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 fibroblast isolation
NOTE: All the procedures described below can be applied to fibroblasts isolated from different mammalian species, including mouse, porcine, and human. Murine cells were isolated from 7-week-old male mice and porcine skin tissue were collected at local slaughterhouse. Human cells were isolated from adult patients, after written informed consent.
<ol>
	<li>Prepare 0.1% porcine gelatin solution.
	<ol>
		<li>Weigh 0.1 g of porcine gelatin and dissolve it in 100 mL of water. Sterilize gelatin solution by autoclaving before use.</li>
		<li>Coat 35 mm Petri dish with 0.1% porcine gelatin by adding 1.5 mL of the prepared solution. Incubate for 2 h at room temperature.</li>
	</ol>
	</li>
	<li>Cut mammalian (mouse, porcine, and human) skin biopsies of approximately 2-5 cm in length and place them in Dulbecco&#39;s Phosphate Buffered Saline (PBS) containing 2% antibiotic antimycotic solution. Store at + 4 &#176;C until use. 
	NOTE: Biopsy collections must be carried out in agreement and after the Ethical Committee&#8217;s approval, in accordance with the established guidelines.</li>
	<li>Extensively wash the collected biopsies three times&#160;in fresh sterile PBS containing 2% antibiotic antimycotic solution.</li>
	<li>Collect biopsies from the last wash and place them into a sterile 100 mm Petri dish. Use a sterile scalpel to cut them into pieces of approximately 2 mm3 size.</li>
	<li>At the end of 2 h incubation, remove the excess of gelatin solution from the 35 mm Petri dish (described in step 1.1.2) and, using a sterile surgical tweezer, immediately place 5-6 skin fragments into each pre-coated culture dish.</li>
	<li>Wet the fragments by adding 100 &#181;L droplets of fibroblast isolation medium (Table 1) over each of them. Culture at 37 &#176;C in 5% CO2 incubator. 
	NOTE: To prevent the medium from evaporation, place the 35 mm Petri dish within a 100 mm or bigger Petri dish containing sterile water. Ensure to cap both Petri dishes.</li>
	<li>After 24 h of culture, check the quantity of the medium in the 35 mm culture Petri dish. If needed, add 500 &#181;L of fibroblast isolation medium to keep wet the fragments.</li>
	<li>Carefully remove the medium and refresh it at least every 2 days of culture using a pipette.</li>
	<li>When fibroblasts start to grow out of the skin fragments placed in the 35 mm Petri dish (described in step 1.5.) and begin to form a cell monolayer (usually 6 days). Remove skin pieces using a sterile surgical tweezer and culture in 2 mL of fibroblast isolation medium.</li>
	<li>Continue to culture the cell monolayer at 37 &#176;C in 5% CO2 incubator until 80% confluence and refresh medium every other day.</li>
</ol>
2. Fibroblast primary cell line culture
<ol>
	<li>When fibroblasts reach 80% confluence, carefully remove fibroblast isolation medium and wash cells three times with 3 mL of PBS containing 1% antibiotic antimycotic solution.</li>
	<li>For cell detaching, add 600 &#181;L of 0.25% trypsin-EDTA solution in the culture dish and incubate at 37 &#176;C for 3-5 min.</li>
	<li>Add 5.4 mL of fibroblast culture medium to neutralize trypsin when cells start to detach from the culture dish (Table 1).</li>
