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 border="1" fo:keep-together.within-page="1" fo:keep-with-next.within-page="always">
	<tbody>
		<tr>
			<td>10 mL serological pipet</td>
			<td>Falcon</td>
			<td>357551</td>
			<td>Sterile, polystyrene</td>
		</tr>
		<tr>
			<td>100 mm plates</td>
			<td>Falcon</td>
			<td>351029</td>
			<td>Treated, sterile cell culture dish</td>
		</tr>
		<tr>
			<td>15 mL sterile tubes</td>
			<td>Falcon</td>
			<td>352097</td>
			<td>Centrifuge sterile tubes, polypropylene</td>
		</tr>
		<tr>
			<td>24-well plates</td>
			<td>Falcon</td>
			<td>353935</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>357525</td>
			<td>Sterile, polystyrene</td>
		</tr>
		<tr>
			<td>35 mm plates</td>
			<td>Falcon</td>
			<td>353001</td>
			<td>Treated, sterile cell culture dish</td>
		</tr>
		<tr>
			<td>5 mL serological pipet</td>
			<td>Falcon</td>
			<td>357543</td>
			<td>Sterile, polystyrene</td>
		</tr>
		<tr>
			<td>50 mL sterile tubes</td>
			<td>Falcon</td>
			<td>352098</td>
			<td>Centrifuge sterile tubes, polypropylene</td>
		</tr>
		<tr>
			<td>Advanced DMEM (Dulbecco&#39;s Modified Eagle Medium)</td>
			<td>Gibco</td>
			<td>12491-015</td>
			<td>Store at 2-8 &#176;C; avoid exposure to light</td>
		</tr>
		<tr>
			<td>DMEM (Dulbecco&#39;s Modified Eagle Medium)</td>
			<td>Sigma- Aldrich</td>
			<td>D6429-500ml</td>
			<td>Store at 2-8 &#176;C; avoid exposure to light</td>
		</tr>
		<tr>
			<td>Fetal Bovine Serum</td>
			<td>Sigma- Aldrich</td>
			<td>F9665-500ml</td>
			<td>Store at -20 &#176;C. The serum should be aliquoted into smaller working volumes to avoid repeated freeze/thaw cycles</td>
		</tr>
		<tr>
			<td>Hank&#39;s Balanced Salt Solution</td>
			<td>Sigma- Aldrich</td>
			<td>H1387-1L</td>
			<td>Powder</td>
		</tr>
		<tr>
			<td>L-glutamine</td>
			<td>Lonza</td>
			<td>BE17-605E</td>
			<td>Store at -20 &#176;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>13778-030</td>
			<td>Synthetic siRNA Transfection Reagent; store at 2-8 &#176;C</td>
		</tr>
		<tr>
			<td>Matrigel</td>
			<td>CORNING</td>
			<td>354234</td>
			<td>Basement Membrane Matrix, store at -20 &#176;C. Avoid multiple freeze-thaws.</td>
		</tr>
		<tr>
			<td>Neubauer Chamber</td>
			<td>VWR</td>
			<td>631-1116</td>
			<td>Hemocytometer</td>
		</tr>
		<tr>
			<td>NutriStem XF Culture Medium</td>
			<td>Biological Industries</td>
			<td>05-100-1A-500ml</td>
			<td>Xeno-free, serum-free, low growth factor human ESC/iPSC culture medium. Store at -20 &#176;C. Upon thawing, the medium may be stored at 2-8 &#176;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>31985-062-100ml</td>
			<td>Reduced-Serum Medium; store at 2-8 &#176;C; avoid exposure to light</td>
		</tr>
		<tr>
			<td>Penicillin and Streptomycin</td>
			<td>Sigma- Aldrich</td>
			<td>P4333-100ml</td>
			<td>Store at -20 &#176;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>P9333</td>
			<td>Powder</td>
		</tr>
		<tr>
			<td>Potassium Phosphate Monobasic</td>
			<td>Sigma- Aldrich</td>
			<td>P5665</td>
			<td>Powder</td>
		</tr>
		<tr>
			<td>RNase-free 0.5 mL tubes</td>
			<td>Eppendorf</td>
			<td>H0030124537</td>
			<td>RNase-free sterile, microfuge tubes, polypropylene</td>
		</tr>
		<tr>
			<td>RNase-free 1.5 mL tubes</td>
			<td>Eppendorf</td>
			<td>H0030120086</td>
			<td>RNase-free sterile, microfuge tubes, polypropylene</td>
		</tr>
		<tr>
			<td>RNaseZAP</td>
			<td>INVITROGEN</td>
			<td>AM9780</td>
			<td>Cleaning agent for removing RNase</td>
		</tr>
		<tr>
			<td>Sodium Bicarbonate</td>
			<td>Sigma- Aldrich</td>
			<td>S5761</td>
			<td>Powder</td>
		</tr>
		<tr>
			<td>Sodium Chloride</td>
			<td>Sigma- Aldrich</td>
			<td>S7653</td>
			<td>Powder</td>
		</tr>
		<tr>
			<td>Sodium Phosphate Dibasic</td>
			<td>Sigma- Aldrich</td>
			<td>94046</td>
			<td>Powder</td>
		</tr>
		<tr>
			<td>StemRNA 3rd Gen Reprogramming Kit</td>
			<td>Reprocell</td>
			<td>00-0076</td>
			<td>Third Generation NM-RNAs-based Reprogramming Kit for Cellular Reprogramming of Fibroblasts, Blood, and Urine. Store at or below -70 &#176;C.</td>
		</tr>
		<tr>
			<td>Trypsin-EDTA</td>
			<td>Sigma- Aldrich</td>
			<td>T4049-100ml</td>
			<td>Store at -20 &#176;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

