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>

<ol>
	<li>Takahashi, K., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Induction+of+pluripotent+stem+cells+from+adult+human+fibroblasts+by+defined+factors.">Induction of pluripotent stem cells from adult human fibroblasts by defined factors.</a> Cell. 131 (5), 861-872 (2007).</li><li>Hentze, H., Graichen, R., Colman, A. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Cell+therapy+and+the+safety+of+embryonic+stem+cell-derived+grafts.">Cell therapy and the safety of embryonic stem cell-derived grafts.</a> Trends Biotechnol. 25 (1), 24-32 (2007).</li><li>Smith, J. R., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Robust,+persistent+transgene+expression+in+human+embryonic+stem+cells+is+achieved+with+AAVS1-targeted+integration.">Robust, persistent transgene expression in human embryonic stem cells is achieved with AAVS1-targeted integration.</a> Stem Cells. 26 (2), 496-504 (2008).</li><li>Lombardo, A., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Site-specific+integration+and+tailoring+of+cassette+design+for+sustainable+gene+transfer.">Site-specific integration and tailoring of cassette design for sustainable gene transfer.</a> Nat Methods. 8 (10), 861-869 (2011).</li><li>Ogata, T., Kozuka, T., Kanda, T. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Identification+of+an+insulator+in+AAVS1,+a+preferred+region+for+integration+of+adeno-associated+virus+DNA.">Identification of an insulator in AAVS1, a preferred region for integration of adeno-associated virus DNA.</a> J Virol. 77 (16), 9000-9007 (2003).</li><li>Zwaka, T. 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E., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=A+transcription+activator-like+effector+toolbox+for+genome+engineering.">A transcription activator-like effector toolbox for genome engineering.</a> Nature protocols. 7 (1), 171-192 (2012).</li><li>Mali, P., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=RNA-guided+human+genome+engineering+via+Cas9.">RNA-guided human genome engineering via Cas9.</a> Science. 339 (6121), 823-826 (2013).</li><li>Ding, Q., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Enhanced+efficiency+of+human+pluripotent+stem+cell+genome+editing+through+replacing+TALENs+with+CRISPRs.">Enhanced efficiency of human pluripotent stem cell genome editing through replacing TALENs with CRISPRs.</a> Cell stem cell. 12 (4), 393-394 (2013).</li><li>Luo, Y., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Stable+Enhanced+Green+Fluorescent+Protein+Expression+After+Differentiation+and+Transplantation+of+Reporter+Human.">Stable Enhanced Green Fluorescent Protein Expression After Differentiation and Transplantation of Reporter Human.</a> Induced Pluripotent Stem Cells Generated by AAVS1 Transcription Activator-Like Effector Nucleases. Stem Cells Transl Med. 3 (7), 821-835 (2014).</li><li>Zou, J., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Oxidase-deficient+neutrophils+from+X-linked+chronic+granulomatous+disease+iPS+cells:+functional+correction+by+zinc+finger+nuclease-mediated+safe+harbor+targeting.">Oxidase-deficient neutrophils from X-linked chronic granulomatous disease iPS cells: functional correction by zinc finger nuclease-mediated safe harbor targeting.</a> Blood. 117 (21), 5561-5572 (2011).</li><li>Luo, Y., Rao, M., Zou, J. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Generation+of+GFP+Reporter+Human+Induced+Pluripotent+Stem+Cells+Using+AAVS1+Safe+Harbor+Transcription+Activator-Like+Effector+Nuclease.">Generation of GFP Reporter Human Induced Pluripotent Stem Cells Using AAVS1 Safe Harbor Transcription Activator-Like Effector Nuclease.</a> Curr Protoc Stem Cell Biol. 29, 5A.7.1-5A.7.18 (2014).</li><li>Ran, F. 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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 border="0" cellpadding="0" cellspacing="0" width="1437">
	<tbody>
		<tr height="21">
			<td height="21" style="height:21px;width:588px;">NAME OF MATERIAL/EQUIPMENT</td>
			<td style="width:123px;">COMPANY</td>
			<td style="width:123px;">CATALOG #</td>
