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 gelatin to 35 mm Petri dishes. Wait 2 hr to coat, maintaining them at room temperature. 
		Note: Human skin biopsies are collected by excision under local anesthesia from an avascular area of the anterior aspect of the forearm and stored in Dulbecco&#39;s Phosphate Buffered Saline (PBS) supplemented with 2% antibiotic antimycotic solution at +4 &#176;C prior to use.</li>
	</ol>
	</li>
	<li>Wash biopsies with new PBS supplemented with 2% antibiotic antimycotic solution.</li>
	<li>Place biopsies in a 100 mm Petri dish and cut into approximately 2 mm3 fragments with sterile scalpels.</li>
	<li>Remove the coating solution excess immediately prior to plating fragments.</li>
	<li>Place 5-6 skin fragments into the pre-coated 35 mm Petri dish.</li>
	<li>Prepare fibroblast culture medium: 77% Dulbecco&#39;s Modified Eagle Medium (DMEM) high glucose, 20% Fetal Bovine Serum (FBS), 1% L-Glutamine solution and 2% antibiotic antimycotic solution.</li>
	<li>Add a droplet of fibroblast medium over each fragment (usually 100 &#181;l per fragment) and culture them at 37 &#176;C in 5% CO2.</li>
	<li>After 24 hr, add 500 &#181;l of fibroblast culture medium over the fragments to keep them wet at 37 &#176;C in 5% CO2.</li>
	<li>Change the medium with a pipette at least once every 48 hr.</li>
	<li>After 6 days of incubation, remove tissue fragments carefully and discard them. 
	Note: After 6 days, fibroblasts start to grow out of the tissue fragments and begin to form a cell monolayer.</li>
	<li>Refresh medium, add 2 ml of fibroblast culture medium and continue cell monolayer culture at 37 &#176;C in 5% CO2 incubator.</li>
</ol>
2. Fibroblast Culture
<ol>
	<li>Culture fibroblasts at 37 &#176;C in 5% CO2 until 80% confluence</li>
	<li>For passaging, aspirate fibroblast culture medium from tissue culture dishes. Wash cells three times with 4 ml of PBS supplemented with 1% antibiotic antimycotic solution.</li>
	<li>Add a thin layer (10% of the culture medium volume) of trypsin-EDTA solution (0.5 g/L porcine trypsin and 0.2 g/L EDTA) and incubate at 37 &#176;C until cell monolayer begins to detach from the bottom of the tissue culture dish and cells dissociate.</li>
	<li>Dilute cell suspension with 9 parts of fibroblast culture medium to neutralize trypsin action. Centrifugation is not necessary.</li>
	<li>Plate cells in new culture dishes (without gelatin) and culture at 37 &#176;C in 5% CO2 incubator. Keep the passage ratio between 1:2 and 1:4, depending on growth rate.</li>
	<li>When cells reach around 80% confluence, passage them (usually twice per week).</li>
</ol>
3. Fibroblast Plating for Epigenetic Conversion
<ol>
	<li>Add 0.26 ml/cm2 of 0.1% porcine gelatin (prepare as described in 1.1) to cell culture dishes. Wait 2 hr to coat.</li>
	<li>Remove the coating solution excess 10-30 min prior to plating fibroblasts.</li>
	<li>Remove fibroblast culture medium from culture dishes. Wash cells three times with PBS supplemented with 1% antibiotic antimycotic solution.</li>
	<li>Add a thin layer (10% of the culture medium volume) of trypsin-EDTA solution (0.5 g/L porcine trypsin and 0.2 g/L EDTA) and incubate at 37 &#176;C until cell monolayer begins to detach from the bottom of the tissue culture dish and cells dissociate.</li>
	<li>Dilute cell suspension with 9 parts of fibroblast culture medium to neutralize trypsin action.</li>
