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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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 vector mediated random integration. This protocol describes a universal and efficient transgene targeted addition platform in human iPSCs based on utilization of validated open-source TALENs and a gene-trap-like donor to deliver transgenes into a safe harbor locus. Importantly, effective gene editing is rate-limited by the delivery efficiency of gene editing vectors. Therefore, this protocol first focuses on preparation of iPSCs for transfection to achieve high nuclear delivery efficiency. When iPSCs are dissociated into single cells using a gentle-cell dissociation reagent and transfected using an optimized program, >50% cells can be induced to take up the large gene editing vectors. Because the AAVS1 locus is located in the intron of an active gene (PPP1R12C), a splicing acceptor (SA)-linked puromycin resistant gene (PAC) was used to select targeted iPSCs while excluding random integration-only and untransfected cells. This strategy greatly increases the chance of obtaining targeted clones, and can be used in other active gene targeting experiments as well. Two weeks after puromycin selection at the dose adjusted for the specific iPSC line, clones are ready to be picked by manual dissection of large, isolated colonies into smaller pieces that are transferred to fresh medium in a smaller well for further expansion and genetic and functional screening. One can follow this protocol to readily obtain multiple GFP reporter iPSC lines that are useful for in vivo and in vitro imaging and cell isolation.

TALEN-mediated gene editing at the safe harbor AAVS1 locus enables high-efficiency transgene addition in human iPSCs. This protocol describes the procedures for preparing iPSCs for TALEN and donor vector delivery, transfecting iPSCs, and selecting and isolating iPSC clones to achieve targeted integration of a GFP gene to generate reporter lines.

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 therapeutically, in the near future, to restore function to damaged organs. Nevertheless, several technical issues, including the highly invasive procedure of isolating MSCs and the inefficiency surrounding their amplification, currently hamper the potential clinical use of these therapeutic modalities. Herein, we introduce a highly efficient method for the generation of dedifferentiated fat cells (DFAT), MSC-like cells. Interestingly, DFAT cells can be differentiated into several cell types including adipogenic, osteogenic, and chondrogenic cells. Although other groups have previously presented various methods for generating DFAT cells from mature adipose tissue, our method allows us to produce DFAT cells more efficiently. In this regard, we demonstrate that DFAT culture medium (DCM), supplemented with 20% FBS, is more effective in generating DFAT cells than DMEM, supplemented with 20% FBS. Additionally, the DFAT cells produced by our cell culture method can be redifferentiated into several tissue types. As such, a very interesting and useful model for the study of tissue dedifferentiation is presented.

We have modified the conditions for DFAT cell generation and provide herein information regarding the use of an improved growth medium for the production of these 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 the ability to self-renew and to differentiate into all subtypes of the trophoblast lineage, equivalent to the in vivo stem cell population giving rise to the fetal portion of the placenta. Therefore, TSCs offer a unique model to study placental development and embryonic versus extra-embryonic cell fate decision in vitro. From the blastocyst stage onwards, a distinct epigenetic barrier consisting of DNA methylation and histone modifications tightly separates both lineages. Here, we describe a protocol to fully overcome this lineage barrier by transient over-expression of trophoblast key regulators Tfap2c, Gata3, Eomes and Ets2 in murine embryonic fibroblasts. The induced trophoblast stem cells are able to self-renew and are almost identical to blastocyst derived trophoblast stem cells in terms of morphology, marker gene expression and methylation pattern. Functional in vitro and in vivo assays confirm that these cells are able to differentiate along the trophoblast lineage generating polyploid trophoblast giant cells and chimerizing the placenta when injected into blastocysts. The induction of trophoblast stem cells from somatic tissue opens new avenues to study genetic and epigenetic characteristics of this extra-embryonic lineage and offers the possibility to generate trophoblast stem cell lines without destroying the respective embryo.

Here we present a protocol for the direct conversion of murine embryonic fibroblasts into fully functional and stable trophoblast stem cells by ten day over-expression of Tfap2c, Gata3, Eomes and Ets2.

