Transfection, Selection, and Colony-picking of Human Induced Pluripotent Stem Cells TALEN-targeted with a GFP Gene into the AAVS1 Safe Harbor

Podsumowanie

TALEN-mediated gene editing at the safe harbor AAVS1 locus enables high-efficiency transgene addition in human iPSCs. This protocol describes the procedures for preparing iPSCs for TALEN and donor vector delivery, transfecting iPSCs, and selecting and isolating iPSC clones to achieve targeted integration of a GFP gene to generate reporter lines.

Streszczenie

Targeted transgene addition can provide persistent gene expression while circumventing the gene silencing and insertional mutagenesis caused by viral vector mediated random integration. This protocol describes a universal and efficient transgene targeted addition platform in human iPSCs based on utilization of validated open-source TALENs and a gene-trap-like donor to deliver transgenes into a safe harbor locus. Importantly, effective gene editing is rate-limited by the delivery efficiency of gene editing vectors. Therefore, this protocol first focuses on preparation of iPSCs for transfection to achieve high nuclear delivery efficiency. When iPSCs are dissociated into single cells using a gentle-cell dissociation reagent and transfected using an optimized program, >50% cells can be induced to take up the large gene editing vectors. Because the AAVS1 locus is located in the intron of an active gene (PPP1R12C), a splicing acceptor (SA)-linked puromycin resistant gene (PAC) was used to select targeted iPSCs while excluding random integration-only and untransfected cells. This strategy greatly increases the chance of obtaining targeted clones, and can be used in other active gene targeting experiments as well. Two weeks after puromycin selection at the dose adjusted for the specific iPSC line, clones are ready to be picked by manual dissection of large, isolated colonies into smaller pieces that are transferred to fresh medium in a smaller well for further expansion and genetic and functional screening. One can follow this protocol to readily obtain multiple GFP reporter iPSC lines that are useful for in vivo and in vitro imaging and cell isolation.

Wprowadzenie

The ability to reprogram human somatic cells into embryonic stem cell-like induced pluripotent stem cells (iPSCs) was first discovered by Takahashi et al. in 2007¹. Human dermal fibroblasts transduced with retroviruses expressing four transcription factors (The so-dubbed Yamanaka factors Oct3/4, Sox2, c-Myc, and Klf4) were shown to be highly similar to human embryonic stem cells (hESCs) based on morphology, proliferation, gene expression, and epigenetic status; crucially, iPSCs are also capable of differentiating into cells of all three germ layers¹. The proliferative potential and differentiation capacity of iPSCs makes them very attractive tools; by reprogramming cells from patients suffering from specific diseases, iPSCs can be used both as in vitro disease model systems and as potential therapeutics.

For the latter purpose, several issues must be addressed before the full potential of iPSCs in a clinical setting can be realized; the tumorigenic potential of in vitro cultured hESCs and iPSCs, the use of xenogenic derivatives during reprogramming and cell maintenance, and the need to track transplanted cells in vivo are all crucial hurdles to the clinical application of pluripotent stem cells (Reviewed by Hentze et at.²). An ideal solution to the need for tracking differentiated cells post-transplantation would involve a visually detectable marker that resists silencing and variegation regardless of the application. Robust and sustained expression of integrated transgenes is most readily achievable when exogenous DNA is introduced into safe-harbor loci; that is, genomic sites that enable sufficient transcription of an integrated vector while at the same time mitigating perturbations of expression in neighboring genes³. One such site that has been very well characterized since its discovery is the adeno-associated virus integration site 1 (AAVS1), in the first intron of the protein phosphatase 1 regulatory subunit 12C (PPP1R12C) gene. This locus has been shown not only to permit sustained and robust expression of integrated transgenes through extended time in culture and in vitro differentiation³, but also to protect surrounding genes from transcriptional perturbation⁴; both features are thought to be due to the presence of endogenous chromatin insulator elements flanking the AAVS1 site⁵.

