This work was performed in part at the Center for Nanoscale Systems (CNS), a member of the National Nanotechnology Infrastructure Network (NNIN), which is supported by the National Science Foundation under NSF award no. ECS-0335765. CNS is part of Harvard University.

The protocol to generate aligned functional myocardial tissue can be divided into three major parts. The fabrication of the micropatterned master using soft lithography techniques is not considered part of the following protocol but can be made based on established method6.
1. Microcontact Printing of Fibronectin onto PDMS Substrates
<ol>
	<li>Mix Sylgard 184 (Dow Corning) PDMS elastomer at a 10:1 base to curing agent ratio and degas the assembly using a desiccator to eliminate...

<ol>
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The authors declare that they have no competing financial interests.

In this protocol, we presented a method to isolate purified populations of cardiogenic progenitors and to seed them on micropatterned Fibronectin substrates what allows them align and take a cardiac myocyte-like rod shape. Normal cellular organization is critical for normal tissue function8,10 , in particular for myocardial tissue. Cardiac myocytes have been shown to develop improved mechanical11,12 and electrophysiological properties11,13 when forming anisotropic cell arrays. Sinc...

Despite recent advances in medical therapy, advanced heart failure remains a leading cause of mortality and morbidity in the developed world. The clinical syndrome arises from a loss of functional myocardial tissue and subsequently an inability of the failing heart to meet the metabolic demands of affected individuals. Since the heart has a limited regenerative capacity, autologous heart transplantation is the only current clinically accepted therapy directly aimed at replenishing lost functional heart tissue. Significant drawbacks of heart transplantation, including a limited number of donor hearts and the need for long-term immunosuppressive therapy, preclude the wide spread use of this therapy. As a result, medical therapy has been the mainstay of treatment for patients with heart disease. A major challenge in drug development is the identification of cellular assays that accurately recapitulate normal and diseased myocardial physiology in vitro.
The recent convergence of stem cell biology and tissue engineering technology raises new promising avenues for cardiac regeneration. Generating functional myocardial tissue from a renewable patient-specific source would be a major advance in the field. This would allow for the development of disease specific cellular assays for drug development and discovery and would lay the foundation for cardiac regenerative medicine. Human embryonic stem (ES) cells and, more significantly, human induced pluripotent stem (iPS) cells represent a potentially renewable patient-specific source of ventricular progenitor cells and mature ventricular myocytes. The combination of stem cell biology and tissue engineering strategies addresses this problem by generating functional heart tissue in vitro that can be used in the development of cellular assays for drug discovery or in cardiac regenerative approaches for the treatment of advanced heart failure.
A central challenge in cardiac regeneration has been the identification of an optimal cell type. A wide array of cells have been studied1, however to date, although different multipotent cardiogenic precursors from various sources have been discovered, the identification of a cell source that meets critical requirements such as commitment to the myogenic cell fate, maintenance of the capacity to expand in vivo or in vitro and composition to a functional myocardial tissue has proven to be a central problem2. We have previously described a double transgenic murine system that enables the isolation of highly purified populations of committed ventricular progenitors (CVP) from ES cells differentiating in vitro. We generated a novel double transgenic mouse that expresses the red fluorescent protein dsRed under the control of an Isl1-dependent enhancer of the MEF2c gene and the enhanced green fluorescent protein under the control of the cardiac-specific Nkx2.5 enhancer. Based on this two-colored fluorescent reporter system, first and second heart field progenitors from developing ES cells can be isolated by means of Fluorescence Activated Cell Sorting (FACS) according to their expression of MEF2c and Nkx2.5 genes. CVP resemble cardiac myocytes based on the expression patterns of myocardial markers and structural and functional qualities3, what offers great promise for cardiac tissue regenerative purposes.
Although there have been many advances in the field of tissue engineering, mimicking native cellular architecture remains a key challenge. Conventional methods to address this challenge include seeding biological or synthetic scaffolds with cells in vitro. Due to a number of disadvantages of scaffolds, including rapid degradation, limited physical and mechanical stability and low cell density1,4,5 , we have attempted to engineer scaffold-less tissue. Although cardiac cells can modify their local microenvironment by the secretion of extracellular matrix proteins, they have a more limited capacity to organize themselves to rod shaped cardiac myocytes in the absence of extracellular cues. Thus, a template that provides cells appropriate spatial and biological cues to compose functional myocardial tissue is required. Microcontact printing addresses this challenge by providing a simple and inexpensive technique to precisely control cell shape, organization, and function6-8, all of which are crucial for the generation of aligned functional myocardial tissue. It comprises the use of microtextured Polydimethylsiloxane (PDMS) stamps with feature sizes ranging down to 2 &mu;m9 that enable the deposition of extracellular matrix proteins onto PDMS substrates in precise patterns and thus to affect cell adhesion spatially.
Herein, we propose to combine tissue bioengineering technology with stem cell biology to generate anisotropic functional myocardial tissue. Accordingly, we here demonstrate our recently published approach for the following: (1) the generation of micropatterned protein surfaces on PDMS substrates by microcontact printing for the generation of a template for aligned myocardial tissue, (2) the isolation of highly purified cardiac progenitors from ES cells differentiating in vitro, and (3) the combination of both techniques to generate aligned functional myocardial tissue.

