Name: establishment of proliferative tetraploid cells from nontransformed human fibroblasts
Uploaded: 2017-01-08T14:00:00
Description: Watch this Scientific Journal Video about Establishment of Proliferative Tetraploid Cells from Nontransformed Human Fibroblasts at JoVE.com

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 cel...

<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. Histopathol. 19 (1), 229-237 (2004).</li><li>Ohshima, S., Seyama, A. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Formation+of+bipolar+spindles+with+two+centrosomes+in+tetraploid+cells+established+from+normal+human+fibroblasts.">Formation of bipolar spindles with two centrosomes in tetraploid cells established from normal human fibroblasts.</a> Hum. Cell. 25 (3), 78-85 (2012).</li><li>Ohshima, S., Seyama, A. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Establishment+of+proliferative+tetraploid+cells+from+normal+human+fibroblasts.">Establishment of proliferative tetraploid cells from normal human fibroblasts.</a> Front. 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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.

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	<tbody>
		<tr height="21">
			<td height="21" style="height:21px;width:205px;">MEM-&#945;</td>
			<td style="width:101px;">Sigma-Aldrich</td>
			<td style="width:136px;">M8042-500ML</td>
			<td style="width:245px;"></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:205px;">Trypsin-EDTA</td>
			<td style="width:101px;">Sigma-Aldrich</td>
			<td style="width:136px;">T4174</td>
			<td style="width:245px;"></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:205px;">FBS</td>
			<td style="width:101px;">Sigma-Aldrich</td>
			<td style="width:136px;">172012-500ML</td>
			<td style="width:245px;"></td>
		</tr>
		<tr height="42">
			<td height="42" style="height:42px;width:205px;">Demecolcine solution (10 &#956;g/mL in HBSS)</td>
			<td style="width:101px;">Sigma-Aldrich</td>
			<td style="width:136px;">D1925-10ML</td>
			<td></td>
		</tr>
		<tr height="42">
			<td height="42" style="height:42px;width:205px;">BD CycleTES Plus DNA Reagent Kits</td>
			<td style="width:101px;">BD Biosciences</td>
			<td style="width:136px;">#340242</td>
			<td style="width:245px;">For examination of DNA ploidy by flow cytometry</td>
		</tr>
		<tr height="42">
			<td height="42" style="height:43px;width:205px;">Human chromosome multicolor FISH probe 24XCyte</td>
			<td style="width:101px;">MetaSystems</td>
			<td style="width:136px;">#D-0125-060-DI</td>
			<td style="width:245px;">Specialized filter set and software for mFISH analysis are necessary</td>
		</tr>
		<tr height="42">
			<td height="42" style="height:42px;width:205px;">Isis imaging system with mFISH&#160; software&#160;</td>
			<td style="width:101px;">MetaSystems</td>
			<td style="width:136px;"></td>
			<td style="width:245px;">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 ...