			<td style="width:603px;">COMMENTS/DESCRIPTION</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">Matrigel Growth Factor Reduced (GFR) Basement Membrane Matrix, *LDEV-Free, 10mL</td>
			<td>Corning</td>
			<td>354230</td>
			<td>Store at -20&#176;C.</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">DMEM/F-12</td>
			<td>Life Technologies</td>
			<td>11320-033</td>
			<td>Store at 4&#176;C.</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">Costar 6 Well Clear TC-Treated Multiple Well Plates</td>
			<td>Corning</td>
			<td>3506</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">Essential 8 Medium</td>
			<td>Life Technologies</td>
			<td>A1517001</td>
			<td>Store basal medium at 4&#176;C. Store supplement at -20&#176;C.</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">Y-27632 dihydrochloride</td>
			<td>Tocris</td>
			<td>1254</td>
			<td>Store at room temp. Once dissolved in H2O, store at -20&#176;C.</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">Sodium Chloride</td>
			<td>Sigma</td>
			<td>S5886-500G</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">UltraPure 0.5M EDTA, pH 8.0</td>
			<td>Life Technologies</td>
			<td>15575-020</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">DPBS, no calcium, no magnesium</td>
			<td>Life Technologies</td>
			<td>14190-250</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">100mm TC-Treated Culture Dish</td>
			<td>Corning</td>
			<td>430167</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">DR4 MEF 2M IRR - Academic</td>
			<td>GlobalStem</td>
			<td>GSC-6204G</td>
			<td>Store in liquid Nitrogen.</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">DMEM, high glucose, pyruvate</td>
			<td>Life Technologies</td>
			<td>11995-040</td>
			<td>Store at 4&#176;C.</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">Defined Fetal Bovine Serum, US Origin</td>
			<td>HyClone</td>
			<td>SH30070.03</td>
			<td>Store at -20&#176;C. Thaw at 4&#176;C overnight and aliquot. Store aliquots at -20&#176;C until needed.</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">MEM Non-Essential Amino Acids Solution (100X)</td>
			<td>Life Technologies</td>
			<td>11140-050</td>
			<td>Store at 4&#176;C.</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">4D-Nucleofector Core unit&#160;</td>
			<td>Lonza</td>
			<td>AAF-1001B</td>
			<td>part of the electroporation system</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">4D-Nucleofector X unit&#160;</td>
			<td>Lonza</td>
			<td>AAF-1001X</td>
			<td>part of the electroporation system</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">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&#176;C.</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">StemPro Accutase Cell Dissociation Reagent</td>
			<td>Life Technologies</td>
			<td>A1110501</td>
			<td>Store at -20&#176;C. Thaw overnight at 4&#176;C and warm an aliquot in a 37&#176;C water bath before use.</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">NutriStem XF/FF Culture Medium</td>
			<td>Stemgent</td>
			<td>01-0005</td>
			<td>Store at -20&#176;C. Thaw overnight at 4&#176;C</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">AAVS1 TALENs (pZT-AAVS1-L1 and pZT-AAVS1-R1)</td>
			<td>Addgene</td>
			<td>52637 and 52638</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">AAVS1-CAG-EGFP Homologous Recombination donor</td>
			<td>Addgene</td>
			<td>22212</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">Puromycin Dihydrochloride</td>
			<td>Life Technologies</td>
			<td>A11138-03</td>
			<td>Store at -20&#176;C. Prepare working aliquots of 1 mg/mL in ddH2O.</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">Disposable Borosilicate Glass Pasteur Pipets</td>
			<td>Fisher Scientific</td>
			<td>13-678-20A</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">Sorvall Legend XTR (Refrigerated), 120V 60Hz</td>
			<td>Thermo Scientific</td>
			<td>75-004-521</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">TX-750 4 &#215; 750mL Swinging Bucket Rotor</td>
			<td>Thermo Scientific</td>
			<td>75003607</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">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

