Name: efficient generation and editing of feeder-free ipscs from human pancreatic cells using the crispr-cas9 system
Uploaded: 2017-11-08T13:00:00
Description: Watch this Scientific Journal Video about Efficient Generation and Editing of Feeder-free IPSCs from Human Pancreatic Cells Using the CRISPR-Cas9 System at JoVE.com

Arbete i labbet stöddes av postdoktorsstipendium grant till Dr Anjali Nandal och förberedande bidrag från Maryland Stem Cell Research Fund att BT (TEDCO).

1. omprogrammering protokoll <ol><li>Generation av mänskliga iPSC från primära mänskliga bukspottkörtelns celler <ol><li>Coat en 6-väl platta med 1,5 mg/mL kall kollagen och låt det gel vid 37 ° C under 1 h.</li>
		<li>Tallrik tidig passage människans primära bukspottkörtelns celler i Prigrow III medium (~ 1-1,5 × 10 5 celler) på en kollagen belagd 6-väl plattan på dag -2 att uppnå cirka 2,5 × 10 5 celler eller minst 60% confluency per brunn på dagen för transd...

<ol>
	<li>Malik, N., Rao, M. S. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=A+review+of+the+methods+for+human+iPSC+derivation.">A review of the methods for human iPSC derivation.</a> Methods Mol. 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O., 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+cytoplasmic+RNA+vector+derived+from+nontransmissible+Sendai+virus+with+efficient+gene+transfer+and+expression.">A cytoplasmic RNA vector derived from nontransmissible Sendai virus with efficient gene transfer and expression.</a> J Virol. 74, 6564-6569 (2000).</li><li>Fusaki, N., Ban, H., Nishiyama, A., Saeki, K., Hasegawa, M. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Efficient+induction+of+transgene-free+human+pluripotent+stem+cells+using+a+vector+based+on+Sendai+virus,+an+RNA+virus+that+does+not+integrate+into+the+host+genome.">Efficient induction of transgene-free human pluripotent stem cells using a vector based on Sendai virus, an RNA virus that does not integrate into the host genome.</a> Proc. Jpn. Acad. Ser. B, PhysBiol. Sci. 85, 348-362 (2009).</li><li>Nakagawa, M., 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+novel+efficient+feeder-free+culture+system+for+the+derivation+of+human+induced+pluripotent+stem+cells.">A novel efficient feeder-free culture system for the derivation of human induced pluripotent stem cells.</a> Sci. Rep. 4, 3594 (2014).</li><li>Hockemeyer, D., Jaenisch, R. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Induced+Pluripotent+Stem+Cells+Meet+Genome+Editing.">Induced Pluripotent Stem Cells Meet Genome Editing.</a> Cell stem cell. 18, 573-586 (2016).</li><li>Komor, A. C., Badran, A. H., Liu, D. R. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=CRISPR-Based+Technologies+for+the+Manipulation+of+Eukaryotic+Genomes.">CRISPR-Based Technologies for the Manipulation of Eukaryotic Genomes.</a> Cell. 169, 559 (2017).</li><li>Komor, A. C., Badran, A. H., Liu, D. R. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=CRISPR-Based+Technologies+for+the+Manipulation+of+Eukaryotic+Genomes.">CRISPR-Based Technologies for the Manipulation of Eukaryotic Genomes.</a> Cell. 168, 20-36 (2017).</li><li>Sander, J. D., Joung, J. K. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=CRISPR-Cas+systems+for+editing,+regulating+and+targeting+genomes.">CRISPR-Cas systems for editing, regulating and targeting genomes.</a> Nat. Biotech. 32, 347-355 (2014).</li><li>McElroy, S. L., Reijo Pera, R. A. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Preparation+of+mouse+embryonic+fibroblast+feeder+cells+for+human+embryonic+stem+cell+culture.">Preparation of mouse embryonic fibroblast feeder cells for human embryonic stem cell culture.</a> CSH Protoc. 2008, (2008).</li><li>Lian, X., 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+cardiomyocyte+differentiation+from+human+pluripotent+stem+cells+via+temporal+modulation+of+canonical+Wnt+signaling.">Robust cardiomyocyte differentiation from human pluripotent stem cells via temporal modulation of canonical Wnt signaling.</a> Proc. Natl. Acad. Sci. U.S.A. 109, E1848-E1857 (2012).</li><li>Rezania, 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=Reversal+of+diabetes+with+insulin-producing+cells+derived+in+vitro+from+human+pluripotent+stem+cells.">Reversal of diabetes with insulin-producing cells derived in vitro from human pluripotent stem cells.</a> Nat. Biotech. 32, 1121-1133 (2014).</li><li>Shi, Y., Inoue, H., Wu, J. C., Yamanaka, S. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Induced+pluripotent+stem+cell+technology:+a+decade+of+progress.">Induced pluripotent stem cell technology: a decade of progress.</a> Nat. Rev. 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Omprogrammering av mänskliga kroppsceller att iPSCs har gett ett stort uppsving i fälten grundläggande biologi forskning, personlig medicin, sjukdom modellering, läkemedelsutveckling och regenerativ medicin16. Många nuvarande och allmänt använda metoder för mänskliga iPSC generation kräver användning av virus med risk för integrering i den mottagande arvsmassan eller episomal vektorer med låg omprogrammering effektivitet. Här presenterar vi en effektiv metod för att generera feeder-...

