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 ...

Research

JoVE Journal

Developmental Biology

Transfection, Selection, and Colony-picking of Human Induced Pluripotent Stem Cells TALEN-targeted with a GFP Gene into the AAVS1 Safe Harbor

Targeted transgene addition can provide persistent gene expression while circumventing the gene silencing and insertional mutagenesis caused by viral ...

An Efficient Method to Obtain Dedifferentiated Fat Cells

Tissue engineering and cell therapy hold great promise clinically. In this regard, multipotent cells, such as mesenchymal stem cells (MSCs), may be used ...

Protocol for the Direct Conversion of Murine Embryonic Fibroblasts into Trophoblast Stem Cells

Trophoblast stem cells (TSCs) arise as a consequence of the first cell fate decision in mammalian development. They can be cultured in vitro, retaining ...

Modified MicroSecure Vitrification: A Safe, Simple and Highly Effective Cryopreservation Procedure for Human Blastocysts

Clinical embryo vitrification evolved with the development of unique vitrification devices in the 21st century and with the misconception that ultra-rapid ...

Establishment of Proliferative Tetraploid Cells from Nontransformed Human Fibroblasts

Polyploid (mostly tetraploid) cells are often observed in preneoplastic lesions of human tissues and their chromosomal instability has been considered to ...

Horizontal Whole Mount: A Novel Processing and Imaging Protocol for Thick, Three-dimensional Tissue Cross-sections of Skin

Processing a tissue of interest to generate a microscopic image that supports a scientific argument can be challenging. The acquisition of high-quality ...

Generation and Culturing of Primary Human Keratinocytes from Adult Skin

The main function of keratinocytes is to provide the structural integrity of the epidermis, thereby maintaining a mechanical barrier to the outside world. ...

Identification of Skeletal Muscle Satellite Cells by Immunofluorescence with Pax7 and Laminin Antibodies

Immunofluorescence is an effective method that helps to identify different cell types on tissue sections. In order to study the desired cell population, ...

A Simplified and Efficient Method to Isolate Primary Human Keratinocytes from Adult Skin Tissue

Primary human keratinocytes isolated from fresh skin tissues and their expansion in vitro have been widely used for laboratory research and for clinical ...

Adult Mouse Digit Amputation and Regeneration: A Simple Model to Investigate Mammalian Blastema Formation and Intramembranous Ossification

Here, we present a protocol of adult mouse distal terminal phalanx (P3) amputation, a procedurally simple and reproducible mammalian model of epimorphic ...