Name: chemical reversion of conventional human pluripotent stem cells to a na&#239;ve-like state with improved multilineage differentiation potency
Uploaded: 2018-06-10T15:00:00
Description: Watch this Scientific Journal Video about Chemical Reversion of Conventional Human Pluripotent Stem Cells to a NaÃƒÂ¯ve-like State with Improved Multilineage Differentiation Potency at JoVE.com

Dette arbejde blev støttet af tilskud fra NIH/NEI (R01EY023962), NIH/NICHD (R01HD082098), RPB Stein Innovation Award, The Maryland Stem Cell Research Fund (2018-MSCRFV-4048, 2014-MSCRFE-0742), Novo Nordisk Science Forum Award og The Moseley Foundation.

Alle dyr procedurer blev udført i overensstemmelse med dyrs pleje retningslinjer og protokoller godkendes af det Johns Hopkins School af medicin Institut for dyrs pleje og brug udvalg (IACUC).
1. forberedelse af mus embryonale fibroblaster (MEF) for Feeder-afhængige konventionel (menneskelige stamceller medium/MEF) eller naive gendannes (LIF-3i medium/MEF) hPSC kultur
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Køb eller forberede in-house lav-passage forsyninger af MEF foderautomater fra CF1 eller CF1 x DR4 hybrid...

<ol>
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LIF-3i system gælder en modificeret version af den klassiske murine 2i naive reversion cocktail1 humane pluripotente stamceller. Self-fornyelse af hPSC (som ikke kan ekspandere i 2i alene) er stabiliseret i LIF-2i ved at supplere denne cocktail med tankyrase hæmmer XAV939. LIF-3i kultur tillader bulk og effektiv reversion af den konventionelle hPSC til en pluripotente tilstand ligner den menneskelige præimplantations epiblast3. Selvom vi...

