Clinically compatible differentiation protocol for human pluripotent stem cell-derived dopaminergic progenitor cells

Cell replacement therapy with human pluripotent stem cells has the potential to be a new therapy for Parkinson’s disease (PD). This protocol induces human induced pluripotent stem cells (iPSCs) to dopaminergic progenitor cells (DAPs) as clinically compatible donor cells in 30 days. The protocol includes starting with high density culture, cell sorting by using a cell surface marker for oor plate, and a maturation culture to form oating aggregates. The DAPs differentiated with this protocol were used in a pre-clinical tumorigenicity and ecacy study aiming for approval to start a clinical trial in Japan.


Introduction
Parkinson's disease (PD) is a neurodegenerative disease caused by a loss of dopaminergic (DA) neurons in the substantia nigra-striatal pathway. The neuronal cell death results in mainly motor symptoms including tremors, rigid muscles, slower movement, and impaired posture and balance. Cell therapy for PD is a concept in which the lost dopaminergic neurons are replaced by surgery. As donor cells, authentic neural progenitors with ventral mesencephalic (VM) phenotype are needed. Developmentally, dopaminergic neurons in substantia nigra are derived from fetal VM. Accordingly, aborted fetal midbrain tissues were used in clinical trials in the 1990s and showed some clinical bene t. Because fetal tissue is in short supply and its use has ethical issues, human pluripotent stem cells (PSCs) are being investigated as an alternative cell source for transplantation. There have been many reported protocols for mesencephalic DA induction from human PSCs at the laboratory level [1][2][3][4] . Most of them cannot be applied to the clinic as they are, however. To meet the criteria of clinical application, xeno-materials and genetic modi cations to the cells must be avoided, the prevention of contamination and GMP compatibility must be con rmed, and regulations with respective countries must be met. We have established a differentiation protocol for dopaminergic progenitor cells (DAPs) from human PSCs 5, 6 and modi ed it for the purpose of a preclinical study and clinical application. The reagents and equipment are changed to clinical grade products to comply with the requirements by a Japanese regulatory agency.

2.
Add 1 mL of 8GMK media and mix, and then aspirate the media and iMatrix solution. Do not allow the plate to dry.

6.
Remove the media from two con uent* 90-mm dishes of undifferentiated iPSCs and wash the cells two times with 4 mL of D-PBS (-) per dish. *con uent means "state that is suitable for passage", not the state that cells cover all area of the wells.

8.
During the incubation, prepare 12 mL of 8GMK+LAY media in a new 50 mL tube.

9.
Remove the dishes from the incubator and pipette the cells 10 times to fully dissociate.
10. Collect the cells in 50 mL tubes with the 12 mL of 8GMK+LAY in step 8 and mix.
13. Add 1.5 mL of 8GMK+LAY media and mix.
14. Count the cells with a sample diluted 10 times with D-PBS (-).
15. Remove the 6-well plate coated with iMatrix from the incubator.
16. Add 5×10 6 cells per well to the 6-well plate and culture the plate at 37 °C, 5% CO 2 in an incubator. 23. Aspirate the media from the well, add 6 mL of the 8GMK+LAFP media, and culture the plate at 37 °C, 5% CO 2 in an incubator.

Troubleshooting
Step 9: if the cells do not detach from the dish, use a cell scraper.  Bright eld images of a) undifferentiated iPSCs, b) differentiation day 1, c) day 12, and day 30. Bars=200 μm.