Name: isolation of adult human dermal fibroblasts from abdominal skin and generation of induced pluripotent stem cells using a non-integrating method
Uploaded: 2020-01-19T16:00:00
Description: 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

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 m...

<ol>
	<li>Lo, B., Parham, L. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Ethical+Issues+in+Stem+Cell+Research.">Ethical Issues in Stem Cell Research.</a> Endocrine Reviews. 30 (3), 204-213 (2009).</li><li>Wong, W. T., Sayed, N., Cooke, J. P. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Induced+Pluripotent+Stem+Cells:+How+They+Will+Change+the+Practice+of+Cardiovascular+Medicine.">Induced Pluripotent Stem Cells: How They Will Change the Practice of Cardiovascular Medicine.</a> Methodist Debakey Cardiovasc Journal. 9 (4), 206-209 (2013).</li><li>Singh, V. K., Kalsan, M., Kumar, N., Saini, A., Chandra, R. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Induced+pluripotent+stem+cells:+applications+in+regenerative+medicine,+disease+modeling,+and+drug+discovery.">Induced pluripotent stem cells: applications in regenerative medicine, disease modeling, and drug discovery.</a> Frontiers in Cell and Developmental Biology. 3, 2 (2015).</li><li>Zacharias, D. G., Nelson, T. J., Mueller, P. S., Hook, C. C. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=The+science+and+ethics+of+induced+pluripotency:+what+will+become+of+embryonic+stem+cells.">The science and ethics of induced pluripotency: what will become of embryonic stem cells.</a> Mayo Clinic Proceedings. 86 (7), 634-640 (2011).</li><li>Takahashi, K., Yamanaka, S. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Induction+of+pluripotent+stem+cells+from+mouse+embryonic+and+adult+fibroblast+cultures+by+defined+factors.">Induction of pluripotent stem cells from mouse embryonic and adult fibroblast cultures by defined factors.</a> Cell. 126 (4), 663-676 (2006).</li><li>Yu, J., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Induced+pluripotent+stem+cell+lines+derived+from+human+somatic+cells.">Induced pluripotent stem cell lines derived from human somatic cells.</a> Science. 318 (5858), 1917-1920 (2007).</li><li>Yu, Y., Wang, X., Scott, L., Nyberg, S. L. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Application+of+Induced+Pluripotent+Stem+Cells+in+Liver+Diseases.">Application of Induced Pluripotent Stem Cells in Liver Diseases.</a> Journal Cell Medicine. 3 (3), 997-1017 (2014).</li><li>Ebert, A. D., Liang, P., Wu, J. C. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Induced+pluripotent+stem+cells+as+a+disease+modeling+and+drug+screening+platform.">Induced pluripotent stem cells as a disease modeling and drug screening platform.</a> Journal Cardiovascular Pharmacology. 60 (4), 408-416 (2012).</li><li>Gupta, S., Sharma, V., Verma, R. S. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Recent+advances+in+induced+pluripotent+stem+cell+(ipsc)+based+therapeutics.">Recent advances in induced pluripotent stem cell (ipsc) based therapeutics.</a> Journal of Stem Cell Research and Therapy. 3 (3), 263-270 (2017).</li><li>Andargie, A., Tadesse, F., Shibbiru, T. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Review+on+Cell+Reprogramming:+Methods+and+Applications.">Review on Cell Reprogramming: Methods and Applications.</a> Journal of Medicine, Physiology and Biophysics. 25, 2422 (2016).</li><li>Warren, L., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Highly+efficient+reprogramming+to+pluripotency+and+directed+differentiation+of+human+cells+using+synthetic+modified+mRNA.">Highly efficient reprogramming to pluripotency and directed differentiation of human cells using synthetic modified mRNA.</a> Cell Stem Cell. 7 (5), 618-630 (2010).</li><li>Malik, N., Rao, M. R. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=A+Review+of+the+Methods+for+Human+iPSC+Derivation.">A Review of the Methods for Human iPSC Derivation.</a> Methods in Molecular Biology. 997, 23-33 (2013).</li><li>Poleganov, M. A., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Efficient+Reprogramming+of+Human+Fibroblasts+and+Blood-Derived+Endothelial+Progenitor+Cells+Using+Nonmodified+RNA+for+Reprogramming+and+Immune+Evasion.">Efficient Reprogramming of Human Fibroblasts and Blood-Derived Endothelial Progenitor Cells Using Nonmodified RNA for Reprogramming and Immune Evasion.</a> Human Gene Therapy. 26 (11), 751-766 (2015).</li><li>Brouwer, M., Zhou, H., Kasri, N. N. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Choices+for+Induction+of+Pluripotency:+Recent+Developments+in+Human+Induced+Pluripotent+Stem+Cell+Reprogramming+Strategies.">Choices for Induction of Pluripotency: Recent Developments in Human Induced Pluripotent Stem Cell Reprogramming Strategies.