Name: a two-step strategy that combines epigenetic modification and biomechanical cues to generate mammalian pluripotent cells
Uploaded: 2020-08-29T13:00:00
Description: Watch this Scientific Journal Video about A Two-Step Strategy that Combines Epigenetic Modification and Biomechanical Cues to Generate Mammalian Pluripotent Cells at JoVE.com

Dette arbejde blev finansieret af Carraresi Foundation og MiND FoodS Hub ID: 1176436. Alle forfatterne er medlemmer af COST Action CA16119 In vitro 3-D total cell guidance and fitness (CellFit).

Alle undersøgelser blev gennemgået og godkendt af det etiske udvalg ved universitetet i Milano. Alle dyreforsøg blev udført i overensstemmelse med Guide for the Care and Use of Laboratory Animals, udgivet af US National Institutes of Health (NIH). Humane celler isolation fra raske voksne individer blev godkendt af den etiske komité for Ospedale Maggiore Policlinico, Milano. Alle metoderne i vores undersøgelse blev udført i overensstemmelse med de godkendte retningslinjer.
1. Isolering af ...

<ol>
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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=5-azacytidine+affects+TET2+and+histone+transcription+and+reshapes+morphology+of+human+skin+fibroblasts.">5-azacytidine affects TET2 and histone transcription and reshapes morphology of human skin fibroblasts.</a> Scientific Reports. 6, 37017 (2016).</li><li>Pennarossa, G., 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=Epigenetic+erasing+and+pancreatic+differentiation+of+dermal+fibroblasts+into+insulin-producing+cells+are+boosted+by+the+use+of+low-stiffness+substrate.">Epigenetic erasing and pancreatic differentiation of dermal fibroblasts into insulin-producing cells are boosted by the use of low-stiffness substrate.</a> Stem Cell reviews and reports. 14 (3), 398-411 (2018).</li><li>Pennarossa, G., 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=Use+of+a+PTFE+micro-bioreactor+to+promote+3D+cell+rearrangement+and+maintain+high+plasticity+in+epigenetically+erased+fibroblasts.">Use of a PTFE micro-bioreactor to promote 3D cell rearrangement and maintain high plasticity in epigenetically erased fibroblasts.</a> Stem Cell Reviews and Reports. 15 (1), 82-92 (2019).</li><li>Constantinides, P. G., Jones, P. A., Gevers, W. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Functional+striated+muscle+cells+from+non-myoblast+precursors+following+5-azacytidine+treatment.">Functional striated muscle cells from non-myoblast precursors following 5-azacytidine treatment.</a> Nature. 267 (5609), 364-366 (1977).</li><li>Taylor, S. M., Jones, P. A. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Multiple+new+phenotypes+induced+in+10T1/2+and+3T3+cells+treated+with+5-azacytidine.">Multiple new phenotypes induced in 10T1/2 and 3T3 cells treated with 5-azacytidine.</a> Cell. 17 (4), 771-779 (1979).</li><li>Taylor, S. M., Constantinides, P. A., Jones, P. A. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=5-Azacytidine,+DNA+methylation,+and+differentiation.">5-Azacytidine, DNA methylation, and differentiation.</a> Current Topics in Microbiology and Immunology. 108, 115-127 (1984).</li><li>Jones, P. A. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Effects+of+5-azacytidine+and+its+2'-deoxyderivative+on+cell+differentiation+and+DNA+methylation.">Effects of 5-azacytidine and its 2'-deoxyderivative on cell differentiation and DNA methylation.</a> Pharmacology &amp; Therapeutics. 28 (1), 17-27 (1985).</li><li>Palii, S. S., Van Emburgh, B. O., Sankpal, U. T., Brown, K. D., Robertson, K. D. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=DNA+methylation+inhibitor+5-Aza-2'-deoxycytidine+induces+reversible+genome-wide+DNA+damage+that+is+distinctly+influenced+by+DNA+methyltransferases+1+and+3B.">DNA methylation inhibitor 5-Aza-2'-deoxycytidine induces reversible genome-wide DNA damage that is distinctly influenced by DNA methyltransferases 1 and 3B.</a> Molecular and Cellular Biology. 28 (2), 752-771 (2008).</li><li>Vining, K. H., Mooney, D. J. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Mechanical+forces+direct+stem+cell+behaviour+in+development+and+regeneration.">Mechanical forces direct stem cell behaviour in development and regeneration.</a> Nature Reviews. Molecular Cell Biology. 18 (12), 728 (2017).</li><li>Matamoro-Vidal, A., Levayer, R. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Multiple+influences+of+mechanical+forces+on+cell+competition.">Multiple influences of mechanical forces on cell competition.</a> Current Biology. 