	<li>Dislodge cells by repeated and gentle pipetting. Plate cells in new culture dishes (without gelatin), keeping the passage ratio between 1:2 and 1:4 (depending on growth rate). 
	NOTE: Centrifugation is not necessary.</li>
	<li>Maintain cells in culture and change medium every 2 days, until they have reached 80% confluency and passage them. 
	NOTE: Propagate fibroblasts twice a week to maintain vigorous growth.</li>
</ol>
3. Fibroblast exposure to 5-aza-CR
<ol>
	<li>Prepare fresh 1 mM 5-aza-CR stock solution.
	<ol>
		<li>Weigh 2.44 mg of 5-aza-CR and dissolve it in 10 mL of DMEM high glucose. Resuspend the powder by vortexing. Sterilize the solution with 0.22 &#181;m filter. 
		NOTE: 5-aza-CR stock solution must be prepared immediately before use.</li>
		<li>Prepare 5-aza-CR working solution by diluting 1 &#181;L of 5-aza-CR stock solution (3.1.1.) in 1 mL of fibroblast culture medium. 
		NOTE: The concentration of 5-aza-CR working solution is 1 &#956;M1,2,3,8,9.</li>
	</ol>
	</li>
	<li>Trypsinize cells as previously described (2.1.-2.3.) and dislodge cells by repeatedly and gently pipetting.</li>
	<li>Collect the cell suspension and transfer it into a conical tube.</li>
	<li>Count cells using a counting chamber under an optical microscope at room temperature. Calculate the volume of medium needed to re-suspend cells to obtain 4 x 104 cells in 30 &#956;L of fibroblast culture medium supplemented with 1 &#956;M 5-aza-CR (see step 3.1.2.). 
	NOTE: The formula to be used depends on the specific type of chamber. 
	<img alt="Equation 1" src="/files/ftp_upload/61655/61655equ01v3.jpg" /></li>
	<li>Centrifuge the cell suspension at 150 x g for 5 min at room temperature. Remove the supernatant and resuspend pellet with the fibroblast culture medium supplemented with 1 &#956;M 5-aza-CR (see step 3.1.2.). For the volume of the fibroblast culture medium to be used see step 3.4. 
	NOTE: As a negative control, resuspend cells ad the same concentration in fibroblast culture medium without 5-aza-CR and proceed with cell encapsulation in PTFE powder (step 4.1.-4.13.).</li>
</ol>
4. Fibroblast encapsulation in PTFE micro-bioreactors
<ol>
	<li>Fill a 35 mm Petri dish with polytetrafluoroethylene (PTFE) powder to produce a bed (Figure 1A). 
	NOTE: Use 35 mm bacteriology Petri dishes to avoid liquid marble adhesion. In order to obtain a thin hydrophobic and porous shell, use a PTFE powder with an average particle size of 1&#8201;&#956;m and produced with a maximum grind of 2.0 NPIRI. This allows for the creation of gas-permeable liquid marbles. Furthermore, the translucent coating facilitates the observation of cell aggregation processes in real-time Larger particle size leads to high polydispersity that can cause elevated evaporation, deformity and loss of the spherical shape, and the premature dissolution of the micro-bioreactors.</li>
	<li>Dispense 30 &#956;L single droplet containing 4 x 104 cells (see steps 3.4.- 3.5.) onto the powder bed (Figure 1B).</li>
	<li>Gently rotate the 35 mm Petri dish in a circular motion to ensure that PFTE powder entirely cover the surface of the liquid drop to form a liquid marble micro-bioreactor (Figure 1C).</li>
	<li>Pick up the liquid marble micro-bioreactor using a 1,000&#8201;&#956;L pipette tip, cut at the edge, to accommodate the diameter of the marble (Figure 1D,E). Plate the liquid marble micro-bioreactor onto a clean bacteriology Petri dish to stabilize it (Figure 1F). 