2i (MEK/ERK og GSK3β hæmmer) kultur-systemet blev oprindeligt udviklet for at forfine heterogene serum-baserede mus embryonale stamceller (mESC) kulturer til en ensartet grundtilstand for pluripotency beslægtet med musen præimplantations epiblast1. 2i understøtter dog ikke stabil vedligeholdelse af humane pluripotente stamceller (hPSC) linjer2. De forskellige komplekse lille molekyle, vækstfaktor-suppleret, og transgene tilgange er for nylig blevet rapporteret at fange derfor lignende menneskelige naiv-lignende pluripotente molekylære hedder2. Men mange af de "naiv-lignende" stater lavet med disse metoder også udstillet karyotypic ustabilitet, epigenomiske defekter (fx globale tab af forældrenes genomisk prægning), eller forringet differentiering potentielle.
I kontrast, Cocktailen af tredobbelt kemiske hæmning af GSK3β, ERK og tankyrase signalering og leukæmi hæmmende faktor (LIF-3i) var tilstrækkelig for et bredt repertoire af konventionelle hPSC linjer3stabile naiv-lignende tilbagevenden. LIF-3i-vendt naiv hPSC (N-hPSC) opretholdes normal karyotypes og øget deres udtryk for naiv-specifikke menneskelige præimplantations epiblast gener (e.g.,NANOG, KLF2, NR5A2, DNMT3L, HERVH, Stella (DPPA3), KLF17 , TFCP2L1). LIF-3i reversion også tillægges hPSC med et array af molekylære og biokemiske egenskaber enestående til mESC-lignende naive pluripotency, der omfattede øget fosforyleres STAT3 signalering, faldt ERK fosforylering, globale 5-methylcytosine CpG hypomethylation, genome-wide CpG demetylering på embryonale stamceller (ESC)-specifikt gen initiativtagere og dominerende distale OCT4 forstærker skik. Desuden, i forhold til andre naive reversion metoder, som resulterede i en anderledes hypomethylated præget genomisk loci, LIF-3i-vendt N-hPSC var blottet for systematisk påtrykt CpG mønstre eller tab af DNA methyltransferase udtryk (f.eks. DNMT1, DNMT3A, DNMT3B)3.
En direkte LIF-3i kultur i en bred vifte af konventionelle humane embryonale stamceller (menneskelige stamceller) og menneskelige inducerede pluripotente stamceller (hiPSC) dyrkes på enten foderautomater eller E8 feeder-fri betingelser opnået hurtige og bulk tilbagevenden til en naiv epiblast-lignende tilstand. Direkte LIF-3i naive reversion kan dog være ineffektiv i nogle ustabile konventionel hPSC linjer på grund af de iboende genomisk og afstamning-pulverlak variabilitet som følge af genetisk forskellige donor baggrunde.
Således, for at udvide nytten af metoden LIF-3i, en trinvis optimering blev udviklet og præsenteres heri, der giver universel naive reversion med næsten alle konventionelle menneskelige stamceller eller transgen-fri hiPSC linje kulturperler på foderautomater. Denne universalized naive reversion metode beskæftiger et forbigående oprindelige kultur skridt i konventionelle hPSC, der supplerer LIF-3i cocktailen med to ekstra små molekyler (LIF-5i) at forstærke protein kinase en (forskolin) og sonic hedgehog (sHH) ( purmorphamine) signalering. En indledende passage af konventionelle hPSC i LIF-5i tilpasser dem til efterfølgende stabil LIF-3i reversion som bulkvarer. Indledende LIF-5i tilpasning øger betydeligt indledende enkelt celle klonede spredning af konventionelle hPSC dyrkes på E8 eller foderautomater (før deres efterfølgende stabil og kontinuerlig passage i LIF-3i alene). Konventionelle hPSC linjer tilpasset først tolerere en passage i LIF-5i efterfølgende bulk klonede passaging naive gendannes celler i LIF-3i betingelser, som overflødiggør behovet for pluk- og subcloning af de sjældne stabile kolonier eller den rutinemæssige anvendelse af anti-apoptotiske molekyler eller Rho-forbundet protein kinase (ROCK) hæmmere.
LIF-3i metode er blevet med held ansat til stabilt udvide og opretholde et bredt repertoire af > 30 uafhængige, genetisk forskellige konventionelle hPSC linjer for > 10 – 30 passager ved hjælp af enten ikke-enzymatiske eller enzymatisk dissociation metoder, og uden beviser af induktion af kromosomale eller epigenomiske abnormiteter, herunder abnormaliteter på påtrykt gen loci. Derudover sekventielle LIF-5i/LIF-3i kultur er den eneste naive reversion metode der hidtil er blevet rapporteret at forbedrer den funktionelle pluripotency af et bredt repertoire af konventionelle hPSC linjer ved at reducere deres slægt-pulverlak genekspression og dramatisk forbedre deres Multipotente differentiering potens. LIF-3i naive reversion metoden sletter den iboende interline variation af differentiering af afstamning-pulverlak, konventionelle hPSC linjer, og vil have en stor nytte af ansøgning i regenerativ medicin og cellulære behandlingsformer.

