To fully realize the potential of human pluripotent stem cells (hPSCs) for both therapeutic and research purposes, it is critical to follow an efficient and reliable in vitro differentiation method that is based on optimal physical, chemical and developmental cues. This highly reproducible protocol describes how to grow hPSCs such as human induced pluripotent and embryonic stem cells in a physically confined area (‘spot’) and efficiently differentiate them into a highly enriched population of healthy and functional midbrain dopamine progenitors (mDAPs) and midbrain dopamine neurons (mDANs). The protocol takes 28 d, during which cells first grow and differentiate in spots for 14 d and then are replated and further differentiated for a further 14 d as a monolayer culture. We describe how to produce mDAPs, control the quality of cells and cryopreserve mDAPs without loss of viability. Previously we showed that mDANs generated by this ‘spotting’-based method exhibit gene expression and (electro)physiological properties typical of A9 mDANs lost in Parkinson’s disease brains and can rescue motor defects when transplanted into the striatum of 6-hydroxydopamine-lesioned rats. This protocol is scalable for production of mDAPs under good manufacturing practice conditions and was also previously successfully used to generate cells for the first autologous cell replacement therapy of a patient with Parkinson’s disease without the need for immune suppression. We anticipate this protocol could also be readily adapted to use spotting-based culture to further optimize the differentiation of hPSC to alternative differentiated cell types.
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This work was supported by NIH grants (NS070577 and OD024622) (to K.S.K.) and NRF grants 2017R1A2B4008456, 2020R1H1A2013386, IITP grant from MSIT 2020-0-01343 (to H.S.), as well as the Parkinson’s Cell Therapy Research Fund at McLean Hospital.
The authors declare no competing interests.
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Key references using this protocol
Song, B. et al. J. Clin. Invest. 130, 904–920 (2020): https://doi.org/10.1172/JCI130767
Schweitzer, J. S. et al. N. Engl. J. Med. 382, 1926–1932 (2020): https://doi.org/10.1056/NEJMoa1915872
The effects of cell number per spot and the number of spots per plate were determined for optimization of spotting condition. Cell loss, cell harvest, the percentage of dead cells in the final harvest and the ratio of cell loss per cell yield are shown.
Extended Data Fig. 5 Characterization of H9-derived mDAPs by immunofluorescence on day 28 following spotting and monolayer culture.
The antibodies for FOXA2, LMX1A and TH were used to stain mDAPs, and the antibodies for cleaved caspase-3 were used to stain apoptotic cells. Scale bars, 100 µm.
Percent success rate of cells differentiating to Day 28.
Source data for a) cell yield vs input; b) ratio of cell loss/Cell yield; c) dead cell percent in final harvested cell; d) percent of TUNEL-positive cells; e) percent of cleaved Caspase 3-positive cells; f) relative mRNA levels expression of BCL, BCL-XL, BAX, p53, and PUMA for H9 and C4 cells.
Source data for: Cell loss; Total Cell yield; Dead Cell percent in final harvested cells; for H9 and C4 cells.
Source data for H9 cells spotting data using 5K, 10k, 20k, and monolayers looking at: Cell Loss; Cell yield/input; Cell harvest; percent dead cells in final harvest; ratio of cell loss/cell yield.
Source data for C4 cells spotting data using 5K, 10k, 20k, and monolayers looking at: Cell Loss; Cell yield/input; Cell harvest; percent dead cells in final harvest; ratio of cell loss/cell yield.
Source data for H9 cells a) TUNEL-positive cells using 10k and 20k cells per spot looking at: 4 spots, 6 spots, 9 spots, 12 spots, and monolayer. b) percent cleaved Caspase 3-positive cells using 10k and 20k cells per spot looking at: 4 spots, 6 spots, 9 spots, 12 spots, and monolayer.
Source data for C4 cells a) TUNEL-positive cells using 10k and 20k cells per spot looking at: 4 spots, 6 spots, 9 spots, 12 spots, and monolayer. b) percent cleaved Caspase 3-positive cells using 10k and 20k cells per spot looking at: 4 spots, 6 spots, 9 spots, 12 spots, and monolayer.
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Kim, J., Jeon, J., Song, B. et al. Spotting-based differentiation of functional dopaminergic progenitors from human pluripotent stem cells. Nat Protoc 17, 890–909 (2022). https://doi.org/10.1038/s41596-021-00673-4