	<li>Count cells using a counting chamber under a microscope at room temperature. Calculate the required volume of fibroblast culture medium to resuspend cells, to obtain a cell concentration of 7.8 x 104 fibroblasts/cm2. This will depend on the specific type of chamber used. 
	Cells/&#181;l = Average number of cell per small grid x 90 (multiplication factor) x dilution</li>
	<li>Centrifuge cell suspension at 150 g for 5 min at room temperature. Remove supernatant and resuspend cells with the previously calculated volume of fibroblast culture medium.</li>
	<li>Plate cells on 0.1% gelatin pre-coated dishes and culture them for 24 hr at 37 &#176;C in 5% CO2 incubator.</li>
</ol>
4. Increase Cell Plasticity Using the De-methylating Agent 5-aza-CR
<ol>
	<li>Day 0
	<ol>
		<li>Prepare 5-aza-CR stock solution by dissolving 2.44 mg of 5-aza-CR in 10 ml of DMEM high glucose medium. Sterilize by filtration. Prepare 5-aza-CR stock immediately prior to use.</li>
		<li>Dilute 1 &#181;l of 5-aza-CR stock solution in 1 ml of fibroblast culture medium (final concentration 1 &#181;M).</li>
		<li>To increase cell plasticity, 24 hr after cell plating (subheading 3.8), remove culture medium from seeded fibroblasts and add 1 &#181;M 5-aza-CR stock solution and culture for 18 hr at 37 &#176;C in 5% CO2 incubator.</li>
	</ol>
	</li>
	<li>Day 1
	<ol>
		<li>Prepare fresh Human Pluripotent (HP) medium as described in Table 1.</li>
		<li>After incubation with 1 &#181;M 5-aza-CR, remove medium and wash cells three times with PBS to ensure that 5-aza-CR is rinsed away.</li>
		<li>Incubate 5-aza-CR treated fibroblasts with HP medium for 3 hr (recovery period) at 37 &#176;C in 5% CO2.</li>
		<li>After the recovery period, remove medium, wash three times with PBS.</li>
		<li>To monitor the efficiency of 5-aza-CR treatment, check in this step for the presence of morphological changes (detailed in Results section). Cells lose the typical elongated morphology of fibroblasts and acquire a round or oval shape, becoming smaller in size, with enlarged nuclei.</li>
		<li>Proceed with pancreatic differentiation.</li>
	</ol>
	</li>
</ol>
5. Pancreatic Differentiation Protocol
<ol>
	<li>Days 1-6
	<ol>
		<li>Prepare Pancreatic Basal Medium as described in Table 2.</li>
		<li>Prepare activin A stock solution: dissolve 5 &#181;g of activin A recombinant human protein in 166.6 &#181;l of sterile water.</li>
		<li>Culture 5-aza-CR treated fibroblasts in 0.26 ml/cm2 of Pancreatic Basal Medium, supplemented with 1 &#181;l/ml activin A stock solution (see 5.1.2) for 6 days at 37 &#176;C in 5% CO2 incubator. Change medium daily.</li>
	</ol>
	</li>
	<li>Days 7-8
	<ol>
		<li>Prepare retinoic acid stock solution by adding 16.6 ml of dimethyl sulfoxide (DMSO) to 50 mg of retinoic acid.</li>
		<li>Culture cells in 0.26 ml/cm2 of Pancreatic Basal Medium supplemented with 1 &#181;l/ml activin A stock solution (see 5.1.2) and 1 &#181;l/ml retinoic acid stock solution (see 5.2.1) for 2 days at 37 &#176;C in 5% CO2. Change medium daily.</li>
	</ol>
	</li>
	<li>Days 9-36
	<ol>
		<li>Culture cells in 0.26 ml/cm2 of Pancreatic Basal Medium supplemented with 1% (v/v) Insulin-Transferrin-Selenium (ITS), 2% (v/v) B27 and 0.1% (v/v) Recombinant Human FGF basic (bFGF) stock solution (see Table 1) at 37 &#176;C in 5% CO2 incubator. Change the medium daily for the first 15 days.</li>
		<li>From day 16 onward, refresh medium every other day, under a microscope, since forming aggregate cells may detach from the bottom of the culture dish.</li>
	</ol>
	</li>
</ol>

