Name: epigenetic conversion as a safe and simple method to obtain insulin-secreting cells from adult skin fibroblasts
Uploaded: 2016-03-18T15:00:00
Description: Watch this Scientific Journal Video about Epigenetic Conversion as a Safe and Simple Method to Obtain Insulin-secreting Cells from Adult Skin Fibroblasts at JoVE.com

This work was funded by Carraresi Foundation and European Foundation for the Study of Diabetes (EFSD). GP is supported by a post-doc fellowship of the University of Milan. The Authors are members of the COST Action FA1201 Epiconcept: Epigenetics and Periconception environment and the COST Action BM1308 Sharing advances on large animal models (SALAAM). TALB is member of the COST Action CM1406 Epigenetic Chemical Biology (EPICHEM).

Note: All the procedures described below must be performed under laminar flow hood in sterile conditions. Make sure that all culture procedures are carried out on thermostatically controlled stages and cells are maintained at 37 &#176;C throughout their handling.
1. Skin Fibroblast Isolation
<ol>
	<li>Prepare Culture Dish Coating Solution
	<ol>
		<li>Dissolve 0.1 g of porcine gelatin in 100 ml of water (final concentration 0.1%). Sterilize solution with autoclave.</li>
		<li>Add 1.5 ml of sterile 0.1% porcine ge...

<ol>
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The present manuscript describes a method that allows the conversion of human skin fibroblasts into insulin-producing cells, through a transient and brief exposure to 5-aza-CR, followed by a tissue specific induction protocol. This approach allows a switch from mesoderm to endoderm related cells, without the forced expression of transcription factors or microRNAs nor the acquisition of a stable pluripotent state, that makes cells more unstable and prone to mistakes 14.
In the first ...

A fundamental objective of regenerative medicine is the generation of new, functional cells that can be used to repair or replace damaged, degenerated tissues. Remaking easily available adult cells into new ones, by converting them from one cell type to another, is a particularly appealing approach, especially when the required cell population is not abundant or difficult to access. However, adult cells are remarkably stable. They acquire their differentiated state through a gradual restriction in their options and, once they reach the mature terminal specialization, they stably retain it 1.
In the last years a number of protocols have been developed, that enable the reprogramming to pluripotency of a somatic cell (iPS) achieved through the forced expression of a set of transcription factors 2,3. Alternatively, cell conversion can be obtained by direct lineage transdifferentiation, introducing a single 4 or a combination of transcription factors 5-7. This strategy does not involve the transition through a de-differentiated state but requires high expression of the specific transcription factors 8.
We have recently developed a conversion protocol based on the brief exposure of adult cells to the demethylating properties of the cytidine analog 5-azacytidine (5-aza-CR), a well-characterized DNA methyltransferase inhibitor. The demethylation step is immediately followed by a specific differentiation protocol 9-11 that allows to obtain the required terminal phenotype. This method is able to convert mature, differentiated cells into cells of a different lineage and has the substantial advantage to avoid both the use of viral vectors and the transfection of any exogenous transcription factors. The acquisition of a stable pluripotent state, and the related increased susceptibility to cell instability is also avoided.
The detailed protocol that allows the conversion of adult human skin fibroblasts into fully functional insulin-secreting cells is presented here. However, it is worth noting that the technique has been applied to different cell types and has generated positive results, when addressing cells towards various differentiation pathways. Furthermore, epigenetic conversion has been successfully used in the human and porcine species 9-13 as well as in the dog (manuscript submitted) suggesting a wide efficacy and robustness of the approach.

