Three-dimensional human neural tissues for CRISPR-mediated perturbation of disease genes
AssigneesThe Broad Institute, Inc., Massachusetts Institutes of Technology
InventorsHalil Tekin, Feng Zhang
Life of this patent
Estimating the term…
Abstract
Described herein are tissue cultures and methods for culturing stem-cell derived neurons in three-dimensional culture. Also provided are methods for screening therapeutic agents, developing cell line models, identifying genes associated with a neurological disease, and identifying genes associated with neuronal cell phenotypes.
Claims34
A tractable neural tissue culture, comprising a three-dimensional (3D) matrix, the 3D matrix comprising neuronal and/or glial cells cultured from a stem cell, a biological supporting material comprising a proliferation inhibitor, and one or more crosslinker,
- wherein the biological supporting material is a composite hydrogel comprising an basement membrane matrix and alginate;
- wherein the concentration of the basement membrane matrix is about 1 mg/mL to about 10 mg/mL;
- wherein the concentration of alginate is about 1 mg/mL to about 10 mg/mL;
- wherein the concentration of the one or more crosslinker is about 1 mM to about 30 mM;
- and
- wherein the basement membrane matrix consists of laminin, collagen IV, growth factors, heparin sulfate proteoglycans, entactin, and nidogen.
A tractable neural tissue culture, comprising a 3D matrix, the 3D matrix comprising neuronal and/or glial cells cultured from a stem cell, a biological supporting material comprising a proliferation inhibitor, and one or more calcium crosslinkers,
- wherein the biological supporting material is a composite hydrogel comprising an basement membrane matrix and alginate;
- wherein the concentration of the basement membrane matrix is about 1 mg/mL to about 10;
- wherein the concentration of alginate is about 1 mg/mL to about 10 mg/mL;
- wherein the concentration of the one or more crosslinker is about 1 mM to about 30 mM;
- wherein the 3D matrix comprises hyaluronic acid;
- wherein the basement membrane matrix consists of laminin, collagen IV, growth factors, heparin sulfate proteoglycans, entactin, and nidogen, and
- wherein one or more genes in the neuronal and/or glial cells have increased or decreased expression relative to cells in a two-dimensional or another 3D matrix.
A method of generating a tractable neural tissue culture, comprising:
- (a) culturing a stem cell on a two-dimensional plate,
- (b) inducing differentiation of the stem cell into neuronal and/or glial cells, and
- (c) detaching and encapsulating the neuronal and/or glial cells in a three dimensional (3D) matrix comprising decreasing the distance of a gene expression profile of the neuronal tissue culture as compared to a target tissue by modulating the density or stiffness of the three dimensional (3D) matrix,
- wherein the 3D matrix comprises neuronal and/or glial cells cultured from a stem cell, a biological supporting material comprising a proliferation inhibitor, and one or more crosslinker;
- wherein the biological supporting material is a composite hydrogel comprising an basement membrane matrix and alginate;
- wherein the concentration of the basement membrane matrix is about 1 mg/mL to about 10;
- wherein the concentration of alginate is about 1 mg/mL to about 10 mg/mL;
- wherein the concentration of the one or more crosslinker is about 1 mM to about 30 mM;
- and
- wherein the basement membrane matrix consists of laminin, collagen IV, growth factors, heparin sulfate proteoglycans, entactin, and nidogen.
A method for identifying a candidate agent for treating a neurological disease, comprising:
- (a) providing a tractable neural tissue culture comprising a three-dimensional (3D) matrix, the 3D matrix comprising a biological supporting material comprising a proliferation inhibitor, and one or more crosslinker,
- (b) contacting the tractable neural tissue culture with a test compound, and
- (c) detecting the expression and/or activity of one or more genes associated with the neurological disease, wherein an increase or decrease of the one or more genes associated with the neurological disease indicates that the agent is effective for treating the neurological disease;
- wherein the biological supporting material is a composite hydrogel comprising an basement membrane matrix and alginate;
- wherein the concentration of the basement membrane matrix is about 1 mg/mL to about 10;
- wherein the concentration of alginate is about 1 mg/mL to about 10 mg/mL;
- wherein the concentration of the one or more crosslinker is about 1 mM to about 30 mM;
- and
- wherein the basement membrane matrix consists of laminin, collagen IV, growth factors, heparin sulfate proteoglