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      Differentiation of cardiomyocytes and generation of human engineered heart tissue

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          Abstract

          This protocol describes how to generate defined embryoid bodies and subsequent standardized beating engineered heart tissue from human iPSCs using small molecules.

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          Most cited references25

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          Derivation of human embryonic stem cells in defined conditions.

          Tenneille E. Ludwig, Mark Levenstein, Jeffrey M Jones … (2006)
          We have previously reported that high concentrations of basic fibroblast growth factor (bFGF) support feeder-independent growth of human embryonic stem (ES) cells, but those conditions included poorly defined serum and matrix components. Here we report feeder-independent human ES cell culture that includes protein components solely derived from recombinant sources or purified from human material. We describe the derivation of two new human ES cell lines in these defined culture conditions.
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            Activin/Nodal and FGF pathways cooperate to maintain pluripotency of human embryonic stem cells.

            Ludovic Vallier, Morgan Alexander, Roger Pedersen (2005)
            Maintenance of pluripotency is crucial to the mammalian embryo's ability to generate the extra-embryonic and embryonic tissues that are needed for intrauterine survival and foetal development. The recent establishment of embryonic stem cells from human blastocysts (hESCs) provides an opportunity to identify the factors supporting pluripotency at early stages of human development. Using this in vitro model, we have recently shown that Nodal can block neuronal differentiation, suggesting that TGFbeta family members are involved in cell fate decisions of hESCs, including preservation of their pluripotency. Here, we report that Activin/Nodal signalling through Smad2/3 activation is necessary to maintain the pluripotent status of hESCs. Inhibition of Activin/Nodal signalling by follistatin and by overexpression of Lefty or Cerberus-Short, or by the Activin receptor inhibitor SB431542, precipitates hESC differentiation. Nevertheless, neither Nodal nor Activin is sufficient to sustain long-term hESC growth in a chemically defined medium without serum. Recent studies have shown that FGF2 can also maintain long-term expression of pluripotency markers, and we find that inhibition of the FGF signalling pathway by the tyrosine kinase inhibitor SU5402 causes hESC differentiation. However, this effect of FGF on hESC pluripotency depends on Activin/Nodal signalling, because it is blocked by SB431542. Finally, long-term maintenance of in-vitro pluripotency can be achieved with a combination of Activin or Nodal plus FGF2 in the absence of feeder-cell layers, conditioned medium or Serum Replacer. These findings suggest that the Activin/Nodal pathway maintains pluripotency through mechanism(s) in which FGF acts as a competence factor and therefore provide further evidence of distinct mechanisms for preservation of pluripotency in mouse and human ESCs.
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              The role of FGF/Erk signaling in pluripotent cells.

              Fredrik Lanner, Janet Rossant (2010)
              Fibroblast growth factor (FGF) signaling controls fundamental processes such as proliferation, differentiation and migration throughout mammalian development. Here we discuss recent discoveries that implicate FGF/Erk signaling in the control of pluripotency and lineage specification in several different stem cell states, including the separation of pluripotent epiblast and primitive endoderm in the blastocyst, the lineage priming of embryonic stem (ES) cells, and in the stabilization of the metastable state of mouse epiblast and human ES cells. Understanding how extrinsic signals such as FGF regulate different stem cell states will be crucial to harvest the clinical promise of induced pluripotent and embryo-derived stem cells.
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                Author and article information

                Journal
                Title: Nature Protocols
                Abbreviated Title: Nat Protoc
                Publisher: Springer Nature
                ISSN (Print): 1754-2189
                ISSN (Electronic): 1750-2799
                Publication date Created: May 11 2017
                Publication date (Print): May 11 2017
                Volume: 12
                Issue: 6
                Pages: 1177-1197
                Article
                DOI: 10.1038/nprot.2017.033
                PubMed ID: 28492526
                SO-VID: 5257844a-5bbc-4b6d-9c23-cb5ca3e7c970
                Copyright © © 2017
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