<ol>
	<li>Zhou, Q., Brown, J., Kanarek, A., Rajagopal, J., Melton, D. A. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=In+vivo+reprogramming+of+adult+pancreatic+exocrine+cells+to+beta-cells.">In vivo reprogramming of adult pancreatic exocrine cells to beta-cells.</a> Nature. 455 (7213), 627-632 (2008).</li><li>Takahashi, K., Yamanaka, S. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Induction+of+pluripotent+stem+cells+from+mouse+embryonic+and+adult+fibroblast+cultures+by+defined+factors.">Induction of pluripotent stem cells from mouse embryonic and adult fibroblast cultures by defined factors.</a> Cell. 126 (4), 663-676 (2006).</li><li>Takahashi, K., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Induction+of+pluripotent+stem+cells+from+adult+human+fibroblasts+by+defined+factors.">Induction of pluripotent stem cells from adult human fibroblasts by defined factors.</a> Cell. 131, 861-872 (2007).</li><li>Davis, R. L., Weintraub, H., Lassar, A. B. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Expression+of+a+single+transfected+cDNA+converts+fibroblasts+to+myoblasts.">Expression of a single transfected cDNA converts fibroblasts to myoblasts.</a> Cell. 51 (6), 987-1000 (1987).</li><li>Vierbuchen, T., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Direct+conversion+of+fibroblasts+to+functional+neurons+by+defined+factors.">Direct conversion of fibroblasts to functional neurons by defined factors.</a> Nature. 463 (7284), 1035-1041 (2010).</li><li>Caiazzo, 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=Direct+generation+of+functional+dopaminergic+neurons+from+mouse+and+human+fibroblasts.">Direct generation of functional dopaminergic neurons from mouse and human fibroblasts.</a> Nature. 476, 224-227 (2011).</li><li>Huang, P., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Induction+of+functional+hepatocyte-like+cells+from+mouse+fibroblasts+by+defined+factors.">Induction of functional hepatocyte-like cells from mouse fibroblasts by defined factors.</a> Nature. 475, 386-389 (2011).</li><li>Marro, S., 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=Direct+Lineage+Conversion+of+Terminally+Differentiated+Hepatocytes+to+Functional+Neurons.">Direct Lineage Conversion of Terminally Differentiated Hepatocytes to Functional Neurons.</a> Cell Stem Cell. 9 (4), 374-382 (2011).</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=Brief+demethylation+step+allows+the+conversion+of+adult+human+skin+fibroblasts+into+insulin-secreting+cells.">Brief demethylation step allows the conversion of adult human skin fibroblasts into insulin-secreting cells.</a> Proc Natl Acad Sci U S A. 110 (22), 8948-8953 (2013).</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=Reprogramming+of+Pig+Dermal+Fibroblast+into+Insulin+Secreting+Cells+by+a+Brief+Exposure+to+5-aza-cytidine.">Reprogramming of Pig Dermal Fibroblast into Insulin Secreting Cells by a Brief Exposure to 5-aza-cytidine.</a> Stem Cell Rev. 10 (1), 31-43 (2014).</li><li>Brevini, T. A., 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=Morphological+and+Molecular+Changes+of+Human+Granulosa+Cells+Exposed+to+5-Azacytidine+and+Addressed+Toward+Muscular+Differentiation.">Morphological and Molecular Changes of Human Granulosa Cells Exposed to 5-Azacytidine and Addressed Toward Muscular Differentiation.</a> Stem Cell Rev. 10 (5), 633-642 (2014).</li><li>Thoma, E. C., 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=Chemical+conversion+of+human+fibroblasts+into+functional+Schwann+cells.">Chemical conversion of human fibroblasts into functional Schwann cells.</a> Stem Cell Reports. 3 (4), 539-547 (2014).</li><li>Mirakhori, F., Zeynali, B., Kiani, S., Baharvand, H. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Brief+azacytidine+step+allows+the+conversion+of+suspension+human+fibroblasts+into+neural+progenitor-like+cells.">Brief azacytidine step allows the conversion of suspension human fibroblasts into neural progenitor-like cells.</a> Cell J. 17 (1), 153-158 (2015).</li><li>Plath, K., Lowry, W. E. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Progress+in+understanding+reprogramming+to+the+induced+pluripotent+state.">Progress in understanding reprogramming to the induced pluripotent state.</a> Nat Rev Genet. 12 (4), 253-265 (2011).</li><li>Taylor, S. M., Jones, P. A. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Multiple+new+phenotypes+induced+in+10T1/2+and+3T3+cells+treated+with+5-azacytidine.">Multiple new phenotypes induced in 10T1/2 and 3T3 cells treated with 5-azacytidine.</a> Cell. 17 (4), 771-779 (1979).</li><li>Glover, T. W., Coyle-Morris, J., Pearce-Birge, L., Berger, C., Gemmill, R. M. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=DNA+demethylation+induced+by+5-azacytidine+does+not+affect+fragile+X+expression.">DNA demethylation induced by 5-azacytidine does not affect fragile X expression.</a> Am J Hum Genet. 38 (3), 309-318 (1986).</li><li>Do, J. T., Scholer, H. R. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Nuclei+of+embryonic+stem+cells+reprogram+somatic+cells.">Nuclei of embryonic stem cells reprogram somatic cells.</a> Stem Cells. 22 (6), 941-949 (2004).</li><li>Niwa, H. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=How+is+pluripotency+determined+and+maintained?.">How is pluripotency determined and maintained?.</a> Development. 134 (4), 635-646 (2007).</li><li>Kahan, B. W., 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=Pancreatic+precursors+and+differentiated+islet+cell+types+from+murine+embryonic+stem+cells:+an+in+vitro+model+to+study+islet+differentiation.">Pancreatic precursors and differentiated islet cell types from murine embryonic stem cells: an in vitro model to study islet differentiation.</a> Diabetes. 52 (8), 2016-2024 (2003).</li></ol>

