1. Recommendations Before Starting
<ol>
	<li>Do not use PSCs contaminated with mycoplasma because they will not differentiate properly into sinus node cells. Test for mycoplasma contamination before starting the protocol. Do this using a PCR kit for rapid, highly sensitive detection of mycoplasmas and follow the manufacturer`s protocol.</li>
	<li>For each Petri dish (step 2.3.4), coat one 10 cm2 cell culture dish with sterile 7 ml 0.1 % gelatin from cold water fish skin for 1 hr at 37 &#176;C. Remove the gelatin and let the dish dry under sterile conditions in a sterile bench.</li>
	<li>Before you can start the differentiation protocol you need a double transfected stable mouse ES cell line clone containing the following features: i) Constitutive over-expression of TBX3 using a mammalian expression vector. Ii) The G418 resistance gene under the control of the Myh6-promoter5.</li>
	<li>The undifferentiated PSCs cells should be co-cultivated with irradiated feeder cells under standard conditions as described 1.</li>
</ol>
2. Differentiation Procedure
<ol>
	<li>Two Days before Differentiation - Remove PSCs from feeder cells.
	<ol>
		<li>Aspirate the medium, wash the cells with 10 ml phosphate buffered saline PBS, add 7 ml Collagenase IV solution and incubate the cells for 10 min at 37 &#176;C. Place a sterile 40 &#181;m Filter in a 50 ml tube.</li>
		<li>Carefully rinse the PSC colonies (avoid to remove the feeder cells) by pipetting the collagenase solution up and down 5 times.</li>
		<li>Transfer the cell suspension to a 40 &#181;m filter, rinse the filter three times with 8 ml PBS. Turn around the filter and place it upside down in a petri dish. Remove the PSC cell colonies by pipetting 10 ml PBS to the bottom of the filter.</li>
		<li>Transfer the cell suspension to a 15 ml tube and centrifuge for 5 min at 300 x g.</li>
		<li>Remove the PBS, suspend the cells in 1 ml Accutase and incubate at 37 &#176;C for 5 min.</li>
		<li>Add 10 ml PBS, mix the cell solution by pipetting it up and down 5 times and centrifuge for 5 min at 300 x g.</li>
		<li>Remove the PBS, suspend the cells in 10 ml cultivation medium and determine the cell number.</li>
		<li>Seed 15,000 cells/ cm2 on a 75 cm2 filter flask and cultivate them for 2 days at 37 &#176;C, 5% CO2. After 2 days the flask should be 50-70% confluent.</li>
	</ol>
	</li>
	<li>Day 0 - Start of Differentiation
	<ol>
		<li>Remove the medium and wash the cells with 10 ml PBS.</li>
		<li>Aspirate the PBS, add 2 ml Accutase and incubate the cells at 37 &#176;C for 5 min. Place a sterile 40 &#181;m Filter in a 50 ml tube.</li>
		<li>Add 10 ml PBS, transfer the cell suspension to the 40 &#181;m filter, rinse the filter via addition of 10 ml PBS and centrifuge the flow-through for 5 min at 300 x g.</li>
		<li>Suspend the cells in 10 ml differentiation medium and determine the cell number.</li>
		<li>Dilute the cell suspension with differentiation medium to a final concentration of 20,000 cells/ml.</li>
		<li>Pipette 20 ml water and 5 ml hanging drop (HD) solution in a quadratic Petri dish to avoid drying-out of the HDs. 
		NOTE: For each 24 well plate containing iSABs (see section 2.8.6.4/2.8.7) start with 16 Petri dishes.</li>
		<li>Pipette up to 50 ml cell suspension in a tray .</li>
		<li>Turn around the lid of the petri dish. Place 180 HDs each containing 20 &#181;l (400 cells/HD) cell suspension onto the lid using a 12 channel pipette.</li>
		<li>Carefully turn around the lid and place it onto the Petri dish. 
		NOTE: The speed of turning around the lid is very important. If the lid is turned around too slow or too fast, the surface tension of the cell suspension is not high enough to retain the hanging drops on the lid. Before trying to produce HDs for first time practice turning around the lid with 20 &#181;l drops of medium.</li>
		<li>Cultivate the cells for 2 days at 37 &#176;C, 5% CO2 to let them form EBs. 
