This research was supported by the NIH Common Fund and Intramural Research Program of the National Institute of Arthritis, Musculoskeletal, and Skin Diseases.

1. Preparation of Basement Membrane Matrix and Coating of Plasticware
<ol>
	<li>Place the frozen basement membrane matrix stock from -20 &#176;C onto ice and thaw overnight at 4 &#176;C.</li>
	<li>After thawing, pipette 2 mg aliquots of basement membrane matrix into pre-chilled eppendorf tubes. Store these at -20 &#176;C until needed.</li>
	<li>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).</li>
	<li>After thawing, add basement membrane matrix to 12 ml&#160;of cold (4 &#176;C) DMEM/F12 to make basement membrane matrix coating solution.</li>
	<li>Add basement membrane matrix solution to the appropriate culture vessel. For a 6-well plate, dispense 1 ml&#160;per well. Swirl the plate to make sure the basement membrane matrix solution completely covers each well.</li>
	<li>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 &#176;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 &#176;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.</li>
	<li>Aspirate basement membrane matrix completely before the addition of medium and cells.</li>
</ol>
2. Preparation of E8 medium
<ol>
	<li>Prepare E8 culture medium by thawing E8 supplement overnight at 4 &#176;C.</li>
	<li>Remove 10 ml&#160;of E8 basal medium from the 500 ml&#160;stock and discard.</li>
	<li>Pipette the entire 10 ml&#160;vial of E8 supplement directly into 490 ml&#160;of E8 basal medium. Do not warm complete E8 medium in a 37 &#176;C water bath, as repeated temperature fluctuations can degrade the bFGF in the complete E8 medium.</li>
	<li>Use complete E8 medium within 2 weeks of addition of the supplement.</li>
</ol>
3. Thawing of iPSCs
<ol>
	<li>Remove a vial of frozen iPSCs from liquid nitrogen and place on dry ice.</li>
	<li>Rapidly thaw the vial in a 37 &#176;C water bath; swirl the vial in the water bath until only a tiny fragment of ice remains.</li>
	<li>Spray the vial with 70% ethanol and transfer to a biological safety cabinet.</li>
	<li>Add 1 ml&#160;of room temperature E8 medium dropwise directly to the vial.</li>
	<li>Using a 2 ml pipette, transfer the cell suspension dropwise into 9 ml&#160;of E8 medium in a 15 ml conical tube. Swirl the tube frequently to ensure that the cells and medium mix well quickly.</li>
	<li>Centrifuge the cells at 200 x g for 5 min.</li>
	<li>Aspirate the supernatant, and resuspend the cell pellet in an appropriate volume of E8 supplemented with 10 &#956;M Y-27632.</li>
	<li>Add the cells to an appropriate number of basement membrane matrix-coated wells, and place in a 37 &#176;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.</li>
</ol>
4. Maintenance and Routine Passaging of iPSCs
<ol>
	<li>Refresh E8 medium daily.</li>
	<li>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 &#8220;cobblestone-like&#8221; appearance.</li>
	<li>Passage the iPSCs cells when they reach ~70% confluency.</li>
	<li>Prepare an EDTA passaging solution by adding 0.9 g NaCl and 500 &#956;l&#160;0.5 M EDTA to 500 ml&#160;DPBS. Mix well to dissolve NaCl, and vacuum filter to sterilize. Warm an aliquot of the passaging solution in a 37 &#176;C water bath prior to passaging.</li>
	<li>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&#160;per well of a 6-well plate).</li>
	<li>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.</li>
	<li>Carefully aspirate the EDTA passaging solution.</li>
	<li>Using a 10 ml&#160;pipette, dispense 4 ml&#160;of E8 medium (if using a 6-well plate) under high pressure directly into each well to be passaged.</li>
	<li>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.</li>
	<li>Place the plate in an incubator, and rock the plate back-and-forth and side-to-side several times to disperse the cells.</li>
</ol>
5. Preparation of MEFs and iPSCs for Tansfection
<ol>
	<li>48 hr&#160;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.</li>
	<li>The next day, thaw DR4 MEFs into MEF medium consisting of DMEM (high glucose) supplemented with 10% FBS and 1x MEM-NEAA.</li>
	<li>Plate DR4 MEFs into two 10-cm dishes at ~ 2 x 104 cells/cm2 and incubate overnight at 37 &#176;C.</li>
	<li>On the day of transfection, change MEF medium to E8 supplemented with 10 &#956;M Y-27632 30 min&#160;before performing transfection on iPSCs.</li>
	<li>Optional: If flow cytometric analysis of iPSCs post-transfection is desired, remove a basement membrane matrix-coated plate from 4 &#176;C and place at room temp.</li>
	<li>Optional: 4 hr&#160;prior to transfection, supplement pre-transfection iPSC culture with Y-27632 at the final concentration of 10 &#956;M.</li>
</ol>
6. Gentle-cell Dissociation Reagent Treatment and Transfection of iPSCs using an Electroporation System
<ol>
	<li>Remove P3 primary cell transfection solution from 4 &#176;C and allow it to come to room temperature for ~30 min. Add the entire 100 &#956;l&#160;supplement to the transfection solution prior to use.</li>
	<li>Warm gentle-cell dissociation reagent in a 37 &#176;C water bath.</li>