19.5K visualizações.  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.

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

Three-dimensional Quantification of Dendritic Spines from Pyramidal Neurons Derived from Human Induced Pluripotent Stem Cells

Dendritic spines are small protrusions that correspond to the post-synaptic compartments of excitatory synapses in the central nervous system. They are distributed along the dendrites. Their morphology is largely dependent on neuronal activity, and they are dynamic. Dendritic spines express glutamatergic receptors (AMPA and NMDA receptors) on their surface and at the levels of postsynaptic densities. Each spine allows the neuron to control its state and local activity independently. Spine morphologies have been extensively studied in glutamatergic pyramidal cells of the brain cortex, using both in vivo approaches and neuronal cultures obtained from rodent tissues. Neuropathological conditions can be associated to altered spine induction and maturation, as shown in rodent cultured neurons and one-dimensional quantitative analysis 1. The present study describes a protocol for the 3D quantitative analysis of spine morphologies using human cortical neurons derived from neural stem cells (late cortical progenitors). These cells were initially obtained from induced&#160;pluripotent stem cells. This protocol allows the analysis of spine morphologies at different culture periods, and with possible comparison between induced&#160;pluripotent stem cells obtained from control individuals with those obtained from patients with psychiatric diseases.

Dendritic spines of pyramidal neurons are the sites of most excitatory synapses in mammalian brain cortex. This method describes a 3D quantitative analysis of spine morphologies in human cortical pyramidal glutamatergic neurons derived from induced&#160;pluripotent stem cells.

Dendritic spines are small protrusions that correspond to the post-synaptic compartments of excitatory synapses in the central nervous system. They are ...

Cultivate Primary Nasal Epithelial Cells from Children and Reprogram into Induced Pluripotent Stem Cells

Nasal epithelial cells (NECs) are the part of the airways that respond to air pollutants and are the first cells infected with respiratory viruses. They are also involved in many airway diseases through their innate immune response and interaction with immune and airway stromal cells. NECs are of particular interest for studies in children due to their accessibility during clinical visits. Human induced pluripotent stem cells (iPSCs) have been generated from multiple cell types and are a powerful tool for modeling human development and disease, as well as for their potential applications in regenerative medicine. This is the first protocol to lay out methods for successful generation of iPSCs from NECs derived from pediatric participants for research purposes. It describes how to obtain nasal epithelial cells from children, how to generate primary NEC cultures from these samples, and how to reprogram primary NECs into well-characterized iPSCs. Nasal mucosa samples are useful in epidemiological studies related to the effects of air pollution in children, and provide an important tool for studying airway disease. Primary nasal cells and iPSCs derived from them can be a tool for providing unlimited material for patient-specific research in diverse areas of airway epithelial biology, including asthma and COPD research.

This publication demonstrates methods for successful sampling and culture of nasal epithelial mucosa from children, and reprogramming these cells to induced Pluripotent Stem Cells (iPSCs).

Nasal epithelial cells (NECs) are the part of the airways that respond to air pollutants and are the first cells infected with respiratory viruses. They ...

In Vitro Differentiation of Human Pluripotent Stem Cells into Trophoblastic Cells

The placenta is the first organ to develop during embryogenesis and is required for the survival of the developing embryo. The placenta is comprised of various trophoblastic cells that differentiate from the extra-embryonic trophectoderm cells of the preimplantation blastocyst. As such, our understanding of the early differentiation events of the human placenta is limited because of ethical and legal restrictions on the isolation and manipulation of human embryogenesis. Human pluripotent stem cells (hPSCs) are a robust model system for investigating human development and can also be differentiated in vitro into trophoblastic cells that express markers of the various trophoblast cell types. Here, we present a detailed protocol for differentiating hPSCs into trophoblastic cells using bone morphogenic protein 4 and inhibitors of the Activin/Nodal signaling pathways. This protocol generates various trophoblast cell types that can be transfected with siRNAs for investigating loss-of-function phenotypes or can be infected with pathogens. Additionally, hPSCs can be genetically modified and then differentiated into trophoblast progenitors for gain-of-function analyses. This in vitro differentiation method for generating human trophoblasts starting from hPSCs overcomes the ethical and legal restrictions of working with early human embryos, and this system can be used for a variety of applications, including drug discovery and stem cell research.

