Mesoderm Differentiation
Cardiomyocytes
A fibroblast-derived, integration-free iPSC line generated using episomal plasmids was differentiated to cardiomyocytes. Cells were exposed to RPMI/B27 (insulin-free) containing 5µM CHIR99021 on day 1, and 12µM IWP4 on days 3 and 4 (cells were fed with RPMI/B27 without small molecules on days 2, 5, and 6). On day 7, media was changed to RPMI/B27 (plus insulin) and was changed every 3 days until beating sheets of cardiomyocytes were evident. Immunofluorescence analysis of the cardiomyocyte marker cardiac troponin type 2 (Tnnt2) exhibiting typical striated staining pattern and video of spontaneously contracting cardiomyocytes are shown.
Macrophages
A PBMC-derived iPSC line was generated using a lentiviral reprogramming vector. iPSCs were co-cultured with OP9 cells for 9 days and GM-CSF/M-CSF for 2 days followed by isolation of CD235a-/CD41a-/CD45+ cells. These cells were terminally differentiated into macrophages by exposure to GM-CSF and M-CSF for an additional 7 days. Cytospin and Wright-Geimsa staining was performed to demonstrate generation of cells with macrophage morphology. Macrophage identity of generated cells was confirmed by immunophenotyping and functional analysis.