29 (15), 762-774 (2019).</li><li>Yim, E. K., Sheetz, M. P. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Force-dependent+cell+signaling+in+stem+cell+differentiation.">Force-dependent cell signaling in stem cell differentiation.</a> Stem Cell Research &amp; Therapy. 3 (5), 41 (2012).</li><li>Kumar, A., Placone, J. K., Engler, A. J. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Understanding+the+extracellular+forces+that+determine+cell+fate+and+maintenance.">Understanding the extracellular forces that determine cell fate and maintenance.</a> Development. 144 (23), 4261-4270 (2017).</li><li>Bissell, M. J., Rizki, A., Mian, I. S. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Tissue+architecture:+the+ultimate+regulator+of+breast+epithelial+function.">Tissue architecture: the ultimate regulator of breast epithelial function.</a> Current Opinion in Cell Biology. 15 (6), 753-762 (2003).</li><li>Streuli, C. H., 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=Laminin+mediates+tissue-specific+gene+expression+in+mammary+epithelia.">Laminin mediates tissue-specific gene expression in mammary epithelia.</a> The Journal of Cell Biology. 129 (3), 591-603 (1995).</li><li>Christman, 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=5-Azacytidine+and+5-aza-2[prime]-deoxycytidine+as+inhibitors+of+DNA+methylation:+mechanistic+studies+and+their+implications+for+cancer+therapy.">5-Azacytidine and 5-aza-2[prime]-deoxycytidine as inhibitors of DNA methylation: mechanistic studies and their implications for cancer therapy.</a> Oncogene. 21, 5483-5495 (2002).</li><li>Stresemann, C., Lyko, F. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Modes+of+action+of+the+DNA+methyltransferase+inhibitors+azacytidine+and+decitabine.">Modes of action of the DNA methyltransferase inhibitors azacytidine and decitabine.</a> International Journal of Cancer. 123 (1), 8-13 (2008).</li><li>Ledda, S., Idda, A., Kelly, J., Ariu, F., Bogliolo, L., Bebbere, D. <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+technique+for+in+vitro+maturation+of+sheep+oocytes+in+a+liquid+marble+microbioreactor.">A novel technique for in vitro maturation of sheep oocytes in a liquid marble microbioreactor.</a> Journal of Assisted Reproduction and Genetics. 33 (4), 513-518 (2016).</li><li>Vadivelu, R. 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=Generation+of+three-dimensional+multiple+spheroid+model+of+olfactory+ensheathing+cells+using+floating+liquid+marbles.">Generation of three-dimensional multiple spheroid model of olfactory ensheathing cells using floating liquid marbles.</a> Scientific Reports. 5, 15083 (2015).</li><li>Vadivelu, R. K., Kamble, H., Munaz, A., Nguyen, N. T. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Liquid+Marble+as+bioreactor+for+engineering+three-dimensional+toroid+tissues.">Liquid Marble as bioreactor for engineering three-dimensional toroid tissues.</a> Scientific Reports. 7 (1), 12388 (2017).</li><li>Vadivelu, R. K., Kamble, H., Munaz, A., Nguyen, N. T. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Liquid+Marbles+as+bioreactors+for+the+study+of+three-dimensional+cell+interactions.">Liquid Marbles as bioreactors for the study of three-dimensional cell interactions.</a> Biomedical Microdevices. 19 (2), 31 (2017).</li><li>Varelas, 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=TAZ+controls+Smad+nucleocytoplasmic+shuttling+and+regulates+human+embryonic+stem-cell+self-renewal.">TAZ controls Smad nucleocytoplasmic shuttling and regulates human embryonic stem-cell self-renewal.</a> Nature Cell Biology. 10 (7), 837-848 (2008).</li><li>Panciera, T., 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+Expandable+Tissue-Specific+Stem/Progenitor+Cells+through+Transient+Expression+of+YAP/TAZ.">Induction of Expandable Tissue-Specific Stem/Progenitor Cells through Transient Expression of YAP/TAZ.</a> Cell Stem Cell. 19 (6), 725-737 (2016).</li><li>Pennarossa, G., Paffoni, A., Ragni, G., Gandolfi, F., Brevini, T. 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I løbet af de sidste årtier fokuserede flere undersøgelser på udviklingen af strategier til at vende en terminalt differentieret celle mod en mindre engageret og højere tilladende tilstand. Protokollen her beskrevet tillader generering og langsigtet vedligeholdelse af pluripotente celler startende fra voksne modne terminalt differentierede celler. Metoden kombinerer to uafhængige trin, der involverer induktion af en høj tilladende tilstand, som opnås gennem kemisk epigenetisk sletning og dens efterfølgende vedli...