Advances in genome engineering tools over just the past decade have greatly facilitated the ease and efficiency with which genetic manipulations in any cell type can be achieved. While early successful experiments relied on exceedingly low levels of endogenous homologous recombination (HR) with an introduced donor to achieve gene targeting in ESCs^6,7, the use of site-specific nucleases, such as zinc finger nucleases (ZFNs), that significantly induce homologous recombination through the generation of a double-stranded DNA break has greatly increased the efficiency of such experiments^8,9. The repurposing of both transcription activator-like effectors (TALEs) of plant pathogenic xanthomonas genera and the prokaryotic clustered regularly interspaced short palindromic repeats (CRISPR)/Cas9 system into efficient site-specific designer nucleases has made gene targeting in pluripotent stem cells an accessible and practicable methodology^10-13.

A recent paper described an efficient method for the stable integration of a green fluorescent reporter cassette into the AAVS1 safe-harbor locus in human iPSCs using TALE nucleases (TALENs)¹⁴. These targeted iPSCs maintained their fluorescence even after directed differentiation to cardiomyocytes and transplantation into a mouse model of myocardial infarction (MI), providing strong evidence for the utility of such stably fluorescent pluripotent stem cells¹⁴. To obtain targeted colonies, a gene-trap method was used wherein a splicing-acceptor (SA), 2A self-cleaving peptide sequence places the puromycin N-acetyl-transferase (PAC) gene under the control of the endogenous PPP1R12C promoter; thus, only iPSCs that have incorporated the DNA donor at the AAVS1 locus express PAC, rendering them selectable based on puromycin-resistance; (Figure 1,¹⁵). This protocol details the procedures of generating AAVS1-GFP iPSCs reported in the recent paper¹⁴, including the process of transfecting iPSCs with TALENs and a 9.8 kb donor to integrate a 4.2kb DNA fragment into the AAVS1 safe-harbor locus, selecting iPSCs based on puromycin-resistance, and picking colonies for clonal expansion. The techniques described herein can be applied to many genome engineering experiments.

Protokół

1. Preparation of Basement Membrane Matrix and Coating of Plasticware

1. Place the frozen basement membrane matrix stock from -20 °C onto ice and thaw overnight at 4 °C.
2. After thawing, pipette 2 mg aliquots of basement membrane matrix into pre-chilled eppendorf tubes. Store these at -20 °C until needed.
3. To prepare basement membrane matrix-coated plates, thaw one aliquot on ice until the last piece of ice in the eppendorf tube disappears (usually within ~2 hr).
4. After thawing, add basement membrane matrix to 12 ml of cold (4 °C) DMEM/F12 to make basement membrane matrix coating solution.
5. Add basement membrane matrix solution to the appropriate culture vessel. For a 6-well plate, dispense 1 ml per well. Swirl the plate to make sure the basement membrane matrix solution completely covers each well.
6. Parafilm seal the basement membrane matrix-coated plate/dish, and incubate at room temperature for 1 hour before use. Alternatively, store basement membrane matrix-coated plates/dishes at 4 °C and use within 2 weeks of coating.
   NOTE: Add extra DMEM/F12 to the basement membrane matrix-coated plate/dish to prevent drying. Before using 4 °C stored basement membrane matrix-coated plate/dish, place it in a biological safety cabinet and allow it to come to room temperature for at least 30 min.
7. Aspirate basement membrane matrix completely before the addition of medium and cells.

2. Preparation of E8 medium

1. Prepare E8 culture medium by thawing E8 supplement overnight at 4 °C.
2. Remove 10 ml of E8 basal medium from the 500 ml stock and discard.
3. Pipette the entire 10 ml vial of E8 supplement directly into 490 ml of E8 basal medium. Do not warm complete E8 medium in a 37 °C water bath, as repeated temperature fluctuations can degrade the bFGF in the complete E8 medium.
4. Use complete E8 medium within 2 weeks of addition of the supplement.

3. Thawing of iPSCs

1. Remove a vial of frozen iPSCs from liquid nitrogen and place on dry ice.
2. Rapidly thaw the vial in a 37 °C water bath; swirl the vial in the water bath until only a tiny fragment of ice remains.
3. Spray the vial with 70% ethanol and transfer to a biological safety cabinet.
4. Add 1 ml of room temperature E8 medium dropwise directly to the vial.
5. Using a 2 ml pipette, transfer the cell suspension dropwise into 9 ml of E8 medium in a 15 ml conical tube. Swirl the tube frequently to ensure that the cells and medium mix well quickly.
6. Centrifuge the cells at 200 x g for 5 min.
7. Aspirate the supernatant, and resuspend the cell pellet in an appropriate volume of E8 supplemented with 10 μM Y-27632.
8. Add the cells to an appropriate number of basement membrane matrix-coated wells, and place in a 37 °C, 5% CO₂ incubator overnight. It is recommended to plate at least 0.2 x 10⁶ iPSC per well of a 6-well plate to enable quick recovery after thawing.

