This is an 18-day protocol, starting from cell expansion to
cardiomyocytes differentiation. Through the differentiation pro-
cess, take 1 mL of sample from the MC spinner culture daily to
measure cell number, viability, and aggregate sizes, as described in
Subheading 3.3.3.
1. Expand hiPSCs in MC spinner culture as described in Subhead-
ing 3.3.
2. After 6 days of expansion, wash the MC suspension twice with
20 mL of DPBS() using a serological pipet in the spinner flask
(see Note 11).
3. Add 50 mL of RPMI/B27-IN containing the optimal concen-
tration of CHIR determined in Subheading 3.2. Incubate the
culture for 24 h in static conditions.
4. Day 1: After 24 h, wash the culture two times with 20 mL of
fresh RPMI/B27-IN, using a 25-mL serological pipet (see Note
11), and incubate for another 24 h under agitation on the
magnetic stirrer with 25–30 rpm in CO2 incubator.
5. Day 2: Aspirate the spent media and gently add 50 mL of
RPMI/B27-IN containing 2.5 μM of IWR-1 [3, 7], then incu-
bate for 48 h in static conditions until day 4.
8. Day 4: Aspirate the spent media and gently add 50 mL of fresh
RPMI/B27-IN. Thereafter, conduct 50% media change (i.e.,
25 mL) with RPMI/B27-IN daily until day 12. As mentioned
earlier, remember to take 1 mL of sample from the MC spinner
culture daily to measure cell number, viability, and aggregate
sizes (see Note 17).
9. Day 12: Apart from taking 1 mL of sample for cell counting and
aggregates imaging, take another 10 mL of sample from the
spinner culture for flow cytometry to determine the expression
of cTnT cardiac marker (see Table 1). Briefly, dissociate the cells
from MC as described in Subheading 3.3.4 (see Note 18) and
perform flow cytometry with anti-human cTnT antibody, as
Fig. 4 The cardiac differentiation process in a spinner culture
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Valerie Ho et al.