|Title of Invention||
DOPAMINERGIC NEURONS AND PROLIFERATION-COMPETENT PRECURSOR CELLS FOR TREATING PARKINSON''S DISEASE
|Abstract||This disclosure provides improved methods for obtaining populations of neural progenitor cells and differentiated neurons from pluripotent stem cells. The technology can be used to produce progenitors that proliferate through at least 40 doublings, while maintaining the ability to differentiate into a variety of different neural phenotypes. Cell populations have been obtained that contain a high proportion of cells staining for tyrosine hydroxylase, which is a feature of dopaminergic neurons. The neural progenitors and terminally differentiated neurons of this invention can be generated in large quantities for use in drug screening and the treatment of clinically important neurological disorders, such as Parkinson’s disease.|
|Full Text||FIELD OF THE INVENTION:
The present invention provides a method of producing neuronal precursor cells. More spccitkalh, the present invention provides a system for efficient production of primate cells that have differentiated from pluripotent cells into cells of the neural lineage. The precursor and tenninals-differentiated cells of this invention can be used in a number of important applications, including dmg testing and the production of medicaments to restore nervous system function.
STATEMENT OF INVENTION:
The present invention provides a method of producing neuronal precursor cells, said method comprising the steps of:
e. culturing pluripotent stem cells or their progeny or neural cell progenitor in a
suitable substrate and nutrient medium;
f. adding suitable differentiation agents and/or mintage’s or growth factors to the
medium of step (a);
g. identifying differentiation agents and combination thereof effective for
generating desired neural phenotypes;
harvesting the desired neural cells.
/'Tofu neurons upon terminal differentiation, consistent with the higher proportion of neural precursors before differentiation.
Figure 4(A) shows the proportion of cells staining positively for tyrosine hydroxylase. Again, the combination of BONF, MT-3, EGF and t)FGF provided optimal yield amongst the combinations tested.
Figure 4(B) shows that even more TIH-positive neurons can be generated by inducing terminal differentiation not by BDNF and NT-3 alone, but also including additional factors such as NT-4, nerve growth factor, ascorbic add, cavil and dopamine (at the concentrations shown in Table 3). Up to 5% of the total ceil number in the population displayed the phenotype of dopaminergic markers.
1. A differentiated cell population cultured in vitro, wherein at least -30% of
MAP-2 positive cells have the characteristic that they are progeny of a line of
primate pluripotent stem (pPS) cells, and have one or more of the following
they express tyrosine hydroxylase; they release dopamine upon activation.
2. A differentiated cell population cultured in vitro, wherein at least -5% of all
the cells in the population have the characteristic that they are progeny of a
line of primate pluripotent stem (pPS) cells, and have one or more of the
they express tyrosine hydroxylase;
they release dopamine upon activation;
they provide clinical improvement in a nigrostriatal lesion model of
3. A neuronal precursor cell population cultured in vitro, in which at least -60% of the cells express A2B5, polysialylated NCAM, or Nestin, and which upon culturing for 7 days with added neurotrophin 3 (NT-3), brain-derived neurotrophic factor (BDNF), neurotrophin 4 (NT-4) and nerve growth factor (NGF), but no added mitogens, generates a population of differentiated cells according to claim 1 or claim 2.
4. The neuronal precursor cell population according to claim 3, which is capable of at least 20 population doublings in culture, and which after 20 doublings maintains an ability to form differentiated cell populations according to claim 1 or 2 upon culturing with NT-3, BDNF, NT-4 and NGP, but no added mitogens.
5. A system for producing neural cells, comprising the cell population according to any of claims 1-4, and the undifferentiated pPS cell line from which they were obtained.
6. The cell population of any of claims 1-4, which belongs to a set of cell
populations according to claim 5.
7. A method of screening a compound for its effect on neural cells or a neural
cell activity, comprising:
a) combining the compound with the differentiated cell population of claims
b) determining any change to phenotype or activity of cells in the population
that results from being combined with the compound; and
c) correlating the change with an effect of the compound on neural cells or a
neural cell activity.
8. The screening method of claim 7, comprising one or more of the following:
• determining whether the compound is toxic to the cells;
• determining whether the compound affects ability of the cells to be
maintained in culture;
• determining whether the compound changes neurotransmitter
synthesis, release, or uptake by the cells, or
• determining whether the compound changes electro physiology of the
9. A differentiated cell population, a neural precursor cell population, a system
for producing neural cells and a method of screening substantially such as
herein described with reference to the accompanying drawings and as
illustrated in forgoing examples.
|Indian Patent Application Number||5529/CHENP/2007|
|PG Journal Number||16/2011|
|Date of Filing||03-Dec-2007|
|Name of Patentee||GERON CORPORATION|
|Applicant Address||230 CONSTITUTION DRIVE, MENLO PARK, CA 94025, USA|
|PCT International Classification Number||C12N5/00|
|PCT International Application Number||N/A|
|PCT International Filing date|