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	<tbody>
		<tr height="21">
			<td height="21" style="height:21px;width:373px;">anti- SSEA-1/CD15 antibody, APC conjugated</td>
			<td style="width:161px;">BD Biosciences</td>
			<td style="width:119px;">561716</td>
			<td style="width:371px;">use 5&#181;L per assay (FACS)</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:373px;">anti- TFCP2L1 antibody</td>
			<td style="width:161px;">Sigma Aldrich</td>
			<td style="width:119px;">HPA029708</td>
			<td>use at a 1:100 dilution (immunostainings)</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:373px;">anti-beta-Actin antibody</td>
			<td style="width:161px;">Abcam</td>
			<td style="width:119px;">ab6276</td>
			<td>use at 1:5000 (Western blot)</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:373px;">anti-CD146 antibody,&#160; PE conjugated&#160;</td>
			<td style="width:161px;">BD Biosciences</td>
			<td style="width:119px;">550315</td>
			<td>use 5&#181;L per assay (FACS)</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:373px;">anti-CD31 antibody, APC conjugated</td>
			<td style="width:161px;">eBioscience</td>
			<td style="width:119px;">17-0319-42</td>
			<td>use 2&#181;L per assay (FACS)</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:373px;">anti-CD31 microbead kit</td>
			<td style="width:161px;">Miltenyi Biotec</td>
			<td style="width:119px;">130-091-935</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:373px;">anti-NANOG antibody</td>
			<td style="width:161px;">Abcam</td>
			<td style="width:119px;">ab109250</td>
			<td>use at a 1:100 dilution (immunostainings)</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:373px;">anti-NR5A2 antibody</td>
			<td style="width:161px;">Sigma Aldrich</td>
			<td style="width:119px;">HPA005455</td>
			<td>use at a 1:100 dilution (immunostainings)</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:373px;">anti-p44/42 MAPK (Erk1/2) antibody</td>
			<td style="width:161px;">Cell Signaling</td>
			<td style="width:119px;">4695</td>
			<td>use at 1:1000 (Western blot), for detection of total protein</td>
		</tr>
		<tr height="42">
			<td height="42" style="height:42px;width:373px;">anti-phospho-p44/42 MAPK (Erk1/2) (Thr202/Tyr204 antibody</td>
			<td style="width:161px;">Cell Signaling</td>
			<td style="width:119px;">4370</td>
			<td>use at 1:1000 (Western blot)</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:373px;">anti-phospho-STAT3 (Tyr705) antibody</td>
			<td style="width:161px;">Cell Signaling</td>
			<td style="width:119px;">9145</td>
			<td>use at 1:1000 (Western blot)</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:373px;">anti-rabbit immunoglobulin antibody, biotinylated</td>
			<td style="width:161px;">Agilent</td>
			<td style="width:119px;">E0432</td>
			<td>use at a 1:500-1:1000 dilution (immunostainings)</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:373px;">anti-SSEA-4 antibody, APC conjugated&#160;</td>
			<td style="width:161px;">R&#38;D System</td>
			<td style="width:119px;">FAB1435A</td>
			<td>use 5&#181;L per assay (FACS)</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:373px;">anti-SSEA-4 GloLIVE antibody, NL493 conjugated</td>
			<td style="width:161px;">R&#38;D System</td>
			<td style="width:119px;">NLLC1435G</td>
			<td>use at 1:50 dilution (live and fixed immunostainings)</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:373px;">anti-STAT3 antibody</td>
			<td style="width:161px;">Cell Signaling</td>
			<td style="width:119px;">9139</td>
			<td>use at 1:1000 (Western blot), for detection of total protein</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:373px;">anti-STELLA/DPPA3 antibody</td>
			<td style="width:161px;">Millipore</td>
			<td style="width:119px;">MAB4388</td>
			<td>use at a 1:50 dilution (immunostainings)</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:373px;">anti-TRA-1-60 GloLIVE antibody, NL557 conjugated&#160;</td>
			<td style="width:161px;">R&#38;D System</td>
			<td style="width:119px;">NLLC4770R</td>
			<td>use at&#160; a 1:50 dilution (live and fixed immunostainings)</td>
		</tr>
		<tr height="42">
			<td height="42" style="height:42px;width:373px;">anti-TRA-1-60 StainAlive Antibody, DyLight 488 conjugated</td>
			<td style="width:161px;">Stemgent</td>
			<td style="width:119px;">09-0068</td>
			<td>use at a 1:100 dilution (live and fixed immunostainings)</td>
		</tr>
		<tr height="42">
			<td height="42" style="height:42px;width:373px;">anti-TRA-1-81 StainAlive Antibody, DyLight 488 conjugated</td>
			<td style="width:161px;">Stemgent</td>
			<td style="width:119px;">09-0069</td>
			<td>use at a 1:100 dilution (live and fixed immunostainings)</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:373px;">anti-TRA1-60 antibody, PE conjugated</td>
			<td style="width:161px;">BD Biosciences</td>
			<td style="width:119px;">560193</td>
			<td>use 10&#181;L per assay (FACS)</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:373px;">anti-TRA1-81 antibody, PE conjugated</td>
			<td style="width:161px;">BD Biosciences</td>
			<td style="width:119px;">560161</td>
			<td>use 10&#181;L per assay (FACS)</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:373px;">APEL2-Li</td>
			<td style="width:161px;">StemCell Technologies</td>
			<td style="width:119px;">5271</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:373px;">Bovine Serum Albumin</td>
			<td style="width:161px;">Sigma Aldrich</td>
			<td style="width:119px;">A3311</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:373px;">CellAdhere dilution buffer</td>
			<td style="width:161px;">StemCell Technologies</td>
			<td style="width:119px;">7183</td>
			<td>dilutent for Vitronectin XF&#8482; matrix</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:373px;">CF1 mouse</td>
			<td style="width:161px;">Charles river</td>
			<td style="width:119px;">023</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:373px;">CHIR99021</td>
			<td style="width:161px;">R&#38;D System</td>
			<td style="width:119px;">L5283</td>
			<td>reconstitute at 100mM in DMSO</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:373px;">confocal microscope system</td>
			<td style="width:161px;">Zeiss</td>
			<td style="width:119px;">LSM 510</td>
			<td></td>
		</tr>
		<tr height="42">
			<td height="42" style="height:42px;width:373px;">cord blood CD34+ derived iPSC line</td>
			<td style="width:161px;">Thermo Fisher Scientific</td>
			<td style="width:119px;">A18945</td>
			<td>also referred as 6.2 line</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:373px;">Corning Costar tissue culture-treated 6-well plates</td>
			<td style="width:161px;">Corning</td>
			<td style="width:119px;">3506</td>
			<td></td>
		</tr>
		<tr height="42">
			<td height="42" style="height:42px;width:373px;">Countess&#160; cell counting chamber slide</td>
			<td style="width:161px;">Thermo Fisher Scientific</td>
			<td style="width:119px;">C10228</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:373px;">Countess automated cell counter</td>
