15. Stage 1 medium: high-glucose (4500 mg/L) DMEM supplemented with 1% ITS-G (Thermo Fisher Scientific), 2.5 mg/
mL AlbuMAX II, 100 μM NEAAs, 2 mM L-Gln, 50 U/mL
penicillin, 50 μg/mL streptomycin, 100 μM β-ME, 10 ng/mL
Activin A, and 5 ng/mL bFGF. Store at 4
C (see Note 3).
16. Stage 2 medium: RPMI supplemented with 100 μM NEAAs,
2 mM L-Gln, 50 U/mL penicillin, 50 μg/mL streptomycin,
100 μM β-ME, 2% B27 supplement (Thermo Fisher Scientific),
50 ng/mL FGF10, 250 nM KAAD-cyclopamine, and 1 μM
RA. Store at 4
C (see Note 3).
17. Stage 3 medium: low-glucose (1000 mg/L) DMEM supplemented with 1% ITS-G, 2.5 mg/mL AlbuMAX II, 100 μM
NEAAs, 2 mM L-Gln, 50 U/mL penicillin, 50 μg/mL streptomycin, 100 μM β-ME, 10 mM NAM, and 10 nM GLP-1.
Store at 4
C (see Note 3).
2.4 C-peptide
Secretion Assay
1. Krebs–Ringer solution containing bicarbonate and HEPES
(KRBH) with low glucose: 129 mM NaCl, 4.8 mM KCl,
2.5 mM CaCl 2 , 1.2 mM KH 2 PO 4 , 1.2 mM MgSO 4 , 5 mM
NaHCO 3 , 10 mM HEPES-NaOH (pH 7.4), 0.1% BSA, and
2.5 mM D-glucose. Sterilize through a 0.22-μm filter. Store at
4
C.
2. Krebs–Ringer solution containing bicarbonate and HEPES
(KRBH) with high glucose: 129 mM NaCl, 4.8 mM KCl,
2.5 mM CaCl 2 , 1.2 mM KH 2 PO 4 , 1.2 mM MgSO 4 , 5 mM
NaHCO 3 , 10 mM HEPES-NaOH (pH 7.4), 0.1% BSA, and
20 mM D-glucose. Sterilize through a 0.22-μm filter. Store at
4
C.
3 Methods
3.1 Purification of
Recombinant Proteins
(His-tag)
1. Culture the BL21 (DE3) bacteria carrying the desired plasmid
(rat Pdx1-pET21a or rat NeuroD-pET21a) in 100 mL of LB
medium with 100 μg/mL ampicillin overnight at 37
C.
2. Decant the cultured bacteria into 900 mL of LB medium with
100 μg/mL ampicillin. Culture them again until the OD600
becomes approximately 0.8 at 37
C (see Note 4).
3. Add 0.1 M IPTG at a final concentration of 0.2 mM to induce
the expression of the desired protein. Continue the culture by
incubating at 32
C for 6–8 h (see Note 5).
4. Harvest the bacteria through centrifugation at 6000 Â g for
10 min at 4
C.
5. Add 100 mL of cold lysis buffer to the bacterial pellet and lyse
the bacterial cells with a vortex mixer. Incubate the samples for
15 min on ice (see Note 6).
Pancreatic Differentiation Through Protein Transduction
89
mL AlbuMAX II, 100 μM NEAAs, 2 mM L-Gln, 50 U/mL
penicillin, 50 μg/mL streptomycin, 100 μM β-ME, 10 ng/mL
Activin A, and 5 ng/mL bFGF. Store at 4
C (see Note 3).
16. Stage 2 medium: RPMI supplemented with 100 μM NEAAs,
2 mM L-Gln, 50 U/mL penicillin, 50 μg/mL streptomycin,
100 μM β-ME, 2% B27 supplement (Thermo Fisher Scientific),
50 ng/mL FGF10, 250 nM KAAD-cyclopamine, and 1 μM
RA. Store at 4
C (see Note 3).
17. Stage 3 medium: low-glucose (1000 mg/L) DMEM supplemented with 1% ITS-G, 2.5 mg/mL AlbuMAX II, 100 μM
NEAAs, 2 mM L-Gln, 50 U/mL penicillin, 50 μg/mL streptomycin, 100 μM β-ME, 10 mM NAM, and 10 nM GLP-1.
Store at 4
C (see Note 3).
2.4 C-peptide
Secretion Assay
1. Krebs–Ringer solution containing bicarbonate and HEPES
(KRBH) with low glucose: 129 mM NaCl, 4.8 mM KCl,
2.5 mM CaCl 2 , 1.2 mM KH 2 PO 4 , 1.2 mM MgSO 4 , 5 mM
NaHCO 3 , 10 mM HEPES-NaOH (pH 7.4), 0.1% BSA, and
2.5 mM D-glucose. Sterilize through a 0.22-μm filter. Store at
4
C.
2. Krebs–Ringer solution containing bicarbonate and HEPES
(KRBH) with high glucose: 129 mM NaCl, 4.8 mM KCl,
2.5 mM CaCl 2 , 1.2 mM KH 2 PO 4 , 1.2 mM MgSO 4 , 5 mM
NaHCO 3 , 10 mM HEPES-NaOH (pH 7.4), 0.1% BSA, and
20 mM D-glucose. Sterilize through a 0.22-μm filter. Store at
4
C.
3 Methods
3.1 Purification of
Recombinant Proteins
(His-tag)
1. Culture the BL21 (DE3) bacteria carrying the desired plasmid
(rat Pdx1-pET21a or rat NeuroD-pET21a) in 100 mL of LB
medium with 100 μg/mL ampicillin overnight at 37
C.
2. Decant the cultured bacteria into 900 mL of LB medium with
100 μg/mL ampicillin. Culture them again until the OD600
becomes approximately 0.8 at 37
C (see Note 4).
3. Add 0.1 M IPTG at a final concentration of 0.2 mM to induce
the expression of the desired protein. Continue the culture by
incubating at 32
C for 6–8 h (see Note 5).
4. Harvest the bacteria through centrifugation at 6000 Â g for
10 min at 4
C.
5. Add 100 mL of cold lysis buffer to the bacterial pellet and lyse
the bacterial cells with a vortex mixer. Incubate the samples for
15 min on ice (see Note 6).
Pancreatic Differentiation Through Protein Transduction
89