		NOTE: Place one petri dish filled with water on the top of a stack of 5 Petri dishes. Otherwise the HD in the upper petri dish will dry.</li>
	</ol>
	</li>
	<li>Day 2
	<ol>
		<li>Carefully turn around the lid of the quadratic Petri dish and transfer the EBs derived from two Petri dishes (360 EBs) to a 50 ml tube.</li>
		<li>Wait 10 min to let the EBs settle at the bottom of the tube (Do not centrifuge the EBs).</li>
		<li>Aspirate as much medium as possible, suspend the EBs in 10 ml differentiation medium and transfer the suspension to a 10 cm Petri dish.</li>
	</ol>
	</li>
	<li>Day 2-6 Suspension culture
	<ol>
		<li>Cultivate the EBs in suspension for 4 days at 37 &#176;C, 5% CO2, change medium after 2 days. 
		NOTE: Even though the petri dish is not coated (as opposed to a cell culture dish) the EBs often attach to the surface. Control the petri dishes for attached EBs every day and if necessary detach the EBs from the surface by gentle pipetting. Optional: Shake the petri dishes continuously during the suspension culture period. Be careful with the speed of the shaker. The EBs should neither accumulate in the middle nor float to the border of the Petri dishes.</li>
	</ol>
	</li>
	<li>Day 6
	<ol>
		<li>Transfer the EBs from one Petri dish to one gelatin coated 10 cm2 cell culture dish.</li>
	</ol>
	</li>
	<li>Day 7-12
	<ol>
		<li>The cells should start to beat after 8-12 days. Check for beating foci of the cells daily under a microscope. The cells start to form a layer.</li>
		<li>Change medium daily. 
		NOTE: After changing medium, the cells require 3-4 hr to recover and to start beating again.</li>
	</ol>
	</li>
	<li>Day 12-15 Selection of sinus nodal cells.
	<ol>
		<li>Three days after the cells have started beating (around day 12-15), aspirate the medium and pipette 10 ml of fresh differentiation medium containing 400 &#181;g/ ml G418 to the cells.</li>
	</ol>
	</li>
	<li>Four days after the beginning of the selection dissociate the cell layer. 
	NOTE: It is possible that the cell layer is already detached from the surface.
	<ol>
		<li>Remove the medium carefully without aspirating the cell layer.</li>
		<li>Add 10 ml of PBS and remove the PBS carefully without aspirating the cell layer.</li>
		<li>Add 10 ml of Collagenase IV solution, transfer the cells to a 50 ml tube and incubate the cells at 37 &#176;C for 5 min.</li>
		<li>Vigorously mix the suspension by pipetting up and down 10 times and incubate the cells at 37 &#176;C for 5 min. A suspension of small clusters should occur. If there are still huge parts of the layer left, then repeat step 2.8.4 two more times.</li>
		<li>Add 20 ml of PBS and centrifuge for 10 min at 300 x g.</li>
		<li>Aspirate the PBS carefully. 
		NOTE: If iSABs are to be generated proceed with step 2.8.7. If iSAB derived sinus nodal single cells are to be generated go on with step 2.9.</li>
		<li>Suspend the cells in 12 ml differentiation medium containing 400 &#181;g/ml G418 and seed them on 6 wells of gelatin coated 24 wells (2 ml/well).</li>
	</ol>
	</li>
	<li>Generation of single nodal cell.
	<ol>
		<li>Suspend the cells from step 2.8.6 in 5 ml Accutase and incubate at 37 &#176;C for 5 min.</li>
		<li>Vigorously mix the suspension by pipetting up and down 10 times and incubate the cells at 37 &#176;C for 5 min.</li>
		<li>Add 20 ml of PBS and centrifuge for 5 min at 300 x g.</li>
		<li>Suspend the cells in 12 ml differentiation medium containing 400 &#181;g/ml G418 and seed them on 6 wells of a gelatin coated 24 wells (2 ml/well).</li>
	</ol>
	</li>
	<li>Day 2 after dissociation, aspirate the medium and wash the cells three times with PBS. Add 1 ml differentiation medium/well.</li>
	<li>Day 4-8 after dissociation, change Medium every 2nd day. On day 10 after Dissociation use iSABs or iSAB derived sinus nodal single cells are available for further analysis.</li>
</ol>

