We thank Mrs. Matsumoto for the technical assistance.

1. Cell Culture
<ol>
	<li>Obtain the cells to induce tetraploidy. To date it has been confirmed that this technique can be applied to the human fibroblast cell lines TIG-1, BJ, IMR-90 and telomerase-immortalized TIG-1 (TIG-hT).</li>
	<li>Grow cells in minimum essential medium with &#945; modification or any other cell culture medium suited for the cell type to be studied supplemented with 10% (v/v) heat-inactivated fetal bovine serum (FBS) by incubating in a 5% (v/v) CO2 atmosphere at 37 &#176;C. Passage cells every 3 or 4 days not to exceed subconfluent density. 
	NOTE: Population doubling level (PDL) should be calculated each time at passaging to evaluate cell growth and cell age. PDL can be calculated from the cell number seeded into a dish (N1) and the cell number harvested at the next passaging (N2) using the following formula (X is the initial PDL). PDL = X + (log N1 - log N2) / log 2</li>
</ol>
2. Induction and Establishment of Tetraploid Cells from Diploid Fibroblasts
<ol>
	<li>Induce tetraploidy without shake-off. 
	NOTE: Some cell strains (e.g. TIG-1) can become almost completely tetraploid by continuous treatment with demecolcine (DC) as follows.
	<ol>
		<li>Passage cells into one or two 100 mm dishes on the day before treatment to induce tetraploidy. Usually, prepare 5 x 105 to 1 x 106 cells per dish for induction of tetraploid cells.</li>
		<li>Treat exponentially growing cells with medium containing 0.1 &#181;g/mL DC for 4 days.</li>
		<li>Replace DC-containing medium with drug-free medium and incubate cells in a 5% (v/v) CO2 atmosphere at 37 &#176;C. Cells usually resume proliferation within 1 week.</li>
	</ol>
	</li>
	<li>Induce tetraploidy with shake-off. 
	NOTE: Most cells become a mixture of diploid and tetraploid populations as a result of the simple treatment with DC described above (2.1). Therefore, some modifications of the procedure to eliminate a diploid population are necessary as follows (Figure 1).
	<ol>
		<li>Passage cells into several T75 flasks the day before treatment to induce tetraploidy. Usually, prepare at least 5 x 106 cells (1 x 106 cells per flask) for induction of tetraploid cells.</li>
		<li>Treat exponentially growing cells with medium containing 0.1 &#181;g/mL DC for 16 - 18 h to arrest cells in mitosis. The time required to arrest cells in mitosis may depend on target cells and should be determined by preliminary experiments to obtain as many mitotic cells as possible. For example, slower growing cells should be treated with DC for longer time than faster growing cells. 
		NOTE: If cells are treated for too long in this step, they might undergo mitotic slippage and adhere to the dish surface, making it impossible to collect mitotic cells by shake-off in the next step.</li>
		<li>Collect DC-arrested mitotic cells by the shake-off method, in which loosely adhered mitotic cells are collected by gentle shaking of flasks and washing with medium followed by centrifugation (300 x g, 5 min). Count cell number by an appropriate method in this step.</li>
		<li>Reseed collected mitotic cells into 60 mm culture dishes and treat cells with 0.1 &#181;g/mL DC for an additional 3 days. Seed more than 1 x 106 mitotic cells per dish, because less than 10% of cells can survive this treatment.</li>
		<li>Replace DC-containing medium with drug-free medium and wait for cells to resume proliferation, which usually occurs within 1 week.</li>
	</ol>
	</li>
	<li>Passage cells similarly to original diploid cells.</li>
</ol>
3. Examination of DNA Ploidy
NOTE: This is a well-established procedure and a technical manual should be referred to for detailed procedures and technical tips.
<ol>
	<li>Harvest cells (5 x 105 - 1 x 106) by incubating cells with a solution containing 0.05% trypsin and 0.02% EDTA for 10 min at 37 &#176;C, and neutralize trypsin by medium containing 10% FBS.</li>
	<li>Centrifuge cells in medium and wash them by resuspending in 5 mL of phosphate-buffered saline (PBS) followed by centrifugation (300 x g, 5 min).</li>
	<li>Prepare cells for DNA analysis by one of the following two methods
	<ol>
		<li>A method for analyzing unfixed cells10 
