The authors have no acknowledgments.

The specimens from human tissue were collected according to the Declaration of Helsinki while observing University Hospital Federico II guidelines. All patients involved in this study provided written consent.
1. Preparation of Supplies and Culture Media
<ol>
	<li>Clean and autoclave one large pair of surgical scissors, two sets of fine forceps, two pairs of microdissecting scissors, 1 L sterile bottle, 500 mL sterile bottle, and a 250 mL sterile bottle.</li>
	<li>Prepare 100 mm plates, 60 mm plates, 35 mm plates, disposable scalpels, 50 mL sterile tubes, 15 mL sterile tubes, and a 100 mm glass plate. Store instruments under sterile conditions until ready for use.</li>
	<li>Clean and sterilize 22 mm x 22 mm cover glasses before use. For this, place cover glasses in a glass plate, cover them with 70% ethanol and wait for a few seconds before aspirating the ethanol. Let cover glasses dry in an oven at 37 &#176;C and then autoclave them.</li>
	<li>Prepare 1 L of Hank&#39;s balanced salt solution (HBSS) at pH 7.4 by dissolving commercially available powdered salts in double-distilled sterile water and adding 0.35 g of sodium bicarbonate. Sterilize by filtration under a sterile hood and store at +4 &#176;C until use.</li>
	<li>Prepare 500 mL of sterile 1x phosphate-buffered-saline (PBS) by dissolving 0.1 g of potassium phosphate monobasic, 0.1 g of potassium chloride, 4.0 g of sodium chloride and 0.575 g of sodium phosphate dibasic in sterile double-distilled water. Check the pH value (7.4) and sterilize under a sterile hood by filtration. Store at +4 &#176;C until use.</li>
	<li>Prepare 50 mL of trypsin stop solution (TSS) by adding 5 mL of 10% fetal bovine serum (FBS) to 45 mL of HBSS under a sterile hood. Store at +4 &#176;C until use.</li>
	<li>Prepare the Dulbecco&#39;s modified Eagle medium for fibroblast isolation (I-DMEM) using 250 mL of Dulbecco&#39;s modified Eagle medium (DMEM) supplemented with 10% FBS and 0.5% penicillin and streptomycin (pen/strep) under a sterile hood. Store at +4 &#176;C until use.</li>
	<li>Prepare the DMEM for fibroblasts synchronization (S-DMEM) using 250 mL of DMEM supplemented with 0.1% FBS and 0.5% pen/strep under a sterile hood. Store at +4 &#176;C until use.</li>
</ol>
2. Isolation of Human Dermal Fibroblasts
NOTE: Steps 2.1 to 2.3 reported below must be performed under a sterile hood. A cylindrical sample measuring about 0.8 cm in diameter yields 2 x 106 fibroblasts at passage 1.
<ol>
	<li>Wash the freshly obtained sample of human skin from abdomen in a 100 mm dish with HBSS solution. Gently shake to remove blood and other biological fluids. Repeat the step three times, changing the HBSS solution and the plate at each wash.</li>
	<li>Place the sample in a 100 mm plate, remove hair and fat using fine forceps and scissors, and dissect it with a scalpel to obtain 2 mm x 1 mm fragments (for a total of 16 fragments) avoiding scars, dirty (e.g., as resulting from drawings with dermographic pen) or burned areas of the tissue.</li>
	<li>Place 4 small fragments in each 35 mm dish, cover with a sterile 22 mm x 22 mm cover glass. Adjust by the means of fine forceps. Add 1.5 mL of I-DMEM.</li>
	<li>Incubate the plates at 37 &#176;C in 5% CO2 for about 15 days or until cells reach 85% confluence. Change the culture medium every 3 days and check the outgrowth of cells using an inverted phase-contrast microscope daily.</li>
</ol>
3. Expansion of Human Skin Fibroblasts
NOTE: The steps reported below must be performed under a sterile hood except the steps performed in the incubator.
<ol>
	<li>Lift the cover glasses with fine forceps, place them upside-down in one 100 mm dish and wash with 1x sterile PBS.</li>
	<li>Remove fragments of the samples and discard them, wash plates with 1x sterile PBS.</li>
	<li>Remove 1x PBS and add 3 mL and 1 mL of trypsin-EDTA (Ethylenediaminetetraacetic acid) to cover glasses placed in the 100 mm dish and 35 mm dishes, respectively. Incubate for 5 min at 37 &#176;C with 5% CO2.</li>
	<li>Block the trypsinization by adding 5 mL of TSS to the 100 mm dish and 2 mL of TSS to each 35 mm dish. Collect the suspension into 15 mL sterile tubes, centrifuge at 400 x g at 4 &#176;C for 5 min.</li>
	<li>Aspirate the supernatant and resuspend the pellet in 12 mL of I-DMEM. Split the cell suspension into 3 mL aliquots for each 60 mm plate.</li>
	<li>Incubate at 37 &#176;C with 5% CO2. Change the medium daily. Keep cells in culture until they reach a confluence of 75%.</li>
	<li>Aspirate the medium from the plates and quickly wash in 1x PBS.</li>
	<li>Repeat trypsinization (steps 3.3 and 3.4) three times to obtain cells at passage 4. Use 1.5 mL of trypsin-EDTA and 3 mL of TSS in each 60 mm dish. Split the cells 1:3.</li>
	<li>Synchronize the cells by replacing the I-DMEM with S-DMEM and incubate for 48 h at 37 &#176;C with 5% CO2.</li>
	<li>Prepare the following (during the cell synchronization).