Omprogrammering av mänskliga kroppsceller till pluripotenta staten av överuttryck av omprogrammering faktorer har revolutionerat stamcellsforskning med tillämpningar i sjukdom modellering, regenerativ medicin och läkemedelsutveckling. Flera icke-virala omplanering metoder finns tillgängliga för leverans av omprogrammering faktorer och generera iPSCs, men processen är labor intensiv och inte mycket effektiv1. De virala metoderna, är men effektiva, associerade med problem med virus integrationen och tumorigenicitetsprov2,3,4. I detta manuskript rapportera vi användning av cytoplasmisk Sendai virus för att leverera omprogrammering faktorer och upprätta fotavtryck-fri iPSC rader som saknar integrering av alla virala vektorn sekvenser i deras genomen5. Sendai virus är ett RNA-virus som späds ut ur cellen cytoplasman ~ 10 passager efter infektion och producerar omplanering faktorer i överflöd, vilket leder till snabb och effektiv omprogrammering6,7. Den etablerade iPSCs kan sedan flyttas lätt över till feeder-fri medium att undvika användning av mus embryonala fibroblaster (MEFs) som mataren celler8.
I denna publikation, förutom beskriver Sendai viruset medierad omprogrammering, beskriver vi också ett förbättrat protokoll för redigering iPSCs, som har potential att leverera obegränsad mänskliga celler med önskad genetiska modifieringar för forskning. Vi har använt CRISPR/Cas9-teknik för modifiering av iPSCs, som nu används för ett brett spektrum av tillämpningar, inklusive knock-ins och knockouts, storskalig genomisk borttagningar, poolade bibliotek screening för gen upptäckt, genteknik av talrika modellorganismer och gene therapy9,10,11. Denna teknik innebär bildandet av komplex av Streptococcus pyogenes-härledda Cas9 nuclease och 20-mer guide RNAs som uppnå målet erkännande via base-ihopkoppling med genomiska mål sekvens intill 3' nukleotid protospacer intill motiv (PAM) sekvens. Den Cas9 nuclease inducerar en dubbel icke-återvinningsbara break ~ 3 nukleotider från PAM platsen, som är därefter reparerats huvudsakligen av icke-homolog slutet att gå (NHEJ) väg leder till infogningar och borttagningar i ramen öppen läsning, och därmed funktionell knockout gener12.
Våra förbättrat protokoll innehåller Detaljer för kultur av mänskliga bukspottskörteln celler, deras omprogrammering på mitotiskt inaktiverat mus embryonala fibroblaster (MEFs) för att uppnå högre effektivitet av omprogrammering, efterföljande anpassning till feeder-fri kultur på Matrigel, karakterisering av etablerade iPSCs, guidade CRISPR RNA design och förberedelse, leverans till iPSCs som RNP komplex, enda cell sortering för att generera klonal rader av redigerade iPSCs, enkel screening och identifiering av redigeringar och karakterisering av enstaka cell kloner. Genomisk borttagningar genererades effektivt i denna studie av införandet av Cas9 protein och två CRISPR sgRNA RNP komplex att inducera dubbel strandsatta raster (DSBs) och borttagning av den ingriper segment. Denna metod kapitaliserar på användningen av två guider för att generera borttagningar i ramen öppen läsning, hög effektivitet av NHEJ leder till lågt antal kloner att behovet att präglas och lätt preliminära screening av kloner av automatiserade kapillären elektrofores enhet, fragment analyzer. Dessa effektiva genomet redigering metoder för att generera mänskliga stamceller-baserade sjukdomsmodeller kommer snart att bli en standard och rutinmässiga strategi i ett laboratorium som stamceller. Slutligen, exakt gen editering gör det möjligt att gå bortom stamceller sjukdom modellering och potentiellt kan bidra till att katalysera cellbaserade terapier.

