BS was supported by University of Milan-Bicocca Summer Student Research Training Program; research was supported from funds of the Italian Ministry of Health and Italian Ministry of Education, University and Research and Fondazione Humanitas to GC; we thanks Michael V.G. Latronico for critically reading the manuscript.

1. Feeder-free Maintenance and Passaging of Human iPS Cell Lines
<ol>
	<li>Prepare the Matrigel-coated dishes. Thaw one vial of human ESC-qualified Matrix on ice for one hour and dilute it in 25 ml DMEM-F12 medium. Add 1 ml to each 35 mm plate (or equivalent amounts per surface area if other dishes are used), keeping everything on ice and ensuring that all plates and tubes are pre-cooled. Cover plates with aluminum foil.</li>
</ol>
Note: Matrigel stock concentrations vary by the batch. Dilution instructions are indicated on the data sheet.
<ol start="2">
	<li>Keep plates on ice overnight and remove from ice the next day. Plates can be kept in the fridge up to one week before using.</li>
	<li>Prepare the mTESR1 complete medium (or thaw a vial of Nutristem medium) and the H-ESM (human ESC medium) according to the table below.
	<table border="1" fo:keep-together.within-page="always">
		<tbody>
			<tr>
				<td>H-ESM</td>
				<td>FINAL CONC</td>
				<td>FOR 500 ml</td>
			</tr>
			<tr>
				<td>Knockout Serum Replacement (KSR)</td>
				<td>20%</td>
				<td>100 ml</td>
			</tr>
			<tr>
				<td>GlutaMAX 100X</td>
				<td>2 mM</td>
				<td>5 ml</td>
			</tr>
			<tr>
				<td>MEM non-essential amino-acids</td>
				<td>0.1 mM</td>
				<td>5 ml</td>
			</tr>
			<tr>
				<td>Penicillin-streptomycin</td>
				<td>100 U/ml - 0.1 mg/ml</td>
				<td>5 ml</td>
			</tr>
			<tr>
				<td>2-mercaptoethanol</td>
				<td>0.1 mM</td>
				<td>900 &mu;l</td>
			</tr>
			<tr>
				<td>B27 Supplement - without VitaminA</td>
				<td>1%</td>
				<td>10 ml</td>
			</tr>
			<tr>
				<td>N2 Supplement</td>
				<td>1%</td>
				<td>5 ml</td>
			</tr>
			<tr>
				<td>Knockout DMEM</td>
				<td>-</td>
				<td>370 ml</td>
			</tr>
		</tbody>
	</table>
	 
	Stock medium can be stored at 4 &deg;C for no more than 2 weeks. To prepare complete growth medium, basic FGF is added just before feeding at a final concentration of 20 ng/ml.</li>
</ol>
Note: Complete H-ESM conditioned on mouse embryonic fibroblasts may be used instead of mTESR1 or Nutristem for iPS cells.
<ol start="4">
	<li>Place coated plates in an incubator at 37 &deg;C for 30 min to 2 hr prior to passaging.</li>
	<li>Cells should be passaged approximately every 5 days, even if they have not reached confluence. Colonies should not be touching.</li>
	<li>Replace growth medium with 1 ml of dispase (1 mg/ml, dissolve from a 10 mg/ml stock in PBS).</li>
	<li>Remove differentiating areas with pulled glass Pasteur pipettes. Areas that should be removed include crater-like or cystic structures in the center of colonies, and any areas where cells have become separated from colonies and flattened and seem to be migrating outwards.</li>
	<li>Incubate at 37 &deg;C under a culture hood until colony borders become brighter and begin to detach (usually around 5-7 min). Discard dispase. Wash with 1 ml H-ESM and discard.</li>
	<li>In 1 ml H-ESM, use a cell lifter (spatula-like scraper) to harvest colonies and collect in a 15 ml Eppendorf tube. Rinse with 1 ml H-ESM and add to the tube. Spin at 1,000 rcf for 4 min and remove the supernatant.</li>
	<li>Resuspend pellet in 1 ml pre-warmed growth medium (either mTESR1 or Nutristem). Avoid breaking up clumps too much - pipette up and down less than 6-8x. Determine how many cells should be plated and remove unnecessary cells (usually 1:4 or 1:5 dilution), e.g. for 2 plates at 1:5, remove 600 &mu;l, then add 1.6 ml fresh medium.</li>
	<li>Remove Matrigel from plates. Place cells drop by drop, distributing evenly on the plate. Avoid shaking the tube excessively, as this will cause cells to move towards the center.</li>
	<li>Wash tube with 1 ml medium and divide between plates. Final volume in each plate should be 1.5 -2 ml.</li>
	<li>Change medium everyday, except for the day after passaging. Although it is ideal to change the medium everyday, this may be occasionally changed at a frequency of once every two days if no more than 2-3 days have passed since the last passage without affecting pluripotency or differentiation potential.</li>
	<li>If the cell line must be frozen, re-suspend instead in 1-2 ml mFreSR freezing medium using a 2 ml pipette and place 1 ml/cryovial. Place vials in a cryobox at -80 &deg;C and transfer to liquid nitrogen in 3-4 days.</li>
</ol>
Note: Manual microdissection of iPS cells must be performed under a stereomicroscope. An in vitro fertilization (IVF) workstation (e.g. an IVF Workstation from KSystem) integrated with a stereomicroscope allows manipulation of the cells in sterile conditions while they are kept on a heated surface area. If this is not available, a stereomicroscope can be moved under a regular laminar flow hood.
2. Embryoid Bodies Aggregation and Cardiac Induction
<ol>
	<li>Prepare EBM-20 medium according to the table below.
	<table border="1" fo:keep-together.within-page="always">
		<tbody>
			<tr>
				<td>EBM-20</td>
				<td>FINAL CONC</td>
				<td>FOR 500 ml</td>
			</tr>
			<tr>
				<td>Fetal Bovine Serum (origin South America)</td>
				<td>20%</td>
				<td>100 ml</td>
			</tr>
			<tr>
				<td>MEM non-essential amino acids</td>
				<td>0.1 mM</td>
				<td>5 ml</td>
			</tr>
			<tr>
				<td>Penicillin - streptomycin</td>
				<td>100U/ml - 0.1 mg/ml</td>
				<td>5 ml</td>
			</tr>
			<tr>
				<td>GlutaMAX 100X</td>
				<td>0.1 mM</td>
				<td>5 ml</td>
			</tr>
			<tr>
				<td>2-mercaptoethanol</td>
				<td>50uM</td>
				<td>450 &mu;l</td>
			</tr>
			<tr>
				<td>DMEM/F12</td>
				<td>-</td>
				<td>385 ml</td>
			</tr>
		</tbody>
	</table>
	 