	<ol>
		<li>Prepare fibroblast expansion medium by adding 5.0 mL of FBS and 0.5 mL of L-glutamine to 44.5 mL of advanced DMEM (A-DMEM), store at +4 &#176;C.</li>
		<li>Prepare total NM-RNA-reprogramming cocktail for fibroblast reprogramming by combining the following in each of 9 sterile RNase-free tubes: 32 &#181;L of OSKMNL NM-RNA, 24 &#181;L of EKB NM-RNA, 5.6 &#181;L of NM-microRNAs (61.6 &#181;L final volume for 9 aliquots).</li>
		<li>Divide each tube of 61.6 &#181;L of total NM-RNA-reprogramming cocktail for fibroblast reprogramming into four 15.4 &#181;L single-use aliquots in sterile, RNase-free tubes and store at -80 &#176;C (15.4 &#181;L final volume for 36 aliquots).</li>
	</ol>
	</li>
</ol>
4. Reprogramming of Dermal Fibroblasts to iPSCs
<ol>
	<li>Day 0: Cell seeding 
	NOTE: Steps 4.1.1 to 4.1.3 and 4.1.8 to 4.1.9 must be performed under a sterile hood. Basement membrane matrix (BMM) gels rapidly at room temperature. Therefore, it is strongly recommended to thaw BMM overnight on ice in a refrigerator, dilute it with ice-cold serum-free medium and use pre-cooled pipets, tips, and tubes.
	<ol>
		<li>Prepare a 24-well plate by coating each well with 100 &#181;L of BMM and incubate for at least 1 h at 37 &#176;C before seeding the cells.</li>
		<li>Aspirate the medium from plates with cells in culture and quickly wash in 1x PBS.</li>
		<li>Repeat trypsinization (steps 3.3 and 3.4) to obtain cells at passage 5. Aspirate the supernatant and resuspend the pellet in an appropriate volume of fibroblasts expansion medium (see step 3.10).</li>
		<li>Prepare and clean the hemocytometer with 70% alcohol.</li>
		<li>Prepare a solution by gently mixing 10 &#181;l of trypan blue and 10 &#181;L of cell suspension in 1.5 mL tube, and incubate for 1&#8211;2 min at room temperature. Be careful not to incubate longer than 5 min.</li>
		<li>Pipette 10 &#181;L of the solution into the hemocytometer place it on the stage of an inverted phase contrast microscope to count. Non-viable cells will be blue, while viable cells will be unstained.</li>
		<li>Count the cells in at least 2 squares of the hemocytometer chamber. Apply the following calculation to obtain the total number of cells: 
		total number of cells = (total number of counted cells/square number) x 2 x 10 x total volume of cell suspension</li>
		<li>Perform a dilution to obtain a density of 2.5 x 104 cell per 500 &#181;L of fibroblasts expansion medium, based on the total number of counted cells. Resuspend the pellet in an appropriate volume of fibroblasts expansion medium.</li>
		<li>Pipette 500 &#181;L of the cell suspension into each well of the 24-well plate. Incubate cells overnight at 37 &#176;C and 5% CO2.</li>
	</ol>
	</li>
	<li>Day 1: Transfection 
	NOTE: All working surfaces and supplies (e.g., gloves, bottles, sterile hood surfaces, pipettors) MUST be cleaned with cleaning reagent for removing RNase before starting the procedure. Perform steps 4.2.2, 4.2.4&#8211;4.2.8 under a sterile hood.
	<ol>
		<li>Warm up xeno-free, serum-free,low growth factor human ESC/iPSC culture medium (XF/FF culture medium) in a 37 &#176;C water bath.</li>
		<li>Remove the old medium from each well and replace it with 500 &#181;L of pre-warmed XF/FF culture medium.</li>
		<li>Incubate at 37 &#176;C under 5% CO2 for at least 6 h.</li>
		<li>Thaw five 15.4 &#181;L aliquots of total NM-RNA reprogramming cocktail at room temperature then place it on ice.</li>
		<li>Add 234.6 &#181;L of reduced-serum medium to each aliquot, gently pipette 3&#8211;5 times and label each one as TUBE A (RNA + reduced-serum medium).</li>
		<li>Label 5 sterile, RNase-free 0.5 mL tubes as TUBE B and mix 6 &#181;L of synthetic siRNA transfection reagent with 244 &#181;L of reduced-serum medium (RNA-transfection reagent + reduced-serum medium).</li>
		<li>Add the contents of each TUBE B to a TUBE A dropwise (to obtain 500 &#181;L final volume of NM-RNA transfection complex solution). Mix by tapping the bottom of the tube. Incubate at room temperature for 15 min.</li>
		<li>Add 125 &#181;L of NM-RNA transfection complex solution from each of the five aliquots of 500 &#181;L to 4 wells (to reach a total of 20 wells), tilting the plate and pipetting dropwise into the medium. Mix by rocking gently. Use the remaining 4 wells as a reference.</li>
		<li>Incubate for 15 h at 37 &#176;C, 5% CO2.</li>
	</ol>
	</li>
	<li>Day 2&#8211;4: Complete the transfection
	<ol>
		<li>Aspirate the medium. Repeat the transfection procedure as on day 1 under the sterile hood.</li>
	</ol>
	</li>
	<li>Day 5&#8211;10: Media changes
	<ol>
		<li>Aspirate the medium and exchange with fresh, pre-warmed XF/FF culture medium under a sterile hood.</li>
		<li>Incubate overnight at 37 &#176;C, 5% CO2.</li>
		<li>Keep in culture until observing the formation of iPSC colonies monitoring daily by a phase-contrast microscope.</li>
	</ol>
	</li>
</ol>

