Methods for controlled induction of bioengineered neuroepithelial tissues and 3-d neuroepithelial tubes
AssigneeWisconsin Alumni Research Foundation
InventorsRandolph Scott Ashton, Gavin Thomas Knight, Benjamin John Knudsen, Nisha Ramdas Iyer, Carlos Marti-Figueroa
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Abstract
Described herein are methods, compositions, and kits for directed differentiation of human pluripotent stem cells, neuromesodermal progenitors, and neural stem cells into bioengineered elliptical neuroepithelial tissues and bioengineered neuroepithelial tubes that contain a single rosette of polarized neuroepithelial cells and have microscale cellular organization similar to that of an in vivo developing human neural tube.
Claims20
A method of producing a biomimetic elliptical neuroepithelial tissue having a singular rosette structure in vitro, the method comprising:
- (a) seeding human pluripotent stem cells (hPSCs) in the presence of a Rho kinase inhibitor onto a micropatterned substrate that is capable of biomimetic neural morphogenesis of cells cultured thereon, wherein the micropatterned substrate comprises at least two circular bounded regions connected by a cell-adhesive bridge;
- (b) culturing the seeded cells of step (a) on the micropatterned substrate for a first culture period of about one to two days in the presence of a pluripotency maintenance medium to obtain a first cell aggregate, wherein the pluripotency maintenance medium comprises a Rho kinase inhibitor;
- and
- (c) culturing the cells obtained in step (b) for a second culture period of about 3 to about 6 days under adherent culture conditions in a neural differentiation base medium,
- whereby a biomimetic elliptical neuroepithelial tissue having a singular rosette structure is obtained, wherein the tissue comprises polarized neuroepithelial cells and has a microscale cellular organization similar to that of a transverse section of an in vivo developing human neural tube.
A composition, comprising:
- one or more micropatterned substrates that is capable of instructing biomimetic neural morphogenesis of cells cultured thereon,
- wherein the one or more micropatterned substrates comprises at least two circular, cell-adhesive microscale regions connected by a cell-adhesive bridge.
A method of producing a bioengineered neuroepithelial tube in vitro, the method comprising:
- (a) seeding human pluripotent stem cells (hPSCs) in the presence of a Rho kinase inhibitor onto a micropatterned substrate that is capable of biomimetic neural morphogenesis of cells cultured thereon, wherein the micropatterned substrate comprises at least two circular bounded regions and connected by a cell-adhesive bridge;
- (b) culturing the seeded cells of step (a) on the micropatterned substrate for a first culture period of about one to two days in the presence of a pluripotency maintenance medium to obtain a first cell aggregate, wherein the pluripotency maintenance medium comprises a Rho kinase inhibitor;
- (c) culturing the cells of step (b) for a second culture period of about 3 to about 6 days under adherent culture conditions in a neural differentiation base medium, whereby a bioengineered neuroepithelial tissue is obtained;
- (d) overlaying the bioengineered neuroepithelial tissue obtained in step (c) with a hydrogel layer;
- (e) culturing the bioengineered neuroepithelial tissue of step (d) for about 24 hours, whereby the tissue morphs into a bioengineered neuroepithelial tube and becomes encased in the hydrogel layer;
- and
- (f) removing the hydrogel comprising the encased neuroepithelial tube from the micropatterned substrate to obtain a bioengineered neuroepithelial tube.
Dependencies are inferred from the claim text.