			<td style="width:161px;">Thermo Fisher Scientific</td>
			<td style="width:119px;">AMQAX1000</td>
			<td></td>
		</tr>
		<tr height="42">
			<td height="42" style="height:42px;width:373px;">DMEM (Dulbecco&#39;s Modified Eagle Medium)&#160;</td>
			<td style="width:161px;">Thermo Fisher Scientific</td>
			<td style="width:119px;">11995-065</td>
			<td></td>
		</tr>
		<tr height="42">
			<td height="42" style="height:42px;width:373px;">DMEM-F12</td>
			<td style="width:161px;">Thermo Fisher Scientific</td>
			<td style="width:119px;">11330-032</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:373px;">DMSO (dimethyl sulfoxide)</td>
			<td style="width:161px;">Sigma Aldrich</td>
			<td style="width:119px;">D2650</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:373px;">DR4 mouse</td>
			<td style="width:161px;">The Jackson Laboratory</td>
			<td align="right" style="width:119px;">3208</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:373px;">Essential 8 (E8) medium</td>
			<td style="width:161px;">StemCell Technologies</td>
			<td style="width:119px;">5940</td>
			<td></td>
		</tr>
		<tr height="42">
			<td height="42" style="height:42px;width:373px;">Fetal bovin serum (FBS)</td>
			<td style="width:161px;">Thermo Fisher Scientific</td>
			<td style="width:119px;">SH30071.03</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:373px;">Forskolin</td>
			<td style="width:161px;">Stemgent</td>
			<td style="width:119px;">04-0025</td>
			<td>reconstitute at 100mM in DMSO</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:373px;">Gelatin (porcine)</td>
			<td style="width:161px;">Sigma Aldrich</td>
			<td style="width:119px;">G1890-100G</td>
			<td>resuspend in water and sterilize with an autoclave</td>
		</tr>
		<tr height="42">
			<td height="42" style="height:42px;width:373px;">KnockOut Serum Replacement</td>
			<td style="width:161px;">Thermo Fisher Scientific</td>
			<td style="width:119px;">10828-028</td>
			<td></td>
		</tr>
		<tr height="42">
			<td height="42" style="height:42px;width:373px;">L-Glutamine (100X)</td>
			<td style="width:161px;">Thermo Fisher Scientific</td>
			<td style="width:119px;">25030-081</td>
			<td></td>
		</tr>
		<tr height="42">
			<td height="42" style="height:42px;width:373px;">MEM Non-essential amino acid (MEM NEAA) (100X)</td>
			<td style="width:161px;">Thermo Fisher Scientific</td>
			<td style="width:119px;">11140-050</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:373px;">mTeSR1 medium</td>
			<td style="width:161px;">StemCell Technologies</td>
			<td style="width:119px;">85850</td>
			<td></td>
		</tr>
		<tr height="42">
			<td height="42" style="height:42px;width:373px;">Nalgene cryogenic vials</td>
			<td style="width:161px;">Thermo Fisher Scientific</td>
			<td style="width:119px;">5000-0020</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:373px;">Nunc Lab-Tek II Chamber Slide System</td>
			<td style="width:161px;">Fisher Scientific</td>
			<td style="width:119px;">154534</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:373px;">Paraformaldehyde (PFA) solution , 4% in PBS</td>
			<td style="width:161px;">USB Corporation&#160;</td>
			<td style="width:119px;">19943</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:373px;">PD0325901</td>
			<td style="width:161px;">Sigma Aldrich</td>
			<td style="width:119px;">PZ0162</td>
			<td>reconstitute at 100mM in DMSO</td>
		</tr>
		<tr height="42">
			<td height="42" style="height:42px;width:373px;">Penicillin/streptomycin (10,000 U/mL)</td>
			<td style="width:161px;">Thermo Fisher Scientific</td>
			<td style="width:119px;">15140-122</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:373px;">Phosphate buffered saline (PBS)</td>
			<td style="width:161px;">Biological Industries</td>
			<td style="width:119px;">02-023-1A</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:373px;">Purmorphamine</td>
			<td style="width:161px;">Stemgent</td>
			<td style="width:119px;">04-0009</td>
			<td>reconstitute at 10mM in DMSO</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:373px;">recombinant human Activin A</td>
			<td style="width:161px;">Peprotech</td>
			<td style="width:119px;">AF-120-14E</td>
			<td></td>
		</tr>
		<tr height="42">
			<td height="42" style="height:42px;width:373px;">recombinant human Bone morphogenetic protein (BMP)-4</td>
			<td style="width:161px;">Peprotech</td>
			<td style="width:119px;">120-05ET</td>
			<td>resupend at 100ug/mL in 0.1% bovine serum albumin in PBS</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:373px;">recombinant human FGF-basic (bFGF)</td>
			<td style="width:161px;">Peprotech</td>
			<td style="width:119px;">100-18B</td>
			<td>resupend at 100ug/mL in 0.1% bovine serum albumin in PBS</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:373px;">recombinant human LIF</td>
			<td style="width:161px;">Peprotech</td>
			<td style="width:119px;">300-05</td>
			<td>resupend at 100ug/mL in 0.1% bovine serum albumin in PBS</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:373px;">SB431542&#160;</td>
			<td style="width:161px;">Stemgent</td>
			<td style="width:119px;">04-0010-05</td>
			<td>reconstitute at 100mM in DMSO</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:373px;">Stemolecule Y27632 in Solution</td>
			<td style="width:161px;">Stemgent</td>
			<td style="width:119px;">04-0012-02</td>
			<td>ROCK inhibitor in solution (10mM)</td>
		</tr>
		<tr height="42">
			<td height="42" style="height:42px;width:373px;">StemPro Accutase Cell Dissociation Reagent</td>
			<td style="width:161px;">Thermo Fisher Scientific</td>
			<td style="width:119px;">A11105-01</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:373px;">Streptavidin-Cy3 conjugate</td>
			<td style="width:161px;">Sigma Aldrich</td>
			<td style="width:119px;">S6402</td>
			<td>use at 1:500-1:1000 dilution (immunostainings)</td>
		</tr>
		<tr height="42">
			<td height="42" style="height:42px;width:373px;">Thermo Scientific Mr. Frosty Freezing Container</td>
			<td style="width:161px;">Thermo Fisher Scientific</td>
			<td style="width:119px;">5100-0001</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:373px;">Vascular endothelial growth factor (VEGF)-165</td>
			<td style="width:161px;">Peprotech</td>
			<td style="width:119px;">100-21</td>
			<td>resupend at 100ug/mL in 0.1% bovine serum albumin in PBS</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:373px;">Vitronectin XF matrix</td>
			<td style="width:161px;">StemCell Technologies</td>
			<td style="width:119px;">7180</td>
			<td>dilute at 40&#181;L/mL in CellAdhere&#8482; dilution buffer</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:373px;">XAV939</td>
			<td style="width:161px;">Sigma Aldrich</td>
			<td style="width:119px;">X3004</td>
			<td>reconstitute at 100mM in DMSO</td>
		</tr>
		<tr height="42">
			<td height="42" style="height:42px;width:373px;">&#946;-mercaptoethanol</td>
			<td style="width:161px;">Thermo Fisher Scientific</td>
			<td style="width:119px;">21985-023</td>
			<td>light sensitive</td>
		</tr>
	</tbody>
</table>