		NOTE: This method can be performed using a commercially available kit for cell cycle analysis. Samples prepared with this method should be analyzed immediately, because cells are not fixed in the procedure and stained nuclei will become damaged over time.
		<ol>
			<li>Wash cells with 40 mM citrate buffer containing 250 mM sucrose. Subsequently treat cells with 250 &#181;L of 0.1% (v/v) Nonidet P40 and 30 &#181;g/mL trypsin in 200 &#181;L of 40 mM citrate buffer containing 250 mM sucrose for 10 min at room temperature.</li>
			<li>Add 200 &#181;L of 0.5 &#181;g/mL trypsin inhibitor and 0.1 &#181;g/mL RNase A in 40 mM citrate buffer containing 250 mM sucrose, and incubate cells for 10 min at room temperature.</li>
			<li>Add 200 &#181;L of 0.4 mg/mL propidium iodide (PI) in 40mM citrate buffer containing 250 mM sucrose, and incubate cells for 10 min at room temperature to stain cell nuclei.</li>
		</ol>
		</li>
		<li>A method for analyzing fixed cells
		<ol>
			<li>Fix cells with 80% ethanol by suspending cells in 1 mL of PBS, adding 4 mL of 100% EtOH dropwise while mixing the cell suspension and leaving it at room temperature for 30 min. Cells in this fixative can be stored at -20 &#176;C for several weeks.</li>
			<li>Wash cells by resuspending them in 5 mL of PBS followed by centrifugation (300 x g, 5 min). Treat cells with 0.5 mg/mL RNase A and stain them with 50 &#181;g/mL propidium iodide (PI) (475 &#181;L of 0.5 mg/mL RNase A and 25 &#181;L of 1 mg/mL PI) for 30 min at room temperature.</li>
		</ol>
		</li>
	</ol>
	</li>
	<li>Analyze DNA content of the cells using a flow cytometer with a 488 nm laser and red channel emission filter (long pass &#62; 610 nm). Analyze a reference sample prepared from genuine diploid cells at the same time to assess ploidy of the target cells.
	<ol>
		<li>Alternatively, add fluorescence standard beads to assess the fluorescence intensity ratio of diploid cells vs. the beads. Use the &#39;pulse height vs. pulse width&#39; option of the flow cytometer to discriminate between single tetraploid cells and clumps of diploid cells11.</li>
	</ol>
	</li>
</ol>
4. Examination of Chromosome Counts and Karyotype Analysis
NOTE: These are all well-established procedures and a technical manual or the manufacturer&#39;s protocols should be referred to for detailed procedures and technical tips.
<ol>
	<li>Treat exponentially growing cells with 0.1 &#181;g/mL DC for 4 h to arrest cells in metaphase.</li>
	<li>Prepare chromosome slides.
	<ol>
		<li>Harvest cells (at least 5 x 105 cells) by trypsinization and suspend in medium containing FBS.</li>
		<li>Centrifuge cells in medium (300 x g, 5 min) and treat with hypotonic solution (5 mL of 0.075 M KCl) at 37 &#176;C for 10 min.</li>
		<li>Fix and suspend cells in Carnoy&#39;s fixative (methanol:acetic acid = 3:1).</li>
		<li>Spread cells onto a glass slide by dropping a small volume (25 - 35 &#181;L) of cell suspension. Additional steps such as exposure to hot steam might be necessary to improve chromosome spreading.</li>
	</ol>
	</li>
	<li>Stain cells for chromosome counts or karyotype analysis or multicolor fluorescence in situ hybridization (mFISH).
	<ol>
		<li>Chromosome counts.
		<ol>
			<li>Stain cells with 5% Giemsa solution (or 5 &#181;g/mL PI containing 0.5 mg/mL RNase A for fluorescence microscopy) for 15 - 20 min.</li>
			<li>Digitally photograph at least 50 metaphase cells.</li>
			<li>Count chromosome number per cell using an image analysis software after manual separation of touching and overlapping chromosomes using a photo editing software. Although chromosome number can be scored manually, computerized scoring is recommended.</li>
		</ol>
		</li>
		<li>Karyotype analysis 
		NOTE: This analysis should be performed by a trained technician or a professional company.
		<ol>
			<li>Stain cells according to a standard G-banding technique12.</li>
			<li>Analyze chromosomes to make standard karyograms12.</li>
		</ol>
		</li>
		<li>mFISH 
		NOTE: This step is optional and should be performed if necessary.
		<ol>
			<li>Stain cells using multicolor FISH probes for human chromosomes according to the manufacturer&#39;s protocol13.</li>
			<li>Analyze chromosomes using a fluorescence microscope with suitable filters and a software for mFISH analysis13.</li>
		</ol>
		</li>
	</ol>
	</li>
</ol>