	NOTE: To create a friction to grip the marble inside the tip, cut the pipette tips with a diameter approximately slightly less than the liquid marble diameter.</li>
	<li>Transfer the liquid marble micro-bioreactor from the Petri dish into a 96 well plate (one marble/well) (Figure 1G).</li>
	<li>Slowly add 100&#8201;&#956;L of media from the margin of the well. The micro-bioreactor starts to float on top of the media (Figure 1H). 
	NOTE: The micro-bioreactor breaks in direct liquid contact, due to the disruption of PTFE hydrophobicity. As an alternative approach, the liquid marble micro-bioreactors can be individually placed in a 35 mm bacteriology culture dish. In this case, in order to prevent liquid marble evaporation, the 35 mm Petri dish containing the micro-bioreactor must be inserted in a 100 mm Petri dish, previously aliquoted with sterile water</li>
	<li>Incubate liquid marble micro-bioreactor for 18 h at 37 &#176;C in 5% CO2 incubator1,2,3,8,9. 
	NOTE: The PTFE particle size of 1 &#956;m can ensure an optimal gas exchange between the interior liquid and the surrounding environment.</li>
	<li>After 5-aza-CR incubation for 18 h, collect the liquid marble micro-bioreactor using a 1,000&#8201;&#956;L pipette tip cut at the edge (see step 4.5).</li>
	<li>Place the micro-bioreactor in a new 35 mm bacteriology Petri dish (Figure 1D-F).</li>
	<li>Use a needle to puncture the liquid marble and break it.</li>
	<li>Recover formed spheroids with a 200&#8201;&#956;L pipette tip, cut at the edge, under a stereomicroscope (Figure 1I,J). 
	NOTE: Epigenetically erased cells encapsulated in PTFE form a 3D spherical structure (one aggregate in each liquid marble).</li>
	<li>To assess the acquisition of pluripotent state in response to 5-aza-CR, check the onset of the pluripotency- related gene expression, OCT4, NANOG, REX1, and SOX2, by qualitative PCR (Table 2).</li>
	<li>Proceed with the second step of the protocol as described below.</li>
</ol>
5. Culture in PTFE micro-bioreactors of epigenetically erased cells
<ol>
	<li>Prepare fresh ESC culture medium (Table 1).</li>
	<li>Transfer organoids in a Petri dish containing ESC medium for washing 5-aza-CR residuals (see steps 5.1.-5.2.).</li>
	<li>Prepare a new 35 mm bacteriology Petri dish containing PTFE powder bed (see also step 4.1.).</li>
	<li>Dispense a single organoid in a droplet of 30 &#956;L ESC culture medium onto the powder bed using a 200&#8201;&#956;L pipette tip, cut at the edge (see steps 4.9.; 5.3.).</li>
	<li>Gently rotate the 35 mm Petri dish in a circular motion to form a new liquid marble micro-bioreactor, pick up it using a 1,000&#8201;&#956;L pipette tip, cut at the edge, and place the newly formed micro-bioreactor into a well of 96-well plate (one marble/well) (see steps 4.3.-4.6.).</li>
	<li>To float the micro-bioreactors, add 100&#8201;&#956;L of media from the margin of the well to slowly bathe the marble (see note 4.7.).</li>
	<li>Culture liquid marble micro-bioreactors at 37 &#176;C in 5% CO2 incubator for as long as required. Change medium every other day, following the procedure described in 5.3.-5.7. 
	NOTE: In the present manuscript, results obtained with organoids culture for 28 days are provided. However, if needed longer culture period can be performed.</li>
</ol>