<table border="1">
 <tbody>
 <tr>
 <td>Name of Material</td>
 <td>Company</td>
 <td>Catalogue Number</td>
 <td>Comments</td>
 </tr>
 <tr>
 <td>L-Ascorbic Acid</td>
 <td>Sigma-Aldrich</td>
 <td>A4544</td>
 <td>Prepare 0.1M solution in ddH2O</td>
 </tr>
 <tr>
 <td>Desiccator, Vacuum 10 inch</td>
 <td>Nova-Tech International, Inc.</td>
 <td>55206</td>
 <td></td>
 </tr>
 <tr>
 <td>4',6-Diamidino-2-Phenylindole, Dihydrochloride (DAPI)</td>
 <td>Life Technologies</td>
 <td>D1306</td>
 <td></td>
 </tr>
 <tr>
 <td>Dulbecco's Modified Eagle Medium</td>
 <td>Thermo Scientific</td>
 <td>SH30022.01</td>
 <td></td>
 </tr>
 <tr>
 <td>DPBS</td>
 <td>Gibco</td>
 <td>14190</td>
 <td></td>
 </tr>
 <tr>
 <td>FACSAria II Flow Cytometer</td>
 <td>BD Biosciences</td>
 <td></td>
 <td></td>
 </tr>
 <tr>
 <td>Fetal Bovine Serum ES cell-grade</td>
 <td>Gemini Bio-Products</td>
 <td>100-106</td>
 <td></td>
 </tr>
 <tr>
 <td>Fetal Bovine Serum Differentiation-grade</td>
 <td>Gemini Bio-Products</td>
 <td>100-500</td>
 <td></td>
 </tr>
 <tr>
 <td>Fibronectin from bovine plasma</td>
 <td>Sigma-Aldrich</td>
 <td>F4759</td>
 <td>Solubilize to 1 mg/ml in ddH2O</td>
 </tr>
 <tr>
 <td>Fisherfinest Premium Cover Glass 22x22mm</td>
 <td>Fisher Scientific</td>
 <td>12-548-B</td>
 <td></td>
 </tr>
 <tr>
 <td>Gelatin from porcine skin</td>
 <td>Sigma-Aldrich</td>
 <td>G1890</td>
 <td>Prepare 0.1% solution in ddH2O</td>
 </tr>
 <tr>
 <td>Headway Spin Coater</td>
 <td>Headway Research, Inc.</td>
 <td>PWM32-PS-CB15</td>
 <td></td>
 </tr>
 <tr>
 <td>Iscove's Modified Dulbecco's Medium</td>
 <td>Thermo Scientific</td>
 <td>SH30228.01</td>
 <td></td>
 </tr>
 <tr>
 <td>Leukemia Inhibitory Factor</td>
 <td>Self-prepared from LIF-secreting cell lines</td>
 <td></td>
 <td>Prepare 500x stock solution</td>
 </tr>
 <tr>
 <td>MEM Non-Essential Amino Acids</td>
 <td>Gibco</td>
 <td>11140-050</td>
 <td></td>
 </tr>
 <tr>
 <td>2-Mercaptoethanol</td>
 <td>Sigma-Aldrich</td>
 <td>M6250</td>
 <td></td>
 </tr>
 <tr>
 <td>Mouse Embryonic Fibroblasts</td>
 <td>Harvested from day 12-15 mouse embryos</td>
 <td></td>
 <td></td>
 </tr>
 <tr>
 <td>Penicillin-Streptomycin</td>
 <td>Gibco</td>
 <td>15140-122</td>
 <td></td>
 </tr>
 <tr>
 <td>Pluronic F-127 </td>
 <td>Sigma-Aldrich</td>
 <td>P2443</td>
 <td>Prepare 1% solution in ddH2O </td>
 </tr>
 <tr>
 <td>Round-Bottom Tube with 35 &mu;m cell strainer</td>
 <td>BD Biosciences</td>
 <td>352235</td>
 <td></td>
 </tr>
 <tr>
 <td>SPI Plasma-Prep II Plasma Cleaner </td>
 <td>SPI Supplies</td>
 <td>11005-AB</td>
 <td></td>
 </tr>
 <tr>
 <td>Sylgard 184 Silicone Elastomer Kit</td>
 <td>Dow Corning</td>
 <td>184 SIL ELAST KIT 0.5KG</td>
 <td></td>
 </tr>
 <tr>
 <td>0.25% Trypsin-EDTA</td>
 <td>Gibco</td>
 <td>25200-056</td>
 <td></td>
 </tr>
 <tr>
 <td>Vacuum Gauge</td>
 <td>SPI Supplies</td>
 <td>11019-AB</td>
 <td></td>
 </tr>
 </tbody>
</table>

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.

FACS-purification of in vitro differentiated ES cells revealed four distinct populations of progenitors (Figure 1). The presence of micropatterned Fibronectin was confirmed by immunofluorescence microscopy that displayed a complete transfer of continuous Fibronectin lines (Figure 2). Plating of FACS-isolated progenitors onto Fibronectin micropatterns resulted in alignment of the R+G+ and part of the R-G+ populations. Although Pl...

Watch this Scientific Journal Video about Generation of Aligned Functional Myocardial Tissue Through Microcontact Printing at JoVE.com

Generation of Aligned Functional Myocardial Tissue Through Microcontact Printing

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. ...

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Bioengineering

11.1K vistas.  Massachusetts General Hospital and Harvard Medical School. La generación de tejido alineado de miocardio es un requisito clave para la adaptación de los recientes avances en la biología de células madre para fines de utilidad clínica. Aquí se describe un enfoque para la impresión por microcontacto el control preciso de la forma y la función celular. Usando poblaciones altamente purificadas de células madre embrionarias derivadas progenitores cardíacos, a continuación, generar anisotrópico tejido miocárdico funcional....