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BRAIN Publication-derived

High-throughput generation of midbrain dopaminergic neuron organoids from reporter human pluripotent stem cells

Source Sarrafha et al., 2021 · Nash Family Department of Neuroscience at Mount Sinai, New York, NY · 10.1016/j.xpro.2021.100463

👤 Lily Sarrafha, Gustavo M. Parfitt, Ricardo Reyes, Camille Goldman, Elena Coccia, Tatyana Kareva, Tim Ahfeldt ⏱ 200 days 📋 4 phases 🧫 Human pluripotent stem cells (hPSCs: WA01/H1 ESC and BJ SiPS-D iPSC)

Abstract

This protocol describes high-throughput 3D differentiation of midbrain dopaminergic neurons from human pluripotent stem cells using spinner flask culture. The approach produces relatively uniform midbrain organoids containing dopaminergic neurons that can be maintained long-term and used for disease modeling, cell sorting, and downstream applications including transplantation studies.

Cell source
Human pluripotent stem cells (hPSCs: WA01/H1 ESC and BJ SiPS-D iPSC)
Application
Disease modeling, drug screening, and developmental study of midbrain dopaminergic neurons

Protocol overview

68 steps across 4 phases

Generation of TH-TdTomato reporter hPSC lines (optional) D-21 to D0 (approximately 3–4 weeks)
  1. 1 Coat 10-cm dishes with Geltrex solution
  2. 2 Aspirate and wash hPSCs
  3. 3 Dissociate cells with Accutase
  4. 4 Prepare wash buffer
  5. 5 Transfer cells to wash buffer
  6. 6 Mix and count cells
  7. 7 Centrifuge and collect cell suspension
  8. 8 Spin down cells
  9. 9 Prepare nucleofection mix with DNA
  10. 10 Resuspend cell pellet with nucleofection mix
  11. 11 Transfer cells to cuvette and place on ice
  12. 12 Nucleofect cells
  13. 13 Replace media in dishes with StemFlex + Thiazovivin
  14. 14 Plate nucleofected cells
  15. 15 Incubate plates
  16. 16 Check plates on day 1 post-nucleofection
  17. 17 Begin puromycin selection on day 2
  18. 18 Replace media without puromycin on day 4
  19. 19 Change media every other day until colonies are visible
  20. 20 Passage colonies and prepare for Cre-GFP targeting
  21. 21 Pause point: Freeze pre-Cre targeted hPSCs
  22. 22 Cre-GFP targeting for selection cassette removal
  23. 23 Prepare cells for FACS on day 1 post-nucleofection
  24. 24 Perform FACS and plate sorted cells
  25. 25 Replace media on day 3 post-nucleofection
  26. 26 Change media every other day until post-Cre colonies visible
  27. 27 Prepare for colony picking
  28. 28 Prepare collection plates
  29. 29 Coat plates with Geltrex
  30. 30 Pick and plate individual colonies
  31. 31 Genotype colonies using touchdown PCR
  32. 32 Validate positive colonies by Sanger sequencing
  33. 33 Expand selected colonies and prepare frozen stocks
Seeding and maintenance of hPSC lines in spinner flasks D-5 to D0
  1. 1 Distribute the hPSC lines across four 10-cm dishes.
  2. 2 Prepare StemFlex stock for seeding
  3. 3 Prepare spinner flasks
  4. 4 Verify hPSC health and confluency
  5. 5 Aspirate and wash hPSCs
  6. 6 Dissociate cells with Accutase
  7. 7 Prepare wash buffer for spinner seeding
  8. 8 Transfer cells to wash buffer
  9. 9 Centrifuge cells
  10. 10 Resuspend cells in StemFlex stock
  11. 11 Filter and add cells to spinner flasks
  12. 12 Monitor sphere formation
  13. 13 First media change
  14. 14 Second media change and begin differentiation prep
Differentiation of pluripotent spheres into midbrain progenitor organoids D0–D12 (2 weeks patterning stage)
  1. 1 Begin differentiation
  2. 2 Media change day 1
  3. 3 Media change day 2
  4. 4 Media change day 3
  5. 5 Media change day 4
  6. 6 Media change day 5
  7. 7 Media change day 6
  8. 8 Media change day 7
  9. 9 Major media change day 8
  10. 10 Media change day 9
  11. 11 Media change day 10
  12. 12 Media change day 11
  13. 13 Major media change day 12 - transition to terminal differentiation
  14. 14 Optional: Transfer to low-attachment plates
  15. 15 Quality control at day 15+
Maturation of midbrain progenitor organoids into dopaminergic neurons D12–D200+ (3+ weeks maturation; long-term culture possible)
  1. 1 Monitoring TH and neuronal marker expression (D20+)
  2. 2 Switch to D22-35 terminal medium on day 22
  3. 3 Transition to D36+ medium on day 36
  4. 4 Quality control for mature DN markers (D30+)
  5. 5 Long-term culture in flasks or low-attachment plates
  6. 6 Neuromelanin expression in mature cultures (D60+)

Full SOP

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Attribution

This SOP was authored by Organthis based on the published method in Sarrafha et al., 2021. The originating laboratory holds no rights in this SOP and has not endorsed it unless marked Verified.

This wording is awaiting legal review.

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