	<li>Obtain AAVS1 TALENs (pZT-AAVS1-L1 and pZT-AAVS1-R1) and AAVS1-CAG-EGFP donor from -20 &#176;C.</li>
	<li>Remove iPSCs from the incubator and wash once with DPBS.</li>
	<li>Add 1 ml&#160;of gentle-cell dissociation reagent per well, and incubate iPSCs at 37 &#176;C for 5 min, or until greater than 50% of the cells have dissociated from the culture vessel.</li>
	<li>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.</li>
	<li>Add 2 ml&#160;of E8 medium to each well, and pipette up and down several times using a 10 ml&#160;pipette to further disaggregate cell clumps into single cells 
	NOTE: Transfection efficiency declines significantly if cell clumps are not sufficiently disaggregated.</li>
	<li>Collect iPSCs in a 15 ml&#160;conical tube, and centrifuge at 100 x g for 3 min.</li>
	<li>Aspirate supernatant, and resuspend cells in a minimal amount of E8 medium.</li>
	<li>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 &#8220;clump&#8221;).</li>
	<li>Dispense 3 x 106 cells into each of two 15 ml&#160;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.</li>
	<li>Set the electroporation system to the cell-type specific program for the human embryonic stem cell line H9 (Program CB-150).</li>
	<li>After centrifugation, aspirate the supernatant from the cell pellets. To one pellet, add 10 &#956;g of the HR donor as control sample. To the other pellet add 10 &#956;g of the HR donor, along with 5 &#956;g of each TALEN (pZT-AAVS1-L1 and pZT-AAVS1-R1) as experimental sample.</li>
	<li>Resuspend each cell pellet in 100 &#956;l&#160;of P3 primary cell transfection solution, and transfer to a cuvette.</li>
	<li>Perform the transfection and immediately add 500 &#956;l&#160;of room temperature E8 medium to each cuvette.</li>
	<li>Transfer the transfected iPSCs dropwise to one 10-cm dish containing DR4 MEFs prepared in step 5.4.</li>
	<li>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.</li>
	<li>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.</li>
	<li>Transient EGFP expression peaks at 48 to 72 hr&#160;post-transfection; assess transfection efficiency as desired.</li>
</ol>
7. Puromycin Selection of Targeted iPSCs
<ol>
	<li>Begin puromycin selection 72 hr&#160;after transfection when the iPSCs reach 70% confluency.</li>
	<li>For NCRM5 iPSCs, begin puromycin selection at 0.5 &#956;g/ml&#160;puromycin (1/2 the full dose) diluted into NutriStem culture medium. The optimal puromycin concentration may vary from 0.25 to 1 &#956;g/mlL for some iPSC lines.</li>
	<li>Culture iPSCs in NutriStem + 0.5 &#956;g/ml&#160;puromycin for 3 days, refreshing the medium daily.</li>
	<li>After 3 days, increase the concentration of puromycin to 1 &#956;g/ml.</li>
	<li>Culture iPSCs under 1 &#956;g/ml&#160;puromycin selection for another 7 to 8 days, or until distinct colonies appear large enough for colony picking.</li>
</ol>
8. Colony Picking and Expanding of Targeted iPSCs
<ol>
	<li>Use basement membrane matrix to coat a 96-well plate by dispensing 50 &#956;l&#160;of basement membrane matrix coating solution (2 mg basement membrane matrix diluted into 12 ml&#160;DMEM/F12) into each well. Store the plate at 4 &#176;C overnight before use.</li>
	<li>After allowing the basement membrane matrix-coated plate to come to room temperature, aspirate the basement membrane matrix and dispense 100 &#956;l&#160;E8 supplemented with 10 &#956;M Y-27632 into each well.</li>
	<li>Place an inverted microscope into a biological safety cabinet, and spray lightly with 70% EtOH to sterilize.</li>
	<li>Pull a glass Pasteur pipette over a Bunsen burner to obtain an ideal colony-picking tool that has a tiny &#8220;hook&#8221; at the tip. Sterilize with 70% EtOH, and place in the hood to dry.</li>
	<li>Remove the dish containing targeted iPSC colonies from the incubator, and place into the hood under the microscope.</li>
	<li>Pick iPSCs by tracing a circle around the colony border with the colony-picking tool.</li>
	<li>Use the &#8220;hook&#8221; 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.</li>
	<li>Once cell clumps are detached and freely floating, use a p200 pipette set to ~30 &#956;l&#160;to collect the iPSCs. Plate cells directly into the 96-well plate.</li>
	<li>Optional: use a p20 pipette set to ~5 &#956;l&#160;to collect the iPSCs into a eppendorf tube, then add 50 &#956;l&#160;gentle-cell dissociation reagent to digest for 5 min&#160;at 37 &#176;C. After digestion, add 500 &#956;l&#160;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 &#956;l&#160;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.</li>
	<li>Continue colony picking until a sufficient number of iPSC colonies have been collected (roughly 20-30 colonies per experiment is recommended).</li>
	<li>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.</li>
	<li>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.</li>
	<li>Assess targeting success by junction PCR and Southern blot analysis to confirm targeted cassette integration and absence of randomly inserted vectors16.</li>
</ol>