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Gastroenterol. 96 (11), 3071-3083 (2001).</li><li>Galipeau, 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=NSAIDs+modulate+CDKN2A,+TP53,+and+DNA+content+risk+for+progression+to+esophageal+adenocarcinoma.">NSAIDs modulate CDKN2A, TP53, and DNA content risk for progression to esophageal adenocarcinoma.</a> PLoS Med. 4 (2), e67 (2007).</li><li>Olaharski, 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=Tetraploidy+and+chromosomal+instability+are+early+events+during+cervical+carcinogenesis.">Tetraploidy and chromosomal instability are early events during cervical carcinogenesis.</a> Carcinogenesis. 27, 337-343 (2006).</li><li>Liu, 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=p53-independent+abrogation+of+a+postmitotic+checkpoint+contributes+to+human+papillomavirus+E6-induced+polyploidy.">p53-independent abrogation of a postmitotic checkpoint contributes to human papillomavirus E6-induced polyploidy.</a> Cancer Res. 67, 2603-2610 (2007).</li><li>Davoli, T., de Lange, T. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=The+causes+and+consequences+of+polyploidy+in+normal+development+and+cancer.">The causes and consequences of polyploidy in normal development and cancer.</a> Annu Rev Cell Dev Biol. 27, 585-610 (2011).</li><li>Fox, D., Duronio, R. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Endoreplication+and+polyploidy:+insights+into+development+and+disease.">Endoreplication and polyploidy: insights into development and disease.</a> Development. 140, 3-12 (2013).</li><li>Fujiwara, 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=Cytokinesis+failure+generating+tetraploids+promotes+tumorigenesis+in+p53-null+cells.">Cytokinesis failure generating tetraploids promotes tumorigenesis in p53-null cells.</a> Nature. 437, 1043-1047 (2005).</li><li>Ganem, N., 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=Cytokinesis+failure+triggers+hippo+tumor+suppressor+pathway+activation.">Cytokinesis failure triggers hippo tumor suppressor pathway activation.</a> Cell. 158 (4), 833-848 (2014).</li><li>Kuznetsova, 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=Chromosomal+instability,+tolerance+of+mitotic+errors+and+multidrug+resistance+are+promoted+by+tetraploidization+in+human+cells.">Chromosomal instability, tolerance of mitotic errors and multidrug resistance are promoted by tetraploidization in human cells.</a> Cell cycle. 14 (17), 2810-2820 (2015).</li><li>Vindel&#248;v, L., Christensen, I. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Detergent+and+proteolytic+enzyme-based+techniques+for+nuclear+isolation+and+DNA+content+analysis.">Detergent and proteolytic enzyme-based techniques for nuclear isolation and DNA content analysis.</a> Methods Cell Biol. 41, 219-229 (1994).</li><li>Darzynkiewicz, Z., Juan, G. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=DNA+content+measurement+for+DNA+ploidy+and+cell+cycle+analysis.">DNA content measurement for DNA ploidy and cell cycle analysis.</a> Curr Protoc Cytom. , Chapter 7: Unit 7.5 (2001).</li><li>Knutsen, T., Bixenman, H., Lawce, H., Martin, P. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Chromosome+analysis+guidelines+preliminary+report.">Chromosome analysis guidelines preliminary report.</a> Cancer Genet Cytogenet. 52 (1), 11-17 (1991).</li><li>Liehr, 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=Multicolor+FISH+probe+sets+and+their+applications.">Multicolor FISH probe sets and their applications.</a> Histol. 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Biol. Cell. 12, 1315-1328 (2001).</li><li>Vogel, C., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Crosstalk+of+the+mitotic+spindle+assembly+checkpoint+with+p53+to+prevent+polyploidy.">Crosstalk of the mitotic spindle assembly checkpoint with p53 to prevent polyploidy.</a> Oncogene. 23, 6845-6853 (2004).</li><li>Aylon, Y., Oren, M. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=p53:+Guardian+of+ploidy.">p53: Guardian of ploidy.</a> Mol. Oncol. 5 (4), 315-323 (2011).</li><li>Uetake, Y., Sluder, G. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Cell+cycle+progression+after+cleavage+failure+:+mammalian+somatic+cells+do+not+possess+a+&amp;#34;tetraploidy+checkpoint&amp;#34.">Cell cycle progression after cleavage failure : mammalian somatic cells do not possess a &amp;#34;tetraploidy checkpoint&amp;#34.</a> J. 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The authors declare that they have no competing financial interest.