<ol>
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M., Wallukat, G., Hescheler, J. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Pluripotent+mouse+embryonic+stem+cells+are+able+to+differentiate+into+cardiomyocytes+expressing+chronotropic+responses+to+adrenergic+and+cholinergic+agents+and+Ca2++channel+blockers.">Pluripotent mouse embryonic stem cells are able to differentiate into cardiomyocytes expressing chronotropic responses to adrenergic and cholinergic agents and Ca2+ channel blockers.</a> Differentiation. 48 (3), 173-182 (1991).</li><li>David, R., 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=MesP1+drives+vertebrate+cardiovascular+differentiation+through+Dkk-1-mediated+blockade+of+Wnt-signalling.">MesP1 drives vertebrate cardiovascular differentiation through Dkk-1-mediated blockade of Wnt-signalling.</a> Nat Cell Biol. 10, 338-345 (2008).</li><li>David, R., 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=Forward+programming+of+pluripotent+stem+cells+towards+distinct+cardiovascular+cell+types.">Forward programming of pluripotent stem cells towards distinct cardiovascular cell types.</a> Cardiovasc Res. 84 (2), 263-272 (2009).</li><li>Hoogaars, W. 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The authors have nothing to disclose.

The ability to produce stem cell derived cardiac pacemaker cells may allow reconstitution of the proper cardiac rhythm in the sense of &#8220;biological pacemakers&#8221;. Likewise, drug testing in vitro will benefit from their availability. PSCs can give rise to any cell type of the mammalian body including cardiomyocytes with pacemaker cell properties8,9,10,11,12,13. However, typically the cell populations within &#8220;Embryoid Bodies&#8221; are highly heterogeneous, which inevitably leads to the requiremen...

The term &#8220;sick sinus syndrome&#8221; summarizes multiple diseases leading to the deterioration of the cardiac pacemaker system. It comprises pathological, symptomatic sinus bradycardia, sinoatrial block, sinus arrest as well as the tachycardia-bradycardia syndrome. Thereby, a &#8220;sick sinus syndrome&#8221; is often accompanied by general cardiac diseases such as an ischemic heart disease, cardiomyopathies or myocarditis. At present, therapeutic approaches are based on the implantation of electrical pacemakers. However, this goes along with a number of risks such as infections and battery failure. Overall, the incidence of complications is still very high in patients having implanted an artificial pacemaker. Furthermore, as opposed to the endogenous pacemaker, these devices do not respond to hormone stimulation.
A future alternative may rely on the availability of &#8220;biological pacemakers&#8221; for which PSCs could serve as a suitable cellular source and which would also be highly valuable for in vitro drug testing. Yet, a major problem lies in the very rare appearance of sinus nodal cells within embryoid bodies (EBs) - this typically does not exceed ~0.5%1.
Previously, it was shown that &#8220;forward programming&#8221; towards specific cardiomyocyte subtypes is feasible via overexpression of distinct early cardiovascular transcription factors such as Mesoderm-specific-posterior 1 (MesP1) and NK2 transcription factor related, locus 5 (Nkx2.5)2,3. For normal size and function of the sinoatrial node (SAN), the T-box transcription factor Tbx3 is crucial, which has been shown to initiate the pacemaker gene program and to control differentiation of the SAN4. While this enhanced the appearance of functional pacemaker cells, the content still did not exceed ~40% within the entire cardiomyocytic cell population.
Therefore, an additional Myh6-promoter based antibiotic selection step5 was introduced by us. This ultimately leads to yet unobserved cardiomyocyte aggregates (&#8220;induced sino-atrial bodies; &#8220;iSABs&#8221;) which exhibit highly increased beating frequencies (&#62;400 bpm) in vitro, for the first time approximating those of a murine heart and comparable to in vitro cultivated sinus nodal cells isolated from a murine heart6. Under Isoprenaline administration even beating frequencies of 550 bpm are achieved. Notably, iSABs consist of over 80% functional nodal cells as evident from extensive physiological analyses7. Recently, several approaches to generate sinus nodal cells using direct reprogramming19, surface markers14 or pharmacological treatment with small molecules16,17 were described. Yet, none of these methods led to such a high purity of pacemaker cells and beating frequencies close to the murine heart as observed in iSABs.
Moreover, in an ex vivo model of cultivated adult mouse ventricular slices which have lost their spontaneous beating activity, the iSABs are capable of integrating into the slice tissue, thereby remaining spontaneously active and robustly pacing the heart slices to contractions7. A detailed protocol for the generation of these iSABs is described in this paper.