I løbet af de sidste årtier blev det bredt accepterede koncept om ensrettet progression mod celleengagement og differentiering fuldstændig revideret. Det er blevet påvist, at cellespecifikation kan vendes, og en terminalt differentieret celle kan skubbes mod en mindre engageret og højere tilladende tilstand ved hjælp af forskellige metoder.
Blandt de flere foreslåede metoder involverer en af de mest lovende metoder brugen af kemiske forbindelser til at inducere celler i en såkaldt kemisk induceret pluripotens. De små molekyler, der anvendes i denne tilgang, er i stand til at interagere og ændre den epigenetiske signatur af en voksen moden celle, så man undgår behovet for transgen og/eller viral vektor 1,2,3,4,5,6,7,8,9,10 . Talrige undersøgelser har for nylig vist, at det er muligt at skifte celler fra en fænotype til en anden ved at tilvejebringe specifikke biokemiske og biologiske stimuli, der inducerer reaktivering af hypermethylerede gener 11,12,13,14,15. Disse demethyleringshændelser muliggør omdannelse af terminalt differentierede celler til en primitiv stamfader, en multipotent eller en høj plasticitet/pluripotent celle 1,2,3,4,5,6,7,8,9,10.
Parallelt hermed har mange undersøgelser for nylig fokuseret på forståelsen af mekanosensing-relaterede signaler og mere specifikt på muligheden for at anvende mekaniske kræfter til direkte at påvirke celleplasticitet og / eller differentiering 16,17,18,19. Faktisk er det tydeligt blevet påvist, at den ekstracellulære matrix (ECM) spiller en nøglerolle i kontrollen med celleskæbne. Især de biomekaniske og biofysiske signaler produceret af ECM regulerer direkte molekylære mekanismer og signalveje, der påvirker celleadfærd og funktioner20,21. Disse nylige data har banet vejen for udviklingen af nye 3D-kultursystemer, der i tættere grad efterligner in vivo-cellemikromiljøet og replikerer mekaniske og fysiske stimuli, der driver celleadfærd.
Vi beskriver her en totrinsprotokol, der kombinerer brugen af kemisk epigenetisk sletning med mekanosensingrelaterede signaler for at generere pattedyrs pluripotente celler. I det første trin inkuberes celler med demethyleringsmolekylet 5-aza-cytidin (5-aza-CR). Dette middel er i stand til at inducere en signifikant global DNA-demethylering gennem en kombineret virkning af den direkte ti-elleve translokation 2 (TET2)-medierede virkning 8,10 og den indirekte hæmning af DNA-methyltransferaserne (DNMT)22,23. Dette trin inducerer fjernelsen af de epigenetiske blokke med en efterfølgende genaktivering af pluripotensrelateret genekspression og derfor dannelsen af celler med høj plasticitet 1,2,3,8,10, i det følgende benævnt "epigeneetisk slettede celler". I det andet trin indkapsles celler i et 3D-kultursystem. Til dette formål anvendes den ikke-reaktive hydrofobe syntetiske forbindelse polytetrafluorethylen (PTFE; med partikelstørrelse på 1 μm) som mikrobioreaktor, der gør det muligt at skabe et cellulært mikromiljø, der ikke kan opnås ved anvendelse af traditionelle 2D-kultursystemer10. PTFE-pulverpartiklerne klæber til overfladen af væskedråben, hvor cellerne suspenderes igen, og isolerer den flydende kerne fra understøtningsoverfladen, samtidig med at gasudvekslingen mellem den indre væske og det omgivende miljøtillades 24. Den således opnåede "PTFE-mikrobioreaktor", også kendt som "Flydende marmor", tilskynder celler til frit at interagere med hinanden, fremme 3D-celleomlægning25,26,27 og udvider og stabilt opretholder den erhvervede høje plasticitetstilstand gennem biomekanosensing-relaterede signaler10.