	Use of FBS of South America origin is critical for determining the differentiation efficiency: employment of other types of sera may affect the differentiation process. To prepare complete EBM-20, add ascorbic acid (AA) to a final concentration of 50 &mu;g/ml. Complete growth medium can be stored at 4 &deg;C for no more than one week.</li>
	<li>Harvest iPS colonies as described above (steps 1.6 to 1.9).</li>
	<li>Resuspend gently in 1 ml EBM-20 with a 2 ml pipette. Avoid breaking up clumps too much - pipette up and down no more than 2-3x, checking the size of the colonies under the stereomicroscope. Plate on ultra-low attachment plates in 1:1 ratio (e.g. for a 35 mm dish, use 1 well of a 6-well plate). Rinse tube with 1 ml EBM-20 and add to plate.</li>
</ol>
Note: The size of the colonies affect the differentiation process, controlling efficiency and timing of cardiac induction. Aggregation of homogeneously-sized EBs have been shown to reduce the variability observed during differentiation.
<ol start="4">
	<li>On day 3, change EBM-20 once using a microscope to avoid removal of EBs. Keep pipette close to edge of plate, and remove medium.</li>
	<li>On day 7, switch EBs to plates coated with 0.1% gelatin and previously placed at 37 &deg;C for 30 min. If necessary, gelatin can be removed and plates left under a cell culture hood O/N. Add 35-40 EBs per 35 mm dish.</li>
	<li>Check EBs for beating areas and change EBM-20 twice per week. Mark out where beating areas are located on the top of the dish cover, to facilitate they localization under the stereomicroscope. Contracting areas should appear after approximately 10-20 days.</li>
	<li>When areas with spontaneous contraction appear, switch medium to EBM-2 (prepare as EBM-20, with 2% FBS instead) and isolate them as described below in section 3.</li>
	<li>Continue to check other areas for beating and change medium twice per week until beating areas are needed for further experiments.</li>
</ol>
Note: Efficiency of the differentiation process is highly dependent on several factors, the most critical of which are the specific cell lines and the batch of serum used to induce cardiac differentiation. To obtain the highest efficiency, test cell lines for cardiomyogenic potential and various batches of serum.
3. Beating Areas Isolation and Culture
<ol>
	<li>Coat 12-well plates (4 cm2 area) or the plates of interest with 5 &mu;g/cm2 fibronectin, diluted in PBS to make enough volume to cover the entire surface completely (300 &mu;l total per well in 12-well plates). Keep uncovered in cell culture hood and allow to dry. Plates can be wrapped and stored at 4 &deg;C O/N.</li>
	<li>Remove beating area using a glass pulled Pasteur pipette and scalpel, as needed. Transfer to fibronectin-coated plates with a 1 ml pipette.</li>
	<li>Add 1 ml EBM-2.</li>
	<li>Cross off the plate to indicate the areas where cells were removed and change the medium. Plates can be kept for up to 1 month, as other areas may start beating later on.</li>
</ol>
Note: If smaller beating clumps are needed, pipette the explanted area up and down several times under the stereomicroscope, until it breaks into smaller pieces. This will result in clusters of a few beating cells (see movie 3).
<ol start="5">
	<li>Change medium twice a week until ready for the analyses.</li>
</ol>
Note: Cardiomyocytes obtained from this protocol possess fetal-like morphological and functional characteristics; maturation towards an adult-like phenotype may be obtained by increasing the time in culture to further differentiate these cells.
4. Single Beating Cells Isolation and Analysis
<ol>
	<li>Coat 2-chamber slides (4 cm2 area) or other appropriate supports with 5 &mu;g/cm2 fibronectin diluted in sufficient PBS to cover the entire culture surface. If a glass support is used, coat with laminin and fibronectin ( 5 &mu;g/cm2 each).</li>
	<li>Remove beating area with a Pasteur pipette/scalpel from the plates from section 3.</li>
	<li>Using a 1 ml pipette, transfer cells to a 15 ml tube containing 500 &mu;l collagenase II (480 U/ml in PBS with Mg and Ca) and incubate 15 min at 37 &deg;C, shaking the tube once during the incubation.</li>
	<li>Pipette up and down with a 200 &mu;l pipette. If any clumps remain, transfer to fresh collagenase II and repeat step 4.3.</li>
	<li>Repeat step 4.4 until no large clumps are left.</li>
	<li>Inactivate collagenase with 3 ml EBM-2 (no AA). The tube can then be left under the hood in a heated rack until other tubes are ready. Centrifuge at 1,000 rcf for 4 min.</li>
	<li>Resuspend pellet in 1 ml 0.25% trypsin/EDTA (diluted from a 0.5% stock in 1X PBS) and incubate at 37 &deg;C for 5 min. Combine fractions of digestions from the same initial sample.</li>
	<li>Inactivate trypsin with 4 ml EBM-2 (no AA). Pass cells through an 18G needle on a 2.5 ml syringe no more than 7x. Centrifuge at 1,000 rcf for 4 min.</li>
	<li>Resuspend cell pellet in 1 ml EBM-2 per well and plate. Cells can be used for functional and morphological analyses 2-3 days after plating.</li>
</ol>

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No financial interests to disclose.