Research

JoVE Journal

Bioengineering

10.2K 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

Development, Expansion, and In vivo Monitoring of Human NK Cells from Human Embryonic Stem Cells (hESCs) and Induced Pluripotent Stem Cells (iPSCs)

We present a method for deriving natural killer (NK) cells from undifferentiated hESCs and iPSCs using a feeder-free approach. This method gives rise to high levels of NK cells after 4 weeks culture and can undergo further 2-log expansion with artificial antigen presenting cells. hESC- and iPSC-derived NK cells developed in this system have a mature phenotype and function. The production of large numbers of genetically modifiable NK cells is applicable for both basic mechanistic as well as anti-tumor studies. Expression of firefly luciferase in hESC-derived NK cells allows a non-invasive approach to follow NK cell engraftment, distribution, and function. We also describe a dual-imaging scheme that allows separate monitoring of two different cell populations to more distinctly characterize their interactions in vivo. This method of derivation, expansion, and dual in vivo imaging provides a reliable approach for producing NK cells and their evaluation which is necessary to improve current NK cell adoptive therapies.

Development, Expansion, and In vivo Monitoring of Human NK Cells from Human Embryonic Stem Cells hESCs and Induced Pluripotent Stem Cells iPSCs

This protocol describes the development, expansion, and in vivo imaging of NK cells derived from hESCs and iPSCs.

We present a method for deriving natural killer (NK) cells from undifferentiated hESCs and iPSCs using a feeder-free approach. This method gives rise to ...

Transplantation of Induced Pluripotent Stem Cell-derived Mesoangioblast-like Myogenic Progenitors in Mouse Models of Muscle Regeneration

Patient-derived iPSCs could be an invaluable source of cells for future autologous cell therapy protocols. iPSC-derived myogenic stem/progenitor cells similar to pericyte-derived mesoangioblasts (iPSC-derived mesoangioblast-like stem/progenitor cells: IDEMs) can be established from iPSCs generated from patients affected by different forms of muscular dystrophy. Patient-specific IDEMs can be genetically corrected with different strategies (e.g. lentiviral vectors, human artificial chromosomes) and enhanced in their myogenic differentiation potential upon overexpression of the myogenesis regulator MyoD. This myogenic potential is then assessed in vitro with specific differentiation assays and analyzed by immunofluorescence. The regenerative potential of IDEMs is further evaluated in vivo, upon intramuscular and intra-arterial transplantation in two representative mouse models displaying acute and chronic muscle regeneration. The contribution of IDEMs to the host skeletal muscle is then confirmed by different functional tests in transplanted mice. In particular, the amelioration of the motor capacity of the animals is studied with treadmill tests. Cell engraftment and differentiation are then assessed by a number of histological and immunofluorescence assays on transplanted muscles. Overall, this paper describes the assays and tools currently utilized to evaluate the differentiation capacity of IDEMs, focusing on the transplantation methods and subsequent outcome measures to analyze the efficacy of cell transplantation.