<table>
	<tbody>
		<tr>
			<td>Sendai viral vectors - CytoTune-iPS 2.0 Kit</td>
			<td>Invitrogen</td>
			<td>A16517</td>
			<td>Thaw on ice; S No: 1</td>
		</tr>
		<tr>
			<td>Trypsin EDTA</td>
			<td>Gibco Life Tech</td>
			<td>25300-054</td>
			<td>0.05%, 100 ml; S No: 2</td>
		</tr>
		<tr>
			<td>Rock inhibitor (Y-27632)</td>
			<td>Milipore</td>
			<td>SCM075</td>
			<td>Use 10 &#956;M; S No: 3</td>
		</tr>
		<tr>
			<td>DMEM/F-12 medium</td>
			<td>Invitrogen</td>
			<td>11330-032</td>
			<td>S No: 4</td>
		</tr>
		<tr>
			<td>Serum replacement (KSR)</td>
			<td>Gibco</td>
			<td>10828028</td>
			<td>S No: 5</td>
		</tr>
		<tr>
			<td>DMEM</td>
			<td>Invitrogen</td>
			<td>11960069</td>
			<td>1X; S No: 6</td>
		</tr>
		<tr>
			<td>Fetal bovine serum</td>
			<td>Thermo Scientific</td>
			<td>SH30071.03</td>
			<td>Aliquot; S No: 7</td>
		</tr>
		<tr>
			<td>L-glutamine (Glutamax, 100X), liquid</td>
			<td>Thermo Scientific</td>
			<td>35050061</td>
			<td>1/100; S No: 8</td>
		</tr>
		<tr>
			<td>Non-Essential Amino Acids</td>
			<td>Gibco</td>
			<td>11140-050</td>
			<td>1/100; S No: 9</td>
		</tr>
		<tr>
			<td>2-Mercaptoethanol</td>
			<td>Gibco</td>
			<td>21985023</td>
			<td>55 mM, 1/1,000; S No: 10</td>
		</tr>
		<tr>
			<td>Hausser Hemacytometers</td>
			<td>Hausser Scientific</td>
			<td>02-671-54</td>
			<td>S No: 11</td>
		</tr>
		<tr>
			<td>&#160;0.1% Gelatin Solution</td>
			<td>STEMCELL Technologies</td>
			<td>7903</td>
			<td>Incubate at 37&#186; C for 1 hour; S No: 12</td>
		</tr>
		<tr>
			<td>SSEA-4 antibody</td>
			<td>Santacruz</td>
			<td>sc-21704</td>
			<td>1/100; S No: 13</td>
		</tr>
		<tr>
			<td>TRA-1-81 antibody</td>
			<td>Cell Signaling</td>
			<td>4745S</td>
			<td>1/200; S No: 14</td>
		</tr>
		<tr>
			<td>&#160;OCT4 antibody</td>
			<td>Santa Cruz</td>
			<td>sc-5279</td>
			<td>1/1,000; S No: 15</td>
		</tr>
		<tr>
			<td>Collagen I, Rat Tail</td>
			<td>Life Technologies</td>
			<td>A10483-01</td>
			<td>Keep cold; S No: 16</td>
		</tr>
		<tr>
			<td>Alexa Fluor fluorescent 488/ 568 (secondary antibodies)</td>
			<td>Invitrogen</td>
			<td>A21202/A10042</td>
			<td>1/2,000; S No: 17</td>
		</tr>
		<tr>
			<td>DPBS</td>
			<td>Hyclone</td>
			<td>SH30028LS</td>
			<td>1X; S No: 18</td>
		</tr>
		<tr>
			<td>100-mm tissue culture dish</td>
			<td>Falcon</td>
			<td>353003</td>
			<td>S No: 20</td>
		</tr>
		<tr>
			<td>96-well tissue culture plate</td>
			<td>Falcon</td>
			<td>353078</td>
			<td>S No: 21</td>
		</tr>
		<tr>
			<td>6-well tissue culture plate</td>
			<td>Falcon</td>
			<td>353046</td>
			<td>S No: 22</td>
		</tr>
		<tr>
			<td>Dissecting scope&#160;</td>
			<td>Nikon</td>
			<td>SMZ745</td>
			<td>S No: 23</td>
		</tr>
		<tr>
			<td>Picking hood</td>
			<td>NuAire</td>
			<td>NU-301</td>
			<td>S No: 24</td>
		</tr>
		<tr>
			<td>15&#160;ml Centrifuge Tube</td>
			<td>Greiner Bio-One</td>
			<td>188271</td>
			<td>S No: 25</td>
		</tr>
		<tr>
			<td>50&#160;ml Centrifuge Tube</td>
			<td>Greiner Bio-One</td>
			<td>227261</td>
			<td>S No: 26</td>
		</tr>
		<tr>
			<td>Sodium pyruvate</td>
			<td>Invitrogen</td>
			<td>11360</td>
			<td>S No: 28</td>
		</tr>
		<tr>
			<td>&#946;-mercaptoethanol</td>
			<td>Sigma</td>
			<td>M7522</td>
			<td>S No: 29</td>
		</tr>
		<tr>
			<td>Prigrow III medium</td>
			<td>ABM</td>
			<td>TM003</td>
			<td>S No: 31</td>
		</tr>
		<tr>
			<td>Countess&#8482; Cell Counter</td>
			<td>Invitrogen</td>
			<td>C10227</td>
			<td>S No: 32</td>
		</tr>
		<tr>
			<td>Faxitron X-ray system</td>
			<td>Faxitron</td>
			<td>CellRad</td>
			<td>S No: 33</td>
		</tr>
		<tr>
			<td>Accutase</td>
			<td>Innovative cell Technologies</td>
			<td>AT-104</td>
			<td>S No: 34</td>
		</tr>
		<tr>
			<td>Collagenase</td>
			<td>Life Technologies</td>
			<td>17104019</td>
			<td>1mg/ml stock; S No: 35</td>
		</tr>
		<tr>
			<td>Dispase</td>
			<td>STEMCELL Technologies</td>
			<td>7923</td>
			<td>S No: 36</td>
		</tr>
		<tr>
			<td>hESC qualified matrigel</td>
			<td>BD Biosciences</td>
			<td>354277</td>
			<td>To dilute, use cold DMEM/F-12; S No: 37</td>
		</tr>
		<tr>
			<td>bFGF</td>
			<td>R &#38; D</td>
			<td>233-FB</td>
			<td>Stock 10 ug/ml; S No: 38</td>
		</tr>
		<tr>
			<td>Paraformaldehyde</td>
			<td>EMS</td>
			<td>15710</td>
			<td>4% stock in PBS; S No: 39</td>
		</tr>
		<tr>
			<td>TRA-1-60</td>
			<td>Santa Cruz</td>
			<td>sc-21705</td>
			<td>1/100; S No: 40</td>
		</tr>
		<tr>
			<td>NANOG</td>
			<td>ReproCELL</td>
			<td>RCAB0004P-F</td>
			<td>1/100; S No: 41</td>
		</tr>
		<tr>
			<td>Tween 20</td>
			<td>Sigma</td>
			<td>P9416-100ML</td>
			<td>S No: 42</td>
		</tr>
		<tr>
			<td>Alkaline Phosphatase kit</td>
			<td>Stemgent</td>
			<td>00-0055</td>
			<td>S No: 43</td>
		</tr>
		<tr>
			<td>Cas9 protein</td>
			<td>PNA Bio</td>
			<td>CP01-50</td>
			<td>Thaw and aliquot; S No: 44</td>
		</tr>
		<tr>
			<td>Goat or donkey serum</td>
			<td>Sigma</td>
			<td>D9663/G9023</td>
			<td>S No: 45</td>
		</tr>
		<tr>
			<td>Triton X-100</td>
			<td>Sigma</td>
			<td>X100-100ML</td>
			<td>S No: 46</td>
		</tr>
		<tr>
			<td>DAPI</td>
			<td>Thermo Scientific</td>
			<td>D1306</td>
			<td>S No: 47</td>
		</tr>
		<tr>
			<td>Tris</td>
			<td>Sigma</td>
			<td>9285-100ML</td>
			<td>S No: 48</td>
		</tr>
		<tr>
			<td>NaCL</td>
			<td>Sigma</td>
			<td>S7653-250G</td>
			<td>S No: 49</td>
		</tr>
		<tr>
			<td>EDTA</td>
			<td>Sigma</td>
			<td>BP2482-500</td>
			<td>S No: 50</td>
		</tr>
		<tr>
			<td>T4 DNA ligase</td>
			<td>NEB</td>
			<td>M0202T</td>
			<td>S No: 51</td>
		</tr>
		<tr>
			<td>Mega Shortscript T7 kit</td>
			<td>Thermo Scientific</td>
			<td>AM1354</td>
			<td>S No: 52</td>
		</tr>
		<tr>
			<td>Mega Clear kit</td>
			<td>Thermo Scientific</td>
			<td>AM1908</td>
			<td>S No: 53</td>
		</tr>
		<tr>
			<td>SMC4</td>
			<td>BD Biosciences</td>
			<td>354357</td>
			<td>S No: 54</td>
		</tr>
		<tr>
			<td>Fibronectin</td>
			<td>STEMCELL Technologies</td>
			<td>7159</td>
			<td>S No: 55</td>
		</tr>
		<tr>
			<td>CloneJET cloning kit</td>
			<td>Thermo Scientific</td>
			<td>K1232</td>
			<td>S No: 56</td>
		</tr>
		<tr>
			<td>Fragment analyzerTM</td>
			<td>Advanced Analytical</td>
			<td></td>
			<td>S No: 57</td>
		</tr>
		<tr>
			<td>mTeSR1 medium kit</td>
			<td>STEMCELL Technologies</td>
			<td>5850</td>
			<td>Warm to room temperature; S No: 58</td>
		</tr>
		<tr>
			<td>Freezing medium mFreSR&#8482;</td>
			<td>STEMCELL Technologies</td>
			<td>5855</td>
			<td>S No: 59</td>
		</tr>
		<tr>
			<td>Freezing medium CryoStor&#174;</td>
			<td>STEMCELL Technologies</td>
			<td>7930</td>
			<td>S No: 60</td>
		</tr>
		<tr>
			<td>MEFs</td>
			<td>Globalstem</td>
			<td>GSC-6301G</td>
			<td>S No: 61</td>
		</tr>
		<tr>
			<td>L-glutamine</td>
			<td>Invitrogen</td>
			<td>25030081</td>
			<td>S No: 62</td>
		</tr>
		<tr>
			<td>Human pancreatic cells</td>
			<td>ABM</td>
			<td>T0159</td>
			<td>S No: 63</td>
		</tr>
		<tr>
			<td>STEMdiff&#8482; Neural Induction Medium</td>
			<td>Stemcell Technologies</td>
			<td>5835</td>
			<td>S No: 64</td>
		</tr>
		<tr>
			<td>RPMI</td>
			<td>Thermofisher</td>
			<td>11875-093</td>
			<td>S No: 65</td>
		</tr>
		<tr>
			<td>2% B27-insulin</td>
			<td>Thermofisher</td>
			<td>A1895601</td>
			<td>S No: 66</td>
		</tr>
		<tr>
			<td>CHIR99021</td>
			<td>Stemcell Technologies</td>
			<td>72052</td>
			<td>S No: 67</td>
		</tr>
		<tr>
			<td>IWP4</td>
			<td>Stemcell Technologies</td>
			<td>72552</td>
			<td>S No: 68</td>
		</tr>
		<tr>
			<td>2% B27</td>
			<td>Thermofisher</td>
			<td>17504044</td>
			<td>S No: 69</td>
		</tr>
		<tr>
			<td>MCDB 131</td>
			<td>Life Technologies</td>
			<td>10372019</td>
			<td>S No: 70</td>
		</tr>
		<tr>
			<td>Sodium bicarbonate</td>
			<td>Sigma-Aldrich</td>
			<td>S8761-100ML</td>
			<td>S No: 71</td>
		</tr>
		<tr>
			<td>Glucose</td>
			<td>Sigma-Aldrich</td>
			<td>G8270-100G</td>
			<td>S No: 72</td>
		</tr>
		<tr>
			<td>BSA</td>
			<td>Proliant</td>
			<td>68700</td>
			<td>S No: 73</td>
		</tr>
		<tr>
			<td>GDF8</td>
			<td>Pepro-Tech</td>
			<td>120-00</td>
			<td>S No: 74</td>
		</tr>
		<tr>
			<td>TUJ1 antibody</td>
			<td>EMD Milipore</td>
			<td>AB9354</td>
			<td>S No: 75</td>
		</tr>
		<tr>
			<td>NKX2-5 antibody</td>
			<td>Santa Cruz</td>
			<td>Sc-14033</td>
			<td>S No: 76</td>
		</tr>
		<tr>
			<td>SOX17 antibody</td>
			<td>R &#38; D systems</td>
			<td>AF1924</td>
			<td>S No: 77</td>
		</tr>
		<tr>
			<td>Propidium iodide</td>
			<td>Thermo Scientific</td>
			<td>P3566</td>
			<td>S No: 78</td>
		</tr>
		<tr>
			<td>Amaxa 4D-nucleofector&#8482;</td>
			<td>Lonza</td>
			<td>AAF-1002</td>
			<td>S No: 79</td>
		</tr>
		<tr>
			<td>FACSAria II (cell sorter)</td>
			<td>BD biosciences</td>
			<td>SORP UV</td>
			<td>S No: 80</td>
		</tr>
	</tbody>
</table>

efficient generation and editing of feeder-free ipscs from human pancreatic cells using the crispr-cas9 system

Embryonala och inducerade pluripotenta stamceller kan själv förnya och differentieras till flera celltyper i kroppen. De pluripotenta cellerna är således eftertraktade för forskning inom regenerativ medicin och är för närvarande i kliniska prövningar för ögonsjukdomar, diabetes, hjärtsjukdomar och andra sjukdomar. Potential att differentieras till specialiserade celltyper som tillsammans med de senaste framstegen i genomet redigering teknik inklusive CRISPR Cas systemet har gett ytterligare möjligheter för att skräddarsy genomet av iPSC för varierande applikationer däribland sjukdom modellering, genterapi, och polarisering vägar av differentiering, för att nämna några. Bland de tillgängliga redigering teknikerna, har den CRISPR/Cas9 från Streptococcus pyogenes uppstått som ett verktyg för val för platsspecifika redigering av eukaryota genomet. CRISPRs är lättillgängligt, billig och mycket effektiv i engineering riktade redigeringar. Systemet kräver en Cas9 nuclease och en guide sekvens (20-mer) specifika för genomisk målet abutting 3-nukleotid ”NGG” protospacer-intilliggande-motiv (PAM) för inriktning Cas9 till det önska genomisk locus, tillsammans med universal Cas9 bindande spårämne () RNA tillsammans kallas enda guide RNA eller sgRNA). Här presenterar vi en stegvisa protokoll för effektiv generering av feeder-oberoende och fotavtryck-gratis iPSC och beskriva metoder för editering av iPSC använder de Cas9 ribonukleoprotein (RNP) komplex. Genomet redigering protokoll är effektiv och kan vara enkelt multiplexed av pre-komplexbildande sgRNAs för mer än ett mål med proteinet Cas9 och samtidigt leverera in i cellerna. Slutligen beskriver vi en förenklad strategi för identifiering och karakterisering av iPSCs med önskade redigeringar. Sammantaget förväntas beskrivs strategierna effektivisera produktion och redigering av iPSC för många applikationer.

I den här publikationen har vi följt ett enkelt men effektivt protokoll för generering av iPSC från mänskliga bukspottkörtelns celler använder integration eller fotavtryck-gratis Sendai virusvektorer. Figur 1A visar en schematisk representation av detta omplanering protokoll. Mänskliga bukspottkörtelns celler köptes kommersiellt odlade i Prigrow III medium och sensorik med Sendai virus som förklaras ovan. Sensorik spindeln formade bukspottkörtelns...

Watch this Scientific Journal Video about Efficient Generation and Editing of Feeder-free IPSCs from Human Pancreatic Cells Using the CRISPR-Cas9 System at JoVE.com

Efficient Generation and Editing of Feeder-free IPSCs from Human Pancreatic Cells Using the CRISPR-Cas9 System

Detta protokollet beskriver i detalj generationen av fotavtryck-fri inducerade pluripotenta stamceller (iPSCs) från mänskliga bukspottkörtelns celler i feeder-fria förhållanden, följt av redigering med CRISPR/Cas9 ribonucleoproteins och karakterisering av de modifierade encelliga kloner.

Effektiv produktion och redigering av Feeder-fri IPSCs från mänsklig bukspottkörtelns celler med hjälp av CRISPR-Cas9-systemet

Embryonic and induced pluripotent stem cells can self-renew and differentiate into multiple cell types of the body. The pluripotent cells are thus coveted ...

