Standardized Reporter Systems for Purification and Imaging of Human Pluripotent Stem Cell-derived Motor Neurons and Other Cholinergic Cells.

Cell Differentiation Cholinergic Agents Homeodomain Proteins Humans Induced Pluripotent Stem Cells Motor Neurons Pluripotent Stem Cells Transcription Factors
CRISPR FACS HB9 MACS VACHT iPSC

Journal

Neuroscience
ISSN: 1873-7544
Titre abrégé: Neuroscience
Pays: United States
ID NLM: 7605074

Informations de publication

Date de publication:
01 12 2020
Historique:
received: 16 02 2020
revised: 11 06 2020
accepted: 19 06 2020
pubmed: 3 7 2020
medline: 15 5 2021
entrez: 3 7 2020
Statut: ppublish

Résumé

Reliable and consistent pluripotent stem cell reporter systems for efficient purification and visualization of motor neurons are essential reagents for the study of normal motor neuron biology and for effective disease modeling. To overcome the inherent noisiness of transgene-based reporters, we developed a new series of human induced pluripotent stem cell lines by knocking in tdTomato, Cre, or CreERT2 recombinase into the HB9 (MNX1) or VACHT (SLC18A3) genomic loci. The new lines were validated by directed differentiation into spinal motor neurons and immunostaining for motor neuron markers HB9 and ISL1. To facilitate efficient purification of spinal motor neurons, we further engineered the VACHT-Cre cell line with a validated, conditional CD14-GFP construct that allows for both fluorescence-based identification of motor neurons, as well as magnetic-activated cell sorting (MACS) to isolate differentiated motor neurons at scale. The targeting strategies developed here offer a standardized platform for reproducible comparison of motor neurons across independently derived pluripotent cell lines.

Identifiants

DOI: 10.1016/j.neuroscience.2020.06.028 PMID: 32615233 PMC: PMC7688562
pubmed: 32615233
pii: S0306-4522(20)30404-8
doi: 10.1016/j.neuroscience.2020.06.028
pmc: PMC7688562
mid: NIHMS1608219
pii:
doi:

Substances chimiques

Cholinergic Agents 0
Homeodomain Proteins 0
MNX1 protein, human 0
Transcription Factors 0

Types de publication

Journal Article Research Support, N.I.H., Extramural Research Support, Non-U.S. Gov't

Langues

eng

Sous-ensembles de citation

IM

Pagination

48-56

Subventions

Organisme : NINDS NIH HHS
ID : R21 NS109661
Pays : United States

Informations de copyright

Copyright © 2020 IBRO. Published by Elsevier Ltd. All rights reserved.

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Auteurs

Alejandro Garcia-Diaz (A)

Departments of Pathology and Cell Biology, Neuroscience, Rehabilitation and Regenerative Medicine (in Neurology), Columbia University Irving Medical Center, New York, NY 10032, USA; The Stem Cell Core Facility, Columbia University Irving Medical Center, New York, NY 10032, USA; Center for Motor Neuron Biology and Disease, Columbia University Irving Medical Center, New York, NY 10032, USA; Columbia Stem Cell Initiative, Columbia University Irving Medical Center, New York, NY 10032, USA; Department of Biological Sciences, Columbia University, New York, NY 10027, USA.

Gizem Efe (G)

Departments of Pathology and Cell Biology, Neuroscience, Rehabilitation and Regenerative Medicine (in Neurology), Columbia University Irving Medical Center, New York, NY 10032, USA; The Stem Cell Core Facility, Columbia University Irving Medical Center, New York, NY 10032, USA.

Khushbu Kabra (K)

The Stem Cell Core Facility, Columbia University Irving Medical Center, New York, NY 10032, USA.

Achchhe Patel (A)

The Stem Cell Core Facility, Columbia University Irving Medical Center, New York, NY 10032, USA; Columbia Stem Cell Initiative, Columbia University Irving Medical Center, New York, NY 10032, USA.

Emily R Lowry (ER)

Departments of Pathology and Cell Biology, Neuroscience, Rehabilitation and Regenerative Medicine (in Neurology), Columbia University Irving Medical Center, New York, NY 10032, USA; Center for Motor Neuron Biology and Disease, Columbia University Irving Medical Center, New York, NY 10032, USA; Columbia Stem Cell Initiative, Columbia University Irving Medical Center, New York, NY 10032, USA.

Neil A Shneider (NA)

Center for Motor Neuron Biology and Disease, Columbia University Irving Medical Center, New York, NY 10032, USA; Department of Neurology, Eleanor and Lou Gehrig ALS Center, Columbia University Irving Medical Center, New York, NY 10032, USA.

Barbara Corneo (B)

The Stem Cell Core Facility, Columbia University Irving Medical Center, New York, NY 10032, USA. Electronic address: bc2599@cumc.columbia.edu.

Hynek Wichterle (H)

Departments of Pathology and Cell Biology, Neuroscience, Rehabilitation and Regenerative Medicine (in Neurology), Columbia University Irving Medical Center, New York, NY 10032, USA; Center for Motor Neuron Biology and Disease, Columbia University Irving Medical Center, New York, NY 10032, USA; Columbia Stem Cell Initiative, Columbia University Irving Medical Center, New York, NY 10032, USA. Electronic address: hw350@columbia.edu.

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Classifications MeSH

questionsmedicales.fr › Phénomènes physiologiques cellulaires › Différenciation cellulaire › Standardized Reporter Systems for Purification...
questionsmedicales.fr › Actions chimiques et utilisations › Actions pharmacologiques › Effets physiologiques des médicaments › Agents neuromédiateurs › Agents cholinergiques › Standardized Reporter Systems for Purification...
questionsmedicales.fr › Acides aminés, peptides et protéines › Protéines › Protéines de liaison à l'ADN › Protéines à homéodomaine › Standardized Reporter Systems for Purification...
questionsmedicales.fr › Eucaryotes › Animaux › Chordés › Vertébrés › Mammifères › Eutheria › Primates › Haplorhini › Catarrhini › Hominidae › Humains › Standardized Reporter Systems for Purification...
questionsmedicales.fr › Cellules › Cellules souches › Cellules souches pluripotentes › Cellules souches pluripotentes induites › Standardized Reporter Systems for Purification...
questionsmedicales.fr › Cellules › Neurones › Neurones efférents › Motoneurones › Standardized Reporter Systems for Purification...
questionsmedicales.fr › Cellules › Cellules souches › Cellules souches pluripotentes › Standardized Reporter Systems for Purification...
questionsmedicales.fr › Acides aminés, peptides et protéines › Protéines › Facteurs de transcription › Standardized Reporter Systems for Purification...