</a> Stem Cell Reviews and Reports. 12, 54-72 (2016).</li><li>Revilla, A., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Current+advances+in+the+generation+of+human+iPS+cells:+implications+in+cell-based+regenerative+medicine.">Current advances in the generation of human iPS cells: implications in cell-based regenerative medicine.</a> Journal of Tissue Engineering and Regenerative Medicine. 10, 893-907 (2016).</li><li>Lee, Y. S., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Exosome-Mediated+Ultra-Effective+Direct+Conversion+of+Human+Fibroblasts+into+Neural+Progenitor-like+Cells.">Exosome-Mediated Ultra-Effective Direct Conversion of Human Fibroblasts into Neural Progenitor-like Cells.</a> ACS Nano. 12 (3), 2531-2538 (2018).</li><li>Bayart, E., Cohen-Haguenauer, O. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Technological+overview+of+iPS+induction+from+human+adult+somatic+cells.">Technological overview of iPS induction from human adult somatic cells.</a> Current Gene Therapy. 13 (2), 73-92 (2013).</li><li>Sacco, A. M., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Diversity+of+dermal+fibroblasts+as+major+determinant+of+variability+in+cell+reprogramming.">Diversity of dermal fibroblasts as major determinant of variability in cell reprogramming.</a> Journal of Cellular and Molecular Medicine. 23 (6), 4256-4268 (2019).</li><li>Shan, Z. Y., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Continuous+passages+accelerate+the+reprogramming+of+mouse+induced+pluripotent+stem+cells.">Continuous passages accelerate the reprogramming of mouse induced pluripotent stem cells.</a> Cellular Reprogramming. 16 (1), 77-83 (2014).</li><li>Schlaeger, T. M., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=A+comparison+of+non-integrating+reprogramming+methods.">A comparison of non-integrating reprogramming methods.</a> Nature Biotechnology. 33 (1), 58-63 (2015).</li><li>Drews, K., Tavernier, G., Demeester, J., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=The+cytotoxic+and+immunogenic+hurdles+associated+with+non-viral+mRNA-mediated+reprogramming+of+human+fibroblasts.">The cytotoxic and immunogenic hurdles associated with non-viral mRNA-mediated reprogramming of human fibroblasts.</a> Biomaterials. 33, 4059-4068 (2012).</li><li>Beissert, T., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Improvement+of+In+Vivo+Expression+of+Genes+Delivered+by+Self-Amplifying+RNA+Using+Vaccinia+Virus+Immune+Evasion+Proteins.">Improvement of In Vivo Expression of Genes Delivered by Self-Amplifying RNA Using Vaccinia Virus Immune Evasion Proteins.</a> Human Gene Therapy. 28 (12), 1138-1146 (2017).</li><li>Mathieu, J., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Hypoxia+induces+re-entry+of+committed+cells+into+pluripotency.">Hypoxia induces re-entry of committed cells into pluripotency.</a> Stem Cells. 31 (9), 1737-1748 (2013).</li><li>Wang, Y., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Hypoxia+Enhances+Direct+Reprogramming+of+Mouse+Fibroblasts+to+Cardiomyocyte-Like+Cells.">Hypoxia Enhances Direct Reprogramming of Mouse Fibroblasts to Cardiomyocyte-Like Cells.</a> Cellular Reprogramming. 18 (1), 1-7 (2016).</li><li>Deng, Y., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Hypoxia+enhances+buffalo+adipose-derived+mesenchymal+stem+cells+proliferation,+stemness,+and+reprogramming+into+induced+pluripotent+stem+cells.">Hypoxia enhances buffalo adipose-derived mesenchymal stem cells proliferation, stemness, and reprogramming into induced pluripotent stem cells.</a> Journal of Cellular Physiology. 234 (10), 17254-17268 (2019).</li><li>Karagiannis, P., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Induced+Pluripotent+Stem+Cells+and+Their+Use+in+Human+Models+of+Disease+and+Development.">Induced Pluripotent Stem Cells and Their Use in Human Models of Disease and Development.</a> Physiological Reviews. 99 (1), 79-114 (2019).</li><li>Maherali, N., Hochedlinger, K. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Guidelines+and+techniques+for+the+generation+of+induced+pluripotent+stem+cells.">Guidelines and techniques for the generation of induced pluripotent stem cells.</a> Cell Stem Cell. 3 (6), 595-605 (2008).</li><li>Asprer, J. S., Lakshmipathy, U. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Current+methods+and+challenges+in+the+comprehensive+characterization+of+human+pluripotent+stem+cells.">Current methods and challenges in the comprehensive characterization of human pluripotent stem cells.</a> Stem Cell Reviews and Reports. 11 (2), 357-372 (2015).</li></ol>