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The authors declare that they have no competing financial interests.

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&#8217;s pap...

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 ESCs6,7, 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 experiments8,9. 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 methodology10-13.
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.

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

transfection, selection, and colony-picking of human induced pluripotent stem cells talen-targeted with a gfp gene into the aavs1 safe harbor

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.

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...

Watch this Scientific Journal Video about Transfection, Selection, and Colony-picking of Human Induced Pluripotent Stem Cells TALEN-targeted with a GFP Gene into the AAVS1 Safe Harbor at JoVE.com

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

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.

Targeted transgene addition can provide persistent gene expression while circumventing the gene silencing and insertional mutagenesis caused by viral ...

transfection-selection-colony-picking-human-induced-pluripotent-stem

Research

JoVE Journal

Developmental Biology

19.7K Views.  National Institutes of Health. 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.

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

Stable and Efficient Genetic Modification of Cells in the Adult Mouse V-SVZ for the Analysis of Neural Stem Cell Autonomous and Non-autonomous Effects

Relatively quiescent somatic stem cells support life-long cell renewal in most adult tissues. Neural stem cells in the adult mammalian brain are restricted to two specific neurogenic niches: the subgranular zone of the dentate gyrus in the hippocampus and the ventricular-subventricular zone (V-SVZ; also called subependymal zone or SEZ) in the walls of the lateral ventricles. The development of in vivo gene transfer strategies for adult stem cell populations (i.e. those of the mammalian brain) resulting in long-term expression of desired transgenes in the stem cells and their derived progeny is a crucial tool in current biomedical and biotechnological research. Here, a direct in vivo method is presented for the stable genetic modification of adult mouse V-SVZ cells that takes advantage of the cell cycle-independent infection by LVs and the highly specialized cytoarchitecture of the V-SVZ niche. Specifically, the current protocol involves the injection of empty LVs (control) or LVs encoding specific transgene expression cassettes into either the V-SVZ itself, for the in vivo targeting of all types of cells in the niche, or into the lateral ventricle lumen, for the targeting of ependymal cells only. Expression cassettes are then integrated into the genome of the transduced cells and fluorescent proteins, also encoded by the LVs, allow the detection of the transduced cells for the analysis of cell autonomous and non-autonomous, niche-dependent effects in the labeled cells and their progeny.