Pluripotent stem cells have the potential to differentiate spontaneously into CMs, albeit with low efficiency and high variability among lines. The development of novel induction methods has therefore focused on improving the efficiency of the process and moving toward more defined protocols. However, most of these new differentiation methods are quite complex, requiring elaborate culture conditions, fine control of timing and concentration of reagents, and treatment with expensive cytokines and growth factors. Also, dif...

Historically, the investigation of the genetic and molecular mechanisms driving human development and disease has been based on the generation of genetically modified animal models. However, numerous human phenotypes fail to be successfully replicated in mice, mainly because of the biological differences existing between the two species. On the other hand, access to human tissues may be limited and often do not allow enough material to be obtained for in-depth experimental studies. The field of cardiovascular biology suffers from both these limitations: the physiology of the human heart is significantly different from that of the mouse, and a significant quantity of heart tissue is accessible only post-mortem or during heart surgery. Finding an optimal source for the differentiation of functional human CMs has therefore become a central topic in cardiovascular biology, and much effort has been made to address this issue. Various cells types have been proposed, including skeletal myoblasts, local cardiac stem cells, bone marrow mononuclear cells, endothelial progenitors, and mesenchymal stem cells. However, data obtained using these cells have not been consistent 4.
The derivation of human embryonic stem cells (ESC) first 5 and the groundbreaking discovery of induced pluripotent stem (iPS) cells by Yamanaka and Thomson 6,7 later seemed to provide solutions: these cells have the ability to grow indefinitely and the potential to give rise to all cell derivatives of the three germ layers, including the CMs. The use of iPS cells offers further advantages: being derived from autologous adult cells, they carry the same genome as the individual or patient from which they are derived. They therefore allow the development of in vitro models that facilitate the investigation of human genetic diseases and mechanisms of development. In virtue of this characteristic, iPS cells can also overcome problems related to immune rejection and ethical issues 8. For these reasons, even though ESCs still represent the gold standard in the field of stem cell biology, researchers worldwide are now moving more toward iPS cell technology.
iPS cells are now being employed to model human disease in vitro for various conditions, including congenital cardiovascular disorders 9,10. Recently, application of iPS cell disease modeling has been performed for monogenic cardiac disorders (i.e. long QT syndromes, catecholaminergic polymorphic ventricular tachycardia) and pathologies in which cardiac defects are part of a complex phenotype (i.e. Leopard and Timothy syndromes, dilated cardiomyopathy). These reports confirmed that patient-specific iPS cells that are differentiated into CMs display similar phenotypic and functional characteristics as the disease in vivo.
However, there are still many challenges to improve the efficacy of inducing the iPS cells into the cardiac lineage. Spontaneous generation of CMs from human ESCs via aggregate formation called embryoid bodies (EBs) have proven successful 17. Since this discovery, many other methods have been proposed. Many groups have greatly enhanced the efficiency of the differentiation protocol and have moved toward chemically defined and animal-product-free culture reagents (see Mummery, C., et al., Circulation Research (2012) for a comprehensive review of all existing methods 3).
Nevertheless, the classical method based on EB aggregation still represents the most commonly employed for performing functional studies and investigating disease mechanisms. Our proposed protocol is based on the aggregation of iPS cells into EBs and culture in the presence of serum and ascorbic acid, which has been shown to enhance the cardiac differentiation process and to positively impact the maturation of these cells 18,19. In this article we will go through this methodology in detail and will show how to use feeder-free iPS cell lines for generating patient-specific iPS-derived CMs.