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...

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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 ...

The following protocol provides instructions to generate autologous human-induced pluripotent stem cells through the fast and efficient reprogramming of human dermal fibroblasts isolated from a skin punch biopsy. The procedures can be carried out by researchers with little or no experience in cell reprogramming and with proper adjustment, allows the product of xeno-free induced pluripotent stem cells. Autologous IPS cells might be used for regenerative medicine applications and for modeling diseases like genetic disorders or cancer to investigate on underlying molecular mechanisms and develop more specific therapies.The workload is high. It is extremely helpful to study the protocol prior to carefully plan the experiment, including the steps during which reagents and materials should be prepared. The visual demonstration of the procedure helps the researches with little or no experience perform the cell isolation and the reprogramming, avoiding common mistakes related to possible microbial and RNA contamination.Under a sterile hood, wash the freshly obtained sample of human skin from the abdomen in a 100 millimeter dish with HPSS solution. Remove hair and fat using fine forceps and scissors and dissect it with a scalpel to obtain fragments of the size of two millimeters by one millimeters, avoiding scars, dirty or burned areas of the tissue. Place four small fragments in each 35 millimeter dish, cover with a sterile cover glass and add 1.5 millimeters of I-DMEM.Incubate the plates at 37 degrees Celsius and 5%carbon dioxide for about 15 days or until cells reach 85%confluence. Change the culture medium every three days and check the outgrowth of cells using an inverted phase contrast microscope daily. Now, lift the cover glasses with fine forceps, place them upside down in a 100 millimeter dish and wash with 1X sterile PBS.Remove the fragments of the samples and discard them. Wash plates with 1X sterile PBS. Remove the 1X PBS and add three millimeters of Trypsin-EDTA to the cover glasses placed in the 100 millimeter dish and add one millimeter of Trypsin-EDTA to the 35 millimeter dishes as well.Incubate for five minutes at 37 degrees Celsius with 5%carbon dioxide. Then, block the trypsinization by adding five millimeters of TSS to the 100 millimeter dish and two millimeters of TSS to each 35 millimeter dish. Collect the suspension into 15 millimeter sterile tubes, centrifuge at 400 times G at four degrees Celsius for five minutes.Aspirate the supernatant and resuspend the pellet in 12 millimeters of I-DMEM. Split the cell suspension into three millimeter aliquots and transfer them each into a 60 millimeter plate. Incubate at 37 degrees Celsius with 5%carbon dioxide.Change the medium daily. Keep cells in culture until they reach a confluence of 75%After that, repeat trypsinization three times to obtain cells that passage four. Then, synchronize the cells by replacing the I-DMEM with S-DMEM and incubate for 48 hours at 37 degrees Celsius with 5%carbon dioxide.During the cell synchronization, prepare fibroblast expansion medium and total non-modified RNA reprogramming cocktail for fibroblast free programming. To simplify the preparation of the nine tubes of non-modified RNA aliquots that are then split into 36, we used to follow a scheme that we attached to the hood wall. Coat a 24-well plate by transferring 100 microliters of basement membrane matrix thawed on ice to each well to uniformly cover the bottom.Incubate for at least one hour at 37 degrees Celsius before seeding the cells. Aspirate the medium from plates with cells in culture and quickly wash in 1X PBS. Repeat trypsinization to obtain cells that passage five.Aspirate the supernatant and resuspend the pellet in an appropriate volume of fibroblast expansion medium. Prepare and clean the hemocytometer with 70%alcohol. Then, prepare a solution by gently mixing 10 microliters of