The overall goal of this procedure is to establish proliferative tetraploid cells, free of a diploid population, from normal human fibroblasts. This method can help answer key questions in the field of carcinogenesis research, such as how aneuploid cells develop in the early stages of carcinogenesis. The main advantage of this technique is that proliferative tetraploid cells, free of a diploid population, can be established by relatively simple procedures.While some strains become completely tetraploid by simple treatment with spindle poison, demecolcine, most need to be handled with the shake-off method during that treatment to emulate a diploid population. To begin the induction of tetraploidy without the shake-off method, passage approximately five times 10 to the 1/5 cultured cells into a 100-millimeter dish. Incubate the dish in a 5%carbon dioxide atmosphere at 37 degrees Celsius for 24 hours.After this, treat the exponentially-growing cells with medium containing 0.1 micrograms per milliliter DC.Incubate in a 5%carbon dioxide atmosphere at 37 degrees Celsius for four days. Then replace the DC-containing medium with drug-free medium. Incubate in 5%carbon dioxide at 37 degrees Celsius until proliferation resumes.To induce tetraploidy utilizing the shake-off method, passage the cultured cells into T-75 flasks, making sure that each flask contains at least one times 10 to the 1/6 cells. Incubate the flasks in a 5%carbon dioxide atmosphere at 37 degrees Celsius for 24 hours. The next day, treat the cells with medium containing DC for 16 to 18 hours to arrest them in mitosis as outlined in the text protocol.After incubation, check the cells in a flask under the inverted phase contrast microscope. And then gently shake each flask to begin the collection of loosely-adhered mitotic cells. Careless handling, such as violent shaking or touching the surface of the flask by the pipette tip should be avoided.Do not contaminate the mitotic cells with adhered interface cells. Gently wash the flasks with warm medium, and transfer the cells to centrifuge tubes. Utilize an atomic cell counter to count the cell number.After cell counting, centrifuge at 300 times g for five minutes. Reseed the collected mitotic cells into 16-millimeter culture dishes. Then treat the plated cells with medium containing 0.1 micrograms per milliliter DC.Incubate in 5%carbon dioxide at 37 degrees Celsius for three days.After this, replace the DC-containing medium with drug-free medium. Incubate in a 5%carbon dioxide atmosphere at 37 degrees Celsius until proliferation resumes. For analysis of DNA ploidy, wash the cells with PBS.Then add a solution containing 0.05%trypsin and 0.02%EDTA to the cells. Incubate for 10 minutes at 37 degrees Celsius. After this, transfer the cell suspension to a centrifuge tube.And neutralize the trypsin by adding medium containing 10%fetal bovine serum. Centrifuge the cells in medium at 300 times g for five minutes. Resuspend the cells in five milliliters of PBS.Then centrifuge again at 300 times g for five minutes. Next, resuspend cells in one milliliter of PBS. Add four milliliters of 100%ethanol drop-wise with mixing.Let the suspension rest at room temperature for 30 minutes to fix the cells. After this, centrifuge the cells in fixative solution at 300 times g for five minutes. And resuspend cells in five milliliters of PBS.Centrifuge the suspension at 300 times g for five minutes. Then treat the cells with 475 microliters of 0.5 milligrams per milliliter RNA solution. Add 25 microliters of one milligram per milliliter propidium iodine solution to stain the cells.And then mix. Allow the suspension to rest at room temperature for 30 minutes. Next, use a flow cytometer with a 488 nanometer laser and red channel emission filter to analyze the DNA content of the cells.Analyze a reference sample prepared from genuine diploid cells as well to assess ploidy of target cells. To begin, treat exponentially-growing cells with medium containing 0.1 micrograms per milliliter DC for four hours to arrest the cells in metaphase. After this, harvest at least five times 10 to the 1/5 cells by trypsinization.Suspend the harvested cells in medium containing FBS. Centrifuge the suspension at 300 times g for five minutes. Then add five milliliters of a 0.075 molar potassium chloride solution.Incubate at 37 degrees Celsius for 10 minutes. After incubation is complete, centrifuge the suspension at 300 times g for five minutes. Resuspend the cells in five milliliters of Carnoy's fixative.And then centrifuge the suspension at 300 times g for five minutes. Then aspirate the supernatant and resuspend cells in 0.5 milliliters of Carnoy's fixative. Drop 30 microliters of the cell suspension onto a glass slide.Finally, stain and analyze the cells via either chromosome counts or karyotype analysis. In this study, proliferative tetraploid cells, free of a diploid population, are cultured from normal human fibroblasts. Although some cell strains can become completely tetraploid by continuous treatment with DC, most cells need to be treated with the shake-off method during the course of DC treatment to eliminate a diploid population.The representative results for the induction of tetraploidy in three fibroblast strains are shown here. The strain TIG-1 becomes almost entirely tetraploid through simple continuous treatment with DC.However, this treatment causes the BJ and TIG-hT strains to become a mixture of diploid and tetrapod populations. Therefore, isolation of mitotic cells by the shake-off method is necessary during the treatment process.A chromosome count is performed in the established tetraploid cells. As can be seen, the modal chromosome number is 92 for both TIG-1 and BJ cells, and 91 for TIG-hT cells. Once mastered, this technique can be done in two weeks if it is performed properly.While attempting this procedure, it's important to remember to gently shake the flasks in the shake-off step, not to cause contamination of mitotic cells by adherent interface cells.