A major problem in induction of tetraploidy from diploid cells by chemical agents, either by cytokinesis inhibitors or by spindle inhibitors, is that cells often become a mixture of diploid and tetraploid populations, and tetraploid cells must be separated from diploid cells. Most common approaches for isolation of a tetraploid population free of diploid cells use FACS or cloning by limiting dilution. However, these procedures are laborious and not easy to perform. In this report, we present a new protocol to establish p...

Polyploidy has been observed not only in specialized tissues of mammalian species but also in a variety of pathological conditions, such as cancer and degenerative diseases. Polyploid (mostly tetraploid) cells are often observed in preneoplastic lesions of human tissues, such as Barrett&#39;s esophagus 1,2 or squamous intraepithelial lesions of the cervix 3,4, and have been considered to be the source of malignant aneuploid cells in those tissues 5,6. Although it is suggested that conversion of tetraploid to aneuploid cells could be a crucial event in the early stages of tumorigenesis, the mechanisms involved in this process are not fully understood. This is partly because no in vitro model has been available where nontransformed polyploid human cells can propagate.
Some researchers have induced tetraploidy in nontransformed human epithelial cells through generation of binucleated cells by inhibiting cytokinesis7-9. In this method, however, unnecessary diploid cells must be eliminated by fluorescence-activated cell sorting (FACS) 7,8 or cloning by limiting dilution 9. Because these procedures are laborious and not easy to perform, simpler methods to establish nontransformed tetraploid cells are desired for research in this field.
In the present report, we describe a protocol to establish proliferative tetraploid cells from normal human fibroblasts or telomerase-immortalized human fibroblasts by relatively simple procedures. The procedures use spindle poison demecolcine (DC) to arrest diploid cells in mitosis, and mitotic cells collected by shaking off are further treated with DC. Diploid mitotic cells treated with DC for prolonged time convert to tetraploid G1 cells, and these cells proliferate as tetraploid cells after growth arrest for several days following drug removal. This protocol provides an efficient method for creating a useful model to study the relationship between chromosome instability and the oncogenic potential of polyploid human cells.

<table><tbody><tr><td>MEM-&amp;alpha;</td><td>Sigma-Aldrich</td><td>M8042-500ML</td><td></td></tr><tr><td>Trypsin-EDTA</td><td>Sigma-Aldrich</td><td>T4174</td><td></td></tr><tr><td>FBS</td><td>Sigma-Aldrich</td><td>172012-500ML</td><td></td></tr><tr><td>Demecolcine solution (10 &amp;mu;g/mL in HBSS)</td><td>Sigma-Aldrich</td><td>D1925-10ML</td><td></td></tr><tr><td>BD CycleTES Plus DNA Reagent Kits</td><td>BD Biosciences</td><td>#340242</td><td>For examination of DNA ploidy by flow cytometry</td></tr><tr><td>Human chromosome multicolor FISH probe 24XCyte</td><td>MetaSystems</td><td>#D-0125-060-DI</td><td>Specialized filter set and software for mFISH analysis are necessary</td></tr><tr><td>Isis imaging system with mFISH&amp;nbsp; software&amp;nbsp;</td><td>MetaSystems</td><td></td><td>Specialized probe kit is necessary</td></tr></tbody></table>

establishment of proliferative tetraploid cells from nontransformed human fibroblasts

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

In our experience, TIG-1 cells can be made almost completely tetraploid by simple continuous treatment with 0.1 &#181;g/mL DC for 4 days (Figure 2A). In contrast, other fibroblast strains, such as BJ or IMR-90, and TIG-hT cells, became a mixture of diploid and tetraploid populations following the same treatment, and isolation of mitotic cells by the shake-off method is necessary during the course of DC treatment (usually 16 - 18 h after the start of treatment) (Fi...

