Figure 1. Cardiomyocyte Differentiation Protocol

Two days before the differentiation protocol, hPSC colonies are harvested and seeded as single cells at 350,000 cells/well in a 12-well format in TeSR™ medium. After one day (Day -1), the medium is replaced with fresh TeSR™ medium. The following day (Day 0), the TeSR™ medium is replaced with Medium A (STEMdiff™ Cardiomyocyte Differentiation Basal Medium containing Supplement A) to begin inducing the cells toward a cardiomyocyte fate. On day 2, a full medium change is performed with fresh Medium B (STEMdiff™ Cardiomyocyte Differentiation Basal Medium containing Supplement B). On days 4 and 6, full medium changes are performed with fresh Medium C (STEMdiff™ Cardiomyocyte Differentiation Basal Medium containing Supplement C). On day 8, medium is switched to STEMdiff™ Cardiomyocyte Maintenance Medium with full medium changes on days 10, 12 and 14, to promote further differentiation into cardiomyocyte cells. Small beating areas of cardiomyocytes can be seen as early as day 8, progressing to a full lawn of beating cardiomyocytes that can be harvested as early as day 15.

Figure 2. Morphology of hPSC-Derived Cardiomyocytes

Representative images of (A) hES (H9) cells and (B) hiPS (WLS-1C) cells on day 15 of differentiation to cardiomyocytes using the STEMdiff™ Ventricular Cardiomyocyte Differentiation Kit. Differentiated cells exhibit typical cardiomyocyte morphology as an adherent, tightly packed web-like monolayer of beating cells. (C) Representative confocal microscopy image of a single hPSC-derived cardiomyocyte generated with the STEMdiff™ Ventricular Cardiomyocyte Differentiation Kit and stained with cTnT (green) and DAPI (blue).

Figure 3. Efficient and Robust Generation of cTnT-Positive Cardiomyocytes

hES and hiPS cells were cultured for 15 days in single wells of 12-well plates using the STEMdiff™ Ventricular Cardiomyocyte Differentiation Kit. At the end of the culture period, cells were harvested and analyzed by flow cytometry for expression of cardiac troponin T (cTnT). (A) Histogram analysis for cardiomyocyte cell marker cTnT for cultures of hES (H9) and hiPS (WLS-1C and STiPS-M001) cells. (Filled = sample; blank = secondary antibody only control) (B,C) Percentages and total numbers of cells expressing cTnT in cultures of hES or hiPS cells are shown. Data shown as mean ± SEM; n=3.

Figure 4. hPSC-Derived Cardiomyocytes Exhibit a Robust and Stable Excitability Profile

Microelectrode array (MEA) voltage recordings of cardiomyocytes (day 27) derived from human pluripotent stem cells generated and maintained with the STEMdiff™ Cardiomyocyte Differentiation and Maintenance Kits. The hPSC-derived cardiomyocytes have a characteristic electrical profile and stable beat rate. A large depolarization spike followed by a smaller repolarization deflection is observed.

Figure 5. Generation of Cardiomyocytes from hPSCs Maintained in mTeSR™ Plus

Human ES (H9) and iPS (WLS-1C) cells were maintained in mTeSR™1 (daily feeds) or mTeSR™ Plus (restricted feeds) and differentiated to cardiomyocytes using the STEMdiff™ Ventricular Cardiomyocyte Differentiation Kit. At the end of the differentiation period, cells were harvested and analyzed by microelectrode array (MEA) and flow cytometry. (A) Representative MEA voltage recordings of cardiomyocytes (day 20) demonstrate a characteristic electrical profile and stable beat rate. (B) Percentages of cells expressing cTNT and (C) total number of viable cells harvested are shown. Data are expressed as the mean (± SEM); n=2.

Figure 6. iPSCdirect™ SCTi003-A Human Pluripotent Stem Cells Can Successfully Differentiate into Ventricular Cardiomyocytes

Ventricular cardiomyocytes were generated from iPSCdirect™ SCTi003-A cells using STEMdiff™ Ventricular Cardiomyocyte Differentiation Kit (Catalog #05010). (A) 48 hours after thawing and plating in mTeSR™ Plus and CloneR™2, iPSCdirect™ cells reached the desired confluency and are ready for Day 0 of differentiation according to the STEMdiff™ Ventricular Cardiomyocyte Product Information Sheet. (B) By Day 15 of differentiation, monolayer cultures show iPSC-derived ventricular cardiomyocytes that (C) exhibit coordinated beating behavior.