4. Maintenance and Routine Passaging of iPSCs

1. Refresh E8 medium daily.
2. Monitor the morphology and confluency of cells with an inverted microscope. iPSCs of high quality grow in flat colonies with distinct borders; individual colonies possess a “cobblestone-like” appearance.
3. Passage the iPSCs cells when they reach ~70% confluency.
4. Prepare an EDTA passaging solution by adding 0.9 g NaCl and 500 μl 0.5 M EDTA to 500 ml DPBS. Mix well to dissolve NaCl, and vacuum filter to sterilize. Warm an aliquot of the passaging solution in a 37 °C water bath prior to passaging.
5. To passage, aspirate spent culture medium and wash cells once with an equal volume of warm passaging solution. Aspirate, and pipette enough EDTA passaging solution to coat the cells (1 ml per well of a 6-well plate).
6. Place the cells under an inverted microscope and observe the iPSC colonies. The appearance of holes within colonies and raised borders should become apparent within 2 to 5 min.
7. Carefully aspirate the EDTA passaging solution.
8. Using a 10 ml pipette, dispense 4 ml of E8 medium (if using a 6-well plate) under high pressure directly into each well to be passaged.
9. Collect the iPSC clumps, and split into an appropriate number of wells depending on the split ratio from 1:8 to 1:12. Do not over-pipette, as disaggregation of cell clumps will result in poor viability.
10. Place the plate in an incubator, and rock the plate back-and-forth and side-to-side several times to disperse the cells.

5. Preparation of MEFs and iPSCs for Tansfection

1. 48 hr prior to transfection, passage iPSCs at ~1:6 into four or more wells of a basement membrane matrix-coated 6-well plate, so that they will be 70% confluent two days hence.
2. The next day, thaw DR4 MEFs into MEF medium consisting of DMEM (high glucose) supplemented with 10% FBS and 1x MEM-NEAA.
3. Plate DR4 MEFs into two 10-cm dishes at ~ 2 x 10⁴ cells/cm² and incubate overnight at 37 °C.
4. On the day of transfection, change MEF medium to E8 supplemented with 10 μM Y-27632 30 min before performing transfection on iPSCs.
5. Optional: If flow cytometric analysis of iPSCs post-transfection is desired, remove a basement membrane matrix-coated plate from 4 °C and place at room temp.
6. Optional: 4 hr prior to transfection, supplement pre-transfection iPSC culture with Y-27632 at the final concentration of 10 μM.

