Reproducible differentiation of pluripotent stem cells (PSCs)

Differentiated cells displayed a high expression level of the cardiomyocyte-specific marker troponin T, as shown by flow cytometry (left). The use of recombinantly engineered REAfinity™ Antibodies against cardiac troponin T, α-actinin, or other cardiomyocyte-specific markers like myosin heavy chain, MLC2a, and MLC2v, allows for a reliable analysis of cardiomyocytes and respective subtypes. Immunofluorescence microscopy revealed the typical cardiomyocyte morphology and sarcomeric structure (right).

The biological activity of Human FGF-2 IS, premium grade, was determined by proliferation assay using 3T3 cells. Activity of Human FGF-2 IS, premium grade, (red line) was compared to wild type Human FGF-2 (black line), and another commercially available product (gray line).

Human iPS cells were differentiated into CXCR4⁺ definitive endoderm (DE) progenitors for 5 days and split into three fractions. The unseparated original fraction was washed with separation buffer only and not subjected to cell separation. The negative fraction represents the flow through after separation with the CD184 (CXCR4) MicroBead Kit. The positive fraction represents magnetically enriched CXCR4⁺ cells. All three fractions were exposed to the hepatogenic factors HGF, FGF-4, oncostatin M, and dexamethasone for 14 days. Hepatogenic potential was determined by immunocytochemical analysis of the hepatoblast marker transthyretin (TTR), and the hepatocyte marker albumin (ALB). Nuclei were counterstained with DAPI. Isolation of CXCR4⁺ DE progenitor cells led to highly enriched hepatocyte-like cells, indicating that the enrichment of differentiation intermediates can increase the overall differentiation efficiency.