chemical reversion of conventional human pluripotent stem cells to a na&#239;ve-like state with improved multilineage differentiation potency

Naive humane pluripotente stamceller (N-hPSC) med forbedret funktionalitet kan have en bred effekt i regenerativ medicin. Målet med denne protokol er effektivt tilbage afstamning-pulverlak, konventionelle humane pluripotente stamceller (hPSC) vedligeholdes på enten feeder-fri eller feeder-afhængige forhold til en naiv-lignende pluripotency med forbedret funktionalitet. Denne kemiske naive reversion metode beskæftiger klassisk leukæmi hæmmende faktor (LIF), GSK3β, og MEK/ERK hæmning cocktail (LIF-2i), suppleret med kun en tankyrase hæmmer XAV939 (LIF-3i). LIF-3i vender konventionel hPSC til en stabil pluripotente stat vedtage biokemiske, transcriptional og epigenetiske funktioner i den menneskelige præ-implantation epiblast. LIF-3i metoden kræver minimal celle kultur manipulation og reproducerbare meget i et bredt repertoire af humane embryonale stamceller (menneskelige stamceller) og transgen-fri menneskelige inducerede pluripotente stamceller (hiPSC) linjer. LIF-3i metode kræver ikke en ny grunding skridt før differentiering; N-hPSC kan differentieres direkte med meget høj effektivitet og vedligeholde karyotypic og epigenomiske stabilitet (herunder på påtrykt loci). For at øge universelle i metoden, konventionelle hPSC er først kulturperler i LIF-3i cocktail suppleres med to yderligere små molekyler, at forstærke protein kinase en (forskolin) og sonic hedgehog (sHH) (purmorphamine) signalering (LIF-5i). Denne korte LIF-5i tilpasning trin betydeligt forbedrer den oprindelige klonal ekspansion af konventionelle hPSC og tillader dem at være efterfølgende naive gendannes med LIF-3i alene i bulk mængder og dermed længere er behov for pluk/subcloning sjældne Nielsen-hPSC kolonier senere. LIF-5i-stabiliseret hPSCs vedligeholdes efterfølgende i LIF-3i alene uden brug af anti-apoptotiske molekyler. Vigtigst, forbedrer LIF-3i reversion markant den funktionelle pluripotency af et bredt repertoire af konventionelle hPSC ved faldende deres slægt-pulverlak genekspression og sletning af interline variabiliteten af styret differentiering almindeligt observeret blandt uafhængige hPSC linjer. Repræsentative beskrivelser af LIF-3i-vendt N-hPSC er forudsat, og eksperimenterende strategier til funktionel sammenligninger af isogene hPSC i slægt-pulverlak vs. naiv-lignende stater er skitseret.