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The authors have nothing to disclose.

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 ...

a-two-step-strategy-that-combines-epigenetic-modification

Research

JoVE Journal

Biology

2.2K Views.  Università degli Studi di Milano. 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.

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

Trypsinizing and Subculturing Mammalian Cells

As cells reach confluency, they must be subcultured or passaged. Failure to subculture confluent cells results in reduced mitotic index and eventually in cell death. The first step in subculturing is to detach cells from the surface of the primary culture vessel by trypsinization or mechanical means. The resultant cell suspension is then subdivided, or reseeded, into fresh cultures. Secondary cultures are checked for growth and fed periodically, and may be subsequently subcultured to produce tertiary cultures. The time between passaging of cells varies with the cell line and depends on the growth rate.

As cells reach confluency, they must be subcultured or passaged. This video will demonstrate a procedure for subculturing both adherent and suspension cells.

As cells reach confluency, they must be subcultured or passaged. Failure to subculture confluent cells results in reduced mitotic index and eventually in ...

Chromatin Immunoprecipitation (ChIP) to Assay Dynamic Histone Modification in Activated Gene Expression in Human Cells

In response to a variety of extracellular ligands, the STAT (signal transducer and activator of transcription) transcription factors are rapidly recruited from their latent state in the cytoplasm to cell surface receptors where they are activated by phosphorylation at a single tyrosine residue1. They then dimerize and translocate to the nucleus to drive the transcription of target genes, affecting growth, differentiation, homeostasis and the immune response. Not surprisingly, given their widespread involvement in normal cell processes, dysregulation of STAT function contributes to human disease, particularly to cancers2 and autoimmune diseases3. It is well established that transcription is regulated by alterations to the chromatin template4,5. These alterations include the activities of ATP-dependent complexes, as well as covalent histone modifications and DNA methylation6. Because STAT activation of gene expression is both rapid and transient, it requires specific mechanisms for modulating the chromatin template at STAT-dependent gene loci. To define these mechanisms, we characterize the histone modifications and the enzymatic activities that generate them at gene loci that respond to STAT signaling. This protocol describes chromatin immunoprecipitation, a method that is valuable for the study of STAT signaling to chromatin in activated gene expression.

Chromatin Immunoprecipitation ChIP to Assay Dynamic Histone Modification in Activated Gene Expression in Human Cells

This protocol describes how chromatin immunoprecipitation (ChIP) is used to study the dynamic alterations to the chromatin template that regulate transcription induced by a signal transduction pathway.

In response to a variety of extracellular ligands, the STAT (signal transducer and activator of transcription) transcription factors are rapidly recruited ...

Mutagenesis and Functional Analysis of Ion Channels Heterologously Expressed in Mammalian Cells

We will demonstrate how to study the functional effects of introducing a point mutation in an ion channel. We study G protein-gated inwardly rectifying potassium (referred to as GIRK) channels, which are important for regulating the excitability of neurons. There are four different mammalian GIRK channel subunits (GIRK1-GIRK4) - we focus on GIRK2 because it forms a homotetramer. Stimulation of different types of G protein-coupled receptors (GPCRs), such as the muscarinic receptor (M2R), leads to activation of GIRK channels. Alcohol also directly activates GIRK channels. We will show how to mutate one amino acid by specifically changing one or more nucleotides in the cDNA for the GIRK channel. This mutated cDNA sequence will be amplified in bacteria, purified, and the presence of the point mutation will be confirmed by DNA sequencing. The cDNAs for the mutated and wild-type GIRK channels will be transfected into human embryonic kidney HEK293T cells cultured in vitro. Lastly, whole-cell patch-clamp electrophysiology will be used to study the macroscopic potassium currents through the ectopically expressed wild-type or mutated GIRK channels. In this experiment, we will examine the effect of a L257W mutation in GIRK2 channels on M2R-dependent and alcohol-dependent activation.

We will demonstrate how to study the effect of a single point mutation on the function of an ion channel.

We will demonstrate how to study the functional effects of introducing a point mutation in an ion channel. We study G protein-gated inwardly rectifying ...

