Abstract
Differentiation of cardiomyocytes (CMs) from human induced pluripotent stem cells (hiPSCs) is critically dependent upon the regulation of the Wnt signaling pathway. The mechanisms remain unclear with regard to the dose and timing of each differentiation inducer, and the interaction of the inducers that regulate the Wnt in mesendoderm specification to cardiac mesoderm. Consequently, it remains far from optimal in differentiation efficiency and consistency from hiPSC lines to CMs. Here, we have carefully deciphered the role of Wnt signaling pathway manipulation on mesoderm specification in a dosage and time dependent manner. To examine the hypothesis of that fate specification of hiPSC-CMs differentiation is dictated by temporal and spatial factors that regulate Wnt, we evaluate hiPSC-CM differentiation with: (1) two-phase modulation of Wnt, (2) dosage variant of GSK3β inhibitors, (3) treatment with insulin, and (4) 3-dimentional suspension culture environment on iPSC-CM differentiation. The results highlight the importance of mesendoderm specification to cardiac mesoderm, which needs precisely regulation of Wnt in a dosage dependent and temporal on/off manner. This temporal regulation dictates the final efficiency and purity of derived cardiomyocytes. After the initial activation of Wnt signaling pathway to generate mesendoderm, the maintenance of Wnt signaling at an appropriate dose is critical to direct the cell fate into cardiac mesoderm. Otherwise, lower Wnt signals lead to definitive endoderm and higher Wnt signals induce presomitic mesoderm differentiation. The precisely specification of cardiac mesoderm results in not only greater than 90% of cTnT+ cardiomyocytes but also high cardiomyocytes yield under both monolayer and suspension culture conditions. Thus, the current findings provide critical insights to decipher the temporal mechanism of Wnt activation in regulation of hiPSC-CMs differentiation, and more importantly provide the guidelines for the consistent and high-yield and high-quality hiPSC-CMs production in cardiovascular research.
Highlights
Wnt inhibition (Wi) treatment groups via flow cytometry; (d) the proportion of cells that expressed cardiac troponin T (cTnT) was calculated and presented as a percentage of the total number of cells (n = 3 different batches of differentiated cells). *P < 0.05 vs. GSK3β inhibitor (Gi)(10/0); #P < 0.05 vs. Gi(10/1); †P < 0.05 vs. Gi(10/2).(e) human induced pluripotent stem cells (hiPSCs) reprogrammed from cardiac or dermal fibroblasts were differentiated via the Gi(I/M)Wi protocol with CHIR initiation www.nature.com/scientificreports and maintenance doses of 10 μM and 2 μM respectively
Our initial results suggested that limiting Wnt activation to just the first 24 hours of the 72-hour Gi period failed to sufficiently induce mesodermal specification before the Wnt inhibition phase was initiated, so we developed a new method, the Gi(I/M)Wi protocol, in which Wnt activation was initiated (I) with a high dose of the glycogen synthase kinase 3β (Gsk3β) inhibitor CHIR99021 (CHIR) for 24 hours and maintained (M) with a lower CHIR dose until the Wnt inhibitor IWR1 was added 48 hours later
The differentiation of hiPSCs into CMs progresses through multiple steps of cell-fate determination, and each stage is accompanied by the expression of identity-specific marker genes. hiPSCs express the pluripotency genes octamer-binding transcription factor 4 (OCT4) and SRY-box 2 (SOX2), and cells in subsequent stages of differentiation can be identified by the expression of Bry and EOMES for mesendodermal cells; MESP1 and kinase insert domain receptor (KDR) for mesodermal cells; KDR, platelet derived growth factor receptor α (PDGFRα), NK2 homeobox 5 (NKX2.5), and heart and neural crest derivative 1 (HAND1) for cells of the cardiac mesoderm and cardiac progenitor-cell lineages; and, cardiac troponin T for cardiomyocytes (Fig. 1a)
Summary
(f,g) hciPSCs and hdiPSCs were passaged 10 (P10) or 50 (P50) times and differentiated via the GiWi or Gi(10/2)Wi protocols; cTnT expression was evaluated via flow cytometry. The efficiency of the conventional GiWi protocol appears to be greater for hiPSCs derived from cardiac (hciPSCs), rather than dermal (hdiPSCs) fibroblasts[12] and may vary depending on how many times the hiPSCs were passaged before differentiation was induced[4,5,6]. We investigated additional adaptable method (i.e. suspension culture) and parameter (i.e. insulin treatment) for the new Gi(I/M) Wi protocol. These results demonstrate a critical temporal mechanism of Wnt activation in fate speciation of hiPSC-CM differentiation. The flexibility of this new protocol established here, enables the magnitude of Wnt activity to be adjusted to optimize hiPSC-CM differentiation across multiple hiPSC lines and culture conditions
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