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

Abstract

Human embryonic stem cells (hESC) can proliferate indefinitely while retaining the capacity to form derivatives of all three germ layers. We have reported previously that hESC differentiate into cardiomyocytes when cocultured 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 threefold increase in the number of beating areas was observed in hESC/ END-2 coculture. In agreement, the 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 reverse transcription-polymerase chain reaction and Western blot analysis showed that insulin inhibited cardiomyogenesis in the early phase of coculture 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 coculture. 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 coculture. Indeed, the inhibition of Akt or insulin-like growth factor-1 receptor reversed the insulin-dependent effects. We conclude that in hESC/END-2 cocultures, insulin does not prevent differentiation but favors the neuroectodermal lineage at the expense of mesendodermal lineages. STEM CELLS 2008;26:724 -733 Disclosure of potential conflicts of interest is found at the end of this article.

We heart cultured hearts. A comparative review of methodologies for targeted differentiation and maintenance of cardiomyocytes derived from pluripotent and multipotent stem cells

“…It exists in two basic variants -B27 containing insulin (B27+insulin) and B27 minus insulin (B27-insulin). The presence of insulin may inhibit the differentiation towards cardiogenic mesoderm and endoderm, favouring differentiation into the neuroectodermal lineage [100]. Thus, B27-insulin is more commonly used when directing pluripotent cells to cardiogenic fate.…”

Differentiation of Human Atrial Myocytes From Endothelial Progenitor Cell-Derived Induced Pluripotent Stem Cells

“…Interestingly, cell to cell contact between pluripotent stem cells and END-2 cell does not appear to be necessary for cardiac differentiation as several studies have shown that exposure of ESCs to END2 conditioned media results in efficient CM generation [234][235][236]. Studies showed that END-2 cells produce soluble cardiac inductive factors and cytokine inhibitory factors.…”

“…Studies showed that END-2 cells produce soluble cardiac inductive factors and cytokine inhibitory factors. These inhibitory factors (i.e., prostaglandin 12 (PG12)) deplete insulin, which acts as a potent inhibitor of cardiogenesis and stimulator of neuroectoderm differentiation [235,236]. Thus CM can be derived in media condition containing insulin-, serum-free supplemented with PG12.…”

“…Cardiac differentiation from pluripotent stem cells can be derived by guided differentiation in adherent culture involving serum free medium supplemented with growth factors Activin A and BMP4 [55,[221][222][223][224][225][226][227][228][229][230][231][232][233][234][235][236][237][238][239]. These growth factors are a member of the TGF b family that plays roles in embryonic development including induction of gastrulation-like events, meso-endoderm development and induction of cardiogenesis [240][241][242][243][244][245][246].…”

Differentiation, Characterization and Applications of Human Embryonic Stem Cell –Derived Cardiomyocytes

“…Systematic testing of END-2 conditioned medium revealed that END-2 cells were able to clear insulin from the medium . Insulin has been shown to inhibit cardiac differentiation by suppressing endoderm and mesoderm formation and favouring ectoderm differentation (Freund, Ward-van Oostwaard et al 2008). Insulin acts via the insulin-like growth factor-1 receptor (IGF-1R) and phosphatidylinositol 3-kinase (PI3K/Akt) pathway and has been suggested to inhibit epithelial-to mesenchymal transition by elevated levels of E-cadherin (Freund, Ward-van Oostwaard et al 2008).…”