Generation of high-purity human ventral midbrain dopaminergic progenitors for in vitro maturation and intracerebral transplantation


Generation of precisely patterned neural cells from human pluripotent stem cells (hPSCs) is instrumental in developing disease models and stem cell therapies. Here, we provide a detailed 16-d protocol for obtaining high-purity ventral midbrain (VM) dopamine (DA) progenitors for intracerebral transplantation into animal models and for in vitro maturation into neurons. We have successfully transplanted such cells into the rat; however, in principle, the cells can be used for transplantation into any animal model, and the protocol is designed to also be compatible with clinical transplantation into humans. We show how to precisely set the balance of patterning factors to obtain specifically the caudal VM progenitors that give rise to DA-rich grafts. By specifying how to perform quality control (QC), troubleshooting and adaptation of the procedure, this protocol will facilitate implementation in different laboratories and with a variety of hPSC lines. To facilitate reproducibility of experiments and enable shipping of cells between centers, we present a method for cryopreservation of the progenitors for subsequent direct transplantation or terminal differentiation into DA neurons. This protocol is free of xeno-derived products and can be performed under good manufacturing practice (GMP) conditions.

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Figure 1: Anatomical patterning of the ventral di/mesencephalon during development.
Figure 2: Overview of protocol steps.
Figure 3: Phenotypic overview of cultures at different differentiation stages.
Figure 4: QC of differentiated cells.
Figure 5: Transplantation outcome of fresh and cryopreserved cells.
Figure 6: Phenotype of terminally differentiated mesDA neurons in vitro.


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We thank U. Jarl, B. Mattsson, M. Sparrenius, S. da Rocha Baez and M.P. Vejgården for excellent technical assistance and U.B. Savukinas for illustrations. The research leading to these results has received funding from the European Community's Seventh Framework Programme through NeuroStemcellRepair (no. 602278), the European Research Council under the European Union's Seventh Framework Programme (FP/2007-2013)/ERC grant agreement no. 309712, the Strong Research Environment at Lund University Multipark (multidisciplinary research in Parkinson's disease), the Swedish Research Council (70862601/Bagadilico and K2014-61X-20391-08-4 and 2015-03444_3), the Swedish Society for Medical Research, the UK Regenerative Medicine Platform and Innovation Fund Denmark (BrainStem). M.P. is a New York Stem Cell Foundation—Robertson Investigator.

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A.K. and M.P. conceptualized the study. S.N., A.K., A.H. and M.P. developed the methodology. S.N., A.K. and A.H. performed investigation and formal analysis. A.K., S.N. and M.P. wrote the original draft of the manuscript. A.K., S.N. and M.P. revised and edited the manuscript. A.K. and S.N. visualized the protocol. A.K. and M.P. performed funding acquisition. A.K. and M.P. supervised the study.

Corresponding authors

Correspondence to Malin Parmar or Agnete Kirkeby.

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Competing interests

A US patent application related to this protocol, listing A.K. and M.P. as inventors, has been filed.

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Nolbrant, S., Heuer, A., Parmar, M. et al. Generation of high-purity human ventral midbrain dopaminergic progenitors for in vitro maturation and intracerebral transplantation. Nat Protoc 12, 1962–1979 (2017).

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