<table><tbody><tr><td>Iscove&#039;s liquid medium with stable glutamine</td><td>Biochrom AG</td><td>FG 0465</td><td></td></tr><tr><td>DMEM liquid medium without Na-pyruvate, with stable glutamine</td><td>Biochrom AG</td><td>FG 0435</td><td></td></tr><tr><td>CLS type IV, CLS IV</td><td>Biochrom AG</td><td>C4-22</td><td></td></tr><tr><td>Non-essential amino acids</td><td>Biochrom AG</td><td>K 0293</td><td></td></tr><tr><td>FBS Superior</td><td>Biochrom AG</td><td>S 0615</td><td></td></tr><tr><td>Sodium pyruvate (100 mM)</td><td>Biochrom AG</td><td>L 0473</td><td></td></tr><tr><td>G 418-BC liquid (ready-to-use solution), sterile</td><td>Biochrom AG</td><td>A 2912</td><td></td></tr><tr><td>Reagent reservoir PP f.multichannel pip. 60ml,sterile</td><td>Brand</td><td>703409</td><td></td></tr><tr><td>Accutase</td><td>eBioscience,Inc.</td><td>00-4555-56</td><td></td></tr><tr><td>Eppendorf Xplorer/Xplorer plus, electronic pipette</td><td>Eppendorf</td><td>4861000155</td><td></td></tr><tr><td>Falcon Cell Strainer 40 &amp;micro;m</td><td>Falcon</td><td>352340</td><td></td></tr><tr><td>Penicillin/Streptomycin&amp;nbsp;</td><td>GE Healthcare</td><td>P11-010</td><td></td></tr><tr><td>Petri Dishes</td><td>Greiner BioOne</td><td>663102</td><td></td></tr><tr><td>DPBS without Ca and Mg</td><td>PAN-Biotech</td><td>P04-36500</td><td></td></tr><tr><td>Fetal bovine serum</td><td>PAN-Biotech</td><td>P30-3302</td><td></td></tr><tr><td>Leukemia inhibitory factor</td><td>Phoenix Europe GmbH</td><td>LIF-250</td><td></td></tr><tr><td>Quadratic petri dishes</td><td>Roth</td><td>PX67.1</td><td></td></tr><tr><td>Gelatin from cold water fish skin</td><td>SigmaAldrich</td><td>G7765</td><td></td></tr><tr><td>2-Mercaptoethanol</td><td>SigmaAldrich</td><td>M3148&amp;nbsp;</td><td></td></tr><tr><td>1-Thioglycerol</td><td>SigmaAldrich</td><td>M6145</td><td></td></tr><tr><td>Tissue culture dishes</td><td>TPP</td><td>93100</td><td></td></tr><tr><td>Tissue culture flask</td><td>TPP</td><td>90076</td><td></td></tr><tr><td>Tissue culture test plates (24 well)</td><td>TPP</td><td>92424</td><td></td></tr></tbody></table>

generation of murine cardiac pacemaker cell aggregates based on es-cell-programming in combination with myh6-promoter-selection