The Authors declare that they have no competing financial interests.

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><tbody><tr><td>Dulbecco&#039;s Phosphate Buffered Saline</td><td>Sigma</td><td>D5652</td><td>PBS; for cell wash and solution preparation</td></tr><tr><td>Antibiotic Antimycotic Solution</td><td>Sigma</td><td>A5955</td><td>Component of Fibroblast, HP and Pancreatic media</td></tr><tr><td>100 mm Petri dish</td><td>Sarstedt</td><td>83.3902</td><td>For Fibroblast isolation</td></tr><tr><td>Porcine Gelatin</td><td>Sigma</td><td>G1890</td><td>For dish coating</td></tr><tr><td>Water</td><td>Sigma</td><td>W3500</td><td>For solution preparation</td></tr><tr><td>35 mm Petri dishes</td><td>Sarstedt</td><td>83.39</td><td>For Fibroblast isolation</td></tr><tr><td>DMEM, high glucose, pyruvate</td><td>Life Technologies</td><td>41966052</td><td>For Fibroblast culture medium</td></tr><tr><td>Fetal Bovine Serum</td><td>Life Technologies</td><td>10500064</td><td>FBS; Component of Fibroblast and HP media</td></tr><tr><td>L-Glutamine solution</td><td>Sigma</td><td>G7513</td><td>Component of Fibroblast, HP and Pancreatic media</td></tr><tr><td>Trypsin-EDTA solution</td><td>Sigma</td><td>T3924</td><td>For Fibroblast dissociation</td></tr><tr><td>KOVA GLASSTIC SLIDE 10 WITH GRIDS</td><td>Hycor Biomedical</td><td>87144</td><td>Cell counting</td></tr><tr><td>5-Azacytidine</td><td>Sigma</td><td>A2385</td><td>5-aza-CR, for increrase cell plasticity in fibroblasts</td></tr><tr><td>Ham&#039;s F-10 Nutrient Mix</td><td>Life Technologies</td><td>31550031</td><td>For HP medium</td></tr><tr><td>DMEM, low glucose, pyruvate</td><td>Life Technologies</td><td>31885023</td><td>For HP medium</td></tr><tr><td>KnockOut Serum Replacement</td><td>Life Technologies</td><td>10828028</td><td>Component of HP medium</td></tr><tr><td>MEM Non-Essential Amino Acids Solution</td><td>Life Technologies</td><td>11140035</td><td>Component of HP and Pancreatic Basal media</td></tr><tr><td>2-Mercaptoethanol</td><td>Sigma</td><td>M7522</td><td>Component of HP and Pancreatic Basal media</td></tr><tr><td>Guanosine</td><td>Sigma</td><td>G6264</td><td>Nucleoside mix stock component of HP medium</td></tr><tr><td>Adenosine</td><td>Sigma</td><td>A4036</td><td>Nucleoside mix stock component of HP medium</td></tr><tr><td>Cytidine</td><td>Sigma</td><td>C4654</td><td>Nucleoside mix stock component of HP medium</td></tr><tr><td>Uridine</td><td>Sigma</td><td>U3003</td><td>Nucleoside mix stock component of HP medium</td></tr><tr><td>Thymidine</td><td>Sigma</td><td>T1895</td><td>Nucleoside mix stock component of HP medium</td></tr><tr><td>Millex-GS 0,22 &amp;micro;m</td><td>Millipore</td><td>SLGS033SB</td><td>For sterilizing of solution</td></tr><tr><td>FGF-Basic (AA 1-155) Recombinant Human Protein</td><td>Life Technologies</td><td>PHG0261</td><td>bFGF; Component of HP and Pancreatic Basal medium</td></tr><tr><td>Bovine Serum Albumin</td><td>Sigma</td><td>A3311</td><td>BSA; Component of Pancreatic Basal medium</td></tr><tr><td>DMEM/F-12</td><td>Life Technologies</td><td>11320074</td><td>For Pancreatic Basal medium</td></tr><tr><td>B-27 Supplement Minus Vitamin A</td><td>Life Technologies</td><td>12587010</td><td>Component of Pancreatic medium</td></tr><tr><td>N-2 Supplement</td><td>Life Technologies</td><td>17502048</td><td>Component of Pancreatic Basal medium</td></tr><tr><td>Activin A Recombinant Human Protein</td><td>Life Technologies</td><td>PHG9014</td><td>For Pancreatic medium</td></tr><tr><td>Retinoic Acid</td><td>Sigma</td><td>R2625</td><td>For Pancreatic medium</td></tr><tr><td>Dimethyl sulfoxide</td><td>Sigma</td><td>D2650</td><td>DMSO; for Retinoic Acid stock preparation</td></tr><tr><td>Insulin-Transferrin-Selenium</td><td>Life Technologies</td><td>41400045</td><td>ITS; for Pancreatic Final medium</td></tr><tr><td>Anti-Vimentin antibody&amp;nbsp;</td><td>Abcam</td><td>ab8069</td><td>For immunocytochemical analisys. Working dilution 1:100</td></tr><tr><td>4&amp;prime;,6-Diamidino-2-phenylindole dihydrochloride</td><td>Sigma</td><td>32670</td><td>DAPI. For immunocytochemical analisys. Working dilution&amp;nbsp; 1 &amp;micro;g/ml</td></tr><tr><td>5-Methylcytidine</td><td>Eurogentec</td><td>MMS-900P-B</td><td>For immunocytochemical analisys. Working dilution 1:500</td></tr><tr><td>Anti-C Peptide antibody&amp;nbsp;</td><td>Abcam</td><td>ab14181</td><td>For immunocytochemical analisys. Working dilution 1:100</td></tr><tr><td>Anti-PDX1 antibody&amp;nbsp;</td><td>Abcam</td><td>ab47267</td><td>For immunocytochemical analisys. Working dilution 1:500</td></tr><tr><td>Mercodia Insulin ELISA</td><td>Mercodia</td><td>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 ...

epigenetic-conversion-as-safe-simple-method-to-obtain-insulin

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6.8K Views.  Università degli Studi di Milano. 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.

