4. It is normal to observe significant cell death after 24 h. How-

ever, if there are little to no cells attached to the well, the cells

had not reached appropriate confluence (95–100%) before

differentiation. Seed cells again and wait the cells to reach

95–100% confluence before CHIR treatment.

5. Record the time when CHIR is added as the medium must be

changed at exactly 24 h later.

6. Cells with transparent and web-like morphology will appear,

and cell death will continue to be observed during this period

as well.

7. Cardiomyocytes require longer incubation times before suc-

cessfully detaching. Using a preferred single-cell dissociation

reagent incubate for longer time compared to normal mono-

layer cultures. After 15 min of incubation, attempt to remove

cells from cell attachment surface by pipetting. However, if cells

remain attached, place plate back into incubator to incubate for

another 15 min. Stop incubation once cells easily detach after

pipetting.

8. Cardiomyocytes can be clumpy despite dissociation, upon

noticing clumps after fixing cells, consider filtering cells before

running flow cytometry to avoid damaging the machine and

producing false positives.

9. As a guideline, successfully differentiated hiPSCs lines have a

low expression of HNF4a (below 40%) and CD44 (below 40%)

and high expression in NKX2-5 (60–80%), cTnT (80–83%),

and MLC2a (65–70%) [3].

10. Work by adding the largest volume reagent (DMEM/F12)

first, followed by the Cytodex^® 1 MCs then Geltrex™. To

prepare

Geltrex™,

refer

to

Gibco’s

user

guide

#MAN0007332 Rev. 3.0. Geltrex™-coated MCs are always

prepared at least a day before use and can be stored in 4 ^C

until ready for use.

11. Use a serological pipet to remove supernatant instead of aspir-

ating, Cytodex^® 1 compacts weakly and will be easily removed

if aspirating.

12. In cases where cell clumps are seen but when resuspension is

not working, filter cells through a 40-μm cell strainer. This is to

ensure a complete single-cell suspension.

13. When observing cells over the course of 6 days, ensure cells are

not over-confluent on MCs. Robust and fast-growing cell lines

can be evaluated earlier after 5 days [3].

14. It is recommended to cut tips when pipetting MC clumps to

ensure clumps are not stuck. When obtaining cell counts, it is

recommended to use a cut tip to properly dissociate cells.

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Valerie Ho et al.