Figure 1. Workflow for the Expansion of Early-Stage hPSC-Derived Cardiomyocytes Using the STEMdiff™ Cardiomyocyte Expansion Kit

Expansion of early-stage human pluripotent stem cell (hPSC)-derived cardiomyocytes begins with the differentiation of hPSCs to cardiomyocytes using the STEMdiff™ Ventricular Cardiomyocyte Differentiation Kit or the STEMdiff™ Atrial Cardiomyocyte Differentiation Kit. On Day 11 of differentiation, a beating, confluent monolayer of early-stage cardiomyocytes can be observed. These cardiomyocytes are harvested and replated at low density to initiate expansion using STEMdiff™ Cardiomyocyte Expansion Kit. Expanding hPSC-derived cardiomyocytes can be passaged up to 5 times before switching to STEMdiff™ Cardiomyocyte Maintenance Medium for downstream assays.

Figure 2. Expanding hPSC-Derived Cardiomyocytes Are Confluent One Week After Replating with STEMdiff™ Cardiomyocyte Expansion Kit

Representative images of expanding hPSC-derived ventricular cardiomyocytes after replating at low density (Day 1) using STEMdiff™ Cardiomyocyte Passaging Medium. Cardiomyocyte confluency increases during the 7 day expansion period. On Day 7, a confluent monolayer of beating hPSC-derived cardiomyocytes is observed and the expanded hPSC-derived cardiomyocytes are ready for harvest.

Figure 3. Expansion of hPSC-Derived Cardiomyocytes Across Multiple Cell Lines with STEMdiff™ Cardiomyocyte Expansion Kit

(A) Expansion of hPSC-derived cardiomyocytes was tracked for 5 passages with 4 hPSC lines. Per passage, fold expansion is typically highest in the first 1 – 2 passages. (B) Cumulative fold expansion over 5 passages ranged from 33- to 103-fold expansion and averaged at 72-fold ± 12 (n = 15, 4 hPSC lines).

Figure 4. Cardiomyocyte Purity Increases During Expansion Using STEMdiff™ Cardiomyocyte Expansion Kit

Cardiomyocyte purity was tracked during expansion using STEMdiff™ Cardiomyocyte Expansion Kit for 5 passages on 4 hPSC lines. Throughout expansion, hPSC-derived cardiomyocytes express high levels of cardiac troponin T, and on average show an increase in cardiomyocyte purity during expansion. After 5 passages, cardiomyocyte cultures show an average cTnT purity of 90% ± 2% (n = 15).

Figure 5. Post-Expanded hPSC-Derived Cardiomyocytes Exhibit Typical Cardiac Electrophysiology and a Stable Beat Profile

Microelectrode Array (MEA) recordings of post-expanded hPSC-derived cardiomyocytes (using STEMdiff™ Cardiomyocyte Expansion Kit) exhibit a stable beat rate across 4 hPSC lines. The post-expanded hPSC-derived cardiomyocytes were replated onto MEA and maintained in STEMdiff™ Cardiomyocyte Maintenance Medium for 1 week. The average beat rate was 12 ± 1 beats per minute (n = 14, 4 hPSC lines).