6. Gentle-cell Dissociation Reagent Treatment and Transfection of iPSCs using an Electroporation System

1. Remove P3 primary cell transfection solution from 4 °C and allow it to come to room temperature for ~30 min. Add the entire 100 μl supplement to the transfection solution prior to use.
2. Warm gentle-cell dissociation reagent in a 37 °C water bath.
3. Obtain AAVS1 TALENs (pZT-AAVS1-L1 and pZT-AAVS1-R1) and AAVS1-CAG-EGFP donor from -20 °C.
4. Remove iPSCs from the incubator and wash once with DPBS.
5. Add 1 ml of gentle-cell dissociation reagent per well, and incubate iPSCs at 37 °C for 5 min, or until greater than 50% of the cells have dissociated from the culture vessel.
6. Pipette the cells up and down a few times using a p1000 pipette to dissociate any remaining cells from the culture vessel, and to break up iPSC clumps.
7. Add 2 ml of E8 medium to each well, and pipette up and down several times using a 10 ml pipette to further disaggregate cell clumps into single cells
   NOTE: Transfection efficiency declines significantly if cell clumps are not sufficiently disaggregated.
8. Collect iPSCs in a 15 ml conical tube, and centrifuge at 100 x g for 3 min.
9. Aspirate supernatant, and resuspend cells in a minimal amount of E8 medium.
10. Count the cells using a hemocytometer after application of a vital stain such as 0.4% Trypan blue. Ensure that cells are sufficiently dissociated while counting (1-3 cells per “clump”).
11. Dispense 3 x 10⁶ cells into each of two 15 ml conical tubes, and spin down again at 100 x g for 3 min.
    NOTE: Low speed centrifugation reduces the cell stress and allows easy re-suspension of iPSCs before electroporation.
12. Set the electroporation system to the cell-type specific program for the human embryonic stem cell line H9 (Program CB-150).
13. After centrifugation, aspirate the supernatant from the cell pellets. To one pellet, add 10 μg of the HR donor as control sample. To the other pellet add 10 μg of the HR donor, along with 5 μg of each TALEN (pZT-AAVS1-L1 and pZT-AAVS1-R1) as experimental sample.
14. Resuspend each cell pellet in 100 μl of P3 primary cell transfection solution, and transfer to a cuvette.
15. Perform the transfection and immediately add 500 μl of room temperature E8 medium to each cuvette.
16. Transfer the transfected iPSCs dropwise to one 10-cm dish containing DR4 MEFs prepared in step 5.4.
17. Optional: Add several drops from each experiment into one well of a basement membrane matrix-coated 6-well plate if flow cytometry anaylsis of transfection efficiency is desired.
18. Repeat steps 6.15-6.17 to finish both samples. The following day, wash cells with DPBS twice and switch culture medium to NutriStem, which appears to support iPSC culture on feeders better than E8.
19. Transient EGFP expression peaks at 48 to 72 hr post-transfection; assess transfection efficiency as desired.

7. Puromycin Selection of Targeted iPSCs

1. Begin puromycin selection 72 hr after transfection when the iPSCs reach 70% confluency.
2. For NCRM5 iPSCs, begin puromycin selection at 0.5 μg/ml puromycin (1/2 the full dose) diluted into NutriStem culture medium. The optimal puromycin concentration may vary from 0.25 to 1 μg/mlL for some iPSC lines.
3. Culture iPSCs in NutriStem + 0.5 μg/ml puromycin for 3 days, refreshing the medium daily.
4. After 3 days, increase the concentration of puromycin to 1 μg/ml.
5. Culture iPSCs under 1 μg/ml puromycin selection for another 7 to 8 days, or until distinct colonies appear large enough for colony picking.

8. Colony Picking and Expanding of Targeted iPSCs

1. Use basement membrane matrix to coat a 96-well plate by dispensing 50 μl of basement membrane matrix coating solution (2 mg basement membrane matrix diluted into 12 ml DMEM/F12) into each well. Store the plate at 4 °C overnight before use.
2. After allowing the basement membrane matrix-coated plate to come to room temperature, aspirate the basement membrane matrix and dispense 100 μl E8 supplemented with 10 μM Y-27632 into each well.
3. Place an inverted microscope into a biological safety cabinet, and spray lightly with 70% EtOH to sterilize.
4. Pull a glass Pasteur pipette over a Bunsen burner to obtain an ideal colony-picking tool that has a tiny “hook” at the tip. Sterilize with 70% EtOH, and place in the hood to dry.
5. Remove the dish containing targeted iPSC colonies from the incubator, and place into the hood under the microscope.
6. Pick iPSCs by tracing a circle around the colony border with the colony-picking tool.
7. Use the “hook” of the colony-picking tool to gently scrape and remove the iPSC colony from surface of the plate. For larger colonies, drawing an X with the colony-picking tool to quarter the colony will facilitate cell growth after re-plating.
8. Once cell clumps are detached and freely floating, use a p200 pipette set to ~30 μl to collect the iPSCs. Plate cells directly into the 96-well plate.
9. Optional: use a p20 pipette set to ~5 μl to collect the iPSCs into a eppendorf tube, then add 50 μl gentle-cell dissociation reagent to digest for 5 min at 37 °C. After digestion, add 500 μl E8 medium into the eppendorf tube to dilute the gentle-cell dissociation reagent and spin down the cells at 200 x g in a standard table-top microcentrifuge for 5 min. Then aspirate most of the medium and leave ~30 μl to resuspend and transfer the cells into the 96-well plate.
   NOTE: This approach will facilitate the dissociation of the cell clump and quick expansion of picked colony.
10. Continue colony picking until a sufficient number of iPSC colonies have been collected (roughly 20-30 colonies per experiment is recommended).
11. After colony picking, place the 96-well plate in an incubator overnight. Refresh with complete E8 medium the following day, and culture until ~70% confluent.
12. Passage cells from the 96-well into a 24-well, then into a 6-well, as described in steps 4.5-4.10.
    NOTE: the volumes of EDTA solution and E8 medium should be proportionally reduced when using wells smaller than a 6-well plate.
13. Assess targeting success by junction PCR and Southern blot analysis to confirm targeted cassette integration and absence of randomly inserted vectors¹⁶.