Induced pluripotent stem cell (iPSC)-derived myogenic progenitors are promising candidates for cell therapy strategies to treat muscular dystrophies. This protocol describes transplantation and functional measurements required to evaluate the engraftment and differentiation of iPSC-derived mesoangioblasts (a type of muscle progenitors) in mouse models of acute and chronic muscle regeneration.

Patient-derived iPSCs could be an invaluable source of cells for future autologous cell therapy protocols. iPSC-derived myogenic stem/progenitor cells ...

Efficient Generation and Editing of Feeder-free IPSCs from Human Pancreatic Cells Using the CRISPR-Cas9 System

Embryonic and induced pluripotent stem cells can self-renew and differentiate into multiple cell types of the body. The pluripotent cells are thus coveted for research in regenerative medicine and are currently in clinical trials for eye diseases, diabetes, heart diseases, and other disorders. The potential to differentiate into specialized cell types coupled with the recent advances in genome editing technologies including the CRISPR/Cas system have provided additional opportunities for tailoring the genome of iPSC for varied applications including disease modeling, gene therapy, and biasing pathways of differentiation, to name a few. Among the available editing technologies, the CRISPR/Cas9 from Streptococcus pyogenes has emerged as a tool of choice for site-specific editing of the eukaryotic genome. The CRISPRs are easily accessible, inexpensive, and highly efficient in engineering targeted edits. The system requires a Cas9 nuclease and a guide sequence (20-mer) specific to the genomic target abutting a 3-nucleotide &#34;NGG&#34; protospacer-adjacent-motif (PAM) for targeting Cas9 to the desired genomic locus, alongside a universal Cas9 binding tracer RNA (together called single guide RNA or sgRNA). Here we present a step-by-step protocol for efficient generation of feeder-independent and footprint-free iPSC and describe methodologies for genome editing of iPSC using the Cas9 ribonucleoprotein (RNP) complexes. The genome editing protocol is effective and can be easily multiplexed by pre-complexing sgRNAs for more than one target with the Cas9 protein and simultaneously delivering into the cells. Finally, we describe a simplified approach for identification and characterization of iPSCs with desired edits. Taken together, the outlined strategies are expected to streamline generation and editing of iPSC for manifold applications.

This protocol describes in detail the generation of footprint-free induced pluripotent stem cells (iPSCs) from human pancreatic cells in feeder-free conditions, followed by editing using CRISPR/Cas9 ribonucleoproteins and characterization of the modified single-cell clones.

Embryonic and induced pluripotent stem cells can self-renew and differentiate into multiple cell types of the body. The pluripotent cells are thus coveted ...

Scalable Generation of Mature Cerebellar Organoids from Human Pluripotent Stem Cells and Characterization by Immunostaining

The cerebellum plays a critical role in the maintenance of balance and motor coordination, and a functional defect in different cerebellar neurons can trigger cerebellar dysfunction. Most of the current knowledge about disease-related neuronal phenotypes is based on postmortem tissues, which makes understanding of disease progression and development difficult. Animal models and immortalized cell lines have also been used as models for neurodegenerative disorders. However, they do not fully recapitulate human disease. Human induced pluripotent stem cells (iPSCs) have great potential for disease modeling and provide a valuable source for regenerative approaches. In recent years, the generation of cerebral organoids from patient-derived iPSCs improved the prospects for neurodegenerative disease modeling. However, protocols that produce large numbers of organoids and a high yield of mature neurons in 3D culture systems are lacking. The protocol presented is a new approach for reproducible and scalable generation of human iPSC-derived organoids under chemically-defined conditions using scalable single-use bioreactors, in which organoids acquire cerebellar identity. The generated organoids are characterized by the expression of specific markers at both mRNA and protein level. The analysis of specific groups of proteins allows the detection of different cerebellar cell populations, whose localization is important for the evaluation of organoid structure. Organoid cryosectioning and further immunostaining of organoid slices are used to evaluate the presence of specific cerebellar cell populations and their spatial organization.

