Table 1.
Methods for neural differentiation of PSCs. The methods have been broken down into 5 stages (illustrated in Figure 1). Each stage is discussed in the text.
| 1) Starting Material | ||||
| Reference | Park, et al.[84] | Iacovitti, et al.[43] | Gerrard et al.[37] | Schultz, et al.[100] |
| Lines | HSF-6, SNU-hes-3, Miz | H9, BG01, HUES7, HUES8 | H1, H7, H9 | SSEA4-enriched BG01 |
| Medium | STD | STD, HUES | MEF-CM | STD |
| Feeders/Substrate | iMEF | iMEF | Matrigel | mmcMEF |
| Passaging | Mechanical | Mechanical, trypsin | Collagenase | Collagenase/Trypsin |
| 2) Induced loss of pluripotency | ||||
| Passage | Collagenase | Collagenase | EDTA | Collagenase+Mechanical |
| Substrate | PA6-SHH Stromal Cells | LA polystyrene | Matrigel or PLL/laminin | LA Polystyrene |
| Media | FGF-free | STD, HUES | DMEM/F12 w/N2B27 | DMEM/F12, 1X N2 |
| Factors | ITS+AA | bFGF | 100ng/mL Noggin | MedII conditioned, BFGF |
| Duration |  |
3 days |  |
 |
| Selection | Bulk passage | |||
| 3) NSC Induction | ||||
| Passage | Pipetted | |||
| Substrate | PA6-SHH Stromal Cells | 100ng/cm2 collagen IV | Matrigel or PLL/laminin | LA Polystyrene |
| Media | FGF-free | DMEM/F12, Glucose, 1% ITS | DMEM/F12 w/N2B27 | DMEM/F12, 1X N2 |
| Factors | ITS+AA | Noggin, fibronectin | 100ng/mL Noggin | MedII conditioned, BFGF |
| Duration | 2 weeks | 4−14 days | 3 passages |  |
| Selection | passive | Rosettes | Bulk enzymatic passage | |
| 4) NSC Maintenance&Expansion | ||||
| Passage | Collagenase | Mechanical. Dispase after 1 week | TrypLE Express | |
| Substrate | FN | TC-treated polystyrene | Matrigel or PLL/laminin | LA Polystyrene |
| Media | DMEM/F12 | DMEM/F12, Glucose, 1% ITS | DMEM/F12 w/N2B27 | DMEM/F12, 1X N2 |
| Factors | N2+AA+bFGF. ITS after 3−4 days | 20ng/mL bFGF | 100ng/mL Noggin+20ng/mL FGF | MedII conditioned, BFGF |
| Duration | 1.5 weeks | 7−10 days | 2+ passages | 2−6 weeks |
| Selection | bulk passage | rosettes | Passive | passive |
| 5) Neural differentiation | ||||
| Passage | Collagenase | Dispase | TrypLE Express | Pipetted |
| Substrate | PLO+FN | PLO | PLL/laminin | PLO/ Laminin |
| Media | DMEM/F12 | DMEM/F12 | N2B27 | MedII/BFGF or Neurobasel.B27 |
| Added Factors | ITS+AA | dbcAMP | I:SHH+FGF8+AA; ii:BDNF+GDNF+AA+Laminin) | 5% FBS+GDNF+BDNF |
| Duration | 2−3 days | 1 week | i:1−2 weeks plus ii: 2 weeks | Undefined |
| Selection | Midbrain DA TuJ1+ neurons | TH+DA B-tubulin III+ neurons | TH positive neurons | TH+MAP2+ and VMAT2+MAP2+ |
| 1) Starting Material | ||||
| Reference | Pankrantz, et al.[81] | Lim et al.[66] | Baharvand, et al.[8] | Park et al.[86] |
| Lines | H1, H9, monkey R366.4 | hES 3.1, 3.2, 3.3 | Royan H5 | MB03 |
| Medium | STD | HES/mod | MOD | MBO3/MBO3 mod |
| Feeders/Substrate | iMEF | iHFF | mmcMEF | mmcSTO |
| Passaging | Bulk enzymatic passage | Bulk enzymatic passage | Mechanical | Trypsin |