Wyniki

A visualization of the protocol is provided in Figure 2, with periods during which iPSCs are cultured in different medium highlighted by either green for E8 or blue for NutriStem. It is important to transfect only high-quality iPSCs; examine culture dishes throughout routine maintenance and verify that iPSC cultures contain mainly distinct colonies bearing a cobblestone-like morphology (Figure 3A); differentiated cells should not occupy more than 10% of the culture. Transfectability of i...

Dyskusje

The most critical steps for the successful generation of AAVS1 safe-harbor targeted human iPSCs are: (1) efficiently delivering TALEN and donor plasmids into iPSCs by transfection; (2) optimizing dissociation of iPSCs into single cells before transfection and plating density after transfection; (3) optimizing dose and time of drug-selection based on the growth of iPSC line; (4) carefully dissecting and picking targeted colonies and transferring to new plate/well. Compared to similar methods used in Hockemeyer’s pap...

Materiały

Name	Company	Catalog Number	Comments
Matrigel Growth Factor Reduced (GFR) Basement Membrane Matrix, *LDEV-Free, 10 ml	Corning	354230	Store at -20 °C.
DMEM/F-12	Life Technologies	11320-033	Store at 4 °C.
Costar 6 Well Clear TC-Treated Multiple Well Plates	Corning	3506
Essential 8 Medium	Life Technologies	A1517001	Store basal medium at 4 °C. Store supplement at -20 °C.
Y-27632 dihydrochloride	Tocris	1254	Store at room temp. Once dissolved in H2O, store at -20 °C.
Sodium Chloride	Sigma	S5886-500G
UltraPure 0.5 M EDTA, pH 8.0	Life Technologies	15575-020
DPBS, no calcium, no magnesium	Life Technologies	14190-250
100 mm TC-Treated Culture Dish	Corning	430167
DR4 MEF 2M IRR - Academic	GlobalStem	GSC-6204G	Store in liquid Nitrogen.
DMEM, high glucose, pyruvate	Life Technologies	11995-040	Store at 4 °C.
Defined Fetal Bovine Serum, US Origin	HyClone	SH30070.03	Store at -20 °C. Thaw at 4 °C overnight and aliquot. Store aliquots at -20 °C until needed.
MEM Non-Essential Amino Acids Solution (100X)	Life Technologies	11140-050	Store at 4 °C.
4D-Nucleofector Core unit	Lonza	AAF-1001B	part of the electroporation system
4D-Nucleofector X unit	Lonza	AAF-1001X	part of the electroporation system
P3 Primary Cell 4D-Nucleofector X Kit L (24 RCT)	Lonza	V4XP-3024	Upon arrival, remove Primary Cell Solution and supplement and store at 4 °C.
StemPro Accutase Cell Dissociation Reagent	Life Technologies	A1110501	Store at -20 °C. Thaw overnight at 4°C and warm an aliquot in a 37 °C water bath before use.
NutriStem XF/FF Culture Medium	Stemgent	01-0005	Store at -20 °C. Thaw overnight at 4 °C
AAVS1 TALENs (pZT-AAVS1-L1 and pZT-AAVS1-R1)	Addgene	52637 and 52638
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AAVS1-CAG-EGFP Homologous Recombination donor	Addgene	22212
Puromycin Dihydrochloride	Life Technologies	A11138-03	Store at -20 °C. Prepare working aliquots of 1 mg/ml in ddH2O.
Disposable Borosilicate Glass Pasteur Pipets	Fisher Scientific	13-678-20A
Sorvall Legend XTR (Refrigerated), 120 V 60 Hz	Thermo Scientific	75-004-521
TX-750 4 × 750 ml Swinging Bucket Rotor	Thermo Scientific	75003607
Trypan Blue Solution, 0.4%	Life Technologies	15250-061