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Abstract

Introduction

Protocol

Representative Results

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Acknowledgements

Materials

References

Biology

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

Published: June 28th, 2013

DOI:

10.3791/50429

1Humanitas Clinical and Research Center, Italy, 2Institute of Genetic and Biomedical Research (IRGB), National Research Council (CNR)

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 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.

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 h....

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1. Feeder-free Maintenance and Passaging of Human iPS Cell Lines

  1. 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.

Note: Matrigel stock concentrations vary by the.......

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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.......

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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.......

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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.

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

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