Treatment of the &#8220;sick sinus syndrome&#8221; is based on artificial pacemakers. These bear hazards such as battery failure and infections. Moreover, they lack hormone responsiveness and the overall procedure is cost-intensive. &#8220;Biological pacemakers&#8221; generated from PSCs may become an alternative, yet the typical content of pacemaker cells in Embryoid Bodies (EBs) is extremely low. The described protocol combines &#8220;forward programming&#8221; of murine PSCs via the sinus node inducer TBX3 with Myh6-promoter based antibiotic selection. This yields cardiomyocyte aggregates consistent of &#62;80% physiologically functional pacemaker cells. These &#8220;induced-sinoatrial-bodies&#8221; (&#8220;iSABs&#8221;) are spontaneously contracting at yet unreached frequencies (400-500 bpm) corresponding to nodal cells isolated from mouse hearts and are able to pace murine myocardium ex vivo. Using the described protocol highly pure sinus nodal single cells can be generated which e.g. can be used for in vitro drug testing. Furthermore, the iSABs generated according to this protocol may become a crucial step towards heart tissue engineering.

The described protocol allows generation of iSABs with a beating frequency of around 450 bpm from PSCs (shown in the movie) which is near to the mouse heart beating frequency. After dissociation of iSABs (step 2.8.8) the observed single cells show the typical shape of cells of the sinus node (spindle and spider cells) as shown in Figure 1. These cells highly express proteins which are known to be essential for the function of the sinus node like hyperpolarization-activated cyclic nucleotide-gated cation ...

Watch this Scientific Journal Video about Generation of Murine Cardiac Pacemaker Cell Aggregates Based on ES-Cell-Programming in Combination with Myh6-Promoter-Selection at JoVE.com

Generation of Murine Cardiac Pacemaker Cell Aggregates Based on ES-Cell-Programming in Combination with Myh6-Promoter-Selection

This protocol describes how to produce functional sinus nodal tissue from murine pluripotent stem cells (PSC). T-Box3 (TBX3) overexpression plus cardiac Myosin-heavy-chain (Myh6) promoter antibiotic selection leads to highly pure pacemaker cell aggregates. These &#8220;Induced-sinoatrial-bodies&#8221; (&#8220;iSABs&#8221;) contain over 80% pacemaker cells, show highly increased beating rates and are able to pace myocardium ex vivo.

Treatment of the &#8220;sick sinus syndrome&#8221; is based on artificial pacemakers. These bear hazards such as battery failure and infections. Moreover, ...

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Universidad de Cartagena UDEC

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9.6K Views.  University of Rostock. This protocol describes how to produce functional sinus nodal tissue from murine pluripotent stem cells (PSC). T-Box3 (TBX3) overexpression plus cardiac Myosin-heavy-chain (Myh6) promoter antibiotic selection leads to highly pure pacemaker cell aggregates. These “Induced-sinoatrial-bodies” (“iSABs”) contain over 80% pacemaker cells, show highly increased beating rates and are able to pace myocardium ex vivo.

Video: Generation of Murine Cardiac Pacemaker Cell Aggregates Based on ES-Cell-Programming in Combination with Myh6-Promoter-Selection

Derivation of Cardiac Progenitor Cells from Embryonic Stem Cells

Cardiac progenitor cells (CPCs) have the capacity to differentiate into cardiomyocytes, smooth muscle cells (SMC), and endothelial cells and hold great promise in cell therapy against heart disease. Among various methods to isolate CPCs, differentiation of embryonic stem cell (ESC) into CPCs attracts great attention in the field since ESCs can provide unlimited cell source. As a result, numerous strategies have been developed to derive CPCs from ESCs. In this protocol, differentiation and purification of embryonic CPCs from both mouse and human ESCs is described. Due to the difficulty of using cell surface markers to isolate embryonic CPCs, ESCs are engineered with fluorescent reporters activated by CPC-specific cre recombinase expression. Thus, CPCs can be enriched by fluorescence-activated cell sorting (FACS). This protocol illustrates procedures to form embryoid bodies (EBs) from ESCs for CPC specification and enrichment. The isolated CPCs can be subsequently cultured for cardiac lineage differentiation and other biological assays. This protocol is optimized for robust and efficient derivation of CPCs from both mouse and human ESCs.

