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Spotting-based differentiation of functional dopaminergic progenitors from human pluripotent stem cells

Nature Protocols volume 17, pages 890–909 (2022)Cite this article

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Abstract

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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Fig. 1: Schematic overview of the spotting method for mDA differentiation from hPSCs.
Fig. 2: Morphological changes in cells during the process of differentiation of mDA from hPSCs.
Fig. 3: Cells’ survival and yield during the mDA differentiation process: comparison between spotting and monolayer methods for H9 hESC and C4 hiPSC.
Fig. 4: Effects of different coating materials on hPSC survival and yield.
Fig. 5: Characterization of mDA cells differentiated from hESCs (H9) in vitro and in vivo.

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Data availability

All data supporting the findings of this study are available within the article and its Supplementary Information files. Source data are provided with this paper.

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Acknowledgements

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.

Author information

Authors and Affiliations

  1. Department of Psychiatry, McLean Hospital, Harvard Medical School, Belmont, MA, USA

    Jisun Kim, Jeha Jeon, Bin Song, Nayeon Lee, Sanghyeok Ko, Young Cha, Pierre Leblanc, Hyemyung Seo & Kwang-Soo Kim

  2. Molecular Neurobiology Laboratory, McLean Hospital and Program in Neuroscience, Harvard Medical School, Belmont, MA, USA

    Jisun Kim, Jeha Jeon, Bin Song, Nayeon Lee, Sanghyeok Ko, Young Cha, Pierre Leblanc, Hyemyung Seo & Kwang-Soo Kim

  3. Department of Molecular and Life Sciences, The Center for Bionano Intelligence Education and Research, Hanyang University, Hanyang, South Korea

    Hyemyung Seo

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Contributions

K.S.K, H.S and J.K. designed the study; J.K., J.J., B.S., N.L., S.K. and Y.C. performed experiments; J.K., H.S. and J.J collected and analyzed data; J.K., J.J, P.L., H.S and K.S.K wrote the manuscript; H.S and K.S.K supervised the study.

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Correspondence to Hyemyung Seo or Kwang-Soo Kim.

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Nature Protocols thanks Asuka Morizane and the other, anonymous, reviewer(s) for their contribution to the peer review of this work.

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Related links

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

Extended data

Extended Data Fig. 1 hPSC spotting condition optimization for H9 hESC cells.

Source data

Extended Data Fig. 2 hPSC spotting condition optimization for H9 hESC cells.

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.

Source data

Extended Data Fig. 3 Effects of H9 hESC spotting conditions on viability and gene expression.

Source data

Extended Data Fig. 4 Effects of C4 hiPSC spotting conditions on viability and gene expression.

a–c, The health of H9 hESCs and C4 hiPSCs were examined by TUNEL staining (a), expression of apoptosis marker cleaved caspase-3 (b) and expression of apoptosis marker genes (c), as described in ref. 10. Scale bars, 100 µm.

Source data

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.

Supplementary information

Supplementary Information

Supplementary Tables 1 and 2.

Source data

Source Data Fig. 2b

Percent success rate of cells differentiating to Day 28.

Source Data Fig. 3

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 Fig. 5

Source data for: Cell loss; Total Cell yield; Dead Cell percent in final harvested cells; for H9 and C4 cells.

Source Data Extended Data Fig. 1

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 Extended Data Fig. 2

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 Extended Data Fig. 3

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 Extended Data Fig. 4

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

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