Thymosin β4 Improves Differentiation and Vascularization of EHTs

Figure 1

Thymosin β4 promotes the generation of cardiomyocytes from murine induced pluripotent stem cells. (a) Undifferentiated iPSCs were treated with Tβ4-peptide and expression of transcription factor encoding mRNAs was measured 4 (Eomes, Brachyury (T)), 6 (Mesp1), or 8 (NKX-2.5) days after Tβ4 treatment (red). In another approach, murine iPSCs were differentiated for 6 days and treated with Tβ4-peptide. 8 days after Tβ4 treatment, expression levels of cardiomyocyte markers (ANF, CX43) were measured and flow cytometric assessment of cardiac specific proteins (αMHC, Troponin, and α-actinin) was performed. Furthermore, the amount of beating areas was measured (blue). (b–g) Relative expression levels of transcripts as indicated ((b–e) without prior differentiation of iPSCs, (f and g) with 6 days of prior differentiation), measured via quantitative real-time PCR, reveal that Tβ4 induces the expression of transcription factors regulating the differentiation of stem cells to cardiomyocytes via mesenchymal and cardiac progenitor stages and the expression of cardiac specific transcripts. (h) Flow cytometry after intracellular staining for αMHC, Troponin, and α-actinin reveals the increased presence of these cardiac specific proteins after Tβ4 stimulation. (i) Tβ4 treated iPSCs show a high rate of spontaneous beating compared to untreated iPSCs in which beating was virtually absent. (, ).

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