Here we describe a procedure based on the use of lentiviral particles for the long-term genetic modification of neural stem cells and/or their adjacent ependymal cells in the adult ventricular-subventricular neurogenic niche which allows the separate analysis of cell autonomous and non-autonomous, niche-dependent effects on neural stem cells.

Relatively quiescent somatic stem cells support life-long cell renewal in most adult tissues. Neural stem cells in the adult mammalian brain are ...

Establishment of Genome-edited Human Pluripotent Stem Cell Lines: From Targeting to Isolation

Genome-editing of human pluripotent stem cells (hPSCs) provides a genetically controlled and clinically relevant platform from which to understand human development and investigate the pathophysiology of disease. By employing site-specific nucleases (SSNs) for genome editing, the rapid derivation of new hPSC lines harboring specific genetic alterations in an otherwise isogenic setting becomes possible. Zinc finger nucleases (ZFNs), transcription activator-like effector nucleases (TALENs) and clustered regularly interspaced short palindromic repeats (CRISPR)/Cas9 are the most commonly used SSNs. All of these nucleases function by introducing a double stranded DNA break at a specified site, thereby promoting precise gene editing at a genomic locus. SSN-meditated genome editing exploits two of the cell&#39;s endogenous DNA repair mechanisms, non-homologous end joining (NHEJ) and homology directed repair (HDR), to either introduce insertion/deletion mutations or alter the genome using a homologous repair template at the site of the double stranded break. Electroporation of hPSCs is an efficient means of transfecting SSNs and repair templates that incorporate transgenes such as fluorescent reporters and antibiotic resistance cassettes. After electroporation, it is possible to isolate only those hPSCs that incorporated the repair construct by selecting for antibiotic resistance. Mechanically separating hPSC colonies and confirming proper integration at the target site through genotyping allows for the isolation of correctly targeted and genetically homogeneous cell lines. The validity of this protocol is demonstrated here by using all three SSN platforms to incorporate EGFP and a puromycin resistance construct into the AAVS1 safe harbor locus in human pluripotent&#160;stem cells.

Genome editing of human pluripotent stem cells (hPSCs) can be done quickly and efficiently. Presented here is a robust experimental procedure to genetically engineer hPSCs as exemplified by editing the AAVS1 safe harbor locus to express EGFP and introduce antibiotic resistance.

Genome-editing of human pluripotent stem cells (hPSCs) provides a genetically controlled and clinically relevant platform from which to understand human ...

Introducing Point Mutations into Human Pluripotent Stem Cells Using Seamless Genome Editing

Custom designed endonucleases, such as RNA-guided Clustered Regularly Interspaced Short Palindromic Repeats (CRISPR)-Cas9, enable efficient genome editing in mammalian cells. Here we describe detailed procedures to seamlessly genome edit the hepatocyte nuclear factor 4 alpha (HNF4&#945;) locus as an example in human pluripotent stem cells. Combining a piggyBac-based donor plasmid and the CRISPR-Cas9 nickase mutant in a two-step genetic selection, we demonstrate correct and efficient targeting of the HNF4&#945; locus.

Here, we describe a detailed method for seamless gene editing in human pluripotent stem cells using a piggyBac-based donor plasmid and the Cas9 nickase mutant. Two point mutations were introduced into exon 8 of the hepatocyte nuclear factor 4 alpha (HNF4&#945;) locus in human embryonic stem cells (hESCs).

Custom designed endonucleases, such as RNA-guided Clustered Regularly Interspaced Short Palindromic Repeats (CRISPR)-Cas9, enable efficient genome editing ...

