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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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 border="0" cellpadding="0" cellspacing="0" width="687">
	<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 ...

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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

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 ...

Direct Induction of Human Neural Stem Cells from Peripheral Blood Hematopoietic Progenitor Cells

Human disease specific neuronal cultures are essential for generating in vitro models for human neurological diseases. However, the lack of access to primary human adult neural cultures raises unique challenges. Recent developments in induced pluripotent stem cells (iPSC) provides an alternative approach to derive neural cultures from skin fibroblasts through patient specific iPSC, but this process is labor intensive, requires special expertise and large amounts of resources, and can take several months. This prevents the wide application of this technology to the study of neurological diseases. To overcome some of these issues, we have developed a method to derive neural stem cells directly from human adult peripheral blood, bypassing the iPSC derivation process. Hematopoietic progenitor cells enriched from human adult peripheral blood were cultured in vitro and transfected with Sendai virus vectors containing transcriptional factors Sox2, Oct3/4, Klf4, and c-Myc. The transfection results in morphological changes in the cells which are further selected by using human neural progenitor medium containing basic fibroblast growth factor (bFGF) and vascular endothelial growth factor (VEGF). The resulting cells are characterized by the expression for neural stem cell markers, such as nestin and SOX2. These neural stem cells could be further differentiated to neurons, astroglia and oligodendrocytes in specified differentiation media. Using easily accessible human peripheral blood samples, this method could be used to derive neural stem cells for further differentiation to neural cells for in vitro modeling of neurological disorders and may advance studies related to the pathogenesis and treatment of those diseases.

A method was developed to directly derive human neural stem cells from hematopoietic progenitor cells enriched from peripheral blood cells.

Human disease specific neuronal cultures are essential for generating in vitro models for human neurological diseases. However, the lack of access to ...

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 ...

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 ...