<table border="0" cellpadding="0" cellspacing="0" width="1019">
	<tbody>
		<tr height="21">
			<td height="21" style="height:21px;width:339px;">Dulbecco&#39;s Phosphate Buffered Saline</td>
			<td style="width:155px;">Sigma</td>
			<td style="width:119px;">D5652</td>
			<td style="width:408px;">PBS; for cell wash and solution preparation</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:339px;">Antibiotic Antimycotic Solution</td>
			<td style="width:155px;">Sigma</td>
			<td style="width:119px;">A5955</td>
			<td style="width:408px;">Component of Fibroblast, HP and Pancreatic media</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:339px;">100 mm petri dish</td>
			<td style="width:155px;">Sarstedt</td>
			<td style="width:119px;">83.3902</td>
			<td style="width:408px;">For Fibroblast isolation</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:339px;">Porcine Gelatin</td>
			<td style="width:155px;">Sigma</td>
			<td style="width:119px;">G1890</td>
			<td style="width:408px;">For dish coating</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:339px;">Water</td>
			<td style="width:155px;">Sigma</td>
			<td style="width:119px;">W3500</td>
			<td style="width:408px;">For solution preparation</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:339px;">35 mm petri dishes</td>
			<td style="width:155px;">Sarstedt</td>
			<td style="width:119px;">83.39</td>
			<td style="width:408px;">For Fibroblast isolation</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:339px;">DMEM, high glucose, pyruvate</td>
			<td style="width:155px;">Life Technologies</td>
			<td style="width:119px;">41966052</td>
			<td style="width:408px;">For Fibroblast culture medium</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:339px;">Fetal Bovine Serum</td>
			<td style="width:155px;">Life Technologies</td>
			<td style="width:119px;">10500064</td>
			<td style="width:408px;">FBS; Component of Fibroblast and HP media</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:339px;">L-Glutamine solution</td>
			<td style="width:155px;">Sigma</td>
			<td style="width:119px;">G7513</td>
			<td style="width:408px;">Component of Fibroblast, HP and Pancreatic media</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:339px;">Trypsin-EDTA solution</td>
			<td style="width:155px;">Sigma</td>
			<td style="width:119px;">T3924</td>
			<td style="width:408px;">For Fibroblast dissociation</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:339px;">KOVA GLASSTIC SLIDE 10 WITH GRIDS</td>
			<td style="width:155px;">Hycor Biomedical</td>
			<td style="width:119px;">87144</td>
			<td style="width:408px;">Cell counting</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:339px;">5-Azacytidine</td>
			<td style="width:155px;">Sigma</td>
			<td style="width:119px;">A2385</td>
			<td style="width:408px;">5-aza-CR, for increrase cell plasticity in fibroblasts</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:339px;">Ham&#39;s F-10 Nutrient Mix</td>
			<td style="width:155px;">Life Technologies</td>
			<td style="width:119px;">31550031</td>
			<td style="width:408px;">For HP medium</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:339px;">DMEM, low glucose, pyruvate</td>
			<td style="width:155px;">Life Technologies</td>
			<td style="width:119px;">31885023</td>
			<td style="width:408px;">For HP medium</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:339px;">KnockOut Serum Replacement</td>
			<td style="width:155px;">Life Technologies</td>
			<td style="width:119px;">10828028</td>
			<td style="width:408px;">Component of HP medium</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:339px;">MEM Non-Essential Amino Acids Solution</td>
			<td style="width:155px;">Life Technologies</td>
			<td style="width:119px;">11140035</td>
			<td style="width:408px;">Component of HP and Pancreatic Basal media</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:339px;">2-Mercaptoethanol</td>
			<td style="width:155px;">Sigma</td>
			<td style="width:119px;">M7522</td>
			<td style="width:408px;">Component of HP and Pancreatic Basal media</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:339px;">Guanosine</td>
			<td style="width:155px;">Sigma</td>
			<td style="width:119px;">G6264</td>
			<td style="width:408px;">Nucleoside mix stock component of HP medium</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:339px;">Adenosine</td>
			<td style="width:155px;">Sigma</td>
			<td style="width:119px;">A4036</td>
			<td style="width:408px;">Nucleoside mix stock component of HP medium</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:339px;">Cytidine</td>
