hVGAT-mCherry: A novel molecular tool for analysis of GABAergic neurons derived from human pluripotent stem cells.
BACKGROUND
GABAergic synaptic transmission is known to play a critical role in the assembly of neuronal circuits during development and is responsible for maintaining the balance between excitatory and inhibitory signaling in the brain during maturation into adulthood. Importantly, defects in GABAergic neuronal function and signaling have been linked to a number of neurological diseases, including autism spectrum disorders, schizophrenia, and epilepsy. With patient-specific induced pluripotent stem cell (iPSC)-based models of neurological disease, it is now possible to investigate the disease mechanisms that underlie deficits in GABAergic function in affected human neurons. To that end, tools that enable the labeling and purification of viable GABAergic neurons from human pluripotent stem cells would be of great value.


RESULTS
To address the need for tools that facilitate the identification and isolation of viable GABAergic neurons from the in vitro differentiation of iPSC lines, a cell type-specific promoter-driven fluorescent reporter construct was developed that utilizes the human vesicular GABA transporter (hVGAT) promoter to drive the expression of mCherry specifically in VGAT-expressing neurons. The transduction of iPSC-derived forebrain neuronal cultures with the hVGAT promoter-mCherry lentiviral reporter construct specifically labeled GABAergic neurons. Immunocytochemical analysis of hVGAT-mCherry expression cells showed significant co-labeling with the GABAergic neuronal markers for endogenous VGAT, GABA, and GAD67. Expression of mCherry from the VGAT promoter showed expression in several cortical interneuron subtypes to similar levels. In addition, an effective and reproducible protocol was developed to facilitate the fluorescent activated cell sorting (FACS)-mediated purification of high yields of viable VGAT-positive cells.


CONCLUSIONS
These studies demonstrate the utility of the hVGAT-mCherry reporter construct as an effective tool for studying GABAergic neurons differentiated in vitro from human pluripotent stem cells. This approach could provide a means of obtaining large quantities of viable GABAergic neurons derived from disease-specific hiPSCs that could be used for functional assays or high-throughput screening of small molecule libraries.
Dr. John T Macdonald Foundation Department of Human Genetics, University of Miami Miller School of Medicine, Miami, FL 33136, United States; John P. Hussman Institute for Human Genomics, University of Miami Miller School of Medicine, Miami, FL 33136, United States. Electronic address: BDeRosa@med.miami.edu.
Brooke A
BA
DeRosa
Dr. John T Macdonald Foundation Department of Human Genetics, University of Miami Miller School of Medicine, Miami, FL 33136, United States; John P. Hussman Institute for Human Genomics, University of Miami Miller School of Medicine, Miami, FL 33136, United States. Electronic address: KBelle@med.miami.edu.
Kinsley C
KC
Belle
Dr. John T Macdonald Foundation Department of Human Genetics, University of Miami Miller School of Medicine, Miami, FL 33136, United States; John P. Hussman Institute for Human Genomics, University of Miami Miller School of Medicine, Miami, FL 33136, United States. Electronic address: thomasb@miamicountryday.info.
Blake J
BJ
Thomas
Dr. John T Macdonald Foundation Department of Human Genetics, University of Miami Miller School of Medicine, Miami, FL 33136, United States; John P. Hussman Institute for Human Genomics, University of Miami Miller School of Medicine, Miami, FL 33136, United States. Electronic address: HCukier@med.miami.edu.
Holly N
HN
Cukier
Dr. John T Macdonald Foundation Department of Human Genetics, University of Miami Miller School of Medicine, Miami, FL 33136, United States; John P. Hussman Institute for Human Genomics, University of Miami Miller School of Medicine, Miami, FL 33136, United States. Electronic address: MPericak@med.miami.edu.
Margaret A
MA
Pericak-Vance
Dr. John T Macdonald Foundation Department of Human Genetics, University of Miami Miller School of Medicine, Miami, FL 33136, United States; John P. Hussman Institute for Human Genomics, University of Miami Miller School of Medicine, Miami, FL 33136, United States. Electronic address: JVance@med.miami.edu.
Jeffery M
JM
Vance
Dr. John T Macdonald Foundation Department of Human Genetics, University of Miami Miller School of Medicine, Miami, FL 33136, United States; John P. Hussman Institute for Human Genomics, University of Miami Miller School of Medicine, Miami, FL 33136, United States. Electronic address: DDykxhoorn@med.miami.edu.
Derek M
DM
Dykxhoorn
D064032:Calbindin 2 / Q000378:metabolism; D002454:Cell Differentiation / Q000187:drug effects / Q000502:physiology; D002470:Cell Survival; D002478:Cells, Cultured; D005434:Flow Cytometry; D059330:GABAergic Neurons / Q000378:metabolism*; D051100:Glial Cell Line-Derived Neurotrophic Factor / Q000494:pharmacology; D005904:Glial Fibrillary Acidic Protein / Q000378:metabolism; D005968:Glutamate Decarboxylase / Q000378:metabolism; D006801:Humans; D008164:Luminescent Proteins / Q000235:genetics / Q000378:metabolism*; D009154:Mutation / Q000235:genetics; D020932:Nerve Growth Factor / Q000494:pharmacology; D010320:Parvalbumins / Q000378:metabolism; D039904:Pluripotent Stem Cells / Q000187:drug effects / Q000502:physiology*; D016548:Prosencephalon / Q000166:cytology; D013004:Somatostatin / Q000378:metabolism; D016704:Synapsins / Q000378:metabolism; D014162:Transfection; D050495:Vesicular Inhibitory Amino Acid Transport Proteins / Q000235:genetics / Q000378:metabolism*; D000097573:Red Fluorescent Protein
D064032:Calbindin 2; D051100:Glial Cell Line-Derived Neurotrophic Factor; D005904:Glial Fibrillary Acidic Protein; D005968:Glutamate Decarboxylase; D008164:Luminescent Proteins; D020932:Nerve Growth Factor; D010320:Parvalbumins; D013004:Somatostatin; D016704:Synapsins; D050495:Vesicular Inhibitory Amino Acid Transport Proteins; D000097573:Red Fluorescent Protein; C492245:SLC32A1 protein, human; C402565:glutamate decarboxylase 1
Fluorescent reporter construct; GABAergic neurons; Human induced pluripotent stem cells; Neural differentiation; Vesicular GABA transporter