Genetics

Engineering Oncogenic Heterozygous Gain-of-Function Mutations in Human Hematopoietic Stem and Progenitor Cells

Throughout their lifetime, hematopoietic stem and progenitor cells (HSPCs) acquire somatic mutations. Some of these mutations alter HSPC functional properties such as proliferation and differentiation, thereby promoting the development of hematologic malignancies. Efficient and precise genetic manipulation of HSPCs is required to model, characterize, and better understand the functional consequences of recurrent somatic mutations. Mutations can have a deleterious effect on a gene and result in loss-of-function (LOF) or, in stark contrast, may enhance function or even lead to novel characteristics of a particular gene, termed gain-of-function (GOF). In contrast to LOF mutations, GOF mutations almost exclusively occur in a heterozygous fashion. Current genome-editing protocols do not allow for the selective targeting of individual alleles, hampering the ability to model heterozygous GOF mutations. Here, we provide a detailed protocol on how to engineer heterozygous GOF hotspot mutations in human HSPCs by combining CRISPR/Cas9-mediated homology-directed repair and recombinant AAV6 technology for efficient DNA donor template transfer. Importantly, this strategy makes use of a dual fluorescent reporter system to allow for the tracking and purification of successfully heterozygously edited HSPCs. This strategy can be employed to precisely investigate how GOF mutations affect HSPC function and their progression toward hematological malignancies.

Novel strategies to faithfully model somatic mutations in hematopoietic stem and progenitor cells (HSPCs) are necessary to better study hematopoietic stem cell biology and hematological malignancies. Here, a protocol to model heterozygous gain-of-function mutations in HSPCs by combining the use of CRISPR/Cas9 and dual rAAV donor transduction is described.

Throughout their lifetime, hematopoietic stem and progenitor cells (HSPCs) acquire somatic mutations. Some of these mutations alter HSPC functional ...

Cancer Research

Transfecting and Nucleofecting Human Induced Pluripotent Stem Cells

Genetic modification is continuing to be an essential tool in studying stem cell biology and in setting forth potential clinical applications of human embryonic stem cells (HESCs)1. While improvements in several gene delivery methods have been described2-9, transfection remains a capricious process for HESCs, and has not yet been reported in human induced pluripotent stem cells (iPSCs). In this video, we demonstrate how our lab routinely transfects and nucleofects human iPSCs using plasmid with an enhanced green fluorescence protein (eGFP) reporter. Human iPSCs are adapted and maintained as feeder-free cultures to eliminate the possibility of feeder cell transfection and to allow efficient selection of stable transgenic iPSC clones following transfection. For nucleofection, human iPSCs are pre-treated with ROCK inhibitor11, trypsinized into small clumps of cells, nucleofected and replated on feeders in feeder cell-conditioned medium to enhance cell recovery. Transgene-expressing human iPSCs can be obtained after 6 hours. Antibiotic selection is applied after 24 hours and stable transgenic lines appear within 1 week. Our protocol is robust and reproducible for human iPSC lines without altering pluripotency of these cells.

Despite recent advancements in genetic modification, transfection of human embryonic stem cells (HESCs) remains a capricious process. To our knowledge, systematic and efficient methods to transfect human induced pluripotent stem cells (iPSCs) have not been reported. Here, we describe robust protocols to efficiently transfect and nucleofect human iPSCs.

Genetic modification is continuing to be an essential tool in studying stem cell biology and in setting forth potential clinical applications of human ...

Biology

Rapid and Efficient Generation of Recombinant Human Pluripotent Stem Cells by Recombinase-mediated Cassette Exchange in the AAVS1 Locus

Even with the revolution of gene-targeting technologies led by CRISPR-Cas9, genetic modification of human pluripotent stem cells&#160;(hPSCs)&#160;is still time consuming. Comparative studies that use recombinant lines with transgenes integrated into safe harbor loci could benefit from approaches that use site-specific targeted recombinases, like Cre or FLPe, which are more rapid and less prone to off-target effects. Such methods have been described, although they do not significantly outperform gene targeting in most aspects. Using Zinc-finger nucleases, we previously created a master cell line in the AAVS1 locus of hPSCs&#160;that contains a GFP-Hygromycin-tk expressing cassette, flanked by heterotypic FRT sequences. Here, we describe the procedures to perform FLPe recombinase-mediated cassette exchange (RMCE) using this line. The master cell line is transfected with a RMCE donor vector, which contains a promoterless Puromycin resistance, and with FLPe recombinase. Application of both a positive (Puromycin) and negative (FIAU) selection program leads to the selection of RMCE without random integrations. RMCE generates fully characterized pluripotent polyclonal transgenic lines in 15 d&#160;with 100% efficiency. Despite the recently described limitations of the AAVS1 locus, the ease of the system paves the way for hPSC transgenesis in isogenic settings, is necessary for comparative studies, and enables semi-high-throughput genetic screens for gain/loss of function analysis that would otherwise be highly time consuming.