			<td style="width:155px;">Sigma</td>
			<td style="width:119px;">C4654</td>
			<td style="width:408px;">Nucleoside mix stock component of HP medium</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:339px;">Uridine</td>
			<td style="width:155px;">Sigma</td>
			<td style="width:119px;">U3003</td>
			<td style="width:408px;">Nucleoside mix stock component of HP medium</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:339px;">Thymidine</td>
			<td style="width:155px;">Sigma</td>
			<td style="width:119px;">T1895</td>
			<td style="width:408px;">Nucleoside mix stock component of HP medium</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:339px;">Millex-GS 0,22 &#181;m</td>
			<td style="width:155px;">Millipore</td>
			<td style="width:119px;">SLGS033SB</td>
			<td style="width:408px;">For sterilizing of solution</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:339px;">FGF-Basic (AA 1-155) Recombinant Human Protein</td>
			<td style="width:155px;">Life Technologies</td>
			<td style="width:119px;">PHG0261</td>
			<td style="width:408px;">bFGF; Component of HP and Pancreatic Basal medium</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:339px;">Bovine Serum Albumin</td>
			<td style="width:155px;">Sigma</td>
			<td style="width:119px;">A3311</td>
			<td style="width:408px;">BSA; Component of Pancreatic Basal medium</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:339px;">DMEM/F-12</td>
			<td style="width:155px;">Life Technologies</td>
			<td style="width:119px;">11320074</td>
			<td style="width:408px;">For Pancreatic Basal medium</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:339px;">B-27 Supplement Minus Vitamin A</td>
			<td style="width:155px;">Life Technologies</td>
			<td style="width:119px;">12587010</td>
			<td style="width:408px;">Component of Pancreatic medium</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:339px;">N-2 Supplement</td>
			<td style="width:155px;">Life Technologies</td>
			<td style="width:119px;">17502048</td>
			<td style="width:408px;">Component of Pancreatic Basal medium</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:339px;">Activin A Recombinant Human Protein</td>
			<td style="width:155px;">Life Technologies</td>
			<td style="width:119px;">PHG9014</td>
			<td style="width:408px;">For Pancreatic medium</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:339px;">Retinoic Acid</td>
			<td style="width:155px;">Sigma</td>
			<td style="width:119px;">R2625</td>
			<td style="width:408px;">For Pancreatic medium</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:339px;">Dimethyl sulfoxide</td>
			<td style="width:155px;">Sigma</td>
			<td style="width:119px;">D2650</td>
			<td style="width:408px;">DMSO; for Retinoic Acid stock preparation</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:339px;">Insulin-Transferrin-Selenium</td>
			<td style="width:155px;">Life Technologies</td>
			<td style="width:119px;">41400045</td>
			<td style="width:408px;">ITS; for Pancreatic Final medium</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:339px;">Anti-Vimentin antibody&#160;</td>
			<td style="width:155px;">Abcam</td>
			<td style="width:119px;">ab8069</td>
			<td>For immunocytochemical analisys. Working dilution 1:100</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:339px;">4&#8242;,6-Diamidino-2-phenylindole dihydrochloride</td>
			<td style="width:155px;">Sigma</td>
			<td style="width:119px;">32670</td>
			<td>DAPI. For immunocytochemical analisys. Working dilution&#160; 1&#181;g/ml</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:339px;">5-Methylcytidine</td>
			<td style="width:155px;">Eurogentec</td>
			<td style="width:119px;">MMS-900P-B</td>
			<td>For immunocytochemical analisys. Working dilution 1:500</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:339px;">Anti-C Peptide antibody&#160;</td>
			<td style="width:155px;">Abcam</td>
			<td style="width:119px;">ab14181</td>
			<td>For immunocytochemical analisys. Working dilution 1:100</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:339px;">Anti-PDX1 antibody&#160;</td>
			<td style="width:155px;">Abcam</td>
			<td style="width:119px;">ab47267</td>
			<td>For immunocytochemical analisys. Working dilution 1:500</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:339px;">Mercodia Insulin ELISA</td>
			<td style="width:155px;">Mercodia</td>
			<td style="width:119px;">10-1113-10</td>
			<td>For insulin release detection</td>
		</tr>
	</tbody>
</table>