In this protocol, derivation of cardiac progenitor cells from both mouse and human embryonic stem cells will be illustrated. A major strategy in this protocol is to enrich cardiac progenitor cells with flow cytometry using fluorescent reporters engineered into the embryonic stem cell lines.

Cardiac progenitor cells (CPCs) have the capacity to differentiate into cardiomyocytes, smooth muscle cells (SMC), and endothelial cells and hold great ...

Improved Generation of Induced Cardiomyocytes Using a Polycistronic Construct Expressing Optimal Ratio of Gata4, Mef2c and Tbx5

Direct conversion of cardiac fibroblasts (CFs) into induced cardiomyocytes (iCMs) holds great potential for regenerative medicine by offering alternative strategies for treatment of heart disease. This conversion has been achieved by forced expression of defined factors such as Gata4 (G), Mef2c (M) and Tbx5 (T). Traditionally, iCMs are generated by a cocktail of viruses expressing these individual factors. However, reprogramming efficiency is relatively low and most of the in vitro G,M,T-transduced fibroblasts do not become fully reprogrammed, making it difficult to study the reprogramming mechanisms. We recently have shown that the stoichiometry of G,M,T is crucial for efficient iCM reprogramming. An optimal stoichiometry of G,M,T with relative high level of M and low levels of G and T achieved by using our polycistronic MGT vector (hereafter referred to as MGT) significantly increased reprogramming efficiency and improved iCM quality in vitro. Here we provide a detailed description of the methodology used to generate iCMs with MGT construct from cardiac fibroblasts. Isolation of cardiac fibroblasts, generation of virus for reprogramming and evaluation of the reprogramming process are also included to provide a platform for efficient and reproducible generation of iCMs.

We describe here a protocol for the generation of iCMs using retrovirus-mediated delivery of Gata4, Tbx5 and Mef2c in a polycistronic construct. This protocol yields a relatively homogeneous population of reprogrammed cells with improved efficiency and quality and is valuable for future studies of iCM reprogramming.

Direct conversion of cardiac fibroblasts (CFs) into induced cardiomyocytes (iCMs) holds great potential for regenerative medicine by offering alternative ...

Differentiation of Atrial Cardiomyocytes from Pluripotent Stem Cells Using the BMP Antagonist Grem2

Protocols for generating populations of cardiomyocytes from pluripotent stem cells have been developed, but these generally yield cells of mixed phenotypes. Researchers interested in pursuing studies involving specific myocyte subtypes require a more directed differentiation approach. By treating mouse embryonic stem (ES) cells with Grem2, a secreted BMP antagonist that is necessary for atrial chamber formation in vivo, a large number of cardiac cells with an atrial phenotype can be generated. Use of the engineered Myh6-DSRed-Nuc pluripotent stem cell line allows for identification, selection, and purification of cardiomyocytes. In this protocol embryoid bodies are generated from Myh6-DSRed-Nuc cells using the hanging drop method and kept in suspension until differentiation day 4 (d4). At d4 cells are treated with Grem2 and plated onto gelatin coated plates. Between d8-d10 large contracting areas are observed in the cultures and continue to expand and mature through d14. Molecular, histological and electrophysiogical analyses indicate cells in Grem2-treated cells acquire atrial-like characteristics providing an in vitro model to study the biology of atrial cardiomyocytes and their response to various pharmacological agents.

Generating cardiomyocytes from pluripotent stem cells in vitro allows access to large amounts of cardiac tissue in vitro for basic science and clinical applications. This protocol uses the atrializing factor Grem2 to both increase the numbers of cardiomyocytes obtained and to generate cardiomyocytes with an atrial phenotype.

Protocols for generating populations of cardiomyocytes from pluripotent stem cells have been developed, but these generally yield cells of mixed ...