Analysis of Cell Cycle Position in Mammalian Cells

The regulation of cell proliferation is central to tissue morphogenesis during the development of multicellular organisms. Furthermore, loss of control of cell proliferation underlies the pathology of diseases like cancer. As such there is great need to be able to investigate cell proliferation and quantitate the proportion of cells in each phase of the cell cycle. It is also of vital importance to indistinguishably identify cells that are replicating their DNA within a larger population. Since a cell&prime;s decision to proliferate is made in the G1 phase immediately before initiating DNA synthesis and progressing through the rest of the cell cycle, detection of DNA synthesis at this stage allows for an unambiguous determination of the status of growth regulation in cell culture experiments.
DNA content in cells can be readily quantitated by flow cytometry of cells stained with propidium iodide, a fluorescent DNA intercalating dye. Similarly, active DNA synthesis can be quantitated by culturing cells in the presence of radioactive thymidine, harvesting the cells, and measuring the incorporation of radioactivity into an acid insoluble fraction. We have considerable expertise with cell cycle analysis and recommend a different approach. We Investigate cell proliferation using bromodeoxyuridine/fluorodeoxyuridine (abbreviated simply as BrdU) staining that detects the incorporation of these thymine analogs into recently synthesized DNA. Labeling and staining cells with BrdU, combined with total DNA staining by propidium iodide and analysis by flow cytometry1 offers the most accurate measure of cells in the various stages of the cell cycle. It is our preferred method because it combines the detection of active DNA synthesis, through antibody based staining of BrdU, with total DNA content from propidium iodide. This allows for the clear separation of cells in G1 from early S phase, or late S phase from G2/M. Furthermore, this approach can be utilized to investigate the effects of many different cell stimuli and pharmacologic agents on the regulation of progression through these different cell cycle phases.
In this report we describe methods for labeling and staining cultured cells, as well as their analysis by flow cytometry. We also include experimental examples of how this method can be used to measure the effects of growth inhibiting signals from cytokines such as TGF-&beta;1, and proliferative inhibitors such as the cyclin dependent kinase inhibitor, p27KIP1. We also include an alternate protocol that allows for the analysis of cell cycle position in a sub-population of cells within a larger culture5. In this case, we demonstrate how to detect a cell cycle arrest in cells transfected with the retinoblastoma gene even when greatly outnumbered by untransfected cells in the same culture. These examples illustrate the many ways that DNA staining and flow cytometry can be utilized and adapted to investigate fundamental questions of mammalian cell cycle control.

Determining the cell cycle position of a population of cells, or understanding how signals affect proliferation, can be readily measured by flow cytometry using this protocol. We report a simple experimental approach to staining cells and quantifying their position in the cell cycle.

The regulation of cell proliferation is central to tissue morphogenesis during the development of multicellular organisms. Furthermore, loss of control of ...

Visualization of Endoplasmic Reticulum Localized mRNAs in Mammalian Cells

In eukaryotes, most of the messenger RNAs (mRNAs) that encode secreted and membrane proteins are localized to the surface of the endoplasmic reticulum (ER). However, the visualization of these mRNAs can be challenging. This is especially true when only a fraction of the mRNA is ER-associated and their distribution to this organelle is obstructed by non-targeted (i.e. "free") transcripts. In order to monitor ER-associated mRNAs, we have developed a method in which cells are treated with a short exposure to a digitonin extraction solution that selectively permeabilizes the plasma membrane, and thus removes the cytoplasmic contents, while simultaneously maintaining the integrity of the ER. When this method is coupled with fluorescent in situ hybridization (FISH), one can clearly visualize ER-bound mRNAs by fluorescent microscopy. Using this protocol the degree of ER-association for either bulk poly(A) transcripts or specific mRNAs can be assessed and even quantified. In the process, one can use this assay to investigate the nature of mRNA-ER interactions.

Here we describe a method to visualize endoplasmic reticulum-associated mRNAs in mammalian tissue culture cells. This technique involves the selective permeabilization of the plasma membrane with digitonin to remove cytoplasmic contents followed by fluorescent in situ hybridization to detect either bulk poly(A) mRNA or specific transcripts.

In eukaryotes, most of the messenger RNAs (mRNAs) that encode secreted and membrane proteins are localized to the surface of the endoplasmic reticulum ...

