region identity or specific neuronal subtypes. For example, Wnt

inhibition can be applied for generating dorsal forebrain neural

cells and sonic hedgehog activation can be used for enriching

ventral forebrain cells.

5. PEG6000 solution is critical for EV isolation via the modified

extraPEG enrichment method, and thus should be prepared

properly. Besides 16% PEG6000 solution, 24% PEG6000 solu-

tion can also be prepared and then the medium:24% PEG6000

solution would be 2:1 to reach a final concentration of 8%

PEG6000 medium mixture.

6. DMSO is a general cryopreservation agent, and is harmful for

cells at room temperature. Thus, the thawing and recovery

process of hiPSCs should be rapid. Moreover, hiPSCs are very

fragile post-thaw and should be taken care in caution, e.g.,

pipetting of cells should be gentle.

7. Passaging hiPSCs with Accutase should be closely monitored.

hiPSC colonies are detached during this process and gentle

pipetting may be required to achieve a single cell suspension.

Treatment time should be minimized to reduce the damage to

the cells.

8. hiPSC-NPC organoids can be collected and replate on the

Geltrex-coated surface for further culture. Neuronal network

can be observed after replating and culturing. Meanwhile,

aggregates or spheroids can be dissociated by enzyme to gen-

erate single cell suspension for further analysis by flow

cytometry.

9. It is recommended to wash hMSCs with PBS several times

before the bioreactor culture using EV-free medium to mini-

mize the EV contamination from the previous culture.

10. During initial seeding phase, the medium volume is reduced to

increase the possibility of hMSCs contacting with microcarriers

for better attachment. The volume can be adjusted as long as it

covers the vertical wheel.

11. It is recommended that collected media should be stored at

4 ^C and processed within 1 week. If immediate processing is

not available, media should be centrifuged at 4 ^C by 500
g

for 5 min, 2000
g for 10 min, and then stored at 50 ^C.

12. After ultracentrifugation, EV pellets can be visualized at the

bottom of the centrifuge tube. It is recommended to circle the

pellets with marker and resuspend the EV pellets to visualize

their complete dissolution. Alternatively, EV pellets can be

directly lysed for protein or microRNA cargo analysis. Sterile,

EV-free milli-Q water can be used to resuspend EVs as well.

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Xuegang Yuan et al.