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The authors have nothing to disclose.

iPSCs are rapidly emerging as the most promising cell candidate for regenerative medicine applications and as a tremendously useful tool for disease modeling and drug testing3,8. The protocol presented here describes the generation of human iPSCs from a sample having the size of a skin punch biopsy with a simple and efficient procedure that does not require any specific equipment or previous experience with reprogramming technology.
It...

Cell reprogramming represents a novel technology to transform every somatic cell of the body into a pluripotent stem cell, known as iPSC1. The possibility of reprogramming an adult somatic cell back to a pluripotent and undifferentiated state has overcome the limits imposed by the poor availability and ethical issues related to the use of pluripotent cells, previously only derivable from human embryos (embryonic stem cells or ESC)2,3,4. In 2006, Kazutoshi Takahashi and Shinya Yamanaka conducted a pioneering study achieving the first conversion of adult somatic cells from skin into pluripotent cells by artificially adding four specific genes (Oct4, Sox2, Klf4, c-Myc)5. A year later, work conducted in Thomson&#39;s laboratory led to the successful reprogramming of somatic cells into iPSCs by transduction of a different combination of four genes (Oct4, Sox2, Nanog, Lin28)6.
iPSCs offer a number of opportunities to scientists and researchers of different fields, such as regenerative medicine and pharmacology, being an excellent platform to study and treat different diseases along with a genotypic reflection of the characteristics of the patient they are derived from. The use of iPSCs provides several advantages including: the reduced risk for immune response due to a completely autologous origin of cells; the possibility of creating a cell library, an important tool to predict response to new drugs and their side effects, as they are able to continuously self-renew and generate different cell types; and the chance to develop a customized approach for drug administration7,8,9.
Diverse techniques are known at present, to induce the expression of the reprogramming factors and they are included in two major categories: non-viral and viral vector-based methods10,11,12,13. Non-viral methods include mRNA transfection, miRNA infection/transfection, PiggyBac, minicircle vectors and episomal plasmids and exosomes10,11,12,13. Viral-based methods include non-integrating viruses, such as Adenovirus, Sendai virus and proteins, and integrating viruses like Retrovirus and Lentivirus10,11,12,13.
According to several studies, no significant differences have been noticed among these methods in terms of effectiveness of cell reprogramming, hence, the choice of the suitable method strictly depends on the cell type used and on the subsequent applications of the iPSCs obtained14,15. All the mentioned methods show disadvantages, for example, the Sendai virus is effective on all cell types, but requires a lot of passages to obtain iPSCs; reprogramming by episomes is excellent for blood cells but needs modification of standard culture conditions for fibroblasts; the PiggyBac method could represent an attractive alternative but studies in human cells are still limited and weak10,11,12,13. Exosomes are nano-vesicles physiologically secreted into all body fluids by cells. According to recent studies, they are responsible for intercellular communication and can have a role in important biological processes, such as cell proliferation, migration and differentiation. Exosomes can transport and transfer mRNA and miRNA to recipient cells with a completely natural mechanism, as they share the same composition of the cell membrane16. Therefore, exosomes are a promising new generation technique for reprogramming, but their potential to reprogram somatic cells by their content is still under investigation. Viral vectors-based methods use viruses modified in order to convey reprogramming genes to recipient cells. This technique, despite the high efficiency of reprogramming, is not considered safe, as the integration of the virus within the cell can be responsible for infection, teratomas and genomic instability17.
The following protocol to generate iPSCs colonies combines the Yamanaka&#39;s and Thompson&#39;s reprogramming cocktail and is based upon the use of a method requiring NM-RNAs and immune evasion factors with the possibility to perform it in xeno-free conditions. The rationale behind the use of this method is to spread, within the scientific community, a protocol allowing a rapid, simple and highly effective reprogramming of adult human fibroblasts from abdominal skin into iPSCs18.
The strengths of the proposed method are, in fact, the ease of performance and the short time needed to obtain iPSCs. Furthermore, the method avoids cellular defense mechanisms and the use of viral vectors, responsible for relevant issues.
With respect to the standard protocol, the following modifications were made: (1) Confluent fibroblasts were synchronized at passage 4 by being placing in 0.1% serum for 48 h before the trypsinization; (2) The cellular density for culture and the volume of reagents were adjusted for the utilization on a 24-well multi-well plate instead of a 6-well plate; (3) The reprogramming experiment was performed using a 5% CO2 incubator instead of an incubator with atmospheric (21% O2) or hypoxic (5% O2) conditions.