epigenetic conversion as a safe and simple method to obtain insulin-secreting cells from adult skin fibroblasts

Regenerative medicine requires new, fully functional cells that are delivered to patients in order to repair degenerated or damaged tissues. When such cells are not readily available, they can be obtained using different approaches that include, among the many, reprogramming and trans-differentiation, with advantages and limitations that are specific of the different techniques. Here a new strategy for the conversion of an adult mature fibroblast into an insulin-secreting cell, arbitrarily designated as epigenetic converted cells (EpiCC), is described. The method has been developed, based on the increasing understanding of the mechanisms controlling epigenetic regulation of cell fate and differentiation. In particular, the first step uses an epigenetic modifier, namely 5-aza-cytidine, to drive adult cells into a "highly permissive" state. It then takes advantage of this brief and reversible window of epigenetic plasticity, to re-address cells toward a different lineage. The approach is designated "epigenetic cell conversion". It is a simple and robust way to obtain an efficient, controlled and stable cellular inter-lineage switch. Since the protocol does not involve the use of any gene transfection, it is free of viral vectors and does not involve a stable pluripotent state, it is highly promising for translational medicine applications.

Establishment of primary culture from skin biopsies 
Skin biopsies were cut in small fragments and placed in gelatin pre-coated dishes. After 6 days, fibroblasts started to grow out of the tissue fragments and formed a cell monolayer (Figure 1A). Cells showed a typical elongated shape and, as expected, displayed a uniform immune-positivity for the fibroblast specific marker vimentin (Vim, Figure 1B).
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Watch this Scientific Journal Video about Epigenetic Conversion as a Safe and Simple Method to Obtain Insulin-secreting Cells from Adult Skin Fibroblasts at JoVE.com

Epigenetic Conversion as a Safe and Simple Method to Obtain Insulin-secreting Cells from Adult Skin Fibroblasts

Here, a new method that allows the conversion of adult skin fibroblasts into insulin-secreting cells is presented. This technique is based on epigenetic conversion, does not involve the use of retroviral vectors nor the acquisition of a stable pluripotent state. It is therefore highly promising for translational medicine applications.

Regenerative medicine requires new, fully functional cells that are delivered to patients in order to repair degenerated or damaged tissues. When such ...

