I/We Claim:
1. A cell culture protocol for obtaining NPCs (neural progenitor cells) from
iPSCs (induced plenipotentiary stem cells) comprising the steps of:
(j) generating embryo id bodies from a confluent culture of iPSCs, wherein the
5 embryoid bodies are in non-adherent suspension culture,
(k) plating the embryoid bodies on a culture dish coated with a suitable
extracellular matrix in neural induction media (NIM) to obtain
neuroectoderm lineage, wherein the NIM comprises at least one WNT
pathway inhibitor and at least two SMAD pathway inhibitors,
10 (l) culturing the neuroectoderm lineage for 6-8 days in neural proliferation
media (NPM) for primary rosette formation, wherein the NPM does not
comprise any inhibitor,
(m) selecting and plating the primary rosettes as suspension culture on ultra-low
attachment culture dish,
15 (n) culturing the primary rosettes of step (d) in suspension culture for 4-5 days
to facilitate primary neurosphere formation,
(o) plating the primary neurospheres of step (e) on a culture dish coated with
suitable extracellular matrix to obtain adherent primary neurospheres,
(p) culturing the adherent primary neurospheres in the NPM for 5-6 days for
20 secondary rosette formation,
(q) selecting and plating the secondary rosettes as suspension culture on ultralow attachment dish,
(r) culturing the secondary rosettes of step (h) in suspension culture for 4-5 days
to facilitate secondary neurosphere formation,
25 (s) plating the secondary neurospheres on a culture dish coated with suitable
extracellular matrix to obtain adherent secondary neurospheres,
(t) culturing the adherent secondary neurospheres in the NPM for 5-7 days for
tertiary rosette formation, and
(u) enzymatically dissociating and plating the tertiary rosettes to obtain the
30 NPCs.
25
2. The cell culture protocol as claimed in claim 1, wherein the confluent culture of
iPSCs have a con fluency in the range of 80% - 90%.
3. The method as claimed in claim 1, wherein generating the embryoid bodies in step
5 (a) comprises:
(a) culturing the confluent iPSCs for 2-4 days in a growth media to form embryoid
bodies, wherein the growth media comprises growth factors, organic molecules, and
lipid concentrates,
(b) gradually subjecting the developing embryoid bodies from the growth media to
10 the NIM by (i) culturing the developing embryoid bodies in a media composition
comprising mTESR and NIM in a ratio of 70:30 for 24 hours, (ii) culturing the
developing embryoid bodies obtained in step (i) in a media composition comprising
mTESR and NIM in a ratio of 50:50 for 24 hours, and (iii) culturing the embryoid
bodies obtained in step (ii) in the NIM for 24 hours followed by plating the embryoid
15 bodies on a tissue culture dish.
4. The method as claimed in claim 1, wherein the primary rosette formation in step
(c) comprises:
(i) maintaining the neuroectoderm lineage in the NIM for 2-4 days;
20 (ii) maintaining the neuroectoderm lineage obtained in step (i) in a media
composition comprising NIM and NPM in a ratio of 50:50 for 24-48 hours for
primary rosette formation.
5. The method as claimed in claim 1, wherein selecting the primary rosettes in step
25 (d) and the secondary rosettes in step (h) is carried out using neural rosette selection
medium.
6. The cell culture protocol as claimed in claim 1, wherein the at least one suitable
extracellular matrix is selected from the group consisting of matrigel, laminin,
30 vitronectin, fibronectin, collagen, poly-L-lysine, poly-L-ornithine, and combinations
thereof.
26
7. The cell culture protocol as claimed in claim 1, wherein the at least one WNT
pathway inhibitor is selected from a group consisting of 4-(1,3,3a,4,7,7a-Hexahydro1,3-dioxo-4,7-methano-2H-isoindol-2-yl)-N-8-quinolinyl- Benzamide, 5-
5 (Phenylsulfonyl)-N-piperidin-4-yl-2-(trifluoromethyl) benzene sulfonamide, 2-
(2′,3-Dimethyl-[2,4′-bipyridin]-5-yl)-N-(5-(pyrazin-2-yl)pyridin-2-yl) acetamide, 2-
(4-(2-methylpyridin-4-yl)phenyl)-N-(4-(pyridin-3-yl)phenyl) acetamide, 8-
Tetrahydro-2-[4-(trifluoromethyl)phenyl]-4H- thiopyrano[4,3-d]pyrimidin-4-one,
and combinations thereof.
10
8. The cell culture protocol as claimed in claim 1, wherein the at least two SMAD
pathway inhibitors are selected from a group consisting of 4-[4-(1,3-benzodioxol-5-
yl)-5-pyridin-2-yl-1H-imidazol-2-yl]benzamide, 4-(6-(4-(piperazin-1-
yl)phenyl)pyrazolo[1,5-a]pyrimidin-3-yl)quinoline, 3-[(1R)-1-(2,6-dichloro-3-
15 fluorophenyl)ethoxy]-5-(1-piperidin-4-ylpyrazol-4-yl)pyridin-2-amine, 5-chloro-2-
N-[2-methoxy-4-[4-(4-methylpiperazin-1-yl)piperidin-1-yl]phenyl]-4-N-(2-propan2-ylsulfonylphenyl)pyrimidine-2,4-diamine, and 9-ethyl-6,6-dimethyl-8-(4-
morpholin-4-ylpiperidin-1-yl)-11-oxo-5H-benzo[b]carbazole-3-carbonitrile, 5-
chloro-2-N-(5-methyl-4-piperidin-4-yl-2-propan-2-yloxyphenyl)-4-N-(2-propan-2-
20 ylsulfonylphenyl) pyrimidine-2,4-diamine, and combinations thereof.
9. The method as claimed in claim 1, wherein the enzymatic dissociation of tertiary
rosette in step (l) is carried out using an enzyme selected from the group consisting
of Accutase, Tryple select, TrypLE, Gentle Cell Dissociation Reagent (GCDR),
25 Dispase.
10. The neural progenitor cells produced by the method as claimed in claim 1.
11. A pharmaceutical composition comprising the neural progenitor cells as claimed
30 in claim 10 and pharmaceutical acceptable excitements.
27
12. A nasal formulation comprising the NPCs as claimed in claim 10 or the
composition as claimed in claim 11, for use in treatment of neurodegenerative
disorders.
5 13. The nasal formulation as claimed in claim 12, wherein the neurodegenerative
disorder is selected from the group consisting of Parkinson’s disease, Alzheimer’s
disease, and prion disease.
14. A method of treating a neurodegenerative disorder in a subject comprising
10 administering the nasal formulation as claimed in claim 12 to the subject