GST-His purification: A Two-step Affinity Purification Protocol Yielding Full-length Purified Proteins

Key assays in enzymology for the biochemical characterization of proteins in vitro necessitate high concentrations of the purified protein of interest. Protein purification protocols should combine efficiency, simplicity and cost effectiveness1. Here, we describe the GST-His method as a new small-scale affinity purification system for recombinant proteins, based on a N-terminal Glutathione Sepharose Tag (GST)2,3 and a C-terminal 10xHis tag4, which are both fused to the protein of interest. The latter construct is used to generate baculoviruses, for infection of Sf9 infected cells for protein expression5. GST is a rather long tag (29 kDa) which serves to ensure purification efficiency. However, it might influence physiological properties of the protein. Hence, it is subsequently cleaved off the protein using the PreScission enzyme6. In order to ensure maximum purity and to remove the cleaved GST, we added a second affinity purification step based on the comparatively small His-Tag. Importantly, our technique is based on two different tags flanking the two ends of the protein, which is an efficient tool to remove degraded proteins and, therefore, enriches full-length proteins. The method presented here does not require an expensive instrumental setup, such as FPLC. Additionally, we incorporated MgCl2 and ATP washes to remove heat shock protein impurities and nuclease treatment to abolish contaminating nucleic acids. In summary, the combination of two different tags flanking the N- and the C-terminal and the capability to cleave off one of the tags, guaranties the recovery of a highly purified and full-length protein of interest.

In the present protocol, we demonstrate a highly efficient and cost-effective small-scale protein purification method, which allows purification of recombinant proteins by uniquely combining a cleavable GST-tag and a small His-tag.

Key assays in enzymology for the biochemical characterization of proteins in vitro necessitate high concentrations of the purified protein of interest. ...

Isolation of Human Hepatocytes by a Two-step Collagenase Perfusion Procedure

The liver, an organ with an exceptional regeneration capacity, carries out a wide range of functions, such as detoxification, metabolism and homeostasis. As such, hepatocytes are an important model for a large variety of research questions. In particular, the use of human hepatocytes is especially important in the fields of pharmacokinetics, toxicology, liver regeneration and translational research. Thus, this method presents a modified version of a two-step collagenase perfusion procedure to isolate hepatocytes as described by Seglen 1.
Previously, hepatocytes have been isolated by mechanical methods. However, enzymatic methods have been shown to be superior as hepatocytes retain their structural integrity and function after isolation. This method presented here adapts the method designed previously for rat livers to human liver pieces and results in a large yield of hepatocytes with a viability of 77&plusmn;10%. The main difference in this procedure is the process of cannulization of the blood vessels. Further, the method described here can also be applied to livers from other species with comparable liver or blood vessel sizes.

A modified two-step collagenase perfusion procedure for isolation of human hepatocytes is described. This method can also be applied to other mammalian livers. The isolated hepatocytes are available in high yield and viability, making them a suitable model for scientific research in areas such as liver regeneration, pharmacokinetics and toxicology.

The liver, an organ with an exceptional regeneration capacity, carries out a wide range of functions, such as detoxification, metabolism and homeostasis. ...

Reverse Yeast Two-hybrid System to Identify Mammalian Nuclear Receptor Residues that Interact with Ligands and/or Antagonists

As a critical regulator of drug metabolism and inflammation, Pregnane X Receptor (PXR), plays an important role in disease pathophysiology linking metabolism and inflammation (e.g.&#160;hepatic steatosis)1,2. There has been much progress in the identification of agonist ligands for PXR, however, there are limited descriptions of drug-like antagonists and their binding sites on PXR3,4,5. A critical barrier has been the inability to efficiently purify full-length protein for structural studies with antagonists despite the fact that PXR was cloned and characterized in 1998. Our laboratory developed a novel high throughput yeast based two-hybrid assay to define an antagonist, ketoconazole&#39;s, binding residues on PXR6. Our method involves creating mutational libraries that would rescue the effect of single mutations on the AF-2 surface of PXR expected to interact with ketoconazole. Rescue or &#34;gain-of-function&#34; second mutations can be made such that conclusions regarding the genetic interaction of ketoconazole and the surface residue(s) on PXR are feasible. Thus, we developed a high throughput two-hybrid yeast screen of PXR mutants interacting with its coactivator, SRC-1. Using this approach, in which the yeast was modified to accommodate the study of the antifungal drug, ketoconazole, we could demonstrate specific mutations on PXR enriched in clones unable to bind to ketoconazole. By reverse logic, we conclude that the original residues are direct interaction residues with ketoconazole. This assay represents a novel, tractable genetic assay to screen for antagonist binding sites on nuclear receptor surfaces. This assay could be applied to any drug regardless of its cytotoxic potential to yeast as well as to cellular protein(s) that cannot be studied using standard structural biology or proteomic based methods. Potential pitfalls include interpretation of data (complementary methods useful), reliance on single Y2H method, expertise in handling yeast or performing yeast two-hybrid assays, and assay optimization.