The overall goal of this procedure is to convert fibroblasts into insulin-secreting cells without inducing pluripotency or using retrovital vectors. This method can help answer key questions in the regenerative medicine field and in particular in cell therapy of diabetes. The main advantage of this technique is that we can obtain a switch in the phenotype without inducing a stable pluripotent state in the converting cells and without retroviral vectors.Demonstrating the procedure will be Georgia Pennarossa, a post-doc in our laboratory. To begin this procedure add 1.5 milliliters of sterile 0.1%porcine gelatin to the 35-millimeter petri dishes and allow coating for two hours at room temperature. Next, wash the human skin biopsies with PBS supplemented with 2%antibiotic antimycotic solution.Afterward, place the biopsies in a 100-millimeter petri dish and cut them into approximately two cubic millimeter fragments with sterile scalpels. Then, place five to six skin fragments into the pre-coated 35-millimeter petri dish. Add a droplet of fibroblast medium over each fragment and culture them at 37 degrees Celsius in 5%carbon dioxide.After six days o f incubation, discard the tissue fragments carefully. The fibroblasts should start to grow out of the tissue fragments and begin to form a cell monolayer. In this procedure, culture the fibroblasts at 37 degrees Celsius in 5%carbon dioxide until 80%confluence.Then plate the cells in new culture dishes. Culture them at 37 degrees Celsius in 5%carbon dioxide and keep the passage ratio between one to two and one to four, depending on growth rate. Next, add 0.1%porcine gelatin to the cell culture dishes and allow coating for two hours.Remove excess coating solution 10 to 30 minutes prior to plating fibroblasts. Then, remove the fibroblast culture medium from the culture dishes. Wash the cells three times with PBS supplemented with 1%antibiotic antimycotic solution.Then, add a thin layer of Trypsin-EDTA solution. Incubate it at 37 degrees Celsius until the cell monolayer begins to detach from the bottom of the tissue culture dish and the cells dissociate. After that, dilute the cell suspension with nine parts of fibroblast culture medium in order to neutralize the trypsin action.Then, count the cells using a counting chamber under the microscope at room temperature. Calculate the volume of fibroblast culture medium required to re-suspend the cells and to obtain the cell concentration of 7.8 times 10 to the 4th fibroblast per square centimeter. Subsequently, centrifuge the cell suspension at 150 g for five minutes at room temperature.Then, remove the supernatant and re-suspend the cells with a calculated volume of fibroblast culture medium. Afterward, plate the cells on the 0.1%gelatin pre-coated dishes and culture them for 24 hours at 37 degrees Celcius in 5%carbon dioxide. On day zero, immediately prior to use, prepare 5-aza-CR stock solution by dissolving 2.44 milligrams of 5-aza-CR in 10 milliliters of DMEM high glucose medium.Sterilize it by filtration. Next, dilute one microliter of 5-aza-CR stock solution in one milliliter of fibroblast culture medium. To increase cell plasticity, 24 hours after cell plating remove culture medium from the seeded fibroblasts.Add one micromolar 5-aza-CR stock solution, and culture for 18 hours at 37 degrees Celsius in 5%carbon dioxide. On day one, prepare fresh HP medium. After incubation with one micromolar 5-aza-CR, remove the medium and wash the cells three times with HP medium to ensure that 5-aza-CR is rinsed away.Then, incuabate the 5-aza-CR treated fibroblasts with HP medium for three hours at 37 degrees Celsius in 5%carbon dioxide. To monitor the efficiency of 5-aza-CR treatment, check for any morphological changes. Cells should lose the typical elongated morphology of fibroblasts and acquire a smaller round or oval shape with enlarged nuclei.On days one through six, prepare the pancreatic Basal Medium and Activin A stock solution. Then, culture the 5-aza-CR treated fibroblasts in pancreatic Basal Medium supplemented with Activin A stock solution for six days at 37 degrees Celsius in 5%carbon dioxide and change the medium daily. On days seven through eight, prepare retinoic acid stock solution by adding 16.6 milliliters of DMSO to 50 milligrams of retinoic acid.Culture the cells in pancreatic Basal Medium supplemented with Activin A stock solution and retinoic acid stock solution for two days at 37 degrees Celsius in 5%carbon dioxide and change the medium daily. On days nine through 36, culture the cells and pancreatic Basal Medium supplemented with ITS, B27, and BFGS stock solution and change the medium daily for the first 15 days and refresh the medium every other day from day 16 onward. Culture the cells at 37 degrees Celsius in 5%carbon dioxide.This figure shows the morphological changes taking place during the endocrine pancreatic differentiation. After seven days of induction human cells gradually organize in clusters. In response to the addition of retinoic acid, they rearrange in a reticular pattern and cluster in distinguishable aggregates.These formations progress with time, recruiting cells and aggregating enlarged 3D colonies. Finally, colonies become spherical structures that tend to detach and flowed freely in the culture medium, reminiscent of typical pancreatic islets in vitro. After 36 days of pancreatic induction global DNA methylation levels of human epigenetic converted cells returned to those observed in the untreated fibroblasts.The co-localization of PDX1 with CPEP is observed at the end of the conversion period while these endocrine pancreatic markers are completely absent in untreated fibroblasts. This graph shows the amount of insulin release from the epigenetic converted cells in response to 20 millimolar D-glucose and 20 millimolar L-glucose exposure. Once mastered, this technique can be done in 38 days if it is performed properly.After its development, this research paved the way for researchers in the area of regenerative medicine to explore the cell therapy of diabetes in human. After watching this video you should have a good understanding on how to convert fibroblasts into insulin-secreting cells without inducing pluripotency nor using retroviral vectors.

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