The overall goal of this procedure is to generate footprint-free induced pluripotent stem cells or IPSCs from human pancreatic cells in feeder-free conditions, then to edit the cells using CRISPR-Cas9 ribonucleoproteins and finally to characterize the modified single cell clones. This method can help answer key questions in the human stem cell genome editing field such as generation of footprint-free IPSCs and editing with CRISPR-Cas9 ribonucleoproteins. The main advantage of this technique is that clonal lines of edited IPSCs can be generated with high reliability and there is no evidence of mosaicism.After coating a six well plate with cold collagen, plate early passage human primary pancreatic cells in Prigrow III medium on day 2 to achieve approximately 2.5 x 10 to the 5 cells or at least 60%confluency per well on the day of transduction or day zero. On the day of transduction after harvesting and counting the cells according to the text protocol, thaw on ice one set of Sendai vector tubes and carefully add the calculated volumes of each tube to 1 mL of prewarmed Prigrow III medium. Gently pipette to mix the solution.Aspirate the Prigrow III from the cells and slowly add the reprogramming virus mixture to the wells. Then incubate the cells in a 37 degree Celsius incubator overnight. Twenty-four hours after transduction, carefully discard the virus mixture on the cells and replace it with fresh Prigrow III medium.On day six, add 1 mL of cell detachment solution to the cells and incubate them for 10 minutes. Then, using Prigrow III with rock inhibitor, plate all the cells on dishes of MEFs prepared the day before. Incubate the cells at 37 degrees Celsius overnight.Observe the plates regularly for the emergence of cell clumps or colonies indicative of reprogrammed cells which will form clonal aggregates with a cobblestone morphology and a big nucleus and nucleoli. Mark the probable IPSC colonies and regularly check them for growth. Almost four weeks following transduction after preparing 24 well MEF plates, prepare matrix membrane by aliquoting it based on the dilution factor on the certificate of analysis.Pick 24-48 colonies and transfer them onto matrix membrane coated 24 well plates with mTeSR1. Incubate the plates for 24 hours and change the medium every day. Refer to the text protocol for additional details.Add 500 mcL of dispase to detach the robust colonies plated on the matrix membrane. After incubating the cells for 20 minutes, plate them again on matrix membrane coated 12 well plates. Expand and characterize the clones using alkaline phosphatase staining, immuno staining and FACS analysis according to the text protocol.To transfect HIPSCs after synthesizing SGRNAs according to the text protocol, prepare nucleofaction master mix for each sample by combining 0.5 mcg of Cas9 protein and 0.5 mcg of each SGRNA in a 22 mcL reaction volume. After aspirating the medium containing rock inhibitor, use 2 mL of RTPBS to wash each well of ISPCs. Then aspirate the PBS and add 1 mL of cell detachment solution.Incubate the plate at 37 degrees Celsius for 10 minutes. Resuspend the cells in 3 mL of mTeSR1 medium and gently pipette up and down to generate a single cell suspension. Transfer the dissociated cells to a 15 mL centrifuge tube containing 5 mL of mTeSR1 medium.To the nucleofection master mix, add 16.4 mcL of P3 primary cell supplement and 3.6 mcL of supplement one from the Nucleofector kit. After counting and spinning the cells, resuspend each unit of 0.5 x 10 to the 6th cells in 22 mcL of the previously prepared transfection master mix. Quickly transfer the cells into the central chamber of one well of a Nucleocuvette strip.Then, place the strip into a Nucleofector device and nucleofect the cells using program CB150. Following nucleofection, quickly add 80 mcL of prewarmed mTeSR1 medium containing 10 micromolar rock inhibitor to each well of the nucleofected cells. Gently mix by pipetting up and down.Gently transfer the cells from the strip to wells of the matrix membrane precoated 12 well plate containing mTeSR1 medium with rock inhibitor. On the second day of incubation after preparing MEF plates, replace the medium on the HIPSCs from mTeSR1 to mTeSR1 supplemented with 1XSMC4 for at least two hours before single cell sorting. Aspirate the medium from HIPSCs and use PBS to gently wash the cells.Then add 500 mcL of cell detachment solution into each well and incubate the cells at 37 degrees Celsius for 10 minutes. Generate a single cell suspension by adding 1 mL of mTeSR1 to each well and gently pipette up and down several times. Sort single cells into individual wells of a previously prepared 96 well plate.Four days after sorting, colony formation should be apparent. At this point, replace the culture medium with HESC medium supplemented with 1X SMC4. After extracting and expanding the target DNA according the text protocol, use a fragment analyzer kit to run the PCR amplified target genomic region of all clones through the gel dye mix per the manufacturer's instructions.Confirm pluripotent IPSC clones according to the text protocol. Before Sendai virus transduction, pancreatic cells show typical spindle-like morphology. Small tight colonies appear on MEFs by day 12 resembling human pluripotent cell colonies.Normally, the fully reprogrammed human IPSC colonies have very clear boundaries and can be picked up by days 23 to 30. A dissociated colony at day 23 is shown here. Presented here is a typical phase contrast image of an IPSC colony after picking and culture in feeder-free conditions after Sendai virus reprogramming of pancreatic cells.Alkaline phosphatase stained red IPSC colonies are on the right. IPSCs were confirmed by immuno staining for the pluripotency markers OCT4 and NANOG as seen in these panels. In this experiment, IPSCs were stained with cell surface markers and FACS analysis was carried out on a single cell suspension.The green peak represents pancreatic IPSCs, and the purple peak contains IPSC negative cells. While attempting this procedure, it is important to remember to maintain aseptic or sterile conditions for cell sorting and cell culture. Following this procedure, other methods like gene targeting in IPSCs and ESCs can be performed, and additional questions can be answered by performing precise genetic modifications.After watching this video, you should have a good understanding of how to reliably generate footprint-free and feeder-free IPSCs and perform genome editing biallelically.

