Large-Scale Production of Mature Neurons from Human Pluripotent Stem Cells in a Three-Dimensional Suspension Culture System

Alessandra Rigamonti; Giuliana G Repetti; Chicheng Sun; Feodor D Price; Danielle C Reny; Francesca Rapino; Karen Weisinger; Chen Benkler; Quinn P Peterson; Lance S Davidow; Emil M Hansson; Lee L Rubin

doi:10.1016/j.stemcr.2016.05.010

Large-Scale Production of Mature Neurons from Human Pluripotent Stem Cells in a Three-Dimensional Suspension Culture System

Stem Cell Reports. 2016 Jun 14;6(6):993-1008. doi: 10.1016/j.stemcr.2016.05.010.

Affiliations

¹ Department of Stem Cell and Regenerative Biology, Harvard University, 7 Divinity Avenue, Cambridge, MA 02138, USA; Harvard Stem Cell Institute, Harvard University, 7 Divinity Avenue, Cambridge, MA 02138, USA.
² Department of Stem Cell and Regenerative Biology, Harvard University, 7 Divinity Avenue, Cambridge, MA 02138, USA.
³ Department of Stem Cell and Regenerative Biology, Harvard University, 7 Divinity Avenue, Cambridge, MA 02138, USA; Department of Medicine, KI-AZ Integrated Cardio Metabolic Centre, Karolinska Institutet, NOVUM, Hälsovägen 7, 141 57 Huddinge, Sweden.
⁴ Department of Stem Cell and Regenerative Biology, Harvard University, 7 Divinity Avenue, Cambridge, MA 02138, USA; Harvard Stem Cell Institute, Harvard University, 7 Divinity Avenue, Cambridge, MA 02138, USA. Electronic address: [email protected].

Abstract

Human pluripotent stem cells (hPSCs) offer a renewable source of cells that can be expanded indefinitely and differentiated into virtually any type of cell in the human body, including neurons. This opens up unprecedented possibilities to study neuronal cell and developmental biology and cellular pathology of the nervous system, provides a platform for the screening of chemical libraries that affect these processes, and offers a potential source of transplantable cells for regenerative approaches to neurological disease. However, defining protocols that permit a large number and high yield of neurons has proved difficult. We present differentiation protocols for the generation of distinct subtypes of neurons in a highly reproducible manner, with minimal experiment-to-experiment variation. These neurons form synapses with neighboring cells, exhibit spontaneous electrical activity, and respond appropriately to depolarization. hPSC-derived neurons exhibit a high degree of maturation and survive in culture for up to 4-5 months, even without astrocyte feeder layers.

MeSH terms

Biomarkers / metabolism
Brain-Derived Neurotrophic Factor / pharmacology
Cell Culture Techniques*
Cell Differentiation / drug effects
Ciliary Neurotrophic Factor / pharmacology
Glial Cell Line-Derived Neurotrophic Factor / pharmacology
Humans
Microtubule-Associated Proteins / genetics
Microtubule-Associated Proteins / metabolism
Nerve Net / cytology*
Nerve Net / physiology
Neurogenesis / drug effects*
Neurogenesis / genetics
Neurons / classification
Neurons / cytology
Neurons / drug effects*
Neurons / metabolism
Observer Variation
Pluripotent Stem Cells / cytology
Pluripotent Stem Cells / drug effects*
Pluripotent Stem Cells / metabolism
Repressor Proteins / genetics
Repressor Proteins / metabolism
Reproducibility of Results
Smad Proteins / antagonists & inhibitors
Smad Proteins / genetics
Smad Proteins / metabolism
Spheroids, Cellular / cytology
Spheroids, Cellular / drug effects
Spheroids, Cellular / metabolism
T-Box Domain Proteins / genetics
T-Box Domain Proteins / metabolism
Tumor Suppressor Proteins / genetics
Tumor Suppressor Proteins / metabolism

Substances

BCL11B protein, human
Biomarkers
Brain-Derived Neurotrophic Factor
Ciliary Neurotrophic Factor
Glial Cell Line-Derived Neurotrophic Factor
MAP2 protein, human
Microtubule-Associated Proteins
Repressor Proteins
Smad Proteins
T-Box Domain Proteins
TBR1 protein, human
Tumor Suppressor Proteins
BDNF protein, human

Grants and funding

P01 NS066888/NS/NINDS NIH HHS/United States