Watch this Scientific Journal Video about Establishment of Proliferative Tetraploid Cells from Nontransformed Human Fibroblasts at JoVE.com

Establishment of Proliferative Tetraploid Cells from Nontransformed Human Fibroblasts

Although proliferative polyploid cells are necessary to analyze chromosomal instability of polyploid cells, creating such cells from nontransformed human cells is not easy. The present report describes relatively simple procedures to establish proliferative tetraploid cells free of a diploid population from normal human fibroblasts.

Polyploid (mostly tetraploid) cells are often observed in preneoplastic lesions of human tissues and their chromosomal instability has been considered to ...

establishment-proliferative-tetraploid-cells-from-nontransformed

Universidad de Cartagena UDEC

Research

JoVE Journal

Developmental Biology

7.2K Views.  Saitama Medical University. Although proliferative polyploid cells are necessary to analyze chromosomal instability of polyploid cells, creating such cells from nontransformed human cells is not easy. The present report describes relatively simple procedures to establish proliferative tetraploid cells free of a diploid population from normal human fibroblasts.

Video: Establishment of Proliferative Tetraploid Cells from Nontransformed Human Fibroblasts

piggyBac Transposon System Modification of Primary Human T Cells

The piggyBac transposon system is naturally active, originally derived from the cabbage looper moth1,2. This non-viral system is plasmid based, most commonly utilizing two plasmids with one expressing the piggyBac transposase enzyme and a transposon plasmid harboring the gene(s) of interest between inverted repeat elements which are required for gene transfer activity. PiggyBac mediates gene transfer through a "cut and paste" mechanism whereby the transposase integrates the transposon segment into the genome of the target cell(s) of interest. PiggyBac has demonstrated efficient gene delivery activity in a wide variety of insect1,2, mammalian3-5, and human cells6 including primary human T cells7,8. Recently, a hyperactive piggyBac transposase was generated improving gene transfer efficiency9,10.
Human T lymphocytes are of clinical interest for adoptive immunotherapy of cancer11. Of note, the first clinical trial involving transposon modification of human T cells using the Sleeping beauty transposon system has been approved12. We have previously evaluated the utility of piggyBac as a non-viral methodology for genetic modification of human T cells. We found piggyBac to be efficient in genetic modification of human T cells with a reporter gene and a non-immunogenic inducible suicide gene7. Analysis of genomic integration sites revealed a lack of preference for integration into or near known proto-oncogenes13. We used piggyBac to gene-modify cytotoxic T lymphocytes to carry a chimeric antigen receptor directed against the tumor antigen HER2, and found that gene-modified T cells mediated targeted killing of HER2-positive tumor cells in vitro and in vivo in an orthotopic mouse model14. We have also used piggyBac to generate human T cells resistant to rapamycin, which should be useful in cancer therapies where rapamycin is utilized15.
Herein, we describe a method for using piggyBac to genetically modify primary human T cells. This includes isolation of peripheral blood mononuclear cells (PBMCs) from human blood followed by culture, gene modification, and activation of T cells. For the purpose of this report, T cells were modified with a reporter gene (eGFP) for analysis and quantification of gene expression by flow cytometry.
PiggyBac can be used to modify human T cells with a variety of genes of interest. Although we have used piggyBac to direct T cells to tumor antigens14, we have also used piggyBac to add an inducible safety switch in order to eliminate gene modified cells if needed7. The large cargo capacity of piggyBac has also enabled gene transfer of a large rapamycin resistant mTOR molecule (15 kb)15. Therefore, we present a non-viral methodology for stable gene-modification of primary human T cells for a wide variety of purposes.

piggyBac Transposon System Modification of Primary Human T Cells

We describe a method to genetically modify primary human T cells with a transgene using the non-viral piggyBac transposon system. T cells modified to using the piggyBac transposon system exhibit stable transgene expression.