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Research

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Developmental Biology

Isolation and Quantitative Immunocytochemical Characterization of Primary Myogenic Cells and Fibroblasts from Human Skeletal Muscle

The repair and regeneration of skeletal muscle requires the action of satellite cells, which are the resident muscle stem cells. These can be isolated from human muscle biopsy samples using enzymatic digestion and their myogenic properties studied in culture. Quantitatively, the two main adherent cell types obtained from enzymatic digestion are: (i) the satellite cells (termed myogenic cells or muscle precursor cells), identified initially as CD56+ and later as CD56+/desmin+ cells and (ii) muscle-derived fibroblasts, identified as CD56&#8211; and TE-7+. Fibroblasts proliferate very efficiently in culture and in mixed cell populations these cells may overrun myogenic cells to dominate the culture. The isolation and purification of different cell types from human muscle is thus an important methodological consideration when trying to investigate the innate behavior of either cell type in culture. Here we describe a system of sorting based on the gentle enzymatic digestion of cells using collagenase and dispase followed by magnetic activated cell sorting (MACS) which gives both a high purity (&#62;95% myogenic cells) and good yield (~2.8 x 106 &#177; 8.87 x 105 cells/g tissue after 7 days in vitro) for experiments in culture. This approach is based on incubating the mixed muscle-derived cell population with magnetic microbeads beads conjugated to an antibody against CD56 and then passing cells though a magnetic field. CD56+ cells bound to microbeads are retained by the field whereas CD56&#8211; cells pass unimpeded through the column. Cell suspensions from any stage of the sorting process can be plated and cultured. Following a given intervention, cell morphology, and the expression and localization of proteins including nuclear transcription factors can be quantified using immunofluorescent labeling with specific antibodies and an image processing and analysis package.

The main adherent cell types derived from human muscle are myogenic cells and fibroblasts. Here, cell populations are enriched using magnetic-activated cell sorting based on the CD56 antigen. Subsequent immunolabelling with specific antibodies and use of image analysis techniques allows quantification of cytoplasmic and nuclear characteristics in individual cells.

The repair and regeneration of skeletal muscle requires the action of satellite cells, which are the resident muscle stem cells. These can be isolated ...

Three-dimensional Quantification of Dendritic Spines from Pyramidal Neurons Derived from Human Induced Pluripotent Stem Cells

Dendritic spines are small protrusions that correspond to the post-synaptic compartments of excitatory synapses in the central nervous system. They are distributed along the dendrites. Their morphology is largely dependent on neuronal activity, and they are dynamic. Dendritic spines express glutamatergic receptors (AMPA and NMDA receptors) on their surface and at the levels of postsynaptic densities. Each spine allows the neuron to control its state and local activity independently. Spine morphologies have been extensively studied in glutamatergic pyramidal cells of the brain cortex, using both in vivo approaches and neuronal cultures obtained from rodent tissues. Neuropathological conditions can be associated to altered spine induction and maturation, as shown in rodent cultured neurons and one-dimensional quantitative analysis 1. The present study describes a protocol for the 3D quantitative analysis of spine morphologies using human cortical neurons derived from neural stem cells (late cortical progenitors). These cells were initially obtained from induced&#160;pluripotent stem cells. This protocol allows the analysis of spine morphologies at different culture periods, and with possible comparison between induced&#160;pluripotent stem cells obtained from control individuals with those obtained from patients with psychiatric diseases.

Dendritic spines of pyramidal neurons are the sites of most excitatory synapses in mammalian brain cortex. This method describes a 3D quantitative analysis of spine morphologies in human cortical pyramidal glutamatergic neurons derived from induced&#160;pluripotent stem cells.

Dendritic spines are small protrusions that correspond to the post-synaptic compartments of excitatory synapses in the central nervous system. They are ...

Establishment of Genome-edited Human Pluripotent Stem Cell Lines: From Targeting to Isolation

Genome-editing of human pluripotent stem cells (hPSCs) provides a genetically controlled and clinically relevant platform from which to understand human development and investigate the pathophysiology of disease. By employing site-specific nucleases (SSNs) for genome editing, the rapid derivation of new hPSC lines harboring specific genetic alterations in an otherwise isogenic setting becomes possible. Zinc finger nucleases (ZFNs), transcription activator-like effector nucleases (TALENs) and clustered regularly interspaced short palindromic repeats (CRISPR)/Cas9 are the most commonly used SSNs. All of these nucleases function by introducing a double stranded DNA break at a specified site, thereby promoting precise gene editing at a genomic locus. SSN-meditated genome editing exploits two of the cell&#39;s endogenous DNA repair mechanisms, non-homologous end joining (NHEJ) and homology directed repair (HDR), to either introduce insertion/deletion mutations or alter the genome using a homologous repair template at the site of the double stranded break. Electroporation of hPSCs is an efficient means of transfecting SSNs and repair templates that incorporate transgenes such as fluorescent reporters and antibiotic resistance cassettes. After electroporation, it is possible to isolate only those hPSCs that incorporated the repair construct by selecting for antibiotic resistance. Mechanically separating hPSC colonies and confirming proper integration at the target site through genotyping allows for the isolation of correctly targeted and genetically homogeneous cell lines. The validity of this protocol is demonstrated here by using all three SSN platforms to incorporate EGFP and a puromycin resistance construct into the AAVS1 safe harbor locus in human pluripotent&#160;stem cells.

