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

TY - JOUR

T1 - Generation of high-purity human ventral midbrain dopaminergic progenitors for in vitro maturation and intracerebral transplantation

AU - Nolbrant, Sara

AU - Heuer, Andreas

AU - Parmar, Malin

AU - Kirkeby, Agnete

PY - 2017/9/1

Y1 - 2017/9/1

N2 - 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 hPSClines. 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 DAneurons. This protocol is free of xeno-derived products and can be performed under good manufacturing practice (GMP) conditions.

AB - 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 hPSClines. 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 DAneurons. This protocol is free of xeno-derived products and can be performed under good manufacturing practice (GMP) conditions.

U2 - 10.1038/nprot.2017.078

DO - 10.1038/nprot.2017.078

M3 - Journal article

C2 - 28858290

SN - 1754-2189

VL - 12

SP - 1962

EP - 1979

JO - Nature Protocols

JF - Nature Protocols

IS - 9

ER -