<table>
	<tbody>
		<tr>
			<td>2-Mercaptoethanol</td>
			<td>Sigma-Aldrich</td>
			<td>M7522</td>
			<td>Component of ESC medium</td>
		</tr>
		<tr>
			<td>5-Azacytidine</td>
			<td>Sigma-Aldrich</td>
			<td>A2385</td>
			<td>5-aza-CR, for fibroblast epigenetic erasing</td>
		</tr>
		<tr>
			<td>Adenosine</td>
			<td>Sigma-Aldrich</td>
			<td>A4036</td>
			<td>Component of nucleoside mix for ESC medium</td>
		</tr>
		<tr>
			<td>Antibiotic Antimycotic Solution (100&#215;)</td>
			<td>Sigma-Aldrich</td>
			<td>A5955</td>
			<td>Component of fibroblast and ESC media</td>
		</tr>
		<tr>
			<td>CFX96 Real-Time PCR</td>
			<td>Bio-Rad Laboratories</td>
			<td>NA</td>
			<td>Thermal cycler for quantitative PCR</td>
		</tr>
		<tr>
			<td>Cytidine</td>
			<td>Sigma-Aldrich</td>
			<td>C4654</td>
			<td>Component of nucleoside mix for ESC medium</td>
		</tr>
		<tr>
			<td>DMEM, high glucose, pyruvate</td>
			<td>Thermo Fisher Scientific</td>
			<td>41966052</td>
			<td>For fibroblast isolation and culture medium</td>
		</tr>
		<tr>
			<td>DMEM, low glucose, pyruvate</td>
			<td>Thermo Fisher Scientific</td>
			<td>31885023</td>
			<td>For ESC medium</td>
		</tr>
		<tr>
			<td>Dulbecco&#8217;s Phosphate Buffered Saline</td>
			<td>Sigma-Aldrich</td>
			<td>D5652</td>
			<td>PBS; for biopsy and cell wash and for solution preparation</td>
		</tr>
		<tr>
			<td>Dynabeads mRNA DIRECT Micro Purification Kit</td>
			<td>Thermo Fisher Scientific</td>
			<td>61021</td>
			<td>mRNA estraction</td>
		</tr>
		<tr>
			<td>ESGRO Recombinant Mouse LIF Protein</td>
			<td>Sigma-Aldrich</td>
			<td>ESG1106</td>
			<td>Component of ESC medium</td>
		</tr>
		<tr>
			<td>Fetal Bovine Serum, qualified, heat inactivated</td>
			<td>Thermo Fisher Scientific</td>
			<td>10500064</td>
			<td>Component of fibroblast and ESC media</td>
		</tr>
		<tr>
			<td>FGF-Basic (AA10-155) Recombinant Human Protein</td>
			<td>Thermo Fisher Scientific</td>
			<td>PHG0024</td>
			<td>Component of ESC medium</td>
		</tr>
		<tr>
			<td>Gelatin from porcine skin</td>
			<td>Sigma-Aldrich</td>
			<td>G1890</td>
			<td>For dish coating</td>
		</tr>
		<tr>
			<td>GeneAmp PCR System 2700</td>
			<td>Applied Biosystems</td>
			<td>NA</td>
			<td>Thermal cycler for qualitative PCR</td>
		</tr>
		<tr>
			<td>Global DNA Methylation ELISA Kit</td>
			<td>CELL BIOLABS</td>
			<td>STA-380</td>
			<td>Methylation study</td>
		</tr>
		<tr>
			<td>GoTaq G2 Flexi DNA Polymerase</td>
			<td>Promega</td>
			<td>M7801</td>
			<td>Qualitative PCR</td>
		</tr>
		<tr>
			<td>Guanosine</td>
			<td>Sigma-Aldrich</td>
			<td>G6264</td>
			<td>Component of nucleoside mix for ESC medium</td>
		</tr>
		<tr>
			<td>Ham&#39;s F-10 Nutrient Mix</td>
			<td>Thermo Fisher Scientific</td>
			<td>31550031</td>
			<td>For ESC medium</td>
		</tr>
		<tr>
			<td>KnockOut Serum Replacement</td>
			<td>Thermo Fisher Scientific</td>
			<td>10828028</td>
			<td>Component of ESC medium</td>
		</tr>
		<tr>