Rapid and Efficient Generation of Recombinant Human Pluripotent Stem Cells by Recombinase-mediated Cassette Exchange in the AAVS1 Locus

Here we report a rapid and efficient gene editing method based on RMCE in the AAVS1 locus of human Pluripotent Stem Cells (hPSCs) that improves upon previously described systems. Using this technique, isogenic lines can be rapidly and reliably generated for proper comparative studies, facilitating transgenesis-mediated research with hPSCs.

Even with the revolution of gene-targeting technologies led by CRISPR-Cas9, genetic modification of human pluripotent stem cells&#160;(hPSCs)&#160;is ...

Medicine

Selecting and Isolating Colonies of Human Induced Pluripotent Stem Cells Reprogrammed from Adult Fibroblasts

Herein we present a protocol of reprogramming human adult fibroblasts into human induced pluripotent stem cells (hiPSC) using retroviral vectors encoding Oct3/4, Sox2, Klf4 and c-myc (OSKM) in the presence of sodium butyrate 1-3. We used this method to reprogram late passage (&gt;p10) human adult fibroblasts derived from Friedreich's ataxia patient (GM03665, Coriell Repository). The reprogramming approach includes highly efficient transduction protocol using repetitive centrifugation of fibroblasts in the presence of virus-containing media. The reprogrammed hiPSC colonies were identified using live immunostaining for Tra-1-81, a surface marker of pluripotent cells, separated from non-reprogrammed fibroblasts and manually passaged 4,5. These hiPSC were then transferred to Matrigel plates and grown in feeder-free conditions, directly from the reprogramming plate. Starting from the first passage, hiPSC colonies demonstrate characteristic hES-like morphology. Using this protocol more than 70% of selected colonies can be successfully expanded and established into cell lines. The established hiPSC lines displayed characteristic pluripotency markers including surface markers TRA-1-60 and SSEA-4, as well as nuclear markers Oct3/4, Sox2 and Nanog. 
The protocol presented here has been established and tested using adult fibroblasts obtained from Friedreich's ataxia patients and control individuals 6, human newborn fibroblasts, as well as human keratinocytes.

We present a protocol for efficient reprogramming of human somatic cells into human induced pluripotent stem cells (hiPSC) using retroviral vectors encoding Oct3/4, Sox2, Klf4 and c-myc (OSKM) and identification of correctly reprogrammed hiPSC by live staining with Tra-1-81 antibody.

Herein we present a protocol of reprogramming human adult fibroblasts into human induced pluripotent stem cells (hiPSC) using retroviral vectors encoding ...

AAV Deployment of Enhancer-Based Expression Constructs In Vivo in Mouse Brain

Enhancers are binding platforms for a diverse array of transcription factors that drive specific expression patterns of tissue- and cell-type-specific genes. Multiple means of assessing non-coding DNA and various chromatin states have proven useful in predicting the presence of enhancer sequences in the genome, but validating the activity of these sequences and finding the organs and developmental stages they are active in is a labor-intensive process. Recent advances in adeno-associated virus (AAV) vectors have enabled the widespread delivery of transgenes to mouse tissues, enabling in vivo enhancer testing without necessitating a transgenic animal. This protocol shows how a reporter construct that expresses EGFP under the control of a minimal promoter, which does not drive significant expression on its own, can be used to study the activity patterns of candidate enhancer sequences in the mouse brain. An AAV-packaged reporter construct is delivered to the mouse brain and incubated for 1-4 weeks, after which the animal is sacrificed, and brain sections are observed under a microscope. EGFP appears in cells in which the tested enhancer is sufficient to initiate gene expression, pinpointing the location and developmental stage in which the enhancer is active in the brain. Standard cloning methods, low-cost AAV packaging, and expanding AAV serotypes and methods for in vivo delivery and standard imaging readout make this an accessible approach for the study of how gene expression is regulated in the brain.