<table border="1" fo:keep-together.within-page="always">
	<tbody>
		<tr>
			<td>Name of the reagent</td>
			<td>Company</td>
			<td>Catalogue number</td>
			<td>Comments (optional)</td>
		</tr>
		<tr>
			<td>&nbsp;</td>
			<td>&nbsp;</td>
			<td>&nbsp;</td>
			<td>Media and Reagents</td>
		</tr>
		<tr>
			<td>Human ESC-qualified Matrix</td>
			<td>BD Biosciences</td>
			<td>354277</td>
			<td>Thaw the 5 ml bottle at 4 &deg;C on ice O/N, aliquot in pre-chilled cryo-tubes on ice, according to the data sheet dilution instructions and store at -20 &deg;C .</td>
		</tr>
		<tr>
			<td>Fibronectin, from human plasma, 0.1% solution</td>
			<td>Sigma</td>
			<td>F0896-2MG</td>
			<td>Working concentration: 5 &mu;g/cm2</td>
		</tr>
		<tr>
			<td>Laminin</td>
			<td>Sigma</td>
			<td>L2020-1MG</td>
			<td>Working concentration: 5 &mu;g/cm2</td>
		</tr>
		<tr>
			<td>PBS (no Mg and Ca), 10X</td>
			<td>Lonza</td>
			<td>BE17-515F</td>
			<td>&nbsp;</td>
		</tr>
		<tr>
			<td>PBS (with Mg and Ca), 10X</td>
			<td>Bio Sera</td>
			<td>XC-S2067/500</td>
			<td>&nbsp;</td>
		</tr>
		<tr>
			<td>Dispase II, neutral protease, grade II</td>
			<td>Roche</td>
			<td>4942078001</td>
			<td>Stock: 10 mg/ml in DMEM-F12 at -20 &deg;C, working solution: 1 mg/ml in PBS (no Mg/Ca)</td>
		</tr>
		<tr>
			<td>Trypsin/EDTA, 0.5%</td>
			<td>Sigma</td>
			<td>T3924</td>
			<td>&nbsp;</td>
		</tr>
		<tr>
			<td>Collagenase type 2</td>
			<td>Worthington</td>
			<td>4176</td>
			<td>Dilute to 480 U/ml (e.g. 1.6 mg/ml) in PBS with Mg and Ca</td>
		</tr>
		<tr>
			<td>DMEM-F12</td>
			<td>Life Technologies</td>
			<td>21331-020</td>
			<td>&nbsp;</td>
		</tr>
		<tr>
			<td>Knockout DMEM</td>
			<td>Life Technologies</td>
			<td>10829-018</td>
			<td>&nbsp;</td>
		</tr>
		<tr>
			<td>Knockout Serum Replacement (KSR)</td>
			<td>Life Technologies</td>
			<td>10828-028</td>
			<td>Thaw at 4 &deg;C, aliquot and store at -20 &deg;C</td>
		</tr>
		<tr>
			<td>Foetal Bovine Serum (origin South America)</td>
			<td>Invitrogen</td>
			<td>10270-106</td>
			<td>Batches should be tested for their cardiogenic potential. Thaw at 4 &deg;C, heat-inactivate at 56 &deg;C for 30 min, aliquot and store at -20 &deg;C</td>
		</tr>
		<tr>
			<td>PenStrep, penicillin-Streptomycin, 100X</td>
			<td>Life Technologies</td>
			<td>15140-122</td>
			<td>&nbsp;</td>
		</tr>
		<tr>
			<td>GlutaMAX, 100X</td>
			<td>Life Technologies</td>
			<td>35050-038</td>
			<td>&nbsp;</td>
		</tr>
		<tr>
			<td>MEM NEAA, 100X</td>
			<td>Invitrogen</td>
			<td>11140-135</td>
			<td>&nbsp;</td>
		</tr>
		<tr>
			<td>N2 supplement, 100X</td>
			<td>Life Technologies</td>
			<td>17502-048</td>
			<td>&nbsp;</td>
		</tr>
		<tr>
			<td>B27 supplement, 50X</td>
			<td>Life Technologies</td>
			<td>12587-010</td>
			<td>&nbsp;</td>
		</tr>
		<tr>
			<td>2-mercaptoethanol</td>
			<td>Invitrogen</td>
			<td>31350-010</td>
			<td>&nbsp;</td>
		</tr>
		<tr>
			<td>Gelatin, from porcine skin</td>
			<td>Sigma</td>
			<td>G1890-100G</td>
			<td>Make stock at 1% in water, store at -20 &deg;C. Dilute to 0.1% in PBS and keep at 4 &deg;C</td>
		</tr>
		<tr>
			<td>mTeSR1</td>
			<td>Stem Cell Technologies</td>
			<td>5850</td>
			<td>Combine Supplement 5X with the basic medium, aliquot and store at -20 &deg;C. Do not keep complete medium at 4 &deg;C for more than 1 week</td>
		</tr>
		<tr>
			<td>Nutristem hESC XF</td>
			<td>Stemgent from Biological Industries</td>
			<td>05-100-1A</td>
			<td>Thaw at 4 &deg;C O/N, aliquot and store at -20 &deg;C until use. Aliquots may be kept at 4 &deg;C for no more than a week.</td>
		</tr>
		<tr>
			<td>mFreSR</td>
			<td>Stem Cell Technologies</td>
			<td>5853</td>
			<td>&nbsp;</td>
		</tr>
		<tr>
			<td>Ascorbic acid</td>
			<td>Sigma</td>
			<td>A4544-25G</td>
			<td>Prepare stocks at 10 mg/ml in water. Store aliquots at -20 &deg;C in the dark. Add to EBM at the final concentration (500X)</td>
		</tr>
		<tr>
			<td>&nbsp;</td>
			<td>&nbsp;</td>
			<td>&nbsp;</td>
			<td>Plasticware</td>
		</tr>
		<tr>
			<td>35 mm culture dishes</td>
			<td>Becton Dickinson</td>
			<td>353001</td>
			<td>&nbsp;</td>
		</tr>
		<tr>
			<td>Cell scraper</td>
			<td>Corning Incorporated</td>
			<td>3008</td>
			<td>&nbsp;</td>
		</tr>
		<tr>
			<td>12-well plates</td>
			<td>Becton Dickinson</td>
			<td>353043</td>
			<td>&nbsp;</td>
		</tr>
		<tr>
			<td>Permanox 2-well chamber slides</td>
			<td>Thermo Fisher Scientific</td>
			<td>177437</td>
			<td>&nbsp;</td>
		</tr>
		<tr>
			<td>Glass 2-well chamber slides</td>
			<td>Thermo Fisher Scientific</td>
			<td>177380</td>
			<td>&nbsp;</td>
		</tr>
		<tr>
			<td>6-well plates, ultra-low-attachment surface</td>
			<td>Corning Incorporated</td>
			<td>3471</td>
			<td>&nbsp;</td>
		</tr>
		<tr>
			<td>18G needle</td>
			<td>Becton Dickinson</td>
			<td>304622</td>
			<td>&nbsp;</td>
		</tr>
		<tr>
			<td>Glass pasteur pipettes, 230 mm</td>
			<td>VWR International</td>
			<td>612-1702</td>
			<td>&nbsp;</td>
		</tr>
		<tr>
			<td>2.5 ml syringe</td>
			<td>Becton Dickinson</td>
			<td>300188</td>
			<td>&nbsp;</td>
		</tr>
		<tr>
			<td>&nbsp;</td>
			<td>&nbsp;</td>
			<td>&nbsp;</td>
			<td>Equipments</td>
		</tr>
		<tr>
			<td>IVF Workstation</td>
			<td>KSystem, by Nikon</td>
			<td>&nbsp;</td>
			<td>&nbsp;</td>
		</tr>
		<tr>
			<td>Nikon SMZ1500 Stereomicroscope</td>
			<td>Nikon</td>
			<td>&nbsp;</td>
			<td>&nbsp;</td>
		</tr>
	</tbody>
</table>

