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

Nolbrant, Sara LU ; Heuer, Andreas LU ; Parmar, Malin LU and Kirkeby, Agnete LU (2017) In Nature Protocols 12(9). p.1962-1979
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... (More)

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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published
subject
in
Nature Protocols
volume
12
issue
9
pages
18 pages
publisher
Nature Publishing Group
external identifiers
  • scopus:85029668287
  • wos:000408997500009
ISSN
1750-2799
DOI
10.1038/nprot.2017.078
language
English
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yes
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e02ddf2a-537b-4a26-8ff1-8d3268aea3a8
date added to LUP
2017-10-06 08:51:33
date last changed
2018-01-16 13:21:13
@article{e02ddf2a-537b-4a26-8ff1-8d3268aea3a8,
  abstract     = {<p>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.</p>},
  author       = {Nolbrant, Sara and Heuer, Andreas and Parmar, Malin and Kirkeby, Agnete},
  issn         = {1750-2799},
  language     = {eng},
  month        = {09},
  number       = {9},
  pages        = {1962--1979},
  publisher    = {Nature Publishing Group},
  series       = {Nature Protocols},
  title        = {Generation of high-purity human ventral midbrain dopaminergic progenitors for in vitro maturation and intracerebral transplantation},
  url          = {http://dx.doi.org/10.1038/nprot.2017.078},
  volume       = {12},
  year         = {2017},
}