In Vitro Differentiation of Human Pluripotent Stem Cells into Trophoblastic Cells

Here, we present a protocol to efficiently generate human trophoblastic cells from human pluripotent stem cells using bone morphogenic protein 4 and inhibitors of the Activin/Nodal pathways. This method is suitable for the efficient differentiation of human pluripotent stem cells and can generate large quantities of cells for genetic manipulation.

The placenta is the first organ to develop during embryogenesis and is required for the survival of the developing embryo. The placenta is comprised of ...

Protocol for the Differentiation of Human Induced Pluripotent Stem Cells into Mixed Cultures of Neurons and Glia for Neurotoxicity Testing

Human pluripotent stem cells can differentiate into various cell types that can be applied to human-based in vitro toxicity assays. One major advantage is that the reprogramming of somatic cells to produce human induced pluripotent stem cells (hiPSCs) avoids the ethical and legislative issues related to the use of human embryonic stem cells (hESCs). HiPSCs can be expanded and efficiently differentiated into different types of neuronal and glial cells, serving as test systems for toxicity testing and, in particular, for the assessment of different pathways involved in neurotoxicity. This work describes a protocol for the differentiation of hiPSCs into mixed cultures of neuronal and glial cells. The signaling pathways that are regulated and/or activated by neuronal differentiation are defined. This information is critical to the application of the cell model to the new toxicity testing paradigm, in which chemicals are assessed based on their ability to perturb biological pathways. As a proof of concept, rotenone, an inhibitor of mitochondrial respiratory complex I, was used to assess the activation of the Nrf2 signaling pathway, a key regulator of the antioxidant-response-element-(ARE)-driven cellular defense mechanism against oxidative stress.

Human induced pluripotent stem cells (hiPSCs) are considered a powerful tool for drug and chemical screening and for the development of new in vitro models for toxicity testing, including neurotoxicity. Here, a detailed protocol for the differentiation of hiPSCs into neurons and glia is described.

Human pluripotent stem cells can differentiate into various cell types that can be applied to human-based in vitro toxicity assays. One major advantage is ...

Differentiating Chondrocytes from Peripheral Blood-derived Human Induced Pluripotent Stem Cells

In this study, we used peripheral blood cells (PBCs) as seed cells to produce chondrocytes via induced pluripotent stem cells (iPSCs) in an integration-free method. Following embryoid body (EB) formation and fibroblastic cell expansion, the iPSCs are induced for chondrogenic differentiation for 21 days under serum-free and xeno-free conditions. After chondrocyte induction, the phenotypes of the cells are evaluated by morphological, immunohistochemical, and biochemical analyses, as well as by the quantitative real-time PCR examination of chondrogenic differentiation markers. The chondrogenic pellets show positive alcian blue and toluidine blue staining. The immunohistochemistry of collagen II and X staining is also positive. The sulfated glycosaminoglycan (sGAG) content and the chondrogenic differentiation markers COLLAGEN 2 (COL2), COLLAGEN 10 (COL10), SOX9, and AGGRECAN are significantly upregulated in chondrogenic pellets compared to hiPSCs and fibroblastic cells. These results suggest that PBCs can be used as seed cells to generate iPSCs for cartilage repair, which is patient-specific and cost-effective.

We present a protocol to generate a chondrogenic lineage from human peripheral blood (PB) via induced pluripotent stem cells (iPSCs) using an integration-free method, which includes embryoid body (EB) formation, fibroblastic cells expansion, and chondrogenic induction.

In this study, we used peripheral blood cells (PBCs) as seed cells to produce chondrocytes via induced pluripotent stem cells (iPSCs) in an ...

Using Human Induced Pluripotent Stem Cell-derived Hepatocyte-like Cells for Drug Discovery

The ability to differentiate human induced pluripotent stem cells (iPSCs) into hepatocyte-like cells (HLCs) provides new opportunities to study inborn errors in hepatic metabolism. However, to provide a platform that supports the identification of small molecules that can potentially be used to treat liver disease, the procedure requires a culture format that is compatible with screening thousands of compounds. Here, we describe a protocol using completely defined culture conditions, which allow the reproducible differentiation of human iPSCs to hepatocyte-like cells in 96-well tissue culture plates. We also provide an example of using the platform to screen compounds for their ability to lower Apolipoprotein B (APOB) produced from iPSC-derived hepatocytes generated from a familial hypercholesterolemia patient. The availability of a platform that is compatible with drug discovery should allow researchers to identify novel therapeutics for diseases that affect the liver.