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 cooling in an &#34;open&#34; system (i.e., direct LN2 contact) was a necessity to optimize vitrification success. The dogma surrounding the importance of cooling rates led to unsafe practices subject to technical variation and to the creation of vitrification devices that disregarded important quality-control factors (e.g., ease of use, repeatability, reliability, labeling security, and storage safety). Understanding the quality-control flaws of other devices allowed for the development of a safe, secure, repeatable, and reliable &#181;S-VTF method aimed to minimize intra- and inter-technician variation. Equally important, it combined the availability of two existing FDA-compliant devices: 1) a 0.3-mL ionomeric resin embryo straw with internalized, dual-colored, tamper-proof labeling with repeatable weld seal potential; and 2) shortened, commonly-used, 300-&#181;m ID sterile flexipettes to directly load the embryo(s) in order to create a highly-effective global vitrification device. Like other aseptic, closed vitrification systems (e.g., High Security Vitrification (HSV), Rapid-i, and VitriSafe) effectively used in reproductive medicine, microSecure Vitrification (&#181;S-VTF) has proven that it can achieve high post-warming survival and pregnancy outcomes with its attention to simplicity, and reduced technical variation. Although the 0.3-mL embryo straw containing an internal hydrophobic plug was commercially replaced with a standard semen straw possessing cotton-polyvinyl pyrrolidone (PVP) plugs, it maintained its ionomeric resin composition to ensure weld sealing. However, the cotton plugs can wick out the fluid-embryo contents of the flexipettes upon contact. A modified &#181;S-VTF method was adapted to include an additional internal weld seal before the plug on the device loading side. The added technical step to the &#181;S-VTF procedure has not affected its successful application, as high survival rates (&#62; 95%) and pregnancy rates continue today.

MicroSecure Vitrification was developed as a non-commercial, aseptic closed vitrification device system that is compliant with FDA good manufacturing and tissue-handling practices. Due to the withdrawal of hydrophobic plugged embryo straws from the industry, the vitrification procedure was modified to include an inner seal before the standard internal cotton plug.

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 be responsible for carcinogenesis in such tissues. Although proliferative polyploid cells are requisite for analyzing chromosomal instability of polyploid cells, creating such cells from nontransformed human cells is rather challenging. Induction of tetraploidy by chemical agents usually results in a mixture of diploid and tetraploid populations, and most studies employed fluorescence-activated cell sorting or cloning by limiting dilution to separate tetraploid from diploid cells. However, these procedures are time-consuming and laborious. The present report describes a relatively simple protocol to induce proliferative tetraploid cells from normal human fibroblasts with minimum contamination by diploid cells. Briefly, the protocol is comprised of the following steps: arresting cells in mitosis by demecolcine (DC), collecting mitotic cells after shaking off, incubating collected cells with DC for an additional 3 days, and incubating cells in drug-free medium (They resume proliferation as tetraploid cells within several days). Depending on cell type, the collection of mitotic cells by shaking off might be omitted. This protocol provides a simple and feasible method to establish proliferative tetraploid cells from normal human fibroblasts. Tetraploid cells established by this method could be a useful model for studying chromosome instability and the oncogenic potential of polyploid human cells.

Although proliferative polyploid cells are necessary to analyze chromosomal instability of polyploid cells, creating such cells from nontransformed human cells is not easy. The present report describes relatively simple procedures to establish proliferative tetraploid cells free of a diploid population from normal 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 microscopic images is not entirely dependent upon the quality of the microscope, but also upon the methods of tissue processing, which often involve multiple critical actions or steps. Furthermore, mesenchymal cell types in the skin and other tissues represent a new challenge for tissue preparation and imaging. Here, we present a complete process, from tissue harvest to microscopy. Our technique, called &#34;horizontal whole mount,&#34; is one that novices can quickly become proficient in and that allows for antigen preservation and detection in 60-300 &#181;m-thick sections cut with a cryostat. Sections of this thickness provide enhanced visualization of tissue microarchitecture in a three-dimensional environment. In addition, the protocol preserves mesenchymal cells in a manner that enhances image quality when compared to standard cryostat or paraffin sections, thereby increasing the efficacy and reliability of immunostaining. We believe that this protocol will benefit all laboratories that visualize skin, and possibly other tissues and organs.

This work presents a novel processing and imaging protocol for thick, three-dimensional tissue cross-section analysis that enables the full exploitation of confocal imaging modalities. This protocol preserves antigenicity and represents a robust system to analyze skin histology and potentially other tissue types.

