 µm Sterile Filtered] (FBS) PAN-Biotech, Aidenbach, Germany P40-37500
Inverted Microscope Leica Mikrosysteme, Wetzlar, Germany Leica DMi8
Microvolume Spectrophotometer (NanoDrop Spectrophotometer) Thermo Fisher Scientific, Waltham, MA
Capillary Transfection System (Neon Transfection System) Thermo Fisher Scientific, Waltham, MA MPK5000
Neon Transfection System 10 µL Kit Thermo Fisher Scientific, Waltham, MA MPK1096
Hemocytometer (Neubauer Chamber) Paul Marienfeld, Lauda-Königshofen, Germany 0640110
PBS Dulbecco w/o Ca2+ w/o Mg2+ Biochrom, Berlin, Germany L182-50
Penicillin [10,000 units/mL] and Streptomycin [10 mg/mL] (Pen/Strep) Merck, Darmstadt, Germany P0781
Pipette Tips (10 µL) Starlab, Hamburg, Germany
Pipette Tips (1000 µL) Starlab, Hamburg, Germany
Pipette Tips (200 µL) Starlab, Hamburg, Germany
Plasmid Maxi Kit Qiagen, Hilden, Germany 12163
Single Channel Pipette (0.1-10 µL) Eppendorf, Hamburg, Germany
Single Channel Pipette (100-1000 µL) Eppendorf, Hamburg, Germany
Single Channel Pipette (10-200 µL) Eppendorf, Hamburg, Germany
Trypan Blue Solution Merck, Darmstadt, Germany T8154
Trypsin-EDTA (0,05 %) Thermo Fisher Scientific, Waltham, MA 25300054
Analyses of transfected primary human RPE cells
10% SDS-Polyacrylamide Gel
1x Incubation Buffer (50 mM NaH2PO4, 300 mM NaCl, 10 mM imidazole, pH 8.0)
2x SDS Sample Buffer
4x Incubation Buffer (200 mM NaH2PO4, 1.2 M NaCl, 40 mM imidazole, pH 8.0)
Amersham Protran Supported 0.2 µm Nitrocellulose Blotting Membrane Cytiva, Marlborough, MA 10600015
Amphotericin B [250 µg/mL] (AmphoB) Merck, Darmstadt, Germany A2942
Anti-PEDF Antibodies (Rabbit Polyclonal) BioProducts, Middletown, MD AB-PEDF1
Anti-Penta-His Antibodies (Mouse Monoclonal) Qiagen, Hilden, Germany 34660
Dulbecco’s Modified Eagle’s Medium/Ham’s F-12 Nutrient Mixture (DMEM/F12) PAN-Biotech, Aidenbach, Germany P04-41150
Elution Buffer (50 mM NaH2PO4, 300 mM NaCl, 250 mM imidazole, pH 8.0) 
Fetal Bovine Serum [0.2 µm Sterile Filtered] (FBS) PAN-Biotech, Aidenbach, Germany P40-37500
Hemocytometer (Neubauer Chamber) Paul Marienfeld, Lauda-Königshofen, Germany 0640110
Horseradish Peroxidase-Conjugated Anti-Mouse Antibodies (Rabbit Polyclonal) Agilent Dako, Santa Clara, CA P0260
Horseradish Peroxidase-Conjugated Anti-Rabbit Antibodies (Goat Polyclonal) Abcam, Cambridge, United Kingdom ab6721
Human PEDF ELISA Kit  BioProducts, Middletown, MD PED613
LAS-3000 Imaging System Fujifilm, Minato, Japan
LightCycler 1.2 Instrument Roche Life Science, Penzberg, Germany
LightCycler FastStart DNA Master SYBR Green I Roche Life Science, Penzberg, Germany 12239264001
LightCycler Capillaries (20 μl) Roche Life Science, Penzberg, Germany 4929292001
Microvolume Spectrophotometer (NanoDrop Spectrophotometer) Thermo Fisher Scientific, Waltham, MA
Mini-PROTEAN Tetra Cell Casting Module Bio-Rad Laboratories, Feldkirchen, Germany 1658015
Mini-PROTEAN Tetra Vertical Electrophoresis Cell for Mini Precast Gels, 4-gel Bio-Rad Laboratories, Feldkirchen, Germany 1658004
Ni-NTA Superflow Qiagen, Hilden, Germany 30410
PageRuler Prestained Protein Ladder Thermo Fisher Scientific, Waltham, MA 26616
Penicillin [10,000 units/mL] and Streptomycin [10 mg/mL] (Pen/Strep) Merck, Darmstadt, Germany P0781
Pipette Tips (10 µL) Starlab, Hamburg, Germany
Pipette Tips (1000 µL) Starlab, Hamburg, Germany
Pipette Tips (200 µL) Starlab, Hamburg, Germany
PowerPac Basic Power Supply Bio-Rad Laboratories, Feldkirchen, Germany 1645050
QIAamp DNA Mini Kit Qiagen, Hilden, Germany 51304
Reverse Transcription System  Promega, Madison, WI A3500
RNase-Free DNase Set Qiagen, Hilden, Germany 79254
RNeasy Mini Kit  Qiagen, Hilden, Germany 74104
Rocking Shaker Cole-Parmer, Staffordshire, United Kingdom SSM3
Safe-Lock Microcentrifuge Tubes (1.5 mL) Eppendorf, Hamburg, Germany
Safe-Lock Microcentrifuge Tubes (2.0 mL) Eppendorf, Hamburg, Germany
Single Channel Pipette (0.1-10 µL) Eppendorf, Hamburg, Germany
Single Channel Pipette (100-1000 µL) Eppendorf, Hamburg, Germany
Single Channel Pipette (10-200 µL) Eppendorf, Hamburg, Germany
Trans-Blot Turbo Transfer System Bio-Rad Laboratories, Feldkirchen, Germany 1704150
Trypan Blue Solution Merck, Darmstadt, Germany T8154
Trypsin-EDTA (0,05 %) Thermo Fisher Scientific, Waltham, MA 25300054

DOWNLOAD MATERIALS LIST

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Tags

Electroporation-based Genetic Modification Primary Human Pigment Epithelial Cells Sleeping Beauty Transposon System Transgene Expression Protein Secretion Cell-based Gene Addition Therapy Retinal Degenerative Diseases Plasmid DNA Microvolume Spectrophotometer SB100X Transposase Plasmid DNA Pigment Epithelium Derived Factor Transposon Plasmid DNA Sterile 1.5 Milliliters Safe Lock Micro Centrifuge Tube Buffer E
Electroporation-Based Genetic Modification of Primary Human Pigment Epithelial Cells Using the Sleeping Beauty Transposon System
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Johnen, S., Harmening, N., Marie,… More

Johnen, S., Harmening, N., Marie, C., Scherman, D., Izsvák, Z., Ivics, Z., Walter, P., Thumann, G. Electroporation-Based Genetic Modification of Primary Human Pigment Epithelial Cells Using the Sleeping Beauty Transposon System. J. Vis. Exp. (168), e61987, doi:10.3791/61987 (2021).

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