Generation of Aligned Functional Myocardial Tissue Through Microcontact Printing

Advanced heart failure represents a major unmet clinical challenge, arising from the loss of viable and/or fully functional cardiac muscle cells. Despite optimum drug therapy, heart failure represents a leading cause of mortality and morbidity in the developed world. A major challenge in drug development is the identification of cellular assays that accurately recapitulate normal and diseased human myocardial physiology in vitro. Likewise, the major challenges in regenerative cardiac biology revolve around the identification and isolation of patient-specific cardiac progenitors in clinically relevant quantities. These cells have to then be assembled into functional tissue that resembles the native heart tissue architecture. Microcontact printing allows for the creation of precise micropatterned protein shapes that resemble structural organization of the heart, thus providing geometric cues to control cell adhesion spatially. Herein we describe our approach for the isolation of highly purified myocardial cells from pluripotent stem cells differentiating in vitro, the generation of cell growth surfaces micropatterned with extracellular matrix proteins, and the assembly of the stem cell-derived cardiac muscle cells into anisotropic myocardial tissue.

The generation of aligned myocardial tissue is a key requirement for adapting the recent advances in stem cell biology to clinically useful purposes. Herein we describe a microcontact printing approach for the precise control of cell shape and function. Using highly purified populations of embryonic stem cell derived cardiac progenitors, we then generate anisotropic functional myocardial tissue.

Advanced heart failure represents a major unmet clinical challenge,  arising from the loss of viable and/or fully functional cardiac muscle  cells. ...

Bioengineering

Suppression of Pro-fibrotic Signaling Potentiates Factor-mediated Reprogramming of Mouse Embryonic Fibroblasts into Induced Cardiomyocytes

Trans-differentiation of one somatic cell type into another has enormous potential to model and treat human diseases. Previous studies have shown that mouse embryonic, dermal, and cardiac fibroblasts can be reprogrammed into functional induced-cardiomyocyte-like cells (iCMs) through overexpression of cardiogenic transcription factors including GATA4, Hand2, Mef2c, and Tbx5 both in vitro and in vivo. However, these previous studies have shown relatively low efficiency. In order to restore heart function following injury, mechanisms governing cardiac reprogramming must be elucidated to increase efficiency and maturation of iCMs.
We previously demonstrated that inhibition of pro-fibrotic signaling dramatically increases reprogramming efficiency. Here, we detail methods to achieve a reprogramming efficiency of up to 60%. Furthermore, we describe several methods including flow cytometry, immunofluorescent imaging, and calcium imaging to quantify reprogramming efficiency and maturation of reprogrammed fibroblasts. Using the protocol detailed here, mechanistic studies can be undertaken to determine positive and negative regulators of cardiac reprogramming. These studies may identify signaling pathways that can be targeted to promote reprogramming efficiency and maturation, which could lead to novel cell therapies to treat human heart disease.

Here we present a robust method to reprogram primary embryonic fibroblasts into functional cardiomyocytes through overexpression of GATA4, Hand2, Mef2c, Tbx5, miR-1, and miR-133 (GHMT2m) alongside inhibition of TGF-&#946; signaling. Our protocol generates beating cardiomyocytes as early as 7 days post-transduction with up to 60% efficiency.

Trans-differentiation of one somatic cell type into another has enormous potential to model and treat human diseases. Previous studies have shown that ...

Construction of Defined Human Engineered Cardiac Tissues to Study Mechanisms of Cardiac Cell Therapy

