In vitro methods of differentiating stem cells into neurons and neurons generated using the same
Inventors
Butts, Jessica • McDevitt, Todd C.
Assignees
Georgia Tech Research Corp • J David Gladstone Institutes • University of California San Diego UCSD
Publication Number
US-11702630-B2
Publication Date
2023-07-18
Expiration Date
2037-05-26
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Abstract
Methods of generating spinal cord glutamatergic interneurons (V2a interneurons) from human pluripotent stem cells (hPSCs) are provided. A method of the present disclosure may include culturing a first population of hPSCs in vitro in a neural induction medium that includes: a retinoic acid signaling pathway activator; a sonic hedgehog (Shh) signaling pathway activator; and a Notch signaling pathway inhibitor, wherein the culturing results in generation of a second population of cultured cells containing CHX10+ V2a interneurons. Also provided are non-human animal models that include the hPSC-derived spinal cord glutamatergic interneurons, and methods of producing the non-human animal models.
Core Innovation
The invention provides methods of generating spinal cord glutamatergic interneurons (V2a interneurons) from human pluripotent stem cells (hPSCs) by culturing the hPSCs in vitro in a neural induction medium containing a retinoic acid signaling pathway activator, a sonic hedgehog (Shh) signaling pathway activator, and a Notch signaling pathway inhibitor. This culturing induces differentiation of the hPSCs into a population of CHX10+ V2a interneurons. The V2a interneurons may further be matured in vitro or in vivo to acquire properties of mature V2a interneurons with functional neuronal characteristics such as electrophysiological activity and expression of neuron-related genes.
The problem being addressed is the lack of a robust source of human V2a interneurons limiting molecular profiling and therapeutic evaluation following injury to the central nervous system. The spinal cord contains multiple neuron types important in motor control, among which excitatory V2a interneurons contribute to locomotion and respiratory function. Existing challenges include generating a sufficient and pure population of human V2a interneurons to study and potentially utilize for regenerative therapies.
The disclosed invention overcomes these limitations by providing an efficient protocol involving specific modulation of developmental signaling pathways—retinoic acid, Shh, and Notch—to differentiate hPSCs into V2a interneurons marked by elevated CHX10 expression. The methods enable the production of large populations of CHX10+ V2a interneurons, which can be matured further to exhibit neuronal functionality, and also supports the generation of non-human animal models by transplantation of these cells.
Claims Coverage
The patent includes two independent claims covering methods of generating spinal cord glutamatergic interneurons from hPSCs using specific culture media compositions and sequences.
Sequential culturing in neural induction media with retinoic acid, Shh activator, and Notch inhibitor to generate CHX10+ V2a interneurons
A method comprising culturing hPSCs first in a neural induction medium containing a retinoic acid signaling pathway activator, then culturing said cells in a neural induction medium containing the retinoic acid signaling pathway activator, a sonic hedgehog (Shh) signaling pathway activator, and a Notch signaling pathway inhibitor so as to generate CHX10+ V2a interneurons.
Stepwise culturing in three neural induction media progressively including retinoic acid activator, Shh activator, and Notch inhibitor for V2a interneuron generation
A method comprising culturing hPSCs consecutively in: (a) a first neural induction medium comprising a retinoic acid signaling pathway activator, (b) a second neural induction medium comprising the retinoic acid activator and a Shh signaling pathway activator, and (c) a third neural induction medium comprising the retinoic acid activator, Shh signaling pathway activator, and a Notch signaling pathway inhibitor to generate CHX10+ V2a interneurons.
Specified concentrations of signaling pathway modulators in neural induction media enhance differentiation
Using retinoic acid signaling pathway activators at about 20 nM to 500 nM, Shh signaling pathway activators at about 50 nM to 500 nM, and Notch signaling pathway inhibitors at about 250 nM to 10 μM in the neural induction media during culturing to produce the cells.
Culturing parameters and substrates for hPSC differentiation into V2a interneurons
Seeding hPSCs on substrates coated with extracellular matrix components at densities from about 5,000 to 120,000 cells/cm2 and culturing in adherent monolayer conditions with appropriate media compositions.
Reseeding and maturation of differentiated cells
After initial differentiation, reseeding at least some of the CHX10+ cell population onto neural maturation substrates and culturing in a neural maturation medium to obtain mature CHX10+ V2a interneurons exhibiting electrical excitability.
The independent claims cover methods for generating CHX10+ V2a interneurons from hPSCs by sequentially culturing cells in neural induction media containing retinoic acid pathway activators, Shh pathway activators, and Notch pathway inhibitors at defined concentrations, optionally including steps of reseeding and maturation to obtain functional neurons. These methods define the inventive features necessary for producing spinal cord glutamatergic interneurons in vitro.
Stated Advantages
Provides an efficient and robust method to generate human V2a interneurons from hPSCs with high purity (up to about 50% CHX10+ cells).
Enables generation of mature V2a interneurons exhibiting neuronal electrophysiological activity and marker expression both in vitro and after transplantation in vivo.
Allows production of non-human animal models for studying human V2a interneuron development and therapeutic potential.
Permits freezing and thawing of differentiated cells without significant loss of CHX10+ cell percentage, supporting scalability and reproducibility.
Use of specific pathway modulators and dosing schedules provides controlled differentiation and neuronal subtype specification including regional identity.
Documented Applications
Use of hPSC-derived V2a interneurons for regenerative cell therapies to treat central nervous system injuries.
Production of non-human animal models by transplanting hPSC-derived V2a interneurons into spinal cords for studying human V2a interneuron growth, maturation, and integration.
Use in molecular profiling and functional characterization of human V2a interneurons.
Studying transplantation methods to establish functional synaptic relay circuits within damaged spinal cords.
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