Genome editing of human pluripotent stem cells (hPSCs) can be done quickly and efficiently. Presented here is a robust experimental procedure to genetically engineer hPSCs as exemplified by editing the AAVS1 safe harbor locus to express EGFP and introduce antibiotic resistance.

Genome-editing of human pluripotent stem cells (hPSCs) provides a genetically controlled and clinically relevant platform from which to understand human ...

Feeder-free Derivation of Melanocytes from Human Pluripotent Stem Cells

Human pluripotent stem cells (hPSCs) represent a platform to study human development in vitro under both normal and disease conditions. Researchers can direct the differentiation of hPSCs into the cell type of interest by manipulating the culture conditions to recapitulate signals seen during development. One such cell type is the melanocyte, a pigment-producing cell of neural crest (NC) origin responsible for protecting the skin against UV irradiation. This protocol presents an extension of a currently available in vitro Neural Crest differentiation protocol from hPSCs to further differentiate NC into fully pigmented melanocytes. Melanocyte precursors can be enriched from the Neural Crest protocol via a timed exposure to activators of WNT, BMP, and EDN3 signaling under dual-SMAD-inhibition conditions. The resultant melanocyte precursors are then purified and matured into fully pigmented melanocytes by culture in a selective medium. The resultant melanocytes are fully pigmented and stain appropriately for proteins characteristic of mature melanocytes.

This work describes an in vitro differentiation protocol to produce pigmented, mature melanocytes from human pluripotent stem cells via a neural crest and melanoblast intermediate stage using a feeder-free, 25 day protocol.

Human pluripotent stem cells (hPSCs) represent a platform to study human development in vitro under both normal and disease conditions. Researchers can ...

Epigenetic Conversion as a Safe and Simple Method to Obtain Insulin-secreting Cells from Adult Skin Fibroblasts

Regenerative medicine requires new, fully functional cells that are delivered to patients in order to repair degenerated or damaged tissues. When such cells are not readily available, they can be obtained using different approaches that include, among the many, reprogramming and trans-differentiation, with advantages and limitations that are specific of the different techniques. Here a new strategy for the conversion of an adult mature fibroblast into an insulin-secreting cell, arbitrarily designated as epigenetic converted cells (EpiCC), is described. The method has been developed, based on the increasing understanding of the mechanisms controlling epigenetic regulation of cell fate and differentiation. In particular, the first step uses an epigenetic modifier, namely 5-aza-cytidine, to drive adult cells into a "highly permissive" state. It then takes advantage of this brief and reversible window of epigenetic plasticity, to re-address cells toward a different lineage. The approach is designated "epigenetic cell conversion". It is a simple and robust way to obtain an efficient, controlled and stable cellular inter-lineage switch. Since the protocol does not involve the use of any gene transfection, it is free of viral vectors and does not involve a stable pluripotent state, it is highly promising for translational medicine applications.

Here, a new method that allows the conversion of adult skin fibroblasts into insulin-secreting cells is presented. This technique is based on epigenetic conversion, does not involve the use of retroviral vectors nor the acquisition of a stable pluripotent state. It is therefore highly promising for translational medicine applications.

Regenerative medicine requires new, fully functional cells that are delivered to patients in order to repair degenerated or damaged tissues. When such ...