The piggyBac transposon system is naturally active, originally derived from the cabbage looper moth1,2. This non-viral system is plasmid based, most ...

Immunology and Infection

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) ...

RNA-based Reprogramming of Human Primary Fibroblasts into Induced Pluripotent Stem Cells

Induced pluripotent stem cells (iPSCs) have proven to be a valuable tool to study human development and disease. Further advancing iPSCs as a regenerative therapeutic requires a safe, robust, and expedient reprogramming protocol. Here, we present a clinically relevant, step-by-step protocol for the extremely high-efficiency reprogramming of human dermal fibroblasts into iPSCs using a non-integrating approach. The core of the protocol consists of expressing pluripotency factors (SOX2, KLF4, cMYC, LIN28A, NANOG, OCT4-MyoD fusion) from in vitro transcribed messenger RNAs synthesized with modified nucleotides (modified mRNAs). The reprogramming modified mRNAs are transfected into primary fibroblasts every 48 h together with mature embryonic stem cell-specific microRNA-367/302 mimics for two weeks. The resulting iPSC colonies can then be isolated and directly expanded in feeder-free conditions. To maximize efficiency and consistency of our reprogramming protocol across fibroblast samples, we have optimized various parameters including the RNA transfection regimen, timing of transfections, culture conditions, and seeding densities. Importantly, our method generates high-quality iPSCs from most fibroblast sources, including difficult-to-reprogram diseased, aged, and/or senescent samples.

Here we describe a clinically relevant, high-efficiency, feeder-free method to reprogram human primary fibroblasts into induced pluripotent stem cells using modified mRNAs encoding reprogramming factors and mature microRNA-367/302 mimics. Also included are methods to assess reprogramming efficiency, expand clonal iPSC colonies, and confirm expression of the pluripotency marker TRA-1-60.

Induced pluripotent stem cells (iPSCs) have proven to be a valuable tool to study human development and disease. Further advancing iPSCs as a regenerative ...

Generation of Induced Pluripotent Stem Cells by Reprogramming Human Fibroblasts with the Stemgent Human TF Lentivirus Set

In 2006, Yamanaka and colleagues first demonstrated that retrovirus-mediated delivery and expression of Oct4, Sox2, c-Myc and Klf4 is capable of inducing the pluripotent state in mouse fibroblasts.1 The same group also reported the successful reprogramming of human somatic cells into induced pluripotent stem (iPS) cells using human versions of the same transcription factors delivered by retroviral vectors.2 Additionally, James Thomson et al. reported that the lentivirus-mediated co-expression of another set of factors (Oct4, Sox2, Nanog and Lin28) was capable of reprogramming human somatic cells into iPS cells.3
iPS cells are similar to ES cells in morphology, proliferation and the ability to differentiate into all tissue types of the body. Human iPS cells have a distinct advantage over ES cells as they exhibit key properties of ES cells without the ethical dilemma of embryo destruction. The generation of patient-specific iPS cells circumvents an important roadblock to personalized regenerative medicine therapies by eliminating the potential for immune rejection of non-autologous transplanted cells.
Here we demonstrate the protocol for reprogramming human fibroblast cells using the Stemgent Human TF Lentivirus Set. We also show that cells reprogrammed with this set begin to show iPS morphology four days post-transduction. Using the Stemolecule Y27632, we selected for iPS cells and observed correct morphology after three sequential rounds of colony picking and passaging. We also demonstrate that after reprogramming cells displayed the pluripotency marker AP, surface markers TRA-1-81, TRA-1-60, SSEA-4, and SSEA-3, and nuclear markers Oct4, Sox2 and Nanog.

We demonstrate the protocol for the generation of induced pluripotent stem cells from human somatic cells using lentivirus-mediated delivery of the human factors Oct4, Sox2, Nanog, and Lin28. Pluripotency was confirmed by morphology and the presence of embryonic stem (ES) cell-specific markers.

In 2006, Yamanaka and colleagues first demonstrated that retrovirus-mediated delivery and expression of Oct4, Sox2, c-Myc and Klf4 is capable of inducing ...