<table><tbody><tr><td>10 mL serological pipet</td><td>Falcon</td><td>&lt;strong&gt;357551&lt;/strong&gt;</td><td>Sterile, polystyrene</td></tr><tr><td>100 mm plates</td><td>Falcon</td><td>&lt;strong&gt;351029&lt;/strong&gt;</td><td>Treated, sterile cell culture dish</td></tr><tr><td>15 mL sterile tubes</td><td>Falcon</td><td>&lt;strong&gt;352097&lt;/strong&gt;</td><td>Centrifuge sterile tubes, polypropylene</td></tr><tr><td>24-well plates</td><td>Falcon</td><td>&lt;strong&gt;353935&lt;/strong&gt;</td><td>Clear, flat bottom, treated multiwell cell culture plate, with lid, sterile</td></tr><tr><td>25 mL serological pipet</td><td>Falcon</td><td>&lt;strong&gt;357525&lt;/strong&gt;</td><td>Sterile, polystyrene</td></tr><tr><td>35 mm plates</td><td>Falcon</td><td>&lt;strong&gt;353001&lt;/strong&gt;</td><td>Treated, sterile cell culture dish</td></tr><tr><td>5 mL serological pipet</td><td>Falcon</td><td>&lt;strong&gt;357543&lt;/strong&gt;</td><td>Sterile, polystyrene</td></tr><tr><td>50 mL sterile tubes</td><td>Falcon</td><td>&lt;strong&gt;352098&lt;/strong&gt;</td><td>Centrifuge sterile tubes, polypropylene</td></tr><tr><td>Advanced DMEM (Dulbecco&#039;s Modified Eagle Medium)</td><td>Gibco</td><td>&lt;strong&gt;12491-015&lt;/strong&gt;</td><td>Store at 2-8 &amp;deg;C; avoid exposure to light</td></tr><tr><td>DMEM (Dulbecco&#039;s Modified Eagle Medium)</td><td>Sigma- Aldrich</td><td>&lt;strong&gt;D6429-500ml&lt;/strong&gt;</td><td>Store at 2-8 &amp;deg;C; avoid exposure to light</td></tr><tr><td>Fetal Bovine Serum</td><td>Sigma- Aldrich</td><td>&lt;strong&gt;F9665-500ml&lt;/strong&gt;</td><td>Store at -20 &amp;deg;C. The serum should be aliquoted into smaller working volumes to avoid repeated freeze/thaw cycles</td></tr><tr><td>Hank&#039;s Balanced Salt Solution</td><td>Sigma- Aldrich</td><td>&lt;strong&gt;H1387-1L&lt;/strong&gt;</td><td>Powder</td></tr><tr><td>L-glutamine</td><td>Lonza</td><td>&lt;strong&gt;BE17-605E&lt;/strong&gt;</td><td>Store at -20 &amp;deg;C. It should be aliquoted into smaller working volumes to avoid repeated freeze/thaw cycles</td></tr><tr><td>Lipofectamine RNAiMAX Transfection Reagent</td><td>INVITROGEN</td><td>&lt;strong&gt;13778-030&lt;/strong&gt;</td><td>Synthetic siRNA Transfection Reagent; store at 2-8 &amp;deg;C</td></tr><tr><td>Matrigel</td><td>CORNING</td><td>&lt;strong&gt;354234&lt;/strong&gt;</td><td>Basement Membrane Matrix, store at -20 &amp;deg;C. Avoid multiple freeze-thaws.</td></tr><tr><td>Neubauer Chamber</td><td>VWR</td><td>&lt;strong&gt;631-1116&lt;/strong&gt;</td><td>Hemocytometer</td></tr><tr><td>NutriStem XF Culture Medium</td><td>Biological Industries</td><td>&lt;strong&gt;05-100-1A-500ml&lt;/strong&gt;</td><td>Xeno-free, serum-free, low growth factor human ESC/iPSC culture medium. Store at -20 &amp;deg;C. Upon thawing, the medium may be stored at 2-8 &amp;deg;C for 14 days. Media should be aliquoted into smaller working volumes to avoid repeated freeze/thaw cycles.</td></tr><tr><td>Opti-MEM</td><td>Gibco</td><td>&lt;strong&gt;31985-062-100ml&lt;/strong&gt;</td><td>Reduced-Serum Medium; store at 2-8 &amp;deg;C; avoid exposure to light</td></tr><tr><td>Penicillin and Streptomycin</td><td>Sigma- Aldrich</td><td>&lt;strong&gt;P4333-100ml&lt;/strong&gt;</td><td>Store at -20 &amp;deg;C. The solution should be aliquoted into smaller working volumes to avoid repeated freeze/thaw cycles</td></tr><tr><td>Potassium Chloride</td><td>Sigma- Aldrich</td><td>&lt;strong&gt;P9333&lt;/strong&gt;</td><td>Powder</td></tr><tr><td>Potassium Phosphate Monobasic</td><td>Sigma- Aldrich</td><td>&lt;strong&gt;P5665&lt;/strong&gt;</td><td>Powder</td></tr><tr><td>RNase-free 0.5 mL tubes</td><td>Eppendorf</td><td>&lt;strong&gt;H0030124537&lt;/strong&gt;</td><td>RNase-free sterile, microfuge tubes, polypropylene</td></tr><tr><td>RNase-free 1.5 mL tubes</td><td>Eppendorf</td><td>&lt;strong&gt;H0030120086&lt;/strong&gt;</td><td>RNase-free sterile, microfuge tubes, polypropylene</td></tr><tr><td>RNaseZAP</td><td>INVITROGEN</td><td>&lt;strong&gt;AM9780&lt;/strong&gt;</td><td>Cleaning agent for removing RNase</td></tr><tr><td>Sodium Bicarbonate</td><td>Sigma- Aldrich</td><td>&lt;strong&gt;S5761&lt;/strong&gt;</td><td>Powder</td></tr><tr><td>Sodium Chloride</td><td>Sigma- Aldrich</td><td>&lt;strong&gt;S7653&lt;/strong&gt;</td><td>Powder</td></tr><tr><td>Sodium Phosphate Dibasic</td><td>Sigma- Aldrich</td><td>&lt;strong&gt;94046&lt;/strong&gt;</td><td>Powder</td></tr><tr><td>StemRNA 3rd Gen Reprogramming Kit</td><td>Reprocell</td><td>&lt;strong&gt;00-0076&lt;/strong&gt;</td><td>Third Generation NM-RNAs-based Reprogramming Kit for Cellular Reprogramming of Fibroblasts, Blood, and Urine. Store at or below -70 &amp;deg;C.</td></tr><tr><td>Trypsin-EDTA</td><td>Sigma- Aldrich</td><td>&lt;strong&gt;T4049-100ml&lt;/strong&gt;</td><td>Store at -20 &amp;deg;C. It should be aliquoted into smaller working volumes to avoid repeated freeze/thaw cycles</td></tr></tbody></table>

