2

Materials

2.1

Materials for

hiPSC Differentiation

in Planar and

Bioreactor Cultures

1. A frozen hiPSC line, iPSK3 cells, was kindly provided by

Dr. Stephen Duncan from Medical College of Wisconsin

[23]. Briefly, the cell line was derived from human foreskin

fibroblasts with transfection of plasmid DNA encoding repro-

gramming factors: OCT4, NANOG, SOX2, and LIN28 (see

Note 1).

2. Prior to hiPSC culture, Lactose Dehydrogenase Elevating

Virus (LDEV)-Free Reduced Growth Factor Basement Mem-

brane Matrix Geltrex (Life Technologies, #A1413202) is

thawed under 4

C overnight, then aliquoted and stored

under 20 ^C. Geltrex is diluted in cold Dulbecco’s Modified

Eagle’s medium (DMEM, Life Technologies, Carlsbad, CA) at

1:100 dilution (1%) and stored under 20 ^C. Tissue culture

surface is coated with 1% Geltrex solution for overnight in

37 ^C incubator (see Note 2).

3. hiPSC complete culture medium (hiPSC-CCM) is prepared by

adding 20% serum-free mTeSR™1 5
supplement (Stem-

Cell™Technologies Inc., #85852) in mTeSR™1 basal

medium

(StemCell™

Technologies

Inc.,

#05850).

The

hiPSC-CCM can be stored under 4 ^C. To dissociate hiPSCs

for passaging, Accutase solution (VWR International, Radnor,

PA., #10210-214) is used for obtaining single cell suspension.

Aliquoted

Accutase

solution

is

stored

under

20

C.

Rho-associated kinase (ROCK) inhibitor Y27632, 2 mg or

10 mM in 624.4 μL dimethyl sulfoxide (DMSO, #MD-

0025), is

purchased

from

iXCells Biotechnologies

(San

Diego, CA). The final working concentration is 10 μM by

adding 1 μL of the stock solution per mL of hiPSC-CCM (see

Note 3).

4. Human NPC differentiation medium is composed of DMEM-

F12 (Gibco™, #125000-062), with 2% B27 serum-free sup-

plement (50
, Life Technologies, #17504044) and stored

under 4 ^C. Dual-SMAD inhibition for neural differentiation

of hiPSCs is achieved by two small molecules: SB431542

(Sigma, #S4317) and LDN193189 (Sigma, #SML0559),

both dissolved in DMSO and stored under 20 ^C. The work-

ing concentrations in differentiation medium are 10 μM

SB431542 and 100 nM LDN193189 (see Note 4).

2.2

Materials for

hMSC Expansion in

Planar and Bioreactor

Cultures

1. Frozen hMSC lines derived from human umbilical cords are

provided by SynergyBiologics (Tallahassee, FL). hMSCs at

passage 0 (P0) are cryopreserved and stored in liquid nitrogen.

2. hMSC complete culture medium (hMSC-CCM) is prepared by

dissolving 10.08 g Minimum Essential Medium Alpha Medium

Human Stem Cell-derived Extracellular Vesicles in Bioreactors

195