Insulin Redirects Differentiation from Cardiogenic Mesoderm and Endoderm to Neuroectoderm in Differentiating Human Embryonic Stem Cells

¹ Hubrecht Institute, Developmental Biology and Stem Cell Research, Utrecht, The Netherlands
² Hubrecht Institute, Developmental Biology and Stem Cell Research, Utrecht, The Netherlands and Interuniversity Cardiology Institute of the Netherlands
³ ES Cell International Pte Ltd, Singapore, Republic of Singapore, Institute of Medical Biology (IMB), Singapore, Republic of Singapore
⁴ Hubrecht Institute, Developmental Biology and Stem Cell Research, Utrecht, The Netherlands and Interuniversity Cardiology Institute of the Netherlands, Utrecht, The Netherlands

^* To whom correspondence should be addressed. E-mail: r.passier{at}niob.knaw.nl.

	Abstract

Human embryonic stem cells (hESC) can proliferate indefinitely whilst retaining the capacity to form derivatives of all three germ layers. We have reported previously that hESC differentiate into cardiomyocytes when co-cultured with a visceral endoderm-like cell line (END-2). Insulin/insulin-like growth factors and their intracellular downstream target protein kinase Akt are known to protect many cell types from apoptosis and to promote proliferation, including hESC-derived cardiomyocytes. Here, we show that in the absence of insulin a 3-fold increase in the number of beating areas was observed in hESC/END-2 co-culture. In agreement, addition of insulin strongly inhibited cardiac differentiation as evidenced by a significant reduction in beating areas as well as in -actinin and -myosin heavy chain (MHC) expressing cells. Real-time RT-PCR and Western blot analysis showed that insulin inhibited cardiomyogenesis in the early phase of co-culture by suppressing the expression of endoderm (Foxa2, GATA-6), mesoderm (brachyury T) and cardiac mesoderm (Nkx2.5, GATA-4). In contrast to previous reports, insulin was not sufficient to maintain hESC in an undifferentiated state since expression of the pluripotency markers Oct3/4 and nanog declined independently of the presence of insulin during co-culture. Instead insulin promoted the expression of neuroectodermal markers. Since insulin triggered sustained phosphorylation of Akt in hESC we analyzed the effect of an Akt inhibitor during co-culture. Indeed the inhibition of Akt or of the IGF-1 receptor reversed the insulin-dependent effects. We conclude that in hESC/END-2 co-cultures insulin does not prevent differentiation but favours the neuroectodermal lineage at the expense of mesendodermal lineages.

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