Ketoconazole binds to and antagonizes Pregnane X Receptor (PXR) activation. Yeast high throughput screens of PXR mutants define a unique region for ketoconazole binding. This yeast-based genetic method discovers novel nuclear receptor interactions with ligands that associate with surface binding sites.

As a critical regulator of drug metabolism and inflammation, Pregnane X Receptor (PXR), plays an important role in disease pathophysiology linking ...

Automating ChIP-seq Experiments to Generate Epigenetic Profiles on 10,000 HeLa Cells

Chromatin immunoprecipitation followed by next generation sequencing (ChIP-seq) is a technique of choice for studying protein-DNA interactions. ChIP-seq has been used for mapping protein-DNA interactions and allocating histones modifications. The procedure is tedious and time consuming, and one of the major limitations is the requirement for high amounts of starting material, usually millions of cells. Automation of chromatin immunoprecipitation assays is possible when the procedure is based on the use of magnetic beads. Successful automated protocols of chromatin immunoprecipitation and library preparation have been specifically designed on a commercially available robotic liquid handling system dedicated mainly to automate epigenetic assays. First, validation of automated ChIP-seq assays using antibodies directed against various histone modifications was shown, followed by optimization of the automated protocols to perform chromatin immunoprecipitation and library preparation starting with low cell numbers. The goal of these experiments is to provide a valuable tool for future epigenetic analysis of specific cell types, sub-populations, and biopsy samples.

Methods for mapping in vivo protein-DNA interactions are becoming crucial for every aspect of genomic research but they are laborious, costly, and time consuming. Here a commercially available robotic liquid handling system that automates chromatin immunoprecipitation for mapping in vivo protein-DNA interactions with limited amounts of cells is presented.

Chromatin immunoprecipitation followed by next generation sequencing (ChIP-seq) is a technique of choice for studying protein-DNA interactions. ChIP-seq ...

Nephrotoxin Microinjection in Zebrafish to Model Acute Kidney Injury

The kidneys are susceptible to harm from exposure to chemicals they filter from the bloodstream. This can lead to organ injury associated with a rapid decline in renal function and development of the clinical syndrome known as acute kidney injury (AKI). Pharmacological agents used to treat medical circumstances ranging from bacterial infection to cancer, when administered individually or in combination with other drugs, can initiate AKI. Zebrafish are a useful animal model to study the chemical effects on renal function in vivo, as they form an embryonic kidney comprised of nephron functional units that are conserved with higher vertebrates, including humans. Further, zebrafish can be utilized to perform genetic and chemical screens, which provide opportunities to elucidate the cellular and molecular facets of AKI and develop therapeutic strategies such as the identification of nephroprotective molecules. Here, we demonstrate how microinjection into the zebrafish embryo can be utilized as a paradigm for nephrotoxin studies.

Renal injuries incurred from nephrotoxins, which include drugs ranging from antibiotics to chemotherapeutics, can result in complex disorders whose pathogenesis remains incompletely understood. This protocol demonstrates how zebrafish can be used for disease modeling of these conditions, which can be applied to the identification of renoprotective measures.

The kidneys are susceptible to harm from exposure to chemicals they filter from the bloodstream. This can lead to organ injury associated with a rapid ...