Methods of differentiating neural cells and related compositions and methods of use
The present disclosure provides methods of lineage specific differentiation of pluripotent stem cells, including induced pluripotent stem cells, into floor plate midbrain progenitor cells, determined dopamine (DA) neuron progenitor cells, and/or DA neurons. Also provided are compositions uses thereof, such as for treating neurodegenerative diseases and conditions, including Parkinson's disease.
1 . A method of differentiating neural cells, the method comprising:
(a) performing a first incubation comprising culturing pluripotent stem cells in a non-adherent culture vessel under conditions to produce a cellular spheroid, wherein beginning at the initiation of the first incubation the cells are exposed to (i) an inhibitor of TGF-β/activin-Nodal signaling; (ii) at least one activator of Sonic Hedgehog (SHH) signaling; (iii) an inhibitor of bone morphogenetic protein (BMP) signaling; and (iv) an inhibitor of glycogen synthase kinase 3β (GSK3β) signaling, wherein day 0 is the first day on which the pluripotent stem cells are exposed to the inhibitor of TGF-β/activin-Nodal signaling and the inhibitor of bone morphogenetic protein (BMP) signaling; and
(b) performing a second incubation comprising culturing cells of the spheroid in a substrate-coated culture vessel under conditions to neurally differentiate the cells.
2 . The method of claim 1 , wherein the second incubation begins on about day 7.
3 . The method of claim 1 , wherein the cells are exposed:
(i) to the inhibitor of TGF-β/activin-Nodal signaling up to a day at or before day 7;
(ii) to the inhibitor of TGF-β/activin-Nodal beginning at day 0 and through day 6, inclusive of each day;
(iii) to the at least one activator of SHH signaling up to a day at or before day 7;
(iv) to the at least one activator of SHH signaling beginning at day 0 and through day 6, inclusive of each day;
(v) to the inhibitor of BMP signaling up to a day at or before day 11;
(vi) to the inhibitor of BMP signaling beginning at day 0 and through day 10, inclusive of each day;
(vii) to the inhibitor of GSK3B signaling up to a day at or before day 13;
(viii) to the inhibitor of GSK3B signaling beginning at day 0 and through day 12, inclusive of each day;
(ix) to transforming growth factor beta-3 (TGFβ3), ascorbic acid, GDNF, and dbcAMP (collectively, “BAGCT”) and an inhibitor of Notch signaling beginning on day 11; and/or
(x) to BAGCT and an inhibitor of Notch signaling beginning at day 11 and until harvest of the neurally differentiated cells, optionally until day 18, optionally until day 25.
4 . The method of claim 1 , wherein culturing the cells under conditions to neurally differentiate the cells comprises exposing the cells to (i) brain-derived neurotrophic factor (BDNF); (ii) ascorbic acid; (iii) glial cell-derived neurotrophic factor (GDNF); (iv) dibutyryl cyclic AMP (dbcAMP); (v) transforming growth factor beta-3 (TGFβ3) (collectively, “BAGCT”); and (vi) an inhibitor of Notch signaling.
5 . The method of claim 1 , wherein the method comprises:
(a) beginning at the initiation of the first incubation day, the cells are exposed to (i) an inhibitor of TGF-β/activin-Nodal signaling through day 6, each day inclusive; (ii) at least one activator of Sonic Hedgehog (SHH) signaling through day 6, each day inclusive; (iii) an inhibitor of bone morphogenetic protein (BMP) signaling through day 6, each day inclusive; and (iv) an inhibitor of glycogen synthase kinase 3β (GSK3β) signaling through day 6, each day inclusive; and
(b) the performing a second incubation begins on day 7, and comprises culturing the cells in a culture vessel coated with a substrate selected from the group consisting of: laminin, collagen, entactin, heparin sulfate proteoglycans, and combinations thereof, wherein beginning on day 7, the cells are exposed to (i) an inhibitor of BMP signaling and (ii) an inhibitor of GSK3β signaling; and beginning on day 11, the cells are exposed to (i) brain-derived neurotrophic factor (BDNF); (ii) ascorbic acid; (iii) glial cell-derived neurotrophic factor (GDNF); (iv) dibutyryl cyclic AMP (dbcAMP); (v) transforming growth factor beta-3 (TGFβ3) (collectively, “BAGCT”); and (vi) an inhibitor of Notch signaling.
6 . The method of claim 1 , further comprising harvesting the neurally differentiated cells, optionally wherein the harvesting is carried out at about day 16 or later, wherein the harvesting is carried out between day 18 and day 25, and/or wherein the harvesting is carried out at or about day 18 or about at day 25.
7 . The method of claim 1 , wherein the neurally differentiated cells are determined dopaminergic neuron progenitor cells, optionally wherein the dopaminergic neuron progenitor cells are capable of innervating host tissue upon transplantation into a subject.
8 . The method of claim 1 , wherein, prior to performing the second incubation, the spheroid is dissociated to produce a cell suspension, and cells of the cell suspension are cultured in the substrate-coated culture vessel, optionally wherein the dissociating is carried out at a time when the spheroid cells express at least one of PAX6 and OTX2, and/or wherein the dissociating is carried out on about day 7, and/or wherein the spheroid is dissociated by enzymatic dissociation.
