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This is intended as a guide only; for full experimental details please read the reference provided.
Kim et al. describe a scalable and reproducible protocol for the generation of midbrain dopaminergic (mDA) neurons from human embryonic stem cells, followed by their cryopreservation, and transplantation in mice and rats. Neurons generated using this protocol have potential for use in regenerative medicine for the treatment of Parkinson’s disease.
On day 0 hESCs (WA09) were plated on basement membrane extract (BME e.g. CultrexTM) in Neurobasal medium supplemented with N2, B27 and L-glutamine, containing SB 431542, LDN 193189, CHIR 99021, SHH and Y-27632. Media was changed daily. On day 1 Y-27632 was withdrawn. On day 4 the concentration of CHIR 99021 was increased to optimize midbrain specification. On day 7 LDN 193189, SB 431542 and SHH were withdrawn. On day 10 media was changed to Neurobasal medium with B27 and L-glutamine supplemented with BDNF, Ascorbic Acid, GDNF, TGF-ß3, dibutyryl cAMP, CHIR 99021 (mDA medium). On day 11 cells were dissociated and replated in mDA medium with the addition of DAPT from day 12. On day 16 cells were dissociated and replated for further study or cryopreservation.
In vitro electrophysiology studies of resulting mDA neurons were conducted from day 25. Cryopreserved cells were thawed and when transplanted into the right striatum of 6-OHDA lesioned rats reduced motor abnormalities.
|Neurobasal medium supplemented with N2/B27/L-Glutamine (Days 1-3)||Neurobasal medium supplemented with N2/B27/L-Glutamine (Days 4-6)||Neurobasal medium supplemented with N2/B27/L-Glutamine (Days 7-9)||mDA differentiation medium (Neurobasal medium supplemented with B27/L-Glutamine) Days 10-12)||mDA differentiation medium (Days 12-16)|
|LDN 193189 (Cat.No. 6053)||250 nM||LDN 193189 (Cat.No. 6053)||250 nM||CHIR 99021 (Cat.No. 4423)||7.5 μM||BDNF (Cat.No. 248-BDB)||20 ng/ml||BDNF (Cat.No. 248-BDB)||20 ng/ml|
|SB 431542 (Cat.No. 1614)||10 μM||SB 431542 (Cat.No. 1614)||10 μM||GDNF (Cat.No. 212-GD)||20 ng/ml||GDNF (Cat.No. 212-GD)||20 ng/ml|
|CHIR 99021 (Cat.No. 4423)||0.7 μM||CHIR 99021 (Cat.No. 4423)||7.5 μM||Ascorbic Acid (Cat.No. 4055)||200 μM||Ascorbic Acid (Cat.No. 4055)||200 μM|
|Y-27632 (Cat.No. 1254)||10 μM (Day 1 only)||Dibutyryl-cAMP (Cat.No. 1141)||500 μM||Dibutyryl-cAMP (Cat.No. 1141)||500 μM|
|SHH25II (Cat.No. 64-SH)||500 ng/ml||SHH25II (Cat.No. 64-SH)||500 ng/ml||TGF-β3 (Cat.No. 8420-B3)||1 ng/ml||TGF-β3 (Cat.No. 8420-B3)||1 ng/ml|
|CHIR 99021 (Cat.No. 4423)||3 μM||DAPT (Cat.No. 2634)||10 μM|