| 2)Induced loss of pluripotency | ||||
| Passage | Collagenase | Collagenase | Mechanical | Trypsin |
| Substrate | LA Polystyrene | LA Polystyrene | Matrigel | LA Polystyrene |
| Media | DMEM/F12 20%KSR | DMEM/HG 20%KSR | DMEM/F12+5%KSR | MB03 |
| Factors | bFGF | 4ng/mL bFGF | bFGF, Noggin | - |
| Duration | 4 days | 6 days | 1 passage | 4 days |
| Selection | bulk | bulk | ePSC colonies | bulk |
| 3) NSC Induction | ||||
| Passage | Pipetted | Pipetted | Mechanical | Pipetted |
| Substrate | suspension for 2 days, laminin for 15 | tissue culture-treated polystyrene | Matrigel | Gelatin |
| Media | (DMEM/F12+N2) | DMEM/HG 20%KSR | DMEM/F12+5%KSR | Serum-free |
| Factors | 20ng/mL bFGF | bFGF, RA, Shh, Wnt3a, BMP-2 | RA first 6 days | ITS+Fibronectin |
| Duration | 15 days |  |
12 days | 8 days |
| Selection | Neural tubes | Rosettes | bulk | |
| 4) NSC Maintenance&Expansion | ||||
| Passage | Enzymatic | Mechanical | Trypsin | |
| Substrate | PO+laminin or suspension | tissue culture-treated polystyrene | Matrigel | Laminin |
| Media | (DMEM/F12+N2) | DMEM/HG 20%KSR | DMDM/F12+5%KSR | N2 |
| Factors | 20ng/mL bFGF or RA | bFGF, RA, Shh, Wnt3a, BMP-2 | bFGF | Laminin, bFGF |
| Duration | 14−16 days | 2 weeks | 6 days | 6 days |
| Selection | Spheres or rosettes | bulk | Neural Tubes | bulk |
| 5) Neural differentiation | ||||
| Passage | Enzymatic | Undefined | Mechanical isol. of Neural Tubes | Trypsin |
| Substrate | Suspension then PLO/Laminin | tissue culture-treated polystyrene | PLO/Laminin | Glass Coverslips |
| Media | Neurobasel, N2, NEAA | Gibco Neurobasal medium+B27 | Neurobasel Medium. 1% N2, 2% B27 | N2 |
| Added Factors | BDNF, GCNF, cAMP, AA and Laminin | 4ng/mL bFGF | bFGF and RA | RA, bFGF, BDNF, TGF-alpha |
| Duration | 8 days | 3 weeks | 14 days | 7, 14, 21 days |
| Selection | b-tubulin+, otx-2, Pax6 | b-tubulin III +, NSE and HB9 | b-tubulin III, Map-2, Synaptophysin, NF | NF200, TH, GAD |
| 1) Starting Material | ||||
| Reference | Joannides, et al.[47] | Nistor, et al.[78] | Sonntag, et al.[109] | Kang, et al.[50] |
| Lines | H9, HUES9 | H7 | H7, H9 | SNUhES1 |
| Medium | STD, HUES | STD-CM | Std/MOD | MOD |
| Feeders/Substrate | iMEF | Matrigel | mmcHFF | mmcSTO |
| Passaging | Mech; collagenase | Collagenase | Collagenase | Mechanical |
| 2) Induced loss of pluripotency | ||||
| Passage | Collagenase+chopping | Collagenase | Collagenase MS5 Stromal Cells | Collagenase LA Polystyrene |
| Substrate | LA Polystyrene | LA Polystyrene | SRM | ePSC media w/o bFGF |
| Media |  |
50%STD, 50%GRM |  |
- |
| Factors | 2 days | 4 days | ||
| Duration | EGF, bFGF, B27, ITS, many hormones | Passive | ||
| Selection | bulk | |||
| 3) NSC Induction | ||||
| Passage | Pipetted | Pipetted | ||
| Substrate | Suspension+Shaking | Suspension | MS5 Stromal Cells | Matrigel |