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

In Vitro Colony Assays for Characterizing Tri-potent Progenitor Cells Isolated from the Adult Murine Pancreas

Stem and progenitor cells from the adult pancreas could be a potential source of therapeutic beta-like cells for treating patients with type 1 diabetes. However, it is still unknown whether stem and progenitor cells exist in the adult pancreas. Research strategies using cre-lox lineage-tracing in adult mice have yielded results that either support or refute the idea that beta cells can be generated from the ducts, the presumed location where adult pancreatic progenitors may reside. These in vivo cre-lox lineage-tracing methods, however, cannot answer the questions of self-renewal and multi-lineage differentiation-two criteria necessary to define a stem cell. To begin addressing this technical gap, we devised 3-dimensional colony assays for pancreatic progenitors. Soon after our initial publication, other laboratories independently developed a similar, but not identical, method called the organoid assay. Compared to the organoid assay, our method employs methylcellulose, which forms viscous solutions that allow the inclusion of extracellular matrix proteins at low concentrations. The methylcellulose-containing assays permit easier detection and analyses of progenitor cells at the single-cell level, which are critical when progenitors constitute a small sub-population, as is the case for many adult organ stem cells. Together, results from several laboratories demonstrate in vitro self-renewal and multi-lineage differentiation of pancreatic progenitor-like cells from mice. The current protocols describe two methylcellulose-based colony assays to characterize mouse pancreatic progenitors; one contains a commercial preparation of murine extracellular matrix proteins and the other an artificial extracellular matrix protein known as a laminin hydrogel. The techniques shown here are 1) dissociation of the pancreas and sorting of CD133+Sox9/EGFP+ ductal cells from adult mice, 2) single cell manipulation of the sorted cells, 3) single colony analyses using microfluidic qRT-PCR and whole-mount immunostaining, and 4) dissociation of primary colonies into single-cell suspensions and re-plating into secondary colony assays to assess self-renewal or differentiation.

In Vitro Colony Assays for Characterizing Tri-potent Progenitor Cells Isolated from the Adult Murine Pancreas

In vitro colony assays to detect self-renewal and differentiation of progenitor cells isolated from adult murine pancreas are devised. In these assays, pancreatic progenitors give rise to cell colonies in 3-dimensional space in methylcellulose-containing semi-solid medium. Protocols for handling single cells and characterization of individual colonies are described.

Stem and progenitor cells from the adult pancreas could be a potential source of therapeutic beta-like cells for treating patients with type 1 diabetes. ...

Efficient Differentiation of Pluripotent Stem Cells to NKX6-1+ Pancreatic Progenitors

Pluripotent stem cells have the ability to self renew and differentiate to multiple lineages, making them an attractive source for the generation of pancreatic progenitor cells that can be used for the study of and future treatment of diabetes. This article outlines a four-stage differentiation protocol designed to generate pancreatic progenitor cells from human embryonic stem cells (hESCs). This protocol can be applied to a number of human pluripotent stem cell (hPSC) lines. The approach taken to generate pancreatic progenitor cells is to differentiate hESCs to accurately model key stages of pancreatic development. This begins with the induction of the definitive endoderm, which is achieved by culturing the cells in the presence of Activin A, basic Fibroblast Growth Factor (bFGF) and CHIR990210. Further differentiation and patterning with Fibroblast Growth Factor 10 (FGF10) and Dorsomorphin generates cells resembling the posterior foregut. The addition of Retinoic Acid, NOGGIN, SANT-1 and FGF10 differentiates posterior foregut cells into cells characteristic of pancreatic endoderm. Finally, the combination of Epidermal Growth Factor (EGF), Nicotinamide and NOGGIN leads to the efficient generation of PDX1+/NKX6-1+ cells. Flow cytometry is performed to confirm the expression of specific markers at key stages of pancreatic development. The PDX1+/NKX6-1+ pancreatic progenitors at the end of stage 4 are capable of generating mature &#946; cells upon transplantation into immunodeficient mice and can be further differentiated to generate insulin-producing cells in vitro. Thus, the efficient generation of PDX1+/NKX6-1+ pancreatic progenitors, as demonstrated in this protocol, is of great importance as it provides a platform to study human pancreatic development in vitro and provides a source of cells with the potential of differentiating to &#946; cells that could eventually be used for the treatment of diabetes.