Denne protokol optimerer effektiv naiv-lignende reversion med LIF-3i i begge feeder-afhængige og feeder-uafhængig afstamning-primet konventionelle hPSC kulturer (figur 1). Den detaljerede protokollen, beskrevet heri konturer fortløbende tilpasning til LIF-3i starter fra enten feeder-afhængige eller feeder-fri konventionelle hPSC betingelser (fx E8 medium).
Repræsentative resultater af ...

Watch this Scientific Journal Video about Chemical Reversion of Conventional Human Pluripotent Stem Cells to a NaÃƒÂ¯ve-like State with Improved Multilineage Differentiation Potency at JoVE.com

Chemical Reversion of Conventional Human Pluripotent Stem Cells to a NaÃƒÆ’Ã†'Ãƒâ€šÃ‚Â¯ve-like State with Improved Multilineage Differentiation Potency

Kemiske Reversion af konventionelle humane pluripotente stamceller til en naiv-lignende tilstand med forbedret Multilineage differentiering potens

Vi præsenterer en protokol for effektiv, bulk og hurtige kemiske reversion af konventionelle afstamning-pulverlak humane pluripotente stamceller (hPSC) ind i en epigenomically-stabil naive præimplantations epiblast-lignende pluripotente tilstand. Denne metode resulterer i nedsat afstamning-pulverlak genekspression og markant forbedring i styret multilineage differentiering på tværs af et bredt repertoire af konventionelle hPSC linjer.