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. In addition, keratinocytes play an essential role in the initiation, maintenance, and regulation of epidermal immune responses by being part of the innate immune system responding to antigenic stimuli in a fast, nonspecific manner. Here, we describe a protocol for isolation of primary human keratinocytes from adult skin, and demonstrate that these cells respond to calcium-induced terminal differentiation, as measured by an increased expression of the differentiation marker involucrin. In addition, we show that the isolated keratinocytes are responsive to IL-1&#946;-induced activation of intracellular signaling pathways as measured by the activation of the p38 MAPK pathway. Taken together, we describe a method for isolation and culturing of primary human keratinocytes from adult skin. Because the keratinocytes are the predominant cell type in the epidermis, this method is useful to study molecular mechanisms in cutaneous biology in vitro.

The human skin acts as a first line of defense against the external environment. We present a method for isolating primary human keratinocytes from adult skin. These isolated keratinocytes are useful in numerous experimental setups, and are a highly suitable model for studying molecular mechanisms in cutaneous biology in vitro.

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, antibodies for specific cell markers are applied on tissue sections. In adult skeletal muscle, satellite cells (SCs) are stem cells that contribute to muscle repair and regeneration. Therefore, it is important to visualize and trace the satellite cell population under different physiological conditions. In resting skeletal muscle, SCs reside between the basal lamina and myofiber plasma membrane. A commonly used marker for identifying SCs on the myofibers or in cell culture is the paired box protein Pax7. In this article, an optimized Pax7 immunofluorescence protocol on skeletal muscle sections is presented that minimizes non-specific staining and background. Another antibody that recognizes a protein (laminin) of the basal lamina was also added to help identify SCs. Similar protocols can also be used to perform double or triple labeling with Pax7 and antibodies for additional proteins of interest.

The precise identification of satellite cells is essential for studying their functions under various physiological and pathological conditions. This article presents a protocol to identify satellite cells on adult skeletal muscle sections by immunofluorescence-based staining.

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 applications. The conventional isolation method of human keratinocytes involves a two-step sequential enzymatic digestion procedure, which has been proven to be inefficient in generating primary cells from adult tissues due to the low cell recovery rate and reduced cell viability. We recently reported an advanced method to isolate human primary epidermal progenitor cells from skin tissues that utilizes the Rho kinase inhibitor Y-27632 in the medium. Compared with the traditional protocol, this new method is simpler, easier, and less time-consuming, and increases epithelial stem cell yield and enhances their stem cell characteristics. Moreover, the new methodology does not require the separation of the epidermis from the dermis, and, therefore, is suitable for isolating cells from different types of adult tissues. This new isolation method overcomes the major shortcomings of conventional methods and is more suitable for producing large numbers of epidermal cells with high potency both for laboratory and for clinical applications. Here, we describe the new method in detail.

Here we present a protocol to efficiently isolate primary human keratinocytes from adult skin tissues. This method simplifies the conventional procedure by using the ROCK Inhibitor Y-27632 in the inoculation medium to spontaneously separate epidermal cells from dermal cells.

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 regeneration, which involves blastema formation and intramembranous ossification analyzed by fluorescence immunohistochemistry and sequential in-vivo microcomputed tomography (&#956;CT). Mammalian regeneration is restricted to amputations transecting the distal region of the terminal phalanx (P3); digits amputated at more proximal levels fail to regenerate and undergo fibrotic healing and scar formation. The regeneration response is mediated by the formation of a proliferative blastema, followed by bone regeneration via intramembranous ossification to restore the amputated skeletal length. P3 amputation is a preclinical model to investigate epimorphic regeneration in mammals, and is a powerful tool for the design of therapeutic strategies to replace fibrotic healing with a successful regenerative response. Our protocol uses fluorescence immunohistochemistry to 1) identify early-and-late blastema cell populations, 2) study revascularization in the context of regeneration, and 3) investigate intramembranous ossification without the need for complex bone stabilization devices. We also demonstrate the use of sequential in vivo &#956;CT to create high resolution images to examine morphological changes after amputation, as well as quantify volume and length changes in the same digit over the course of regeneration. We believe this protocol offers tremendous utility to investigate both epimorphic and tissue regenerative responses in mammals.

Here, we present a protocol of adult mouse terminal phalanx amputation to investigate mammalian blastema formation and intramembranous ossification, analyzed by fluorescent immunohistochemistry and sequential in-vivo microcomputed tomography.

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