The protocol presented here describes a platform for identifying small molecules for the treatment of liver disease. A step-by-step description is presented detailing how to differentiate iPSCs into cells with hepatocyte characteristics in 96-well plates, and to use the cells to screen for small molecules with potential therapeutic activity.

The ability to differentiate human induced pluripotent stem cells (iPSCs) into hepatocyte-like cells (HLCs) provides new opportunities to study inborn ...

A Familial Hypercholesterolemia Human Liver Chimeric Mouse Model Using Induced Pluripotent Stem Cell-derived Hepatocytes

Familial hypercholesterolemia (FH) is mostly caused by low-density lipoprotein receptor (LDLR) mutations and results in an increased risk of early-onset cardiovascular disease due to marked elevation of LDL cholesterol (LDL-C) in blood. Statins are the first line of lipid-lowering drugs for treating FH and other types of hypercholesterolemia, but new approaches are emerging, in particular PCSK9 antibodies, which are now being tested in clinical trials. To explore novel therapeutic approaches for FH, either new drugs or new formulations, we need appropriate in vivo models. However, differences in the lipid metabolic profiles compared to humans are a key problem of the available animal models of FH. To address this issue, we have generated a human liver chimeric mouse model using FH induced pluripotent stem cell (iPSC)-derived hepatocytes (iHeps). We used Ldlr-/-/Rag2-/-/Il2rg-/- (LRG) mice to avoid immune rejection of transplanted human cells and to assess the effect of LDLR-deficient iHeps in an LDLR null background. Transplanted FH iHeps could repopulate 5-10% of the LRG mouse liver based on human albumin staining. Moreover, the engrafted iHeps responded to lipid-lowering drugs and recapitulated clinical observations of increased efficacy of PCSK9 antibodies compared to statins. Our human liver chimeric model could thus be useful for preclinical testing of new therapies to FH. Using the same protocol, similar human liver chimeric mice for other FH genetic variants, or mutations corresponding to other inherited liver diseases, may also be generated.

Here, we present a protocol to generate a human liver chimeric mouse model of familial hypercholesterolemia using human induced pluripotent stem cell-derived hepatocytes. This is a valuable model for testing new therapies for hypercholesterolemia.

Familial hypercholesterolemia (FH) is mostly caused by low-density lipoprotein receptor (LDLR) mutations and results in an increased risk of early-onset ...

RNA-based Reprogramming of Human Primary Fibroblasts into Induced Pluripotent Stem Cells

Induced pluripotent stem cells (iPSCs) have proven to be a valuable tool to study human development and disease. Further advancing iPSCs as a regenerative therapeutic requires a safe, robust, and expedient reprogramming protocol. Here, we present a clinically relevant, step-by-step protocol for the extremely high-efficiency reprogramming of human dermal fibroblasts into iPSCs using a non-integrating approach. The core of the protocol consists of expressing pluripotency factors (SOX2, KLF4, cMYC, LIN28A, NANOG, OCT4-MyoD fusion) from in vitro transcribed messenger RNAs synthesized with modified nucleotides (modified mRNAs). The reprogramming modified mRNAs are transfected into primary fibroblasts every 48 h together with mature embryonic stem cell-specific microRNA-367/302 mimics for two weeks. The resulting iPSC colonies can then be isolated and directly expanded in feeder-free conditions. To maximize efficiency and consistency of our reprogramming protocol across fibroblast samples, we have optimized various parameters including the RNA transfection regimen, timing of transfections, culture conditions, and seeding densities. Importantly, our method generates high-quality iPSCs from most fibroblast sources, including difficult-to-reprogram diseased, aged, and/or senescent samples.