| Media | DMEM+Human Serum | 1 day 50%STD, 50%GRM. 100% GRM 7 days | SRM 14 days, N2 7 days | DMEM/F12 |
| Factors | ITS+EGF+bFGF | EGF, bFGF, B27, hormones + RA | Noggin 300ng/mL 7−21 days | ITS+Fibronectin |
| Duration | 8 days | 8 days | 21 days | 5 days |
| Selection | bulk | Yellow spheres | rosettes |  |
| 4) NSC Maintenance&Expansion | ||||
| Passage | Pipetted+chopping |  |
 |
|
| Substrate | Suspension+Shaking | Matrigel | ||
| Media | DMEM+Human Serum | DMEM/F12 | ||
| Factors | ITS+EGF+bFGF | 1X N2, bFGF | ||
| Duration | >8 days | 5 days | ||
| Selection | bulk | rosettes+neural tubes | ||
| 5) Neural differentiation | ||||
| Passage | Pipetted+chopping | Pipetted | Mechanical | Mechanical |
| Substrate | poly(D-lysine)/Laminin | poly (L-Lysine)/Laminin | PLO/`Laminin | Matrigel |
| Media | DMEM/2% B27 | GRM | N2 | DMEM/F12 |
| Added Factors | none | None | Shh, FGF8, BDNF, bFGF, TGF-B3, GDNF, cAMP, AA | N2 and T3 |
| Duration | 14 days | 1 week | 16 days | 20 days |
| Selection | b-tubulinIII, Map2ab, Synapsin 1, GABA | GAL C, RIP, O4 | NCAM, TuJ1, TH | O4, O1, MBP |
| 1) Starting Material | ||||
| Reference | Banin, et al.[9] | Kim, et al.[54] | Lee, et al[60] | |
| Lines | HES-1, EF1α-GFP | HSF6, Miz-hES4, Miz-hES6 | H9 | |
| Medium | HES | STD | STD | |
| Feeders/Substrate | iMEF | iMEF | mmcMEF | |
| Passaging | Mechanical | Collagenase | Collagenase | |
| 2)Induced loss of pluripotency | ||||
| Passage | Collagenase | Collagenase | Collagenase | |
| Substrate | iMEF | LA Polystyrene | MS-5 Stromal cells | |
| Media | DMEM-glucose w/ 10% FBS | ePSC media w/o bFGF | SRM | |
| Factors | 50ng/mL Noggin | - | - | |
| Duration |  |
8 days | 16 days | |
| Selection | Passive |  |
||
| 3) NSC Induction | ||||
| Passage | Pipetted | |||
| Substrate | iMEFs | TC-treated polystyrene | MS-5 Stromal cells | |
| Media | DMEM-glucose w/ 10% FBS | ITS medium | DMEM, 1X N2 | |
| Factors | 500ng/mL Noggin | 5ug/mL fibronectin | Shh, FGF8, BDNF, AA | |
| Duration | 8 days w/noggin, 5 days w/o | 8 days | 12 days | |
| Selection | “Gray, opaque morphology” | bulk | Rosettes | |
| 4) NSC Maintenance&Expansion | ||||
| Passage | Mechanical |  |
Mechanical | |
| Substrate | LA Polystyrene | PO/Laminin | ||
| Media | DMEM/F12 | DMEM, 1X N2 | ||
| Factors | B27, 20ng/mL EGF, bFGF | Initial passage BFGF, AA, BDNF. Subsequent passages BFGF, EGF. | ||
| Duration | >1 week | 6−7 day passages | ||
| Selection | spheres | p75+, HNK1+ cells for first passage. Bulk for subsequent passages. | ||
| 5) Neural differentiation | ||||
| Passage | HBSS | |||
| Substrate | Poly D-Lysine/Laminin | PO/Laminin | ||
| Media | DMEM/F12. B27 | DMEM, 1X N2 | ||
| Added Factors | NT3, NT4, BDNF | BDNF, GDNF, NGF, dbcAMP | ||
| Duration | 2−3 weeks | >2weeks | ||
| Selection | B-tubulin III, NF 160, MAP2ab, GABA,TH | TuJ1, islet1, TH, GABA, PLP | TH, Peripherin, Brn3a |