Differentiation Human Induced Pluripotent Stem Cells (hiPSCs) into Cardiomyocytes
Cardiovascular diseases are the leading causes of morbidity and mortality in industrialized countries. Adult cardiomyocytes are terminally differentiated, and thus unable to proliferate and replace degenerated tissue in case of an injury. Cardiovascular regenerative medicine aims to develop strategies to produce functional myocardial tissue to be used in cell replacement therapy. The most promising cell types for this are human embryonic stem cells (hESCs) and induced pluripotent stem cells (hiPSCs). They are able to differentiate into any cell type in body and proliferate indefinitely in undifferentiated state, making them a good source for cardiomyocytes (CM).
-into-Cardiomyocytes-1.jpg)
Figure 1. Schematic diagram of hPSC differentiation into cardiomyocytes.
Materials and Equipment
| QualiCell® Induced Pluripotent Stem Cell-XLC095 | Cell counting chamber or automated cell counter |
| QualiStem® IPS Cell Cardiomyocyte Differentiation Kit (containing Differentiation Medium A/B/C) | Recombinant Trypsin (rTrypsin) |
| SuperCult® Serum-free & feeder-free Human Pluripotent Stem Cell Growth Medium | 0.4% Trypan blue solution |
| Centrifuge tubes | DPBS without Ca2+/Mg2+ |
| Water bath | 12-well culture plates |
| Centrifuge | Inverted microscope |
| Incubator | Matrigel |
Differentiation Procedure
Allowing hiPSCs to expand and reach the optimal confluency in different maintenance culture systems. The differentiation procedure is described for a 12-well plate format.
- Differentiation day 0
1) Prewarm 12 mL Differentiation Medium A by incubating 15 min in a water bath of 37°C.
2) Aspirate the medium from all wells, and replace with 1 mL prewarmed Differentiation Medium A per well.
3) Culture the cells in a 37°C, 5% CO2 incubator for 2 days.
- Differentiation day 2
1) Prewarm 12 mL Differentiation Medium B by incubating 15 min in a water bath of 37°C.
2) Aspirate the medium from all wells, and replace with 1 mL prewarmed Differentiation Medium B per well at the same time as day 0.
3) Culture the cells in a 37°C, 5% CO2 incubator for 2 days.
- Differentiation day 4
1) Prewarm 12 mL Differentiation Medium C by incubating 15 min in a water bath of 37°C.
2) Aspirate the medium from all wells, and replace with 1 mL prewarmed Differentiation Medium C per well at the same time as day 0.
3) Culture the cells in a 37°C, 5% CO2 incubator for 2 days.
- Differentiation day 6-14
1) Refresh with Differentiation Medium C every 2 days. Cells should start beating between day 7 and day 11, dependent on the hiPSCs.
Note: It is normal to observe some cell death between day 5 and day 8.
2) Around day 14, cardiomyocytes can be dissociated for further characterization and/or for use in a variety of application.
Table 1. Reagent volumes per well or dish.
| Culture vessel (surface area/well) | DPBS | Recombinant Trypsin (rTrypsin) | SuperCult® |
| 6-well (10 cm2/well) | 2 mL | 1 mL | 2 mL |
| 12-well (4 cm2/well) | 1 mL | 0.5 mL | 1 mL |
| 24-well (2 cm2/well) | 0.5 mL | 0.25 mL | 0.5 mL |
| 35-mm (10 cm2/dish) | 2 mL | 1 mL | 2 mL |
| 60-mm (20 cm2/dish) | 4 mL | 2 mL | 4 mL |
| 100-mm (60 cm2/dish) | 12 mL | 6 mL | 12 mL |
Note
1) For successful differentiations, use karyotypically normal hiPSCs with minimal or no differentiated colonies and that express pluripotency markers.
2) When plating hiPSCs for differentiation, ROCK inhibitor treatment is optional and can be used to improve cell survival and plating consistency. In this case, refresh medium the day after seeding to remove the ROCK inhibitor.
3) Pre-coat cell culture treated plates with appropriate substrate, following manufacturer’s recommended instructions, before seeding cells.
4) The differentiation efficiency of iPSC into cardiomyocytes varies between different lines. To achieve best results, the optimal confluency at day 0 of differentiation needs to be established (Table 2).
5) Rapid media aspiration and addition can be detrimental to culture differentiation efficiency, we recommend slow addition and removal of media, especially during the induction phase.
Table 2. Seeding density VS confluence.
| (surface area/well) | Viable cells/well | % confluence |
12-well (4 cm2/well) | 2 x 104 | 30% |
| 3 x 104 | 50% | |
| 4 x 104 | 70% | |
| 5 x 104 | 90% |
References
- Friedman C. E. et al. Single-cell transcriptomic analysis of cardiac differentiation from human PSCs reveals HOPX-dependent cardiomyocyte maturation. Cell Stem Cell, 2018, 23(4): 586-598.
- Braam S. R. et al. Cardiomyocytes from human pluripotent stem cells in regenerative medicine and drug discovery. Trends in Pharmacological Sciences, 2009, 30(10).