generation of human cardiomyocytes: a differentiation protocol from feeder-free human induced pluripotent stem cells

In order to investigate the events driving heart development and to determine the molecular mechanisms leading to myocardial diseases in humans, it is essential first to generate functional human cardiomyocytes (CMs). The use of these cells in drug discovery and toxicology studies would also be highly beneficial, allowing new pharmacological molecules for the treatment of cardiac disorders to be validated pre-clinically on cells of human origin. Of the possible sources of CMs, induced pluripotent stem (iPS) cells are among the most promising, as they can be derived directly from readily accessible patient tissue and possess an intrinsic capacity to give rise to all cell types of the body 1. Several methods have been proposed for differentiating iPS cells into CMs, ranging from the classical embryoid bodies (EBs) aggregation approach to chemically defined protocols 2,3. In this article we propose an EBs-based protocol and show how this method can be employed to efficiently generate functional CM-like cells from feeder-free iPS cells.

In our studies we generated CMs from several iPS cell lines. These lines were initially generated on an MEF feeder layer but were immediately placed on Matrigel using a defined medium (either mTESR1 or Nutristem). Various assays were used to verify maintenance of the morphological properties, the expression of markers typical of pluripotent cells (OCT-4, SSEA4 and TRA1-60) and their genome stability (Figure 1).
Induction into the cardiac lineage was triggered by aggregation of...

Watch this Scientific Journal Video about Generation of Human Cardiomyocytes: A Differentiation Protocol from Feeder-free Human Induced Pluripotent Stem Cells at JoVE.com

Generation of Human Cardiomyocytes: A Differentiation Protocol from Feeder-free Human Induced Pluripotent Stem Cells

Pluripotent stem cells, either embryonic or induced pluripotent stem (iPS) cells, constitute a valuable source of human differentiated cells, including cardiomyocytes. Here, we will focus on cardiac induction of iPS cells, showing how to use them to obtain functional human cardiomyocytes through an embryoid bodies-based protocol.

In order to investigate the events driving heart development and to determine the molecular mechanisms leading to myocardial diseases in humans, it is ...

generation-human-cardiomyocytes-differentiation-protocol-from-feeder

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Biology

21.2K vistas.  Humanitas Clinical and Research Center, Italy. Las células madre pluripotentes, cualquiera de las células madre pluripotentes embrionarias o inducidas (iPS), constituyen una valiosa fuente de células diferenciadas humanas, incluyendo cardiomiocitos. Aquí, nos centraremos en la inducción cardiaca de las células iPS, que muestra cómo usarlos para obtener cardiomiocitos humanos funcionales a través de un protocolo de los organismos con sede en embrioide.

Video: Generation of Human Cardiomyocytes: A Differentiation Protocol from Feeder-free Human Induced Pluripotent Stem Cells

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 ...

Transfecting and Nucleofecting Human Induced Pluripotent Stem Cells

Genetic modification is continuing to be an essential tool in studying stem cell biology and in setting forth potential clinical applications of human embryonic stem cells (HESCs)1. While improvements in several gene delivery methods have been described2-9, transfection remains a capricious process for HESCs, and has not yet been reported in human induced pluripotent stem cells (iPSCs). In this video, we demonstrate how our lab routinely transfects and nucleofects human iPSCs using plasmid with an enhanced green fluorescence protein (eGFP) reporter. Human iPSCs are adapted and maintained as feeder-free cultures to eliminate the possibility of feeder cell transfection and to allow efficient selection of stable transgenic iPSC clones following transfection. For nucleofection, human iPSCs are pre-treated with ROCK inhibitor11, trypsinized into small clumps of cells, nucleofected and replated on feeders in feeder cell-conditioned medium to enhance cell recovery. Transgene-expressing human iPSCs can be obtained after 6 hours. Antibiotic selection is applied after 24 hours and stable transgenic lines appear within 1 week. Our protocol is robust and reproducible for human iPSC lines without altering pluripotency of these cells.

Despite recent advancements in genetic modification, transfection of human embryonic stem cells (HESCs) remains a capricious process. To our knowledge, systematic and efficient methods to transfect human induced pluripotent stem cells (iPSCs) have not been reported. Here, we describe robust protocols to efficiently transfect and nucleofect human iPSCs.

