title: Embryonic stem cell-derived Pitx3-enhanced green fluorescent protein midbrain dopamine neurons survive enrichment by fluorescence-activated cell sorting and function in an animal model of Parkinson's disease.
abstract: Both fetal ventral mesencephalic (VM) and embryonic stem (ES) cell-derived dopamine neurons have been used successfully to correct behavioral responses in animal models of Parkinson's disease. However, grafts derived from fetal VM cells or from ES cells contain multiple cell types, and the majority of these cells are not dopamine neurons. Isolation of ES cell-derived dopamine neurons and subsequent transplantation would both elucidate the capacity of these neurons to provide functional input and also further explore an efficient and safer use of ES cells for the treatment of Parkinson's disease. Toward this goal, we used a Pitx3-enhanced green fluorescent protein (Pitx3-eGFP) knock-in mouse blastocyst-derived embryonic stem (mES) cell line and fluorescence-activated cell sorting (FACS) to select and purify midbrain dopamine neurons. Initially, the dopaminergic marker profile of intact Pitx3-eGFP mES cultures was evaluated after differentiation in vitro. eGFP expression overlapped closely with that of Pitx3, Nurr1, Engrailed-1, Lmx1a, tyrosine hydroxylase (TH), l-aromatic amino acid decarboxylase (AADC), and vesicular monoamine transporter 2 (VMAT2), demonstrating that these cells were of a midbrain dopamine neuron character. Furthermore, postmitotic Pitx3-eGFP(+) dopamine neurons, which constituted 2%-5% of all live cells in the culture after dissociation, could be highly enriched to >90% purity by FACS, and these isolated neurons were viable, extended neurites, and maintained a dopaminergic profile in vitro. Transplantation to 6-hydroxydopamine-lesioned rats showed that an enriched dopaminergic population could survive and restore both amphetamine- and apomorphine-induced functions, and the grafts contained large numbers of midbrain dopamine neurons, which innervated the host striatum. Disclosure of potential conflicts of interest is found at the end of this article.
authors.affiliation: Udall Parkinson's Disease Research Center for Excellence, McLean Hospital, Harvard Medical School, Belmont, Massachusetts 02478, USA.
authors.forename: Eva
authors.initials: E
authors.lastname: Hedlund
authors.forename: Jan
authors.initials: J
authors.lastname: Pruszak
authors.forename: Thomas
authors.initials: T
authors.lastname: Lardaro
authors.forename: Wesley
authors.initials: W
authors.lastname: Ludwig
authors.forename: Angel
authors.initials: A
authors.lastname: Viñuela
authors.forename: Kwang-Soo
authors.initials: KS
authors.lastname: Kim
authors.forename: Ole
authors.initials: O
authors.lastname: Isacson
mesh_terms: D000661:Amphetamine / Q000494:pharmacology; D000818:Animals; D001058:Apomorphine / Q000494:pharmacology; D002454:Cell Differentiation; D002470:Cell Survival; D053595:Embryonic Stem Cells / Q000166:cytology* / Q000502:physiology*; D005434:Flow Cytometry / Q000379:methods; D017930:Genes, Reporter; D049452:Green Fluorescent Proteins / Q000235:genetics; D018398:Homeodomain Proteins / Q000235:genetics*; D006892:Hydroxydopamines / Q000633:toxicity; D008636:Mesencephalon / Q000166:cytology / Q000502:physiology*; D051379:Mice; D008938:Mitosis; D009043:Motor Activity / Q000187:drug effects / Q000502:physiology; D020734:Parkinsonian Disorders / Q000473:pathology* / Q000503:physiopathology*; D051381:Rats; D033581:Stem Cell Transplantation / Q000379:methods; D014157:Transcription Factors / Q000235:genetics*; D014183:Transplantation, Heterologous
chemical_list: D018398:Homeodomain Proteins; D006892:Hydroxydopamines; D014157:Transcription Factors; C433629:homeobox protein PITX3; D049452:Green Fluorescent Proteins; C027802:5-hydroxydopamine; D000661:Amphetamine; D001058:Apomorphine