			<td>KOVA glasstic slide 10 with grids</td>
			<td>Hycor Biomedical</td>
			<td>87144</td>
			<td>For cell counting</td>
		</tr>
		<tr>
			<td>Leica MZ APO Stereo Microscope</td>
			<td>Leica</td>
			<td>NA</td>
			<td>For organoid observation</td>
		</tr>
		<tr>
			<td>L-Glutamine solution</td>
			<td>Sigma-Aldrich</td>
			<td>G7513</td>
			<td>Component of fibroblast and ESC media</td>
		</tr>
		<tr>
			<td>MEM Non-Essential Amino Acids Solution (100X)</td>
			<td>Thermo Fisher Scientific</td>
			<td>11140035</td>
			<td>Component of ESC medium</td>
		</tr>
		<tr>
			<td>Millex-GS 0.22 &#181;m pore filters</td>
			<td>Millipore</td>
			<td>SLGS033SB</td>
			<td>For solution sterilization</td>
		</tr>
		<tr>
			<td>M-MLV Reverse Transcriptase, RNase H Minus, Point Mutant</td>
			<td>Promega</td>
			<td>M3681</td>
			<td>mRNA reverse transcription</td>
		</tr>
		<tr>
			<td>Multiskan FC Microplate Photometer</td>
			<td>Thermo Fisher Scientific</td>
			<td>51119000</td>
			<td>For ELISA plate reading</td>
		</tr>
		<tr>
			<td>Nikon Eclipse TE300 Inverted Phase Contrast Microscope</td>
			<td>Nikon</td>
			<td>NA</td>
			<td>For cell observation</td>
		</tr>
		<tr>
			<td>Perkin Elmer Thermal Cycler 480</td>
			<td>Perkin Elmer</td>
			<td>NA</td>
			<td>Thermal cycler for reverse transcription</td>
		</tr>
		<tr>
			<td>Poly(tetrafluoroethylene) 1 &#956;m particle size</td>
			<td>Sigma-Aldrich</td>
			<td>430935</td>
			<td>For generating micro-bioreactor</td>
		</tr>
		<tr>
			<td>PureLink Genomic DNA Mini Kit</td>
			<td>Thermo Fisher Scientific</td>
			<td>K182001</td>
			<td>Genomic DNA estraction</td>
		</tr>
		<tr>
			<td>TaqMan Gene Expression Cells-to-CT Kit</td>
			<td>Thermo Fisher Scientific</td>
			<td>AM1728</td>
			<td>Quantitative PCR</td>
		</tr>
		<tr>
			<td>Thymidine</td>
			<td>Sigma-Aldrich</td>
			<td>T1895</td>
			<td>Component of nucleoside mix for ESC medium</td>
		</tr>
		<tr>
			<td>Tissue Culture Dish 100X20 mm, Standard</td>
			<td>Sarstedt</td>
			<td>833902</td>
			<td>For fibroblast isolation</td>
		</tr>
		<tr>
			<td>Tissue Culture Dish 35X10 mm, Standard</td>
			<td>Sarstedt</td>
			<td>833900</td>
			<td>For Fibroblast isolation</td>
		</tr>
		<tr>
			<td>Tissue Culture Dish 35X10 mm, Suspension</td>
			<td>Sarstedt</td>
			<td>833900500</td>
			<td>Bacteriology petri dish for liquid marble culture</td>
		</tr>
		<tr>
			<td>Tissue Culture Plate 96 Well,Standard,F</td>
			<td>Sarstedt</td>
			<td>833924005</td>
			<td>For liquid marble culture</td>
		</tr>
		<tr>
			<td>Trypsin-EDTA solution</td>
			<td>Sigma-Aldrich</td>
			<td>T3924</td>
			<td>For fibroblast dissociation</td>
		</tr>
		<tr>
			<td>Tube 15ml, 120x17mm, PS</td>
			<td>Sarstedt</td>
			<td>62553041</td>
			<td>For cell suspension centrifugation</td>
		</tr>
		<tr>
			<td>Uridine</td>
			<td>Sigma-Aldrich</td>
			<td>U3003</td>
			<td>Component of nucleoside mix for ESC medium</td>
		</tr>
	</tbody>
</table>