Here we describe a clinically relevant, high-efficiency, feeder-free method to reprogram human primary fibroblasts into induced pluripotent stem cells using modified mRNAs encoding reprogramming factors and mature microRNA-367/302 mimics. Also included are methods to assess reprogramming efficiency, expand clonal iPSC colonies, and confirm expression of the pluripotency marker TRA-1-60.

Induced pluripotent stem cells (iPSCs) have proven to be a valuable tool to study human development and disease. Further advancing iPSCs as a regenerative ...

Efficient Differentiation of Human Pluripotent Stem Cells into Liver Cells

The liver detoxifies harmful substances, secretes vital proteins, and executes key metabolic activities, thus sustaining life. Consequently, liver failure&#8212;which can be caused by chronic alcohol intake, hepatitis, acute poisoning, or other insults&#8212;is a severe condition that can culminate in bleeding, jaundice, coma, and eventually death. However, approaches to treat liver failure, as well as studies of liver function and disease, have been stymied in part by the lack of a plentiful supply of human liver cells. To this end, this protocol details the efficient differentiation of human pluripotent stem cells (hPSCs) into hepatocyte-like cells, guided by a developmental roadmap that describes how liver fate is specified across six consecutive differentiation steps. By manipulating developmental signaling pathways to promote liver differentiation and to explicitly suppress the formation of unwanted cell fates, this method efficiently generates populations of human liver bud progenitors and hepatocyte-like cells by days 6 and 18 of PSC differentiation, respectively. This is achieved through the temporally-precise control of developmental signaling pathways, exerted by small molecules and growth factors in a serum-free culture medium. Differentiation in this system occurs in monolayers and yields hepatocyte-like cells that express characteristic hepatocyte enzymes and have the ability to engraft a mouse model of chronic liver failure. The ability to efficiently generate large numbers of human liver cells in vitro has ramifications for treatment of liver failure, for drug screening, and for mechanistic studies of liver disease.

This protocol details a monolayer, serum-free method to efficiently generate hepatocyte-like cells from human pluripotent stem cells (hPSCs) in 18 days. This entails six steps as hPSCs sequentially differentiate into intermediate cell-types such as the primitive streak, definitive endoderm, posterior foregut and liver bud progenitors before forming hepatocyte-like cells.

The liver detoxifies harmful substances, secretes vital proteins, and executes key metabolic activities, thus sustaining life. Consequently, liver ...

Generation of 3D Skin Organoid from Cord Blood-derived Induced Pluripotent Stem Cells

The skin is the body&#8217;s largest organ and has many functions. The skin acts as a physical barrier and protector of the body and regulates bodily functions. Biomimetics is the imitation of the models, systems, and elements of nature for the purpose of solving complex human problems1. Skin biomimetics is a useful tool for in vitro disease research and in vivo regenerative medicine. Human induced pluripotent stem cells (iPSCs) have the characteristic of unlimited proliferation and the ability of differentiation to three germ layers. Human iPSCs are generated from various primary cells, such as blood cells, keratinocytes, fibroblasts, and more. Among them, cord blood mononuclear cells (CBMCs) have emerged as an alternative cell source from the perspective of allogeneic regenerative medicine. CBMCs are useful in regenerative medicine because human leukocyte antigen (HLA) typing is essential to the cell banking system. We provide a method for the differentiation of CBMC-iPSCs into keratinocytes and fibroblasts and for generation of a 3D skin organoid. CBMC-iPSC-derived keratinocytes and fibroblasts have characteristics similar to a primary cell line. The 3D skin organoids are generated by overlaying an epidermal layer onto a dermal layer. By transplanting this 3D skin organoid, a humanized mice model is generated. This study shows that a 3D human iPSC-derived skin organoid may be a novel, alternative tool for dermatologic research in vitro and in vivo.

We propose a protocol that shows how to differentiate induced pluripotent stem cell-derived keratinocytes and fibroblasts and generate a 3D skin organoid, using these keratinocytes and fibroblasts. This protocol contains an additional step of generating a humanized mice model. The technique presented here will improve dermatologic research.

The skin is the body&#8217;s largest organ and has many functions. The skin acts as a physical barrier and protector of the body and regulates bodily ...