Efficient generation of neural progenitors, neurons, and dopaminergic neurons from human embryonic stem cells
Inventors
Cho, Myung Soo • Kim, Myung-Hwa • Moon, Young-II • Moon, Shin Yong • Oh, Sun Kyung • Kim, Hee Sun • Kim, Dong-Wook
Assignees
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Publication Number
US-8492149-B2
Publication Date
2013-07-23
Expiration Date
Abstract
The present invention relates to a method for inducing the differentiation of neural progenitors, neurons, and dopaminergic neurons from human embryonic stem cells with high efficiency, in which neural selection can be performed by the selected media and physical methods. The invention has advantages such as higher efficiency, the effect of lowering cost and time, and maintenance of neural progenitors for a longer period of time, as compared to the known methods for inducing the differentiation into neural progenitors, neurons, and dopaminergic neurons. Accordingly, the method can stably generate cells used for treating Parkinson's disease or other nervous system diseases.
Core Innovation
The invention provides a method of producing spherical neural masses from human embryonic stem cells by progressing through embryoid body formation, selective removal of cystic embryoid bodies, and attachment to a culture dish coated with Matrigel, laminin, or L-polyornithine. Embryoid bodies are cultured in media containing 0.5×N-2 supplement to inhibit growth of cells other than neural progenitors, and then further cultured in media containing N-2 and bFGF.
Neural structures with neural rosette or neural tube morphology are dissected and isolated from the resulting culture. The isolated neural structures are then cultured in suspension in media containing N-2 and bFGF to obtain spherical neural masses characterized by expanded neural structures and spherical morphology, and the spherical neural masses express Nestin, Sox1 and Pax6.
During this stage, spot-forming fibroblast-like cells and cystic structures are generated, and the method removes the spot-forming fibroblast-like cells and cystic structures and isolates only the expanded neural structures in the spherical neural masses. To increase purity and expand the spherical neural masses, the method repeats the suspension culture steps and the removal/enrichment steps at least four times.
Claims Coverage
The document provides one independent method claim supported by multiple dependent claims that refine media composition constraints, isolate dissection tooling, and set repetition bounds. The independent claim contains four inventive features.
Cystic embryoid body removal and neural progenitor enrichment
Culturing human embryonic stem cells to obtain embryoid bodies and removing cystic embryoid bodies having hollow ball morphology from the embryoid bodies.
Matrix attachment with N-2 supplement to inhibit non-neural growth
Attaching the resultant embryoid bodies to the surface of a culture dish coated with Matrigel, laminin, or L-polyornithine, and culturing them in media containing 0.5×N-2 supplement to inhibit the growth of cells other than neural progenitors.
Neural rosette/tube isolation followed by suspension culture to form spherical neural masses expressing Nestin, Sox1 and Pax6
Dissecting and isolating neural structures having neural rosette or neural tube morphology from the resultant culture, and culturing the neural structures in suspension in media containing N-2 and bFGF to obtain spherical neural masses with expanded neural structures and spherical morphology, wherein the spherical neural masses express Nestin, Sox1 and Pax6.
Removal of fibroblast-like cells and cystic structures with iterative repetition to increase purity
Removing spot-forming fibroblast-like cells and cystic structures generated during suspension culture and isolating only the expanded neural structures in the spherical neural masses, and repeating the suspension culture step and the removal step at least four times to increase a purity of the spherical neural masses and expand the spherical neural masses.
Overall, the claims are directed to producing spherical neural masses from hESC by removing cystic embryoid bodies, performing matrix-coated neural progenitor induction in N-2-based media, isolating neural rosette/tube structures, forming Nestin/Sox1/Pax6-expressing spherical neural masses by suspension culture, and repeatedly removing spot-forming fibroblast-like cells and cystic structures to increase purity and expand the spherical neural masses.
Stated Advantages
Increase a purity of the spherical neural masses.
Expand the spherical neural masses.
Documented Applications
Not explicitly described in patent.
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