AAV Deployment of Enhancer-Based Expression Constructs In Vivo in Mouse Brain

This protocol describes a novel rAAV-based transient enhancer-reporter assay. This assay can be used to induce enhancer-driven expression in vivo in the mouse brain.

Enhancers are binding platforms for a diverse array of transcription factors that drive specific expression patterns of tissue- and cell-type-specific ...

Neuroscience

Derivation and Characterization of a Transgene-free Human Induced Pluripotent Stem Cell Line and Conversion into Defined Clinical-grade Conditions

Human induced pluripotent stem cells (hiPSCs) can be generated with lentiviral-based reprogramming methodologies. However, traces of potentially oncogenic genes remaining in actively transcribed regions of the genome, limit their potential for use in human therapeutic applications1. Additionally, non-human antigens derived from stem cell reprogramming or differentiation into therapeutically relevant derivatives preclude these hiPSCs from being used in a human clinical context2. In this video, we present a procedure for reprogramming and analyzing factor-free hiPSCs free of exogenous transgenes. These hiPSCs then can be analyzed for gene expression abnormalities in the specific intron containing the lentivirus. This analysis may be conducted using sensitive quantitative polymerase chain reaction (PCR), which has an advantage over less sensitive techniques previously used to detect gene expression differences3. Full conversion into clinical-grade good manufacturing practice (GMP) conditions, allows human clinical relevance. Our protocol offers another methodology&#8212;provided that current safe-harbor criteria will expand and include factor-free characterized hiPSC-based derivatives for human therapeutic applications&#8212;for deriving GMP-grade hiPSCs, which should eliminate any immunogenicity risk due to non-human antigens. This protocol is broadly applicable to lentiviral reprogrammed cells of any type and provides a reproducible method for converting reprogrammed cells into GMP-grade conditions.

We describe a protocol for deriving lentiviral-based reprogrammed and characterized factor-free human induced pluripotent stem cells and conversion into putative clinical-grade conditions.

Human induced pluripotent stem cells (hiPSCs) can be generated with lentiviral-based reprogramming methodologies. However, traces of potentially oncogenic ...

Crude Vector Production and Efficient DNA Delivery in Cell Cultures

Transgene Expression in Cultured Cells Using Unpurified Recombinant Adeno-Associated Viral Vectors

Recombinant adeno-associated viral vectors (rAAV) can achieve potent and durable transgene expression without integration in a broad range of tissue types, making them a popular choice for gene delivery in animal models and in clinical settings. In addition to therapeutic applications, rAAVs are a useful laboratory tool for delivering transgenes tailored to the researcher&#39;s experimental needs and scientific goals in cultured cells. Some examples include exogenous reporter genes, overexpression cassettes, RNA interference, and CRISPR-based tools, including those for genome-wide screens. rAAV transductions are less harmful to cells than electroporation or chemical transfection and do not require any special equipment or expensive reagents to produce. Crude lysates or conditioned media containing rAAVs can be added directly to cultured cells without further purification to transduce many cell types&#8212;an underappreciated feature of rAAVs. Here, we provide protocols for basic transgene cassette cloning and demonstrate how to produce and apply crude rAAV preparations to cultured cells. As proof of principle, we demonstrate the transduction of three cell types that have not yet been reported in rAAV applications: placental cells, myoblasts, and small intestinal organoids. We discuss appropriate uses for crude rAAV preparations, the limitations of rAAVs for gene delivery, and considerations for capsid choice. This protocol outlines a simple, low-cost, and effective method for researchers to achieve productive DNA delivery in cell culture using rAAV without the need for laborious titration and purification steps.

Author Spotlight: Unveiling the Potential of Unpurified Recombinant AAVs in Cell Culture Research 

Recombinant adeno-associated virus (rAAV) is widely used for clinical and preclinical gene delivery. An underappreciated use for rAAVs is the robust transduction of cultured cells without the need for purification. For researchers new to rAAV, we provide a protocol for transgene cassette cloning, crude vector production, and cell culture transduction.

Recombinant adeno-associated viral vectors (rAAV) can achieve potent and durable transgene expression without integration in a broad range of tissue ...

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

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