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

This work was funded by Carraresi Foundation and MiND FoodS Hub ID: 1176436. All the authors are members&#160;of the COST Action CA16119 In vitro 3-D total cell guidance and fitness (CellFit).

All studies were reviewed and approved by the Ethical Committee of the University of Milan. All animal experiments were performed in accordance with the Guide for the Care and Use of Laboratory Animals, published by the US National Institutes of Health (NIH). Human cells isolation from healthy adult individuals was approved by the Ethical Committee of the Ospedale Maggiore Policlinico, Milano. All the methods in our study were carried out in accordance with the approved guidelines.
1. Skin fibro...

<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. 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During the last decades, several studies focused on the development of strategies to revert a terminally differentiated cell towards a less committed and higher permissive state. The protocol here described allow the generation and long-term maintenance of pluripotent cells starting from adult mature terminally differentiated cells. The method combines two independent steps that involve the induction of a high permissive state which is achieved through chemical epigenetic erasing and its subsequent maintenance ensured us...

During the last decades, the widely accepted concept of unidirectional progression towards cell commitment and differentiation was completely revised. It has been demonstrated that cell specification can be reversed, and a terminally differentiated cell can be pushed towards a less committed and higher permissive state, using different methods.
Among the several methods proposed, one of the most promising method involves the use of chemical compounds to induce cells into a so called chemically induced pluripotency. The small molecules used in this approach are able to interact and modify the epigenetic signature of an adult mature cell, avoiding the need of any transgenic and/or viral vector1,2,3,4,5,6,7,8,9,10. Numerous studies have recently shown that it is possible to switch cells from one phenotype to another by providing specific biochemical and biological stimuli that induce the reactivation of hypermethylated genes11,12,13,14,15. These demethylating events allow for the conversion of terminally differentiated cells into a primitive progenitor, a multipotent or a high plasticity/pluripotent cell1,2,3,4,5,6,7,8,9,10.
In parallel, many studies have been recently focussing on the understanding of mechanosensing-related cues and, more specifically, on the possibility to use mechanical forces to directly influence cell plasticity and/or differentiation16,17,18,19. Indeed, it has been clearly demonstrated that the extracellular matrix (ECM) plays a key role in the control of cell fate. In particular, the biomechanical and biophysical signals produced by ECM directly regulate molecular mechanisms and signaling pathways, influencing cell behavior and functions20,21. These recent data have paved the way to the development of novel 3D culture systems that more closely mimic the in vivo cell microenvironment, replicating mechanical and physical stimuli driving cell behaviour.
We here describe a two-step protocol that combines the use of chemical epigenetic erasing with mechanosensing-related cues, to generate mammalian pluripotent cells. In the first step, cells are incubated with the demethylating molecule 5-aza-cytidine (5-aza-CR). This agent is able to induce a significant global DNA demethylation through a combined effect of the direct ten-eleven translocation 2 (TET2)-mediated action8,10 and the indirect inhibition of the DNA methyltransferases (DNMT)22,23. This step induces the removal of the epigenetic blocks with a subsequent re-activation of pluripotency-related gene expression and, therefore, the generation of high plasticity cells1,2,3,8,10, hereinafter referred as &#8220;epigenetically erased cells&#8221;. In the second step, cells are encapsulated in a 3D culture system. To this end, the non-reactive hydrophobic synthetic compound polytetrafluoroethylene (PTFE; with particle size of 1 &#956;m) is used as micro-bioreactor, that permits the creation of a cellular microenvironment unachievable through the use of traditional 2D culture systems10. The PTFE powder particles adhere to the surface of the liquid drop in which cells are re-suspended and isolate the liquid core from the supporting surface, while allowing gas exchange between the interior liquid and the surrounding environment24. The &#8220;PTFE micro-bioreactor&#8221; thus obtained, also known as &#8220;Liquid Marble&#8221;, encourages cells to freely interact with each other, promoting 3D cell rearrangement25,26,27, and extends and stably maintains the acquired high plasticity state though bio-mechanosensing-related cues10.

<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

Cell phenotype can be reversed or modified with different methods, with advantages and limitations that are specific for each technique. Here we describe a new strategy that combines the use of chemical epigenetic erasing with mechanosensing-related cues, to generate mammalian pluripotent cells. Two main steps are required. In the first step, adult mature (terminally differentiated) cells are exposed to the epigenetic eraser 5-aza-cytidine to drive them into a pluripotent state. This part of the protocol was developed, based on the increasing understanding of the epigenetic mechanisms controlling cell fate and differentiation, and involves the use of the epigenetic modifier to erase cell differentiated state and then drive into a transient high plasticity window.
In the second step, erased cells are encapsulated in polytetrafluoroethylene (PTFE) micro-bioreactors, also known as Liquid Marbles, to promote 3D cell rearrangement to extend and stably maintain the acquired high plasticity. PTFE is a non-reactive hydrophobic synthetic compound and its use permits the creation of a cellular microenvironment, which cannot be achieved in traditional 2D culture systems. This system encourages and boosts the maintenance of pluripotency though bio-mechanosensing-related cues.
The technical procedures described here are simple strategies to allow for the induction and maintenance of a high plasticity state in adult somatic cells. The protocol allowed the derivation of high plasticity cells in all mammalian species tested. Since it does not involve the use of gene transfection and is free of viral vectors, it may represent a notable technological advance for translational medicine applications. Furthermore, the micro-bioreactor system provides a notable advancement in stem cell organoid technology by in vitro re-creating a specific micro-environment that allows for the long-term culture of high plasticity cells, namely as ESCs, iPSCs, epigenetically erased cells and MSCs.