Na&#239;ve human pluripotent stem cells (N-hPSC) with improved functionality may have a wide impact in regenerative medicine. The goal of this protocol is ...

Research

JoVE Journal

Developmental Biology

Chemical Reversion of Conventional Human Pluripotent Stem Cells to a Na&#239;ve-like State with Improved Multilineage Differentiation Potency

Stencil Micropatterning of Human Pluripotent Stem Cells for Probing Spatial Organization of Differentiation Fates

Stencil Micropatterning for Human pluripotente stamceller til Sondering Spatial Organisation af differentiering Fates

Human pluripotent stem cells (hPSCs), including embryonic stem cells and induced pluripotent stem cells, have the intrinsic ability to differentiate into ...

Large-Scale Production of Cardiomyocytes from Human Pluripotent Stem Cells Using a Highly Reproducible Small Molecule-Based Differentiation Protocol

Storstilet produktion af cardiomyocytter fra menneskelige pluripotente stamceller Ved hjælp af en meget reproducerbar lille molekyle-Based Differentiering Protocol

Maximizing the benefit of human pluripotent stem cells (hPSCs) for research, disease modeling, pharmaceutical and clinical applications requires robust ...

Rapid Neuronal Differentiation of Induced Pluripotent Stem Cells for Measuring Network Activity on Micro-electrode Arrays

Hurtig neuronal differentiering af induceret pluripotente stamceller til måling Network Aktivitet på Micro-elektrode Arrays

Neurons derived from human induced Pluripotent Stem Cells (hiPSCs) provide a promising new tool for studying neurological disorders. In the past decade, ...

Directed Differentiation of Primitive and Definitive Hematopoietic Progenitors from Human Pluripotent Stem Cells

Instrueret differentiering af Primitive og endelige hæmatopoietisk ophav fra humane pluripotente stamceller

One of the major goals for regenerative medicine is the generation and maintenance of hematopoietic stem cells (HSCs) derived from human pluripotent stem ...

Efficient Differentiation of Pluripotent Stem Cells to NKX6-1+ Pancreatic Progenitors

Efficient Differentiation of Pluripotent Stem Cells to NKX6-1+ Pancreatic Progenitors

Effektiv Differentiering af pluripotente stamceller til NKX6-1<sup&gt; +</sup&gt; Pankreatisk progenitorer

Pluripotent stem cells have the ability to self renew and differentiate to multiple lineages, making them an attractive source for the generation of ...

In Vitro Differentiation of Human Pluripotent Stem Cells into Trophoblastic Cells

In Vitro Differentiation of Human Pluripotent Stem Cells into Trophoblastic Cells

In vitro differentiering af humane pluripotente stamceller til trofoblastceller

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 ...

Rapid, Directed Differentiation of Retinal Pigment Epithelial Cells from Human Embryonic or Induced Pluripotent Stem Cells

Hurtig, instrueret differentiering af retinale Pigment epitel celler fra menneskelige embryonale eller induceret pluripotente stamceller

We describe a robust method to direct the differentiation of pluripotent stem cells into retinal pigment epithelial cells (RPE). The purpose of providing ...

Efficient and Scalable Directed Differentiation of Clinically Compatible Corneal Limbal Epithelial Stem Cells from Human Pluripotent Stem Cells

Effektiv og skalerbar styret differentiering af klinisk kompatibel hornhinde Limbal epitelial stamceller fra humane pluripotente stamceller

Corneal limbal epithelial stem cells (LESCs) are responsible for continuously renewing the corneal epithelium, and thus maintaining corneal homeostasis ...

Efficient Differentiation of Human Pluripotent Stem Cells into Liver Cells

Effektiv differentiering af humane pluripotente stamceller i leverceller

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

In Vitro Generation of Somite Derivatives from Human Induced Pluripotent Stem Cells

I Vitro Generation af Somite derivater fra menneskelige induceret celler pluripotente stamceller

In response to signals such as WNTs, bone morphogenetic proteins (BMPs), and sonic hedgehog (SHH) secreted from surrounding tissues, somites (SMs) give ...