a two-step strategy that combines epigenetic modification and biomechanical cues to generate mammalian pluripotent cells

Cellefænotype kan vendes eller modificeres med forskellige metoder med fordele og begrænsninger, der er specifikke for hver teknik. Her beskriver vi en ny strategi, der kombinerer brugen af kemisk epigenetisk sletning med mekanosensing-relaterede signaler for at generere pattedyrs pluripotente celler. To hovedtrin er påkrævet. I det første trin udsættes voksne modne (terminalt differentierede) celler for det epigenetiske viskelæder 5-aza-cytidin for at drive dem i en pluripotent tilstand. Denne del af protokollen blev udviklet, baseret på den stigende forståelse af de epigenetiske mekanismer, der styrer celleskæbne og differentiering, og involverer brugen af den epigenetiske modifikator til at slette celledifferentieret tilstand og derefter køre ind i et forbigående vindue med høj plasticitet.
I det andet trin indkapsles slettede celler i polytetrafluorethylen (PTFE) mikrobioreaktorer, også kendt som flydende marmor, for at fremme 3D-celleomlejring for at udvide og stabilt opretholde den erhvervede høje plasticitet. PTFE er en ikke-reaktiv hydrofob syntetisk forbindelse, og dens anvendelse tillader oprettelse af et cellulært mikromiljø, som ikke kan opnås i traditionelle 2D-kultursystemer. Dette system tilskynder til og øger opretholdelsen af pluripotens gennem biomekanosensing-relaterede signaler.
De tekniske procedurer, der er beskrevet her, er enkle strategier til at muliggøre induktion og vedligeholdelse af en høj plasticitetstilstand i voksne somatiske celler. Protokollen tillod afledning af celler med høj plasticitet i alle testede pattedyrarter. Da det ikke involverer anvendelse af gentransfektion og er fri for virale vektorer, kan det repræsentere et bemærkelsesværdigt teknologisk fremskridt for translationelle medicinske applikationer. Desuden giver mikrobioreaktorsystemet et bemærkelsesværdigt fremskridt inden for stamcelleorganoidteknologi ved in vitro-genskabelse af et specifikt mikromiljø, der muliggør langsigtet dyrkning af celler med høj plasticitet, nemlig som ESC'er, iPSC'er, epigenetisk slettede celler og MSC'er.

Den nuværende protokol beskriver alle de trin, der skal udføres for at generere og stabilt opretholde pattedyrs pluripotente celler fra voksne somatiske celler. Denne metode har været vellykket med fibroblaster isoleret fra forskellige pattedyrarter, nemlig mus, svin og mennesker. De repræsentative resultater, der her rapporteres, opnås fra alle cellelinjer, uanset oprindelsesarten.
Morfologiske analyser viser, at fibroblaster indkapslet i PTFE-mikroreaktorer (3D Post 5-aza-CR) efter 18 t...

Watch this Scientific Journal Video about A Two-Step Strategy that Combines Epigenetic Modification and Biomechanical Cues to Generate Mammalian Pluripotent Cells at JoVE.com

A Two-Step Strategy that Combines Epigenetic Modification and Biomechanical Cues to Generate Mammalian Pluripotent Cells

Vi præsenterer her en metode, der kombinerer brugen af kemisk epigenetisk sletning med mekanosensing-relaterede signaler til effektivt at generere pattedyrs pluripotente celler uden behov for gentransfektion eller retrovirale vektorer. Denne strategi er derfor lovende for translationel medicin og repræsenterer et bemærkelsesværdigt fremskridt inden for stamcelleorganoidteknologi.

En to-trins strategi, der kombinerer epigenetisk modifikation og biomekaniske signaler til at generere pattedyrs pluripotente celler

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