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.

The aim of the protocol was to reprogram dermal fibroblasts isolated from abdominal skin using non-integrating reprogramming method based on NM-RNAs to induce the expression of specific factors. To achieve this goal, human dermal fibroblasts were isolated from skin specimens of patients undergoing tummy tuck surgery and iPSCs were generated introducing Oct4, Sox2, Klf4, cMyc, Nanog, Lin28 reprogramming factors and E3, K3, B18 immune evasion factors by a commercial ready-to-use reprogramming kit that combines NM-RNA and m...

Watch this Scientific Journal Video about Isolation of Adult Human Dermal Fibroblasts from Abdominal Skin and Generation of Induced Pluripotent Stem Cells Using a Non-Integrating Method at JoVE.com

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

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 ...

isolation-adult-human-dermal-fibroblasts-from-abdominal-skin

Universidad de Cartagena UDEC

Research

JoVE Journal

Bioengineering

10.3K Views.  University of Naples Federico II. 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.

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

Generation of Integration-free Human Induced Pluripotent Stem Cells Using Hair-derived Keratinocytes

Recent advances in reprogramming allow us to turn somatic cells into human induced pluripotent stem cells (hiPSCs). Disease modeling using patient-specific hiPSCs allows the study of the underlying mechanism for pathogenesis, also providing a platform for the development of in vitro drug screening and gene therapy to improve treatment options. The promising potential of hiPSCs for regenerative medicine is also evident from the increasing number of publications (>7000) on iPSCs in recent years. Various cell types from distinct lineages have been successfully used for hiPSC generation, including skin fibroblasts, hematopoietic cells and epidermal keratinocytes. While skin biopsies and blood collection are routinely performed in many labs as a source of somatic cells for the generation of hiPSCs, the collection and subsequent derivation of hair keratinocytes are less commonly used. Hair-derived keratinocytes represent a non-invasive approach to obtain cell samples from patients. Here we outline a simple non-invasive method for the derivation of keratinocytes from plucked hair. We also provide instructions for maintenance of keratinocytes and subsequent reprogramming to generate integration-free hiPSC using episomal vectors.

This manuscript provides a step-by-step procedure for the derivation and maintenance of human keratinocytes from plucked hair and subsequent generation of integration-free human induced pluripotent stem cells (hiPSCs) by episomal vectors.

Recent advances in reprogramming allow us to turn somatic cells into human induced pluripotent stem cells (hiPSCs). Disease modeling using ...