Human cardiac tissue engineering can fundamentally impact therapeutic discovery through the development of new species-specific screening systems that replicate the biofidelity of three-dimensional native human myocardium, while also enabling a controlled level of biological complexity, and allowing non-destructive longitudinal monitoring of tissue contractile function. Initially, human engineered cardiac tissues (hECT) were created using the entire cell population obtained from directed differentiation of human pluripotent stem cells, which typically yielded less than 50% cardiomyocytes. However, to create reliable predictive models of human myocardium, and to elucidate mechanisms of heterocellular interaction, it is essential to accurately control the biological composition in engineered tissues. 
To address this limitation, we utilize live cell sorting for the cardiac surface marker SIRP&#945; and the fibroblast marker CD90 to create tissues containing a 3:1 ratio of these cell types, respectively, that are then mixed together and added to a collagen-based matrix solution. Resulting hECTs are, thus, completely defined in both their cellular and extracellular matrix composition.
Here we describe the construction of defined hECTs as a model system to understand mechanisms of cell-cell interactions in cell therapies, using an example of human bone marrow-derived mesenchymal stem cells (hMSC) that are currently being used in human clinical trials. The defined tissue composition is imperative to understand how the hMSCs may be interacting with the endogenous cardiac cell types to enhance tissue function. A bioreactor system is also described that simultaneously cultures six hECTs in parallel, permitting more efficient use of the cells after sorting.

This manuscript describes the creation of defined engineered cardiac tissues using surface marker expression and cell sorting. The defined tissues can then be used in a multi-tissue bioreactor to investigate mechanisms of cardiac cell therapy in order to provide a functional, yet controlled, model system of the human heart.

Human cardiac tissue engineering can fundamentally impact therapeutic discovery through the development of new species-specific screening systems that ...

In Vitro Generation of Heart Field-specific Cardiac Progenitor Cells

Pluripotent stem cells offer great potential for understanding heart development and disease and for regenerative medicine. While recent advances in developmental cardiology have led to generating cardiac cells from pluripotent stem cells, it is unclear if the two cardiac fields - the first and second heart fields (FHF and SHF) &#8212; are induced in pluripotent stem cells systems. To address this, we generated a protocol for in vitro specification and isolation of heart field-specific cardiac progenitor cells. We used embryonic stem cells lines carrying Hcn4-GFP and Tbx1-Cre; Rosa-RFP reporters of the FHF and the SHF, respectively, and live cell immunostaining of the cell membrane protein Cxcr4, a SHF marker. With this approach, we generated progenitor cells which recapitulate the functional properties and transcriptome of their in vivo counterparts. Our protocol can be utilized to study early specification and segregation of the two heart fields and to generate chamber-specific cardiac cells for heart disease modelling. Since this is an in vitro organoid system, it may not provide precise anatomical information. However, this system overcomes the poor accessibility of gastrulation-stage embryos and can be upscaled for high-throughput screens.

The purpose of this method is to generate heart field-specific cardiac progenitor cells in vitro in order to study the progenitor cell specification and functional properties, and to generate chamber specific cardiac cells for heart disease modelling.

Pluripotent stem cells offer great potential for understanding heart development and disease and for regenerative medicine. While recent advances in ...

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 ...

Biology

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.

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 of Myospheres From hESCs by Epigenetic Reprogramming

Generation of a homogeneous and abundant population of skeletal muscle cells from human embryonic stem cells (hESCs) is a requirement for cell-based therapies and for a "disease in a dish" model of human neuromuscular diseases. Major hurdles, such as low abundance and heterogeneity of the population of interest, as well as a lack of protocols for the formation of three-dimensional contractile structures, have limited the applications of stem cells for neuromuscular disorders. We have designed a protocol that overcomes these limits by ectopic introduction of defined factors in hESCs - the muscle determination factor MyoD and SWI/SNF chromatin remodeling complex component BAF60C - that are able to reprogram hESCs into skeletal muscle cells. Here we describe the protocol established to generate hESC-derived myoblasts and promote their clustering into tridimensional miniaturized structures (myospheres) that functionally mimic miniaturized skeletal muscles7.

Here, we describe a protocol based on epigenetic reprogramming of human embryonic stem cells (hESCs) toward generating a homogeneous population of skeletal muscle progenitors that under permissive culture conditions form three-dimensional clusters of contractile myofibers (myospheres), which recapitulate biological features of human skeletal muscles.

Generation of a homogeneous and abundant population of skeletal muscle cells from human embryonic stem cells (hESCs) is a requirement for cell-based ...