The present protocol describes all the steps to be performed to generate and stably maintain mammalian pluripotent cells from adult somatic cells. This method has been successful with fibroblasts isolated from different mammalian species, namely mouse, porcine and human. The representative results here reported are obtained from all cell lines, irrespectively of the species of origin.
Morphological analyses show that, after 18 h incubation with the demethylating agent 5-aza-CR, fibroblasts enc...

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

We here present a method that combines the use of chemical epigenetic erasing with mechanosensing-related cues to efficiently generate mammalian pluripotent cells, without the need of gene transfection or retroviral vectors. This strategy is, therefore, promising for translational medicine and represents a notable advancement in stem cell organoid technology.

Cell phenotype can be reversed or modified with different methods, with advantages and limitations that are specific for each technique. Here we describe ...

The protocol shown in this video for the first time combines the use of chemical epigenetic erasing with biomechanical cues to induce and maintain pluripotency in adult terminally differentiated cells. The main strength of this protocol is that it does not require transgenic or viral vectors. Moreover, it is robust, highly reproducible, and flexible.This technique does not require the use of gene transfection, and therefore represents a notable technological advance for translational medicine and cell therapy, while supporting long-term culture of high plasticity cells. Demonstrating the procedure will be Georgia Pennarossa, an assistant professor, and Teresina De Iorio, a PhD student from my laboratory. Begin by preparing fresh one-millimolar 5-aza-CR stock solution.Weigh 2.44 milligrams of 5-aza-CR and dissolve it in 10 milliliters of DMEM with high glucose. Re-suspend the powder by vortexing and sterilize the solution with a 0.22-micrometer filter. Prepare 5-aza-CR working solution by diluting one microliter of the stock solution in one milliliter of fibroblast culture medium.Trypsinize the cells as described in the text manuscript and dislodge them with gentle pipetting. Then collect the cell suspension and transfer it into a conical tube. Count cells using a counting chamber under an optical microscope.Centrifuge the cell suspension at 150 times G for five minutes. Then, remove the supernatant and resuspend the pellet to obtain 40, 000 cells in 30 microliters of fibroblast culture medium supplemented with one-micromolar 5-aza-CR. Fill a 35-millimeter Petri dish with PTFE powder to produce a bed and dispense the 30 microliters of cells onto the powder bed.Gently rotate the Petri dish in a circular motion to ensure that PTFE powder entirely covers the surface of the liquid drop to form a liquid marble microbioreactor. Pick up the liquid marble microbioreactor using a 1000-microliter pipette tip that has been cut to accommodate the diameter of the marble. Plate the microbioreactor onto a clean bacteriology Petri dish to stabilize it.Then, transfer the microbioreactor from the Petri dish into a 96-well plate. Slowly add 100 microliters of media from the margin of the well. The microbioreactor should float on top of the media.Incubate the liquid marble microbioreactor for 18 hours at 37 degrees Celsius in a 5%carbon dioxide incubator. After the incubation, collect the liquid marble microbioreactor using a 1000-microliter pipette tip and place it in a new 35-millimeter bacteriology Petri dish. Use a needle to puncture the liquid marble and break it.Recover the formed spheroids with a 200-microliter pipette tip that has been cut at the edge under a stereomicroscope. To wash off 5-aza-CR residuals, transfer the organoids into a Petri dish containing ESC medium. Prepare a new 35-millimeter Petri dish containing the PTFE powder bed and dispense a single organoid in a 30-microliter droplet of ESC culture medium onto the powder using a 200-microliter cut pipette tip.Gently rotate the dish in a circular motion to form a new liquid marble microbioreactor and transfer it into a well of a 96-well plate. Add 100 microliters of media from the margin of the well to slowly bathe the marble. Culture the liquid marble microbioreactors at 37 degrees Celsius and 5%carbon dioxide.Morphological analyses show that after an 18-hour incubation with the demethylating agent 5-aza-CR, fibroblasts from three mammalian species encapsulated in PTFE microbioreactors aggregated and formed 3D spherical structures with a uniform size geometry. Under 3D conditions, 86.31%of encapsulated cells remarkably modified their phenotype. In contrast, post-5-aza-CR cells cultured in 2D standard conditions were considerably smaller in size, with granulated nuclei and retained a monolayer distribution.The morphological changes were accompanied by the onset of pluripotency-related gene expression, both in 3D and 2D post-5-aza-CR cells. Transcription was observed for POU class five homeobox one, NANOG homeobox, ZFP42 zinc finger protein, and sex-determining region Ybox2, which are absent in untreated fibroblasts. A significant upregulation of the 10-11 translocation 2 epithelial cell adhesion molecule and cadherin 1 genes was also observed in 3D post-5-aza-CR cells compared to cells cultured in 2D standard plastic dishes.In parallel, downregulation of the fibroblast-specific marker Thy1 cell surface antigen was detected in both 3D and 2D post-5-aza-CR cells. A significant decrease of methylation levels in both 3D and 2D post-5-aza-CR cells was confirmed with ELISA. DNA methylation levels were significantly lower in 3D post-5-aza-CR cells compared to the cells cultured in 2D conditions.Furthermore, 3D post-5-aza-CR cells retained the acquired 3D spherical structure, high expression levels of pluripotency-related genes, and low DNA methylation levels for 28 days, at which point culture was arrested. The microbioreactors can also be used to maintain optimal conditions for progenitor cell cultures, as well as to boost terminal differentiation efficiency.

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