Developmental Biology

Murine Dermal Fibroblast Isolation by FACS

Fibroblasts are the principle cell type responsible for secreting extracellular matrix and are a critical component of many organs and tissues. Fibroblast physiology and pathology underlie a spectrum of clinical entities, including fibroses in multiple organs, hypertrophic scarring following burns, loss of cardiac function following ischemia, and the formation of cancer stroma. However, fibroblasts remain a poorly characterized type of cell, largely due to their inherent heterogeneity. Existing methods for the isolation of fibroblasts require time in cell culture that profoundly influences cell phenotype and behavior. Consequently, many studies investigating fibroblast biology rely upon in vitro manipulation and do not accurately capture fibroblast behavior in vivo. To overcome this problem, we developed a FACS-based protocol for the isolation of fibroblasts from the dorsal skin of adult mice that does not require cell culture, thereby preserving the physiologic transcriptional and proteomic profile of each cell. Our strategy allows for exclusion of non-mesenchymal lineages via a lineage negative gate (Lin-) rather than a positive selection strategy to avoid pre-selection or enrichment of a subpopulation of fibroblasts expressing specific surface markers and be as inclusive as possible across this heterogeneous cell type.

Fibroblast behavior underlies a spectrum of clinical entities, but they remain poorly characterized, largely due to their inherent heterogeneity. Traditional fibroblast research relies upon in vitro manipulation, masking in vivo fibroblast behavior. We describe a FACS-based protocol for the isolation of mouse skin fibroblasts that does not require cell culture.

Fibroblasts are the principle cell type responsible for secreting extracellular matrix and are a critical component of many organs and tissues. Fibroblast ...

Generation of Integration-free Induced Pluripotent Stem Cells from Human Peripheral Blood Mononuclear Cells Using Episomal Vectors

Induced Pluripotent Stem Cells (iPSCs) hold great promise for disease modeling and regenerative therapies. We previously reported the use of Episomal Vectors (EV) to generate integration-free iPSCs from peripheral blood mononuclear cells (PB MNCs). The episomal vectors used are DNA plasmids incorporated with oriP and EBNA1 elements from the Epstein-Barr (EB) virus, which&#160;allow for replication and long-term retainment of plasmids in mammalian cells, respectively. With further optimization, thousands of iPSC colonies can be obtained from 1 mL of peripheral blood. Two critical factors for achieving high reprogramming efficiencies&#160;are: 1) the use of a 2A &#34;self-cleavage&#34; peptide to link OCT4 and SOX2, thus achieving equimolar expression of the two factors; 2) the use of two vectors to express MYC and KLF4 individually. Here we describe a step-by-step protocol for generating integration-free iPSCs from adult peripheral blood samples. The generated iPSCs are integration-free as residual episomal plasmids are undetectable after five passages. Although the reprogramming efficiency is comparable to that of Sendai Virus (SV) vectors, EV plasmids are considerably more economical than the commercially available SV vectors. This affordable EV reprogramming system holds potential for clinical applications in regenerative medicine and provides an approach for the direct reprogramming of PB MNCs to integration-free mesenchymal stem cells, neural stem cells, etc.

This protocol describes a detailed method for efficient generation of integration-free iPSCs from human adult peripheral blood cells. With the use of four oriP/EBNA-based episomal vectors to express the reprogramming factors, KLF4, MYC, BCL-XL, or OCT4 and SOX2, thousands of iPSC colonies can be obtained from 1 mL of peripheral blood.

Induced Pluripotent Stem Cells (iPSCs) hold great promise for disease modeling and regenerative therapies. We previously reported the use of Episomal ...

Isolation of Papillary and Reticular Fibroblasts from Human Skin by Fluorescence-activated Cell Sorting

Fibroblasts are a highly heterogeneous cell population implicated in the pathogenesis of many human diseases. In human skin dermis, fibroblasts have traditionally been attributed to the superficial papillary or lower reticular dermis according to their histological localization. In mouse dermis, papillary and reticular fibroblasts originate from two different lineages with diverging functions regarding physiological and pathological processes and a distinct cell surface marker expression profile by which they can be distinguished.
Importantly, evidence from explant cultures from superficial and lower dermal layers suggest that at least two functionally distinct dermal fibroblasts lineages exist in human skin dermis as well. However, unlike for mouse skin, cell surface markers enabling the discrimination of different fibroblast subsets have not yet been established for human skin. We developed a novel protocol for the isolation of human papillary and reticular fibroblast populations via fluorescence-activated cell sorting (FACS) using the two cell surface markers Fibroblast Activation Protein (FAP) and Thymocyte antigen 1 (Thy1)/CD90. This method enables the isolation of pure fibroblast subsets without in vitro manipulation, which was shown to affect gene expression, thus permitting accurate functional analysis of human dermal fibroblast subsets in regard to tissue homeostasis or disease pathology.

This manuscript describes a FACS-based protocol for isolation of papillary and reticular fibroblasts from human skin. It circumvents in vitro culture which was inevitable with the commonly used isolation protocol via explant cultures. The emanating fibroblast subsets are functionally distinct and display differential gene expression and localization within the dermis.