Genetic modification is continuing to be an essential tool in studying stem cell biology and in setting forth potential clinical applications of human ...

Efficient Derivation of Human Cardiac Precursors and Cardiomyocytes from Pluripotent Human Embryonic Stem Cells with Small Molecule Induction

To date, the lack of a suitable human cardiac cell source has been the major setback in regenerating the human myocardium, either by cell-based transplantation or by cardiac tissue engineering1-3. Cardiomyocytes become terminally-differentiated soon after birth and lose their ability to proliferate. There is no evidence that stem/progenitor cells derived from other sources, such as the bone marrow or the cord blood, are able to give rise to the contractile heart muscle cells following transplantation into the heart1-3. The need to regenerate or repair the damaged heart muscle has not been met by adult stem cell therapy, either endogenous or via cell delivery1-3. The genetically stable human embryonic stem cells (hESCs) have unlimited expansion ability and unrestricted plasticity, proffering a pluripotent reservoir for in vitro derivation of large supplies of human somatic cells that are restricted to the lineage in need of repair and regeneration4,5. Due to the prevalence of cardiovascular disease worldwide and acute shortage of donor organs, there is intense interest in developing hESC-based therapies as an alternative approach. However, how to channel the wide differentiation potential of pluripotent hESCs efficiently and predictably to a desired phenotype has been a major challenge for both developmental study and clinical translation. Conventional approaches rely on multi-lineage inclination of pluripotent cells through spontaneous germ layer differentiation, resulting in inefficient and uncontrollable lineage-commitment that is often followed by phenotypic heterogeneity and instability, hence, a high risk of tumorigenicity6-8 (see a schematic in Fig. 1A). In addition, undefined foreign/animal biological supplements and/or feeders that have typically been used for the isolation, expansion, and differentiation of hESCs may make direct use of such cell-specialized grafts in patients problematic9-11. To overcome these obstacles, we have resolved the elements of a defined culture system necessary and sufficient for sustaining the epiblast pluripotence of hESCs, serving as a platform for de novo derivation of clinically-suitable hESCs and effectively directing such hESCs uniformly towards clinically-relevant lineages by small molecules12 (see a schematic in Fig. 1B). After screening a variety of small molecules and growth factors, we found that such defined conditions rendered nicotinamide (NAM) sufficient to induce the specification of cardiomesoderm direct from pluripotent hESCs that further progressed to cardioblasts that generated human beating cardiomyocytes with high efficiency (Fig. 2). We defined conditions for induction of cardioblasts direct from pluripotent hESCs without an intervening multi-lineage embryoid body stage, enabling well-controlled efficient derivation of a large supply of human cardiac cells across the spectrum of developmental stages for cell-based therapeutics.

We have established a protocol for induction of cardioblasts direct from pluripotent human embryonic stem cells maintained under defined conditions with small molecules, which enables derivation of a large supply of human cardiac progenitors and functional cardiomyocytes for cardiovascular repair.

To date, the lack of a suitable human cardiac cell source has been the major setback in regenerating the human myocardium, either by cell-based ...

Generation of Mice Derived from Induced Pluripotent Stem Cells

The production of induced pluripotent stem cells (iPSCs) from somatic cells provides a means to create valuable tools for basic research and may also produce a source of patient-matched cells for regenerative therapies. iPSCs may be generated using multiple protocols and derived from multiple cell sources. Once generated, iPSCs are tested using a variety of assays including immunostaining for pluripotency markers, generation of three germ layers in embryoid bodies and teratomas, comparisons of gene expression with embryonic stem cells (ESCs) and production of chimeric mice with or without germline contribution2. Importantly, iPSC lines that pass these tests still vary in their capacity to produce different differentiated cell types2. This has made it difficult to establish which iPSC derivation protocols, donor cell sources or selection methods are most useful for different applications.
The most stringent test of whether a stem cell line has sufficient developmental potential to generate all tissues required for survival of an organism (termed full pluripotency) is tetraploid embryo complementation (TEC)3-5. Technically, TEC involves electrofusion of two-cell embryos to generate tetraploid (4n) one-cell embryos that can be cultured in vitro to the blastocyst stage6. Diploid (2n) pluripotent stem cells (e.g. ESCs or iPSCs) are then injected into the blastocoel cavity of the tetraploid blastocyst and transferred to a recipient female for gestation (see Figure 1). The tetraploid component of the complemented embryo contributes almost exclusively to the extraembryonic tissues (placenta, yolk sac), whereas the diploid cells constitute the embryo proper, resulting in a fetus derived entirely from the injected stem cell line.
Recently, we reported the derivation of iPSC lines that reproducibly generate adult mice via TEC1. These iPSC lines give rise to viable pups with efficiencies of 5-13%, which is comparable to ESCs3,4,7 and higher than that reported for most other iPSC lines8-12. These reports show that direct reprogramming can produce fully pluripotent iPSCs that match ESCs in their developmental potential and efficiency of generating pups in TEC tests. At present, it is not clear what distinguishes between fully pluripotent iPSCs and less potent lines13-15. Nor is it clear which reprogramming methods will produce these lines with the highest efficiency. Here we describe one method that produces fully pluripotent iPSCs and "all- iPSC" mice, which may be helpful for investigators wishing to compare the pluripotency of iPSC lines or establish the equivalence of different reprogramming methods.

Generating induced pluripotent stem cell (iPSC) lines produces lines of differing developmental potential even when they pass standard tests for pluripotency. Here we describe a protocol to produce mice derived entirely from iPSCs, which defines the iPSC lines as possessing full pluripotency1.

