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

Research output: Contribution to journalArticle

Abstract

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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Authors
Organisations
External organisations
  • University of Copenhagen
Research areas and keywords

Subject classification (UKÄ) – MANDATORY

  • Neurosciences
  • Cell and Molecular Biology
Original languageEnglish
Pages (from-to)1962-1979
Number of pages18
JournalNature Protocols
Volume12
Issue number9
Publication statusPublished - 2017 Sep 1
Publication categoryResearch
Peer-reviewedYes