Fibroblasts are a highly heterogeneous cell population implicated in the pathogenesis of many human diseases. In human skin dermis, fibroblasts have ...

Genetics

Integration Free Derivation of Human Induced Pluripotent Stem Cells Using Laminin 521 Matrix

Xeno-free and fully defined conditions are key parameters for robust and reproducible generation of homogenous human induced pluripotent stem (hiPS) cells. Maintenance of hiPS cells on feeder cells or undefined matrices are susceptible to batch variances, pathogenic contamination and risk of immunogenicity. Utilizing the defined recombinant human laminin 521 (LN-521) matrix in combination with xeno-free and defined media formulations reduces variability and allows for the consistent generation of hiPS cells. The Sendai virus (SeV) vector is a non-integrating RNA-based system, thus circumventing concerns associated with the potential disruptive effect on genome integrity integrating vectors can have. Furthermore, these vectors have demonstrated relatively high efficiency in the reprogramming of dermal fibroblasts. In addition, enzymatic single cell passaging of cells facilitates homogeneous maintenance of hiPS cells without substantial prior experience of stem cell culture. Here we describe a protocol that has been extensively tested and developed with a focus on reproducibility and ease of use, providing a robust and practical way to generate defined and xeno-free human hiPS cells from fibroblasts.

Robust derivation of human induced pluripotent stem (hiPS) cells was achieved by using non-integrating Sendai virus (SeV) vector mediated reprogramming of dermal fibroblasts. hiPS cell maintenance and clonal expansion was performed using xeno-free and chemically defined culture conditions with recombinant human laminin 521 (LN-521) matrix and Essential E8 (E8) Medium.

Xeno-free and fully defined conditions are key parameters for robust and reproducible generation of homogenous human induced pluripotent stem (hiPS) ...

Generation of 3D Skin Organoid from Cord Blood-derived Induced Pluripotent Stem Cells

The skin is the body&#8217;s largest organ and has many functions. The skin acts as a physical barrier and protector of the body and regulates bodily functions. Biomimetics is the imitation of the models, systems, and elements of nature for the purpose of solving complex human problems1. Skin biomimetics is a useful tool for in vitro disease research and in vivo regenerative medicine. Human induced pluripotent stem cells (iPSCs) have the characteristic of unlimited proliferation and the ability of differentiation to three germ layers. Human iPSCs are generated from various primary cells, such as blood cells, keratinocytes, fibroblasts, and more. Among them, cord blood mononuclear cells (CBMCs) have emerged as an alternative cell source from the perspective of allogeneic regenerative medicine. CBMCs are useful in regenerative medicine because human leukocyte antigen (HLA) typing is essential to the cell banking system. We provide a method for the differentiation of CBMC-iPSCs into keratinocytes and fibroblasts and for generation of a 3D skin organoid. CBMC-iPSC-derived keratinocytes and fibroblasts have characteristics similar to a primary cell line. The 3D skin organoids are generated by overlaying an epidermal layer onto a dermal layer. By transplanting this 3D skin organoid, a humanized mice model is generated. This study shows that a 3D human iPSC-derived skin organoid may be a novel, alternative tool for dermatologic research in vitro and in vivo.

We propose a protocol that shows how to differentiate induced pluripotent stem cell-derived keratinocytes and fibroblasts and generate a 3D skin organoid, using these keratinocytes and fibroblasts. This protocol contains an additional step of generating a humanized mice model. The technique presented here will improve dermatologic research.

The skin is the body&#8217;s largest organ and has many functions. The skin acts as a physical barrier and protector of the body and regulates bodily ...

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

Isolation and Culture of Adult Epithelial Stem Cells from Human Skin

The homeostasis of all self-renewing tissues is dependent on adult stem cells. As undifferentiated stem cells undergo asymmetric divisions, they generate daughter cells that retain the stem cell phenotype and transit-amplifying cells (TA cells) that migrate from the stem cell niche, undergo rapid proliferation and terminally differentiate to repopulate the tissue.
Epithelial stem cells have been identified in the epidermis, hair follicle, and intestine as cells with a high in vitro proliferative potential and as slow-cycling label-retaining cells in vivo 1-3. Adult, tissue-specific stem cells are responsible for the regeneration of the tissues in which they reside during normal physiologic turnover as well as during times of stress 4-5. Moreover, stem cells are generally considered to be multi-potent, possessing the capacity to give rise to multiple cell types within the tissue 6. For example, rodent hair follicle stem cells can generate epidermis, sebaceous glands, and hair follicles 7-9. We have shown that stem cells from the human hair follicle bulge region exhibit multi-potentiality 10.
Stem cells have become a valuable tool in biomedical research, due to their utility as an in vitro system for studying developmental biology, differentiation, tumorigenesis and for their possible therapeutic utility. It is likely that adult epithelial stem cells will be useful in the treatment of diseases such as ectodermal dysplasias, monilethrix, Netherton syndrome, Menkes disease, hereditary epidermolysis bullosa and alopecias 11-13. Additionally, other skin problems such as burn wounds, chronic wounds and ulcers will benefit from stem cell related therapies 14,15. Given the potential for reprogramming of adult cells into a pluripotent state (iPS cells)16,17, the readily accessible and expandable adult stem cells in human skin may provide a valuable source of cells for induction and downstream therapy for a wide range of disease including diabetes and Parkinson's disease.