Defined and Scalable Generation of Hepatocyte-like Cells from Human Pluripotent Stem Cells

Human pluripotent stem cells (hPSCs) possess great value for biomedical research. hPSCs can be scaled and differentiated to all cell types found in the human body. The differentiation of hPSCs to human hepatocyte-like cells (HLCs) has been extensively studied, and efficient differentiation protocols have been established. The combination of extracellular matrix and biological stimuli, including growth factors, cytokines, and small molecules, have made it possible to generate HLCs that resemble primary human hepatocytes. However, the majority of procedures still employ undefined components, giving rise to batch-to-batch variation. This serves as a significant barrier to the application of the technology. To tackle this issue, we developed a defined system for hepatocyte differentiation using human recombinant laminins as extracellular matrices in combination with a serum-free differentiation process. Highly efficient hepatocyte specification was achieved, with demonstrated improvements in both HLC function and phenotype. Importantly, this system is easy to scale up using research and GMP-grade hPSC lines&#160;promising advances in cell-based modelling and therapies.

The method presented here describes a scalable and good manufacturing practice (GMP)-ready differentiation system to generate human hepatocyte-like cells from pluripotent stem cells. It serves as a cost-effective and standardized system to generate human hepatocyte-like cells for basic and applied human liver research.

Human pluripotent stem cells (hPSCs) possess great value for biomedical research. hPSCs can be scaled and differentiated to all cell types found in the ...

Isolation of Endothelial Progenitor Cells from Human Umbilical Cord Blood

The existence of endothelial progenitor cells (EPCs) in peripheral blood and its involvement in vasculogenesis was first reported by Ashara and colleagues1. Later, others documented the existence of similar types of EPCs originating from bone marrow2,3. More recently, Yoder and Ingram showed that EPCs derived from umbilical cord blood had a higher proliferative potential compared to ones isolated from adult peripheral blood4,5,6. Apart from being involved in postnatal vasculogenesis, EPCs have also shown promise as a cell source for creating tissue-engineered vascular and heart valve constructs7,8. Various isolation protocols exist, some of which involve the cell sorting of mononuclear cells (MNCs) derived from the sources mentioned earlier with the help of endothelial and hematopoietic markers, or culturing these MNCs with specialized endothelial growth medium, or a combination of these techniques9. Here, we present a protocol for the isolation and culture of EPCs using specialized endothelial medium supplemented with growth factors, without the use of immunosorting, followed by the characterization of the isolated cells using Western blotting and immunostaining.

The goal of this protocol is to isolate endothelial progenitor cells from umbilical cord blood. Some of the applications include using these cells as a biomarker for identifying patients with cardiovascular risk, treating ischemic diseases, and creating tissue-engineered vascular and heart valve constructs.

The existence of endothelial progenitor cells (EPCs) in peripheral blood and its involvement in vasculogenesis was first reported by Ashara and ...

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 ...

In Vitro Generation of Somite Derivatives from Human Induced Pluripotent Stem Cells

In response to signals such as WNTs, bone morphogenetic proteins (BMPs), and sonic hedgehog (SHH) secreted from surrounding tissues, somites (SMs) give rise to multiple cell types, including the myotome (MYO), sclerotome (SCL), dermatome (D), and syndetome (SYN), which in turn develop into skeletal muscle, axial skeleton, dorsal dermis, and axial tendon/ligament, respectively. Therefore, the generation of SMs and their derivatives from human induced pluripotent stem cells (iPSCs) is critical to obtain pluripotent stem cells (PSCs) for application in regenerative medicine and for disease research in the field of orthopedic surgery. Although the induction protocols for MYO and SCL from PSCs have been previously reported by several researchers, no study has yet demonstrated the induction of SYN and D from iPSCs. Therefore, efficient induction of fully competent SMs remains a major challenge. Here, we recapitulate human SM patterning with human iPSCs in vitro by mimicking the signaling environment during chick/mouse SM development, and report on methods of systematic induction of SM derivatives (MYO, SCL, D, and SYN) from human iPSCs under chemically defined conditions through the presomitic mesoderm (PSM) and SM states. Knowledge regarding chick/mouse&#160;SM development was successfully applied to the induction of SMs with human iPSCs. This method could be a novel tool for studying human somitogenesis and patterning without the use of embryos and for cell-based therapy and disease modeling.

We present here a protocol for the differentiation of human induced pluripotent stem cells into each somite derivative (myotome, sclerotome, dermatome, and syndetome) in chemically defined conditions, which has applications in future disease modeling and cell-based therapies in orthopedic surgery.

In response to signals such as WNTs, bone morphogenetic proteins (BMPs), and sonic hedgehog (SHH) secreted from surrounding tissues, somites (SMs) give ...

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 ...