4. Seed 5  10⁵ cells in each well of the Geltrex^®-coated six-well

plate. Culture the cells at 37^C in CO2 incubator until

95–100% confluency.

3.2.2

CM Differentiation

(Monolayer Method)

The protocol described here is an adaptation of a previously

described CHIR-based monolayer method [3, 7] (see Fig. 1).

1. For start of differentiation, grow cultures to 95–100% conflu-

ence in each well of a six-well tissue culture.

2. Wash the hiPSCs twice with 5 mL of DPBS(
) before

differentiation.

3. To screen the working concentrations of CHIR for CM differ-

entiation, incubate the cells with 5 mL of CM differentiation

basal medium (RPMI/B27^-IN, see Table 3) with 0–14 μM of

CHIR [7] for 24 h (see Notes 4 and 5).

4. Day 1: After 24 h, aspirate the spent media and gently add 5 mL

of fresh RPMI/B27^-IN, incubate for another 24 h.

5. Day 2: Aspirate the spent media and gently add 5 mL of

RPMI/B27^-IN containing 2.5 μM of IWR-1, an optimal con-

centration as previously reported [3, 7], then incubate for 48 h

until day 4.

6. Day 4: Aspirate the spent media and gently add 5 mL of fresh

RPMI/B27^-IN.

Thereafter,

conduct

media

change

with

RPMI/B27^-IN every day until day 14 (see Note 6).

7. Day 14: Dissociate the cells into single-cell suspension by

TrypeLE™-Express, as described in the manufacturer’s user

guide Pub. No. MAN0007321 Rev. 2.0 (see Note 7), for

characterization using flow cytometry.

3.2.3

Characterization of

hiPSC-Derived CM from

Monolayer by Flow

Cytometry (Fig. 2)

1. Count the cell number using NucleoCounter^® automated cell

counter, as described in the manufacturer’s Application Note

No. 0258 (Rev 1.2).

2. Prepare a suspension of at least 1  10⁶ cells/mL and fix the

cells with 500 μL 4% PFA solution for 15–20 min at room

temperature (see Note 8).

3. Remove the PFA by centrifugation (300–400  g for 3 min).

Fig. 2 Preparation of cells for flow cytometry

72

Valerie Ho et al.