Efficient Differentiation of Pluripotent Stem Cells to NKX6-1+ Pancreatic Progenitors

Here we describe a 4-stage protocol to differentiate human embryonic stem cells to NKX6-1+ pancreatic progenitors in vitro. This protocol can be applied to a variety of human pluripotent stem cell lines.

Pluripotent stem cells have the ability to self renew and differentiate to multiple lineages, making them an attractive source for the generation of ...

Isolation, Culture, and Imaging of Human Fetal Pancreatic Cell Clusters

For almost 30 years, scientists have demonstrated that human fetal ICCs transplanted under the kidney capsule of nude mice matured into functioning endocrine cells, as evidenced by a significant increase in circulating human C-peptide following glucose stimulation1-9. However in vitro, genesis of insulin producing cells from human fetal ICCs is low10; results reminiscent of recent experiments performed with human embryonic stem cells (hESC), a renewable source of cells that hold great promise as a potential therapeutic treatment for type 1 diabetes. Like ICCs, transplantation of partially differentiated hESC generate glucose responsive, insulin producing cells, but in vitro genesis of insulin producing cells from hESC is much less robust11-17. A complete understanding of the factors that influence the growth and differentiation of endocrine precursor cells will likely require data generated from both ICCs and hESC. While a number of protocols exist to generate insulin producing cells from hESC in vitro11-22, far fewer exist for ICCs10,23,24. Part of that discrepancy likely comes from the difficulty of working with human fetal pancreas. Towards that end, we have continued to build upon existing methods to isolate fetal islets from human pancreases with gestational ages ranging from 12 to 23 weeks, grow the cells as a monolayer or in suspension, and image for cell proliferation, pancreatic markers and human hormones including glucagon and C-peptide. ICCs generated by the protocol described below result in C-peptide release after transplantation under the kidney capsule of nude mice that are similar to C-peptide levels obtained by transplantation of fresh tissue6. Although the examples presented here focus upon the pancreatic endoderm proliferation and &#946;&#160;cell genesis, the protocol can be employed to study other aspects of pancreatic development, including exocrine, ductal, and other hormone producing cells.

A protocol to isolate, culture, and image islet cell clusters (ICCs) derived from human fetal pancreatic cells is described.&#160; The method details the steps necessary to generate ICCs from tissue, culture as monolayers or in suspension as aggregates, and image for markers of proliferation and pancreatic cell fate decisions.

For almost 30 years, scientists have demonstrated that human fetal ICCs transplanted under the kidney capsule of nude mice matured into functioning ...

Medicine

Generation of Scaffold-free, Three-dimensional Insulin Expressing Pancreatoids from Mouse Pancreatic Progenitors In Vitro

The pancreas is a complex organ composed of many different cell types that work together to regulate blood glucose homeostasis and digestion. These cell types include enzyme-secreting acinar cells, an arborized ductal system responsible for the transportation of enzymes to the gut, and hormone-producing endocrine cells. 
Endocrine beta-cells are the sole cell type in the body that produce insulin to lower blood glucose levels. Diabetes, a disease characterized by a loss or the dysfunction of beta-cells, is reaching epidemic proportions. Thus, it is essential to establish protocols to investigate beta-cell development that can be used for screening purposes to derive the drug and cell-based therapeutics. While the experimental investigation of mouse development is essential, in vivo studies are laborious and time-consuming. Cultured cells provide a more convenient platform for screening; however, they are unable to maintain the cellular diversity, architectural organization, and cellular interactions found in vivo. Thus, it is essential to develop new tools to investigate pancreatic organogenesis and physiology.
Pancreatic epithelial cells develop in the close association with mesenchyme from the onset of organogenesis as cells organize and differentiate into the complex, physiologically competent adult organ. The pancreatic mesenchyme provides important signals for the endocrine development, many of which are not well understood yet, thus difficult to recapitulate during the in vitro culture. Here, we describe a protocol to culture three-dimensional, cellular complex mouse organoids that retain mesenchyme, termed pancreatoids. The e10.5 murine pancreatic bud is dissected, dissociated, and cultured in a scaffold-free environment. These floating cells self-assemble with mesenchyme enveloping the developing pancreatoid and a robust number of endocrine beta-cells developing along with the acinar and the duct cells. This system can be used to study the cell fate determination, structural organization, and morphogenesis, cell-cell interactions during organogenesis, or for the drug, small molecule, or genetic screening.

Generation of Scaffold-free, Three-dimensional Insulin Expressing Pancreatoids from Mouse Pancreatic Progenitors In Vitro

Here, we present a protocol to generate insulin expressing 3D murine pancreatoids from free-floating e10.5 dissociated pancreatic progenitors and the associated mesenchyme.

The pancreas is a complex organ composed of many different cell types that work together to regulate blood glucose homeostasis and digestion. These cell ...

