Figure 5.

Repeated batch and perfusion bioreactor-expanded hCBiPS2 cells differentiate into derivatives of all three germ layers. (A): Immunofluorescence analysis of day 7 bioreactor-derived aggregates from repeated batch as well as perfusion cultures induced for spontaneous differentiation revealed expression of marker proteins representative of all three germ layers (positive staining in red or green): sarcomeric actinin, cardiac Troponin T (mesoderm), α-fetoprotein (AFP) (endoderm), and β3 Tubulin (ectoderm). Nuclei were stained with 4′,6-diamidino-2-phenylindole (blue). Respective isotype controls confirmed specificity of stainings (data not shown). Scale bars = 100 µm. (B): Furthermore, modulation of the Wnt pathway by small molecules resulted in cardiac differentiation of cells from repeated batch as well as perfusion cultures. Expression of cardiac-specific markers sarcomeric actinin and αMHC as well as βMHC could be detected by flow cytometry. Abbreviations: MHC, major histocompatibility complex; sarc., sarcomeric.