Biology

Hemogenic Reprogramming of Human Fibroblasts by Enforced Expression of Transcription Factors

The cellular and molecular mechanisms underlying specification of human hematopoietic stem cells (HSCs) remain elusive. Strategies to recapitulate human HSC emergence in vitro are required to overcome limitations in studying this complex developmental process. Here, we describe a protocol to generate hematopoietic stem and progenitor-like cells from human dermal fibroblasts employing a direct cell reprogramming approach. These cells transit through a hemogenic intermediate cell-type, resembling the endothelial-to-hematopoietic transition (EHT) characteristic of HSC specification. Fibroblasts were reprogrammed to hemogenic cells via transduction with GATA2, GFI1B and FOS transcription factors. This combination of three factors induced morphological changes, expression of hemogenic and hematopoietic markers and dynamic EHT transcriptional programs. Reprogrammed cells generate hematopoietic progeny and repopulate immunodeficient mice for three months. This protocol can be adapted towards the mechanistic dissection of the human EHT process as exemplified here by defining GATA2 targets during the early phases of reprogramming. Thus, human hemogenic reprogramming provides a simple and tractable approach to identify novel markers and regulators of human HSC emergence. In the future, faithful induction of hemogenic fate in fibroblasts may lead to the generation of patient-specific HSCs for transplantation.

This protocol demonstrates the induction of a hemogenic program in human dermal fibroblasts by enforced expression of the transcription factors GATA2, GFI1B and FOS to generate hematopoietic stem and progenitor cells.

The cellular and molecular mechanisms underlying specification of human hematopoietic stem cells (HSCs) remain elusive. Strategies to recapitulate human ...

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

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 ...

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

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

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

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

Bioengineering

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 ...

Derivation and Characterization of a Transgene-free Human Induced Pluripotent Stem Cell Line and Conversion into Defined Clinical-grade Conditions

Human induced pluripotent stem cells (hiPSCs) can be generated with lentiviral-based reprogramming methodologies. However, traces of potentially oncogenic genes remaining in actively transcribed regions of the genome, limit their potential for use in human therapeutic applications1. Additionally, non-human antigens derived from stem cell reprogramming or differentiation into therapeutically relevant derivatives preclude these hiPSCs from being used in a human clinical context2. In this video, we present a procedure for reprogramming and analyzing factor-free hiPSCs free of exogenous transgenes. These hiPSCs then can be analyzed for gene expression abnormalities in the specific intron containing the lentivirus. This analysis may be conducted using sensitive quantitative polymerase chain reaction (PCR), which has an advantage over less sensitive techniques previously used to detect gene expression differences3. Full conversion into clinical-grade good manufacturing practice (GMP) conditions, allows human clinical relevance. Our protocol offers another methodology&#8212;provided that current safe-harbor criteria will expand and include factor-free characterized hiPSC-based derivatives for human therapeutic applications&#8212;for deriving GMP-grade hiPSCs, which should eliminate any immunogenicity risk due to non-human antigens. This protocol is broadly applicable to lentiviral reprogrammed cells of any type and provides a reproducible method for converting reprogrammed cells into GMP-grade conditions.

We describe a protocol for deriving lentiviral-based reprogrammed and characterized factor-free human induced pluripotent stem cells and conversion into putative clinical-grade conditions.

Human induced pluripotent stem cells (hiPSCs) can be generated with lentiviral-based reprogramming methodologies. However, traces of potentially oncogenic ...

Establishment of Proliferative Tetraploid Cells from Nontransformed Human Fibroblasts

Summary

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Chapters in this video

piggyBac Transposon System Modification of Primary Human T Cells

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

RNA-based Reprogramming of Human Primary Fibroblasts into Induced Pluripotent Stem Cells

Generation of Induced Pluripotent Stem Cells by Reprogramming Human Fibroblasts with the Stemgent Human TF Lentivirus Set

Hemogenic Reprogramming of Human Fibroblasts by Enforced Expression of Transcription Factors

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

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

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

Naïve Adult Stem Cells Isolation from Primary Human Fibroblast Cultures

Derivation and Characterization of a Transgene-free Human Induced Pluripotent Stem Cell Line and Conversion into Defined Clinical-grade Conditions