Derivation of Adult Human Fibroblasts and their Direct Conversion into Expandable Neural Progenitor Cells

Generation of&#160;induced pluripotent stem cell (iPSCs) from adult skin fibroblasts and subsequent differentiation into somatic cells provides fascinating prospects for the derivation of autologous transplants that circumvent histocompatibility barriers. However, progression through a pluripotent state and subsequent complete differentiation into desired lineages remains a roadblock for the clinical translation of iPSC technology because of the associated neoplastic potential and genomic instability. Recently, we and others showed that somatic cells cannot only be converted into iPSCs but also into different types of multipotent somatic stem cells by using defined factors, thereby circumventing progression through the pluripotent state. In particular, the direct conversion of human fibroblasts into induced neural progenitor cells (iNPCs) heralds the possibility of a novel autologous cell source for various applications such as cell replacement, disease modeling and drug screening. Here, we describe the isolation of adult human primary fibroblasts by skin biopsy and their efficient direct conversion into iNPCs by timely restricted expression of Oct4, Sox2, Klf4, as well as c-Myc. Sox2-positive neuroepithelial colonies appear after 17 days of induction and iNPC lines can be established efficiently by monoclonal isolation and expansion. Precise adjustment of viral multiplicity of infection and supplementation of leukemia inhibitory factor during the induction phase represent critical factors to achieve conversion efficiencies of up to 0.2%. Thus far, patient-specific iNPC lines could be expanded for more than 12 passages and uniformly display morphological and molecular features of neural stem/progenitor cells, such as the expression of Nestin and Sox2. The iNPC lines can be differentiated into neurons and astrocytes as judged by staining against TUJ1 and GFAP, respectively. In conclusion, we report a robust protocol for the derivation and direct conversion of human fibroblasts into stably expandable neural progenitor cells that might provide a cellular source for biomedical applications such as autologous neural cell replacement and disease modeling.

Generation of induced pluripotent stem cells provides fascinating prospects for the derivation of autologous transplants. However, progression through a pluripotent state and laborious re-differentiation still hinders clinical translation. Here we describe the derivation of adult human fibroblasts and their direct conversion into induced neural progenitor cells and the subsequent differentiation into neural lineages.

Generation of&#160;induced pluripotent stem cell (iPSCs) from adult skin fibroblasts and subsequent differentiation into somatic cells provides ...

Neuroscience

Differentiation of Human Pluripotent Stem Cells into Insulin-Producing Islet Clusters

Differentiation of human pluripotent stem cells (hPSCs) into insulin-secreting beta cells provides material for investigating beta cell function and diabetes treatment. However, challenges remain in obtaining stem cell-derived beta cells that adequately mimic native human beta cells. Building upon previous studies, hPSC-derived islet cells have been generated to create a protocol with improved differentiation outcomes and consistency. The protocol described here utilizes a pancreatic progenitor kit during Stages 1-4, followed by a protocol modified from a paper previously published in 2014 (termed "R-protocol" hereafter) during Stages 5-7. Detailed procedures for using the pancreatic progenitor kit and 400 &#181;m diameter microwell plates to generate pancreatic progenitor clusters, R-protocol for endocrine differentiation in a 96-well static suspension format, and in vitro characterization and functional evaluation of hPSC-derived islets, are included. The complete protocol takes 1 week for initial hPSC expansion followed by ~5 weeks to obtain insulin-producing hPSC islets. Personnel with basic stem cell culture techniques and training in biological assays can reproduce this protocol.

The differentiation of stem cells into islet cells provides an alternative solution to conventional diabetes treatment and disease modeling. We describe a detailed stem cell culture protocol that combines a commercial differentiation kit with a previously validated method to aid in producing insulin-secreting, stem cell-derived islets in a dish.

Differentiation of human pluripotent stem cells (hPSCs) into insulin-secreting beta cells provides material for investigating beta cell function and ...

A High-content In Vitro Pancreatic Islet &#946;-cell Replication Discovery Platform

Loss of insulin-producing &#946;-cells is a central feature of diabetes. While a variety of potential replacement therapies are being explored, expansion of endogenous insulin-producing pancreatic islet &#946;-cells remains an attractive strategy. &#946;-cells have limited spontaneous regenerative activity; consequently, a crucial research effort is to develop a precise understanding of the molecular pathways that restrain &#946;-cell growth and to identify drugs capable of overcoming these restraints. Herein an automated high-content image-based primary-cell screening method to identify &#946;-cell replication-promoting small molecules is presented. Several, limitations of prior methodologies are surmounted. First, use of primary islet cells rather than an immortalized cell-line maximizes retention of in vivo growth restraints. Second, use of mixed-composition islet-cell cultures rather than a &#946;-cell-line allows identification of both lineage-restricted and general growth stimulators. Third, the technique makes practical the use of primary islets, a limiting resource, through use of a 384-well format. Fourth, detrimental experimental variability associated with erratic islet culture quality is overcome through optimization of isolation, dispersion, plating and culture parameters. Fifth, the difficulties of accurately and consistently measuring the low basal replication rate of islet endocrine-cells are surmounted with optimized immunostaining parameters, automated data acquisition and data analysis; automation simultaneously enhances throughput and limits experimenter bias. Notable limitations of this assay are the use of dispersed islet cultures which disrupts islet architecture, the use of rodent rather than human islets and the inherent limitations of throughput and cost associated with the use of primary cells. Importantly, the strategy is easily adapted for human islet replication studies. This assay is well suited for investigating the mitogenic effect of substances on &#946;-cells and the molecular mechanisms that regulate &#946;-cell growth.