The production of induced pluripotent stem cells (iPSCs) from somatic cells provides a means to create valuable tools for basic research and may also ...

Generation of Human Induced Pluripotent Stem Cells from Peripheral Blood Using the STEMCCA Lentiviral Vector

Through the ectopic expression of four transcription factors, Oct4, Klf4, Sox2 and cMyc, human somatic cells can be converted to a pluripotent state, generating so-called induced pluripotent stem cells (iPSCs)1-4. Patient-specific iPSCs lack the ethical concerns that surround embryonic stem cells (ESCs) and would bypass possible immune rejection. Thus, iPSCs have attracted considerable attention for disease modeling studies, the screening of pharmacological compounds, and regenerative therapies5.
We have shown the generation of transgene-free human iPSCs from patients with different lung diseases using a single excisable polycistronic lentiviral Stem Cell Cassette (STEMCCA) encoding the Yamanaka factors6. These iPSC lines were generated from skin fibroblasts, the most common cell type used for reprogramming. Normally, obtaining fibroblasts requires a skin punch biopsy followed by expansion of the cells in culture for a few passages. Importantly, a number of groups have reported the reprogramming of human peripheral blood cells into iPSCs7-9. In one study, a Tet inducible version of the STEMCCA vector was employed9, which required the blood cells to be simultaneously infected with a constitutively active lentivirus encoding the reverse tetracycline transactivator. In contrast to fibroblasts, peripheral blood cells can be collected via minimally invasive procedures, greatly reducing the discomfort and distress of the patient. A simple and effective protocol for reprogramming blood cells using a constitutive single excisable vector may accelerate the application of iPSC technology by making it accessible to a broader research community. Furthermore, reprogramming of peripheral blood cells allows for the generation of iPSCs from individuals in which skin biopsies should be avoided (i.e. aberrant scarring) or due to pre-existing disease conditions preventing access to punch biopsies.
Here we demonstrate a protocol for the generation of human iPSCs from peripheral blood mononuclear cells (PBMCs) using a single floxed-excisable lentiviral vector constitutively expressing the 4 factors. Freshly collected or thawed PBMCs are expanded for 9 days as described10,11 in medium containing ascorbic acid, SCF, IGF-1, IL-3 and EPO before being transduced with the STEMCCA lentivirus. Cells are then plated onto MEFs and ESC-like colonies can be visualized two weeks after infection. Finally, selected clones are expanded and tested for the expression of the pluripotency markers SSEA-4, Tra-1-60 and Tra-1-81. This protocol is simple, robust and highly consistent, providing a reliable methodology for the generation of human iPSCs from readily accessible 4 ml of blood.

Here we show a simple and effective protocol for the generation of human iPSCs from 3-4 ml of peripheral blood using a single lentiviral reprogramming vector. Reprogramming of readily available blood cells promises to accelerate the utilization of iPSC technology by making it accessible to a broader research community.

Through the ectopic expression of four transcription factors, Oct4, Klf4, Sox2 and cMyc, human somatic cells can be converted to a pluripotent state, ...

Rapid and Efficient Generation of Neurons from Human Pluripotent Stem Cells in a Multititre Plate Format

Existing protocols for the generation of neurons from human pluripotent stem cells (hPSCs) are often tedious in that they are multistep procedures involving the isolation and expansion of neural precursor cells, prior to terminal differentiation. In comparison to these time-consuming approaches, we have recently found that combined inhibition of three signaling pathways, TGF&beta;/SMAD2, BMP/SMAD1, and FGF/ERK, promotes rapid induction of neuroectoderm from hPSCs, followed by immediate differentiation into functional neurons. Here, we have adapted our procedure to a novel multititre plate format, to further enhance its reproducibility and to make it compatible with mid-throughput applications. It comprises four days of neuroectoderm formation in floating spheres (embryoid bodies), followed by a further four days of differentiation into neurons under adherent conditions. Most cells obtained with this protocol appear to be bipolar sensory neurons. Moreover, the procedure is highly efficient, does not require particular expert skills, and is based on a simple chemically defined medium with cost-efficient small molecules. Due to these features, the procedure may serve as a useful platform for further functional investigation as well as for cell-based screening approaches requiring human sensory neurons or neurons of any type.

Protocols for neuronal differentiation of pluripotent human stem cells (hPSCs) are often time-consuming and require substantial cell culture skills. Here, we have adapted a small molecule-based differentiation procedure to a multititre plate format, allowing simple, rapid, and efficient generation of human neurons in a controlled manner.

Existing protocols for the generation of neurons from human pluripotent stem cells (hPSCs) are often tedious in that they are  multistep procedures ...

Efficient Generation Human Induced Pluripotent Stem Cells from Human Somatic Cells with Sendai-virus

A few years ago, the establishment of human induced pluripotent stem cells (iPSCs) ushered in a new era in biomedicine. Potential uses of human iPSCs include modeling pathogenesis of human genetic diseases, autologous cell therapy after gene correction, and personalized drug screening by providing a source of patient-specific and symptom relevant cells. However, there are several hurdles to overcome, such as eliminating the remaining reprogramming factor transgene expression after human iPSCs production. More importantly, residual transgene expression in undifferentiated human iPSCs could hamper proper differentiations and misguide the interpretation of disease-relevant in vitro phenotypes. With this reason, integration-free and/or transgene-free human iPSCs have been developed using several methods, such as adenovirus, the piggyBac system, minicircle vector, episomal vectors, direct protein delivery and synthesized mRNA. However, efficiency of reprogramming using integration-free methods is quite low in most cases.
Here, we present a method to isolate human iPSCs by using Sendai-virus (RNA virus) based reprogramming system. This reprogramming method shows consistent results and high efficiency in cost-effective manner.