This protocol describes a dynamic culture system to produce controlled size aggregates of human pluripotent stem cells and further stimulate differentiation in cerebellar organoids under chemically-defined and feeder-free conditions using a single-use bioreactor.

The cerebellum plays a critical role in the maintenance of balance and motor coordination, and a functional defect in different cerebellar neurons can ...

Guided Differentiation of Mature Kidney Podocytes from Human Induced Pluripotent Stem Cells Under Chemically Defined Conditions

Kidney disease affects more than 10% of the global population and costs billions of dollars in federal expenditures. The most severe forms of kidney disease and eventual end-stage renal failure are often caused by the damage to the glomerular podocytes, which are the highly specialized epithelial cells that function together with endothelial cells and the glomerular basement membrane to form the kidney&#8217;s filtration barrier. Advances in renal medicine have been hindered by the limited availability of primary tissues and the lack of robust methods for the derivation of functional human kidney cells, such as podocytes. The ability to derive podocytes from renewable sources, such as stem cells, could help advance current understanding of the mechanisms of human kidney development and disease, as well as provide new tools for therapeutic discovery. The goal of this protocol was to develop a method to derive mature, post-mitotic podocytes from human induced pluripotent stem (hiPS) cells with high efficiency and specificity, and under chemically defined conditions. The hiPS cell-derived podocytes produced by this method express lineage-specific markers (including nephrin, podocin, and Wilm&#8217;s Tumor 1) and exhibit the specialized morphological characteristics (including primary and secondary foot processes) associated with mature and functional podocytes. Intriguingly, these specialized features are notably absent in the immortalized podocyte cell line widely used in the field, which suggests that the protocol described herein produces human kidney podocytes that have a developmentally more mature phenotype than the existing podocyte cell lines typically used to study human kidney biology.

Presented here is a chemically defined protocol for the derivation of human kidney podocytes from induced pluripotent stem cells with high efficiency (&#62;90%) and independent of genetic manipulations or subpopulation selection. This protocol produces the desired cell type within 26 days and could be useful for nephrotoxicity testing and disease modeling.

Kidney disease affects more than 10% of the global population and costs billions of dollars in federal expenditures. The most severe forms of kidney ...

Single-Cell Optical Action Potential Measurement in Human Induced Pluripotent Stem Cell-Derived Cardiomyocytes

Conventional intracellular microelectrode techniques to quantify cardiomyocyte electrophysiology are extremely complex, labor intensive, and typically carried out in low throughput. Rapid and ongoing expansion of induced pluripotent stem cell (iPSC) technology presents a new standard in cardiovascular research and alternate methods are now necessary to increase throughput of electrophysiological data at a single cell level. VF2.1Cl is a recently derived voltage sensitive dye which provides a rapid single channel, high magnitude response to fluctuations in membrane potential. It possesses kinetics superior to those of other existing voltage indicators and makes available functional data equivalent to that of traditional microelectrode techniques. Here, we demonstrate simplified, non-invasive action potential characterization in externally paced human iPSC derived cardiomyocytes using a modular and highly affordable photometry system.

Here we describe optical acquisition and characterization of action potentials from induced pluripotent stem cell derived cardiomyocytes using a high-speed modular photometry system.

Conventional intracellular microelectrode techniques to quantify cardiomyocyte electrophysiology are extremely complex, labor intensive, and typically ...

Isogenic Kidney Glomerulus Chip Engineered from Human Induced Pluripotent Stem Cells

Chronic kidney disease (CKD) affects 15% of the U.S. adult population, but the establishment of targeted therapies has been limited by the lack of functional models that can accurately predict human biological responses and nephrotoxicity. Advancements in kidney precision medicine could help overcome these limitations. However, previously established in vitro models of the human kidney glomerulus-the primary site for blood filtration and a key target of many diseases and drug toxicities-typically employ heterogeneous cell populations with limited functional characteristics and unmatched genetic backgrounds. These characteristics significantly limit their application for patient-specific disease modeling and therapeutic discovery. 
This paper presents a protocol that integrates human induced pluripotent stem (iPS) cell-derived glomerular epithelium (podocytes) and vascular endothelium from a single patient to engineer an isogenic and vascularized microfluidic kidney glomerulus chip. The resulting glomerulus chip is comprised of stem cell-derived endothelial and epithelial cell layers that express lineage-specific markers, produce basement membrane proteins, and form a tissue-tissue interface resembling the kidney's glomerular filtration barrier. The engineered glomerulus chip selectively filters molecules and recapitulates drug-induced kidney injury. The ability to reconstitute the structure and function of the kidney glomerulus using isogenic cell types creates the opportunity to model kidney disease with patient specificity and advance the utility of organs-on-chips for kidney precision medicine and related applications.

Presented here is a protocol to engineer a personalized organ-on-a-chip system that recapitulates the structure and function of the kidney glomerular filtration barrier by integrating genetically matched epithelial and vascular endothelial cells differentiated from human induced pluripotent stem cells. This bioengineered system can advance kidney precision medicine and related applications.

Chronic kidney disease (CKD) affects 15% of the U.S. adult population, but the establishment of targeted therapies has been limited by the lack of ...

Generation of Human Blood Vessel Organoids from Pluripotent Stem Cells

An organoid is defined as an engineered multicellular in vitro tissue that mimics its corresponding in vivo organ such that it can be used to study defined aspects of that organ in a tissue culture dish. The breadth and application of human pluripotent stem cell (hPSC)-derived organoid research have advanced significantly to include the brain, retina, tear duct, heart, lung, intestine, pancreas, kidney, and blood vessels, among several other tissues. The development of methods for the generation of human microvessels, specifically, has opened the way for modeling human blood vessel development and disease in vitro and for the testing and analysis of new drugs or tissue tropism in virus infections, including SARS-CoV-2. Complex and lengthy protocols lacking visual guidance hamper the reproducibility of many stem cell-derived organoids. Additionally, the inherent stochasticity of organoid formation processes and self-organization necessitates the generation of optical protocols to advance the understanding of cell fate acquisition and programming. Here, a visually guided protocol is presented for the generation of 3D human blood vessel organoids (BVOs) engineered from hPSCs. Presenting a continuous basement membrane, vascular endothelial cells, and organized articulation with mural cells, BVOs exhibit the functional, morphological, and molecular features of human microvasculature. BVO formation is initiated through aggregate formation, followed by mesoderm and vascular induction. Vascular maturation and network formation are initiated and supported by embedding aggregates in a 3D collagen and solubilized basement membrane matrix. Human vessel networks form within 2-3 weeks and can be further grown in scalable culture systems. Importantly, BVOs transplanted into immunocompromised mice anastomose with the endogenous mouse circulation and specify into functional arteries, veins, and arterioles. The present visually guided protocol will advance human organoid research, particularly in relation to blood vessels in normal development, tissue vascularization, and disease.

This protocol describes the generation of self-organizing blood vessels from human pluripotent and induced pluripotent stem cells. These blood vessel networks exhibit an extensive and connected endothelial network surrounded by pericytes, smooth muscle actin, and a continuous basement membrane.

An organoid is defined as an engineered multicellular in vitro tissue that mimics its corresponding in vivo organ such that it can be used to study ...

Generation, High-Throughput Screening, and Biobanking of Human-Induced Pluripotent Stem Cell-Derived Cardiac Spheroids

Human-induced pluripotent stem cell-derived cardiomyocytes (hiPSC-CMs) are of paramount importance for human cardiac disease modeling and therapeutics. We recently published a cost-effective strategy for the massive expansion of hiPSC-CMs in two dimensions (2D). Two major limitations are cell immaturity and a lack of three-dimensional (3D) arrangement and scalability in high-throughput screening (HTS) platforms. To overcome these limitations, the expanded cardiomyocytes form an ideal cell source for the generation of 3D cardiac cell culture and tissue engineering techniques. The latter holds great potential in the cardiovascular field, providing more advanced and physiologically relevant HTS. Here, we describe an HTS-compatible workflow with easy scalability for the generation, maintenance, and optical analysis of cardiac spheroids (CSs) in a 96-well-format. These small CSs are essential to fill the gap present in current in vitro disease models and/or generation for 3D tissue engineering platforms. The CSs present a highly structured morphology, size, and cellular composition. Furthermore, hiPSC-CMs cultured as CSs display increased maturation and several functional features of the human heart, such as spontaneous calcium handling and contractile activity. By automatization of the complete workflow, from the generation of CSs to functional analysis, we increase intra- and inter-batch reproducibility as demonstrated by high-throughput (HT) imaging and calcium handling analysis. The described protocol allows modeling of cardiac diseases and assessing drug/therapeutic effects at the single-cell level within a complex 3D cell environment in a fully automated HTS workflow. In addition, the study describes a straightforward procedure for long-term preservation and biobanking of whole-spheroids, thereby providing researchers the opportunity to create next-generation functional tissue storage. HTS combined with long-term storage will substantially contribute to translational research in a wide range of areas, including drug discovery and testing, regenerative medicine, and the development of personalized therapies.

Presented here is a set of protocols for the generation and cryopreservation of cardiac spheroids (CSs) from human-induced pluripotent stem cell-derived cardiomyocytes cultured in a high-throughput, multidimensional format. This three-dimensional model functions as a robust platform for disease modeling, high-throughput screenings, and maintains its functionality after cryopreservation.

Human-induced pluripotent stem cell-derived cardiomyocytes (hiPSC-CMs) are of paramount importance for human cardiac disease modeling and therapeutics. We ...

Differentiation of Human Induced Pluripotent Stem Cells to Brain Microvascular Endothelial Cell-Like Cells with a Mature Immune Phenotype

Blood-brain barrier (BBB) dysfunction is a pathological hallmark of many neurodegenerative and neuroinflammatory diseases affecting the central nervous system (CNS). Due to the limited access to disease-related BBB samples, it is still not well understood whether BBB malfunction is causative for disease development or rather a consequence of the neuroinflammatory or neurodegenerative process. Human induced pluripotent stem cells (hiPSCs) therefore provide a novel opportunity to establish in vitro BBB models from healthy donors and patients, and thus to study disease-specific BBB characteristics from individual patients. Several differentiation protocols have been established for deriving brain microvascular endothelial cell (BMEC)-like cells from hiPSCs. Consideration of the specific research question is mandatory for the correct choice of the respective BMEC-differentiation protocol. Here, we describe the extended endothelial cell culture method (EECM), which is optimized to differentiate hiPSCs into BMEC-like cells with a mature immune phenotype, allowing the study of immune cell-BBB interactions. In this protocol, hiPSCs are first differentiated into endothelial progenitor cells (EPCs) by activating Wnt/&#946;-catenin signaling. The resulting culture, which contains smooth muscle-like cells (SMLCs), is then sequentially passaged to increase the purity of endothelial cells (ECs) and to induce BBB-specific properties. Co-culture of EECM-BMECs with these SMLCs or conditioned medium from SMLCs allows for the reproducible, constitutive, and cytokine-regulated expression of EC adhesion molecules. Importantly, EECM-BMEC-like cells establish barrier properties comparable to primary human BMECs, and due to their expression of all EC adhesion molecules, EECM-BMEC-like cells are different from other hiPSC-derived in vitro BBB models. EECM-BMEC-like cells are thus the model of choice for investigating the potential impact of disease processes at the level of the BBB, with an impact on immune cell interaction in a personalized fashion.

Here, we describe a protocol, the extended endothelial cell culture method (EECM), that allows differentiation of pluripotent stem cells to brain microvascular endothelial cell (BMEC)-like cells. These cells show endothelial cell adhesion molecule expression and are thus a human blood-brain barrier model suitable to study immune cell interactions in vitro.

Blood-brain barrier (BBB) dysfunction is a pathological hallmark of many neurodegenerative and neuroinflammatory diseases affecting the central nervous ...