A High-content In Vitro Pancreatic Islet &#946;-cell Replication Discovery Platform

Critical challenges for the diabetes research field are to understand the molecular mechanisms that regulate islet &#946;-cell replication and to develop methods for stimulating &#946;-cell regeneration. Herein a high-content screening method to identify and assess the &#946;-cell replication-promoting activity of small molecules is presented.

Loss of insulin-producing &#946;-cells is a central feature of diabetes. While a variety of potential replacement therapies are being explored, expansion ...

Isolation of Adult Human Dermal Fibroblasts from Abdominal Skin and Generation of Induced Pluripotent Stem Cells Using a Non-Integrating Method

Induced pluripotent stem cells (iPSCs) could be considered, to date, a promising source of pluripotent cells for the management of currently untreatable diseases, for the reconstitution and regeneration of injured tissues and for the development of new drugs. Despite all the advantages related to the use of iPSCs, such as the low risk of rejection, the lessened ethical issues, and the possibility to obtain them from both young and old patients without any difference in their reprogramming potential, problems to overcome are still numerous. In fact, cell reprogramming conducted with viral and integrating viruses can cause infections and the introduction of required genes can induce a genomic instability of the recipient cell, impairing their use in clinic. In particular, there are many concerns about the use of c-Myc gene, well-known from several studies for its mutation-inducing activity. Fibroblasts have emerged as the suitable cell population for cellular reprogramming as they are easy to isolate and culture and are harvested by a minimally invasive skin punch biopsy. The protocol described here provides a detailed step-by-step description of the whole procedure, from sample processing to obtain cell cultures, choice of reagents and supplies, cleaning and preparation, to cell reprogramming by the means of a commercial non-modified RNAs (NM-RNAs)-based reprogramming kit. The chosen reprogramming kit allows an effective reprogramming of human dermal fibroblast to iPSCs and small colonies can be seen as early as 24 h after the first transfection, even with modifications with the respect to the standard datasheet. The reprogramming procedure used in this protocol offers the advantage of a safe reprogramming, without the risk of infections caused by viral vector-based methods, reduces the cellular defense mechanisms, and allows the generation of xeno-free iPSCs, all critical features that are mandatory for further clinical applications.

Cell reprogramming requires the introduction of key genes, which regulate and maintain the pluripotent cell state. The protocol described enables the formation of induced pluripotent stem cells (iPSCs) colonies from human dermal fibroblasts without viral/integrating methods but using non-modified RNAs (NM-RNAs) combined with immune evasion factors reducing cellular defense mechanisms.

Induced pluripotent stem cells (iPSCs) could be considered, to date, a promising source of pluripotent cells for the management of currently untreatable ...

Bioengineering

Simple Generation of a High Yield Culture of Induced Neurons from Human Adult Skin Fibroblasts

Induced neurons (iNs), the product of somatic cells directly converted to neurons, are a way to obtain patient-derived neurons from tissue that is easily accessible. Through this route, mature neurons can be obtained in a matter of a few weeks. Here, we describe a straightforward and rapid one-step protocol to obtain iNs from dermal fibroblasts obtained through biopsy samples from adult human donors. We explain each step of the process, including the maintenance of the dermal fibroblasts, the freezing procedure to build a stock of the cell line, seeding of the cells for reprogramming, as well as the culture conditions during the conversion process. In addition, we describe the preparation of glass coverslips for electrophysiological recordings, long-term coating conditions, and fluorescence activated cell sorting (FACS). We also illustrate examples of the results to be expected. The protocol described here is easy to perform and can be applied to human fibroblasts derived from human skin biopsies from patients with various different diagnoses and ages. This protocol generates a sufficient amount of iNs which can be used for a wide array of biomedical applications, including disease modeling, drug screening, and target validation.

Direct neuronal reprogramming generates neurons that maintain the age of the starting somatic cell. Here, we describe a single vector-based method to generate induced neurons from dermal fibroblasts obtained from adult human donors.

Induced neurons (iNs), the product of somatic cells directly converted to neurons, are a way to obtain patient-derived neurons from tissue that is easily ...

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.

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 ...

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

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