Here, we present our established method to reprogram human somatic cells into transgene-free human iPSCs with Sendai virus, which shows consistent outcome and enhanced efficiency.

A few years ago, the establishment of human induced pluripotent stem cells (iPSCs) ushered in a new era in biomedicine. Potential uses of human iPSCs ...

High Efficiency Differentiation of Human Pluripotent Stem Cells to Cardiomyocytes and Characterization by Flow Cytometry

There is an urgent need to develop approaches for repairing the damaged heart, discovering new therapeutic drugs that do not have toxic effects on the heart, and improving strategies to accurately model heart disease. The potential of exploiting human induced pluripotent stem cell (hiPSC) technology to generate cardiac muscle &#8220;in a dish&#8221; for these applications continues to generate high enthusiasm. In recent years, the ability to efficiently generate cardiomyogenic cells from human pluripotent stem cells (hPSCs) has greatly improved, offering us new opportunities to model very early stages of human cardiac development not otherwise accessible. In contrast to many previous methods, the cardiomyocyte differentiation protocol described here does not require cell aggregation or the addition of Activin A or BMP4 and robustly generates cultures of cells that are highly positive for cardiac troponin I and T (TNNI3, TNNT2), iroquois-class homeodomain protein IRX-4 (IRX4), myosin regulatory light chain 2, ventricular/cardiac muscle isoform (MLC2v) and myosin regulatory light chain 2, atrial isoform (MLC2a) by day 10 across all human embryonic stem cell (hESC) and hiPSC lines tested to date. Cells can be passaged and maintained for more than 90 days in culture. The strategy is technically simple to implement and cost-effective. Characterization of cardiomyocytes derived from pluripotent cells often includes the analysis of reference markers, both at the mRNA and protein level. For protein analysis, flow cytometry is a powerful analytical tool for assessing quality of cells in culture and determining subpopulation homogeneity. However, technical variation in sample preparation can significantly affect quality of flow cytometry data. Thus, standardization of staining protocols should facilitate comparisons among various differentiation strategies. Accordingly, optimized staining protocols for the analysis of IRX4, MLC2v, MLC2a, TNNI3, and TNNT2 by flow cytometry are described.

The article describes the detailed methodology to efficiently differentiate human pluripotent stem cells into cardiomyocytes by selectively modulating the Wnt pathway, followed by flow cytometry analysis of reference markers to assess homogeneity and identity of the population.

There is an urgent need to develop approaches for repairing the damaged heart, discovering new therapeutic drugs that do not have toxic effects on the ...

Generation and Maintenance of Primate Induced Pluripotent Stem Cells Derived from Urine

Cross-species approaches studying primate pluripotent stem cells and their derivatives are crucial to better understand the molecular and cellular mechanisms of disease, development, and evolution. To make primate induced pluripotent stem cells (iPSCs) more accessible, this paper presents a non-invasive method to generate human and non-human primate iPSCs from urine-derived cells, and their maintenance using a feeder-free culturing method.
The urine can be sampled from a non-sterile environment (e.g., the cage of the animal) and treated with a broad-spectrum antibiotic cocktail during primary cell culture to reduce contamination efficiently. After propagation of the urine-derived cells, iPSCs are generated by a modified transduction method of a commercially available Sendai virus vector system. First&#160;iPSC colonies may already be visible after 5 days, and can be picked after 10 days at the earliest. Routine clump passaging with enzyme-free dissociation buffer supports pluripotency of the generated iPSCs for more than 50 passages.

The present protocol describes a method to isolate, expand, and reprogram human and non-human primate urine-derived cells to induced pluripotent stem cells (iPSCs), as well as instructions for feeder-free maintenance of the newly generated iPSCs.

Cross-species approaches studying primate pluripotent stem cells and their derivatives are crucial to better understand the molecular and cellular ...

Characterizing Surface Antigens and Proliferation Ability in hiPSC-Driving MSCs

Comparison of Two Representative Methods for Differentiation of Human Induced Pluripotent Stem Cells into Mesenchymal Stromal Cells

Mesenchymal stromal cells (MSCs) are adult pluripotent stem cells which have been widely used in regenerative medicine. As somatic tissue-derived MSCs are restricted by limited donation, quality variations, and biosafety, the past 10 years have seen a great rise in efforts to generate MSCs from human induced pluripotent stem cells (hiPSCs). Past and recent efforts in the differentiation of hiPSCs into MSCs have been centered around two culture methodologies: (1) the formation of embryoid bodies (EBs) and (2) the use of monolayer culture. This protocol describes these two representative methods in deriving MSC from hiPSCs. Each method presents its advantages and disadvantages, including time, cost, cell proliferation ability, the expression of MSC markers, and their capability of differentiation in vitro. This protocol demonstrates that both methods can derive mature and functional MSCs from hiPSCs. The monolayer method is characterized by lower cost, simpler operation, and easier osteogenic differentiation, while the EB method is characterized by lower time consumption.

Author Spotlight: Advancements in iPSCs and Genetic Disease Research 

This protocol describes and compares two representative methods for differentiating hiPSCs into mesenchymal stromal cells (MSCs). The monolayer method is characterized by lower cost, simpler operation, and easier osteogenic differentiation. The embryoid bodies (EBs) method is characterized by lower time consumption.

Mesenchymal stromal cells (MSCs) are adult pluripotent stem cells which have been widely used in regenerative medicine. As somatic tissue-derived MSCs are ...