A rapid, robust way of isolating viable adult epithelial stem cells from human skin is described. The method utilizes enzymatic digestion of skin collagen matrix , followed by plucking of hair follicles and isolation of single cell suspensions or tissue fragments for cell culture.

The homeostasis of all self-renewing tissues is dependent on adult stem cells. As undifferentiated stem cells undergo asymmetric divisions, they generate ...

Biology

Selecting and Isolating Colonies of Human Induced Pluripotent Stem Cells Reprogrammed from Adult Fibroblasts

Herein we present a protocol of reprogramming human adult fibroblasts into human induced pluripotent stem cells (hiPSC) using retroviral vectors encoding Oct3/4, Sox2, Klf4 and c-myc (OSKM) in the presence of sodium butyrate 1-3. We used this method to reprogram late passage (&gt;p10) human adult fibroblasts derived from Friedreich's ataxia patient (GM03665, Coriell Repository). The reprogramming approach includes highly efficient transduction protocol using repetitive centrifugation of fibroblasts in the presence of virus-containing media. The reprogrammed hiPSC colonies were identified using live immunostaining for Tra-1-81, a surface marker of pluripotent cells, separated from non-reprogrammed fibroblasts and manually passaged 4,5. These hiPSC were then transferred to Matrigel plates and grown in feeder-free conditions, directly from the reprogramming plate. Starting from the first passage, hiPSC colonies demonstrate characteristic hES-like morphology. Using this protocol more than 70% of selected colonies can be successfully expanded and established into cell lines. The established hiPSC lines displayed characteristic pluripotency markers including surface markers TRA-1-60 and SSEA-4, as well as nuclear markers Oct3/4, Sox2 and Nanog. 
The protocol presented here has been established and tested using adult fibroblasts obtained from Friedreich's ataxia patients and control individuals 6, human newborn fibroblasts, as well as human keratinocytes.

We present a protocol for efficient reprogramming of human somatic cells into human induced pluripotent stem cells (hiPSC) using retroviral vectors encoding Oct3/4, Sox2, Klf4 and c-myc (OSKM) and identification of correctly reprogrammed hiPSC by live staining with Tra-1-81 antibody.

Herein we present a protocol of reprogramming human adult fibroblasts into human induced pluripotent stem cells (hiPSC) using retroviral vectors encoding ...

Na&iuml;ve Adult Stem Cells Isolation from Primary Human Fibroblast Cultures

Over the last decade, several adult stem cell populations have been identified in human skin 1-4. The isolation of multipotent adult dermal precursors was first reported by Miller F. D laboratory 5, 6. These early studies described a multipotent precursor cell population from adult mammalian dermis 5. These cells--termed SKPs, for skin-derived precursors-- were isolated and expanded from rodent and human skin and differentiated into both neural and mesodermal progeny, including cell types never found in skin, such as neurons 5. Immunocytochemical studies on cultured SKPs revealed that cells expressed vimentin and nestin, an intermediate filament protein expressed in neural and skeletal muscle precursors, in addition to fibronectin and multipotent stem cell markers 6. Until now, the adult stem cells population SKPs have been isolated from freshly collected mammalian skin biopsies.
Recently, we have established and reported that a population of skin derived precursor cells could remain present in primary fibroblast cultures established from skin biopsies 7. The assumption that a few somatic stem cells might reside in primary fibroblast cultures at early population doublings was based upon the following observations: (1) SKPs and primary fibroblast cultures are derived from the dermis, and therefore a small number of SKP cells could remain present in primary dermal fibroblast cultures and (2) primary fibroblast cultures grown from frozen aliquots that have been subjected to unfavorable temperature during storage or transfer contained a small number of cells that remained viable 7. These rare cells were able to expand and could be passaged several times. This observation suggested that a small number of cells with high proliferation potency and resistance to stress were present in human fibroblast cultures 7.
We took advantage of these findings to establish a protocol for rapid isolation of adult stem cells from primary fibroblast cultures that are readily available from tissue banks around the world (Figure 1). This method has important significance as it allows the isolation of precursor cells when skin samples are not accessible while fibroblast cultures may be available from tissue banks, thus, opening new opportunities to dissect the molecular mechanisms underlying rare genetic diseases as well as modeling diseases in a dish.

Na&#239;ve Adult Stem Cells Isolation from Primary Human Fibroblast Cultures

We report a method to isolate na&#239;ve multipotent skin-derived precursor (SKP) cells from primary human fibroblast cultures. We show that these SKPs derived from fibroblast cultures share similar stem cell properties to the ones derived directly from human skin biopsies. These cells express the neural crest marker, nestin, in addition to the multipotent markers such as OCT4 and Nanog.

Over the last decade, several adult stem cell  populations have been identified in human skin 1-4.  The isolation of multipotent adult dermal precursors ...