9 . The method of claim 1 , wherein the inhibitor of TGF-β/activin-Nodal signaling is SB431542; wherein the at least one activator of SHH signaling is SHH protein, purmorphamine, C25II SHH protein, or a combination thereof; and/or wherein the inhibitor of BMP signaling is LDN193189, and/or wherein the inhibitor of GSK3β signaling is CHIR99021; and/or wherein the inhibitor of Notch signaling is DAPT.
10 . The method claim 1 , wherein the culturing in the first incubation and/or the second incubation is carried out in media comprising serum or a serum replacement, or wherein the cells are cultured in the absence of serum for the duration of culture.
11 . The method of claim 1 , wherein the cells are exposed to an inhibitor of Rho-associated protein kinase (ROCK) signaling on day 0, day 8, day 16, and/or day 20.
12 . The method of claim 1 , wherein the first incubation is performed in media that comprises the inhibitor of TGF-β/activin-Nodal signaling, the activator of SHH signaling, the inhibitor of BMP signaling, and the inhibitor of GSK3β signaling, and at least about 50% of the media is replaced daily or every other day, or every third day.
13 . The method of claim 6 , further comprising formulating the harvested cells with a cryoprotectant, optionally further comprising cryopreserving the harvested cells, and optionally thawing the cells prior to use.
14 . The method of claim 1 , wherein the pluripotent stem cells are embryonic stem (ES) cells, induced pluripotent stem cells (iPSCs), or a combination thereof.
15 . The method of claim 1 , wherein the pluripotent stem cells are human induced pluripotent stem cells, and/or wherein the pluripotent stem cells are autologous to a subject to be treated with the neurally differentiated cells or allogeneic to a subject to be treated with the neurally differentiated cells.
16 . The method of claim 15 , wherein the pluripotent stem cells are hypoimmunogenic, optionally wherein the pluripotent stem cells are engineered to (a) remove genes encoding one or more of polymorphic HLA-A/-B/-C and HLA class II molecules; and (b) to provide genes encoding one or more of PD-L1, HLA-G, and CD47, optionally into a AAVS1 safe harbor locus.
17 . A method of differentiating neural cells, the method comprising:
(a) performing a first incubation comprising culturing pluripotent stem cells in a culture vessel that is coated with a substrate selected from one or more of laminin, collagen, entactin, heparin sulfate proteoglycans, and combinations thereof, wherein beginning at the initiation of the first incubation the cells are exposed to (i) an inhibitor of TGF-β/activin-Nodal signaling; (ii) at least one activator of Sonic Hedgehog (SHH) signaling; (iii) an inhibitor of bone morphogenetic protein (BMP) signaling; and (iv) an inhibitor of glycogen synthase kinase 3β (GSK3β) signaling, wherein day 0 is the first day on which the pluripotent stem cells are exposed to the inhibitor of TGF-β/activin-Nodal signaling and the inhibitor of bone morphogenetic protein (BMP) signaling, wherein the method does not comprise exposing the cells to fibroblast growth factor 8 (FGF8); and
(b) performing a second incubation comprising culturing cells obtained in step (a) in a substrate-coated culture vessel under conditions to neurally differentiate the cells.
18 . The method of claim 17 , wherein the substrate comprises laminin.
19 . The method of claim 17 , wherein the inhibitor of TGF-β/activin-Nodal signaling is SB431542.
20 . The method of claim 17 , wherein the at least one activator of SHH signaling is SHH protein, purmorphamine, C25II SHH protein, or a combination thereof.
21 . The method of claim 17 , wherein the inhibitor of BMP signaling is LDN193189.
22 . The method of claim 17 , wherein the inhibitor of GSK3B signaling is CHIR99021.
23 . The method of claim 1 , wherein the method does not comprise exposing the cells to fibroblast growth factor 8 (FGF8).
24 . A method of differentiating neural cells, the method comprising:
(a) performing a first incubation comprising culturing pluripotent stem cells in a culture vessel that is coated with a substrate selected from one or more of laminin, collagen, entactin, heparin sulfate proteoglycans, and combinations thereof, wherein beginning at the initiation of the first incubation the cells are exposed to (i) an inhibitor of TGF-β/activin-Nodal signaling; (ii) at least one activator of Sonic Hedgehog (SHH) signaling; (iii) an inhibitor of bone morphogenetic protein (BMP) signaling comprising LDN193189; and (iv) an inhibitor of glycogen synthase kinase 3β (GSK3β) signaling, wherein day 0 is the first day on which the pluripotent stem cells are exposed to the inhibitor of TGF-β/activin-Nodal signaling and the inhibitor of bone morphogenetic protein (BMP) signaling; and
(b) performing a second incubation comprising culturing cells obtained in step (a) in a substrate-coated culture vessel under conditions to neurally differentiate the cells.
25 . The method of claim 24 , wherein the substrate comprises laminin.
26 . The method of claim 24 , wherein the inhibitor of TGF-β/activin-Nodal signaling is SB431542.
27 . The method of claim 24 , wherein the at least one activator of SHH signaling is SHH protein, purmorphamine, C25II SHH protein, or a combination thereof.
28 . The method of claim 24 , wherein the inhibitor of GSK3β signaling is CHIR 99021.
29 . The method of claim 24 , wherein the method does not comprise exposing the cells to fibroblast growth factor 8 (FGF8).