Research

JoVE Journal

Bioengineering

Therapeutic Gene Delivery and Transfection in Human Pancreatic Cancer Cells using Epidermal Growth Factor Receptor-targeted Gelatin Nanoparticles

Gene Delivery och Transfektion i mänskliga celler bukspottkörtelcancer med Epidermal Growth Factor Receptor-riktade Gelatin-Based Engineered Nanovectors

More than 32,000 patients are diagnosed with pancreatic cancer in the United States per year and the disease is associated with very high mortality 1. ...

Development, Expansion, and In vivo Monitoring of Human NK Cells from Human Embryonic Stem Cells (hESCs) and Induced Pluripotent Stem Cells (iPSCs)

Development, Expansion, and In vivo Monitoring of Human NK Cells from Human Embryonic Stem Cells hESCs and Induced Pluripotent Stem Cells iPSCs

Utveckling, expansion och<em&gt; In vivo</em&gt; Övervakning av humana NK celler från mänskliga embryonala stamceller (hESCs) och och inducerad pluripotenta stamceller (iPSCs)

We present a method for deriving natural killer (NK) cells from undifferentiated hESCs and iPSCs using a feeder-free approach. This method gives rise to ...

Production of Genetically Engineered Golden Syrian Hamsters by Pronuclear Injection of the CRISPR/Cas9 Complex

Produktion av genmanipulerade gyllene syriska hamstrar genom pronukleär injektion av CRISPR/Cas9 komplexet

The pronuclear (PN) injection technique was first established in mice to introduce foreign genetic materials into the pronuclei of one-cell stage embryos. ...

Microinjection of Western Corn Rootworm, Diabrotica virgifera virgifera, Embryos for Germline Transformation, or CRISPR/Cas9 Genome Editing

Microinjection of Western Corn Rootworm, Diabrotica virgifera virgifera, Embryos for Germline Transformation, or CRISPR/Cas9 Genome Editing

Mikroskop västra havren Rootworm, Diabrotica virgifera virgifera, embryon för könsceller omvandling eller CRISPR/Cas9 gen editering

The western corn rootworm (WCR) is an important pest of corn and is well known for its ability to rapidly adapt to pest management strategies. Although ...

Generation of Cationic Nanoliposomes for the Efficient Delivery of In Vitro Transcribed Messenger RNA

Generation of Cationic Nanoliposomes for the Efficient Delivery of In Vitro Transcribed Messenger RNA

Generation av katjoniska Nanoliposomes för effektiv leverans av In Vitro transkriberas budbärar-RNA

The development of messenger RNA (mRNA)-based therapeutics for the treatment of various diseases becomes more and more important because of the positive ...

Purification and Analytics of a Monoclonal Antibody from Chinese Hamster Ovary Cells Using an Automated Microbioreactor System

Rening och analys av en monoklonal antikropp från äggstocksceller från kinesisk hamster med hjälp av ett automatiserat Mikrobioreaktor system

Monoclonal antibodies (mAbs) are one of the most popular and well-characterized biological products manufactured today. Most commonly produced using ...

Isolation of Adult Human Dermal Fibroblasts from Abdominal Skin and Generation of Induced Pluripotent Stem Cells Using a Non-Integrating Method

Isolering av vuxna humana dermal fibroblaster från buk hud och generering av inducerade pluripotenta stamceller med en icke-integrerande metod

Induced pluripotent stem cells (iPSCs) could be considered, to date, a promising source of pluripotent cells for the management of currently untreatable ...

Scalable Generation of Mature Cerebellar Organoids from Human Pluripotent Stem Cells and Characterization by Immunostaining

Skalbar Generation av Mogna Cerebellar Organoids från Mänskliga Pluripotenta stamceller och karakterisering av Immunfärgning

The cerebellum plays a critical role in the maintenance of balance and motor coordination, and a functional defect in different cerebellar neurons can ...

Single-Cell Optical Action Potential Measurement in Human Induced Pluripotent Stem Cell-Derived Cardiomyocytes

Encellig optisk åtgärd potentiell mätning hos mänskliga inducerade pluripotenta stamcellsbaserade kardiomyocyter

Conventional intracellular microelectrode techniques to quantify cardiomyocyte electrophysiology are extremely complex, labor intensive, and typically ...