trypan blue and 10 microliters of cell suspension in a 1.5 milliliter tube, and incubate for one to two minutes at room temperature.Pipette 10 microliters of the solution into the hemocytometer and place it on the stage of an inverted phase contrast microscope to count. Non-viable cells are blue while viable cells are unstained. Count the cells in at least two squares of the hemocytometer chamber.Perform a dilution to obtain a density of 2.5 times 10 to the fourth cells per 500 microliters of fibroblast expansion medium and resuspend the cell pellet. Pipette 500 microliters of the cell suspension into each well of the 24-well plate. Incubate cells overnight at 37 degrees Celsius and 5%carbon dioxide.In the morning, remove the old medium from each well and replace it with 500 microliters of pre-zarmed xeno-free factor-free culture medium. Incubate at 37%degrees Celsius under 5%carbon dioxide for at least six hours. Thaw five 15.4 microliter aliquots of total nmRNA reprogramming cocktail at room temperature then place them in ice.Add 234.6 microliters of reduced serum medium to each aliquot, gently pipette three to five times and label each one as tube A.Label five sterile RNase-free 0.5 milliliter tubes as tube B and mix six microliters of synthetic small interfering RNA transfection reagent with 244 microliters of reduced serum medium. Then, use pipettes to add the contents of each tube B to a tube A drop wise. Mix by tapping the bottom of the tube.Incubate at room temperature for 15 minutes. After that, to add 125 microliters of nmRNA transfection complex solution from each of the five aliquots to each well, tilt the plate and pipette drop wise into the medium. Mix the contents in the 20 wells by rocking gently.Use the remaining four wells as a reference. Incubate the plate for 15 hours at 37 degrees Celsius and 5%carbon dioxide. The next day, aspirate the medium.Under the sterile hood, repeat the transfection procedure, as on day one, every day for three more days. On day five, aspirate the medium and exchange with fresh, pre-warmed XF/FF Culture Medium under a sterile hood. Incubate the plate at 37 degrees Celsius and 5%carbon dioxide.Monitor the cell culture daily by a phase contrast microscope, until observing the formation of IPSC colonies. The aim of this protocol was to reprogram dermal fibroblasts isolated from abdominal skin using non-integrating reprogramming method. Human fibroblasts outgrew from samples of abdominal skin within one week of culture and reached 85%confluence within two weeks.Cells were characterized by adhesive growth on plastic culture dishes, and their morphology varied from elongated and spindle-shaped to flattened and star-shaped. Fibroblast morphology and arrangement and culture dramatically changed after seeding on BMM, when they acquired an elongated morphology and arranged to form thin branched structures. Small colonies were already visible in culture at day one from first transfection and their size grew progressively over time, while their number increased til day seven and then remained stable between day seven and day 14, probably as a result of smaller colonies merging to form larger colonies.Since the reprogramming procedure was performed using antibiotic-free media, in the case where sterile conditions were not guaranteed, microbial contamination became a major issue. Proliferation rate and the plating density impact the reprogramming efficiency, so it is important to plan cell passaging and define the plating density on the basis of cell proliferation rate. Substituting animal-derived reagents for appropriate xeno-free reagents, this protocol allows the reprogramming of adult human fibroblasts into IPS cells in a complete xeno-free culture environment that weren't their clinical use.Human cells are a potential source of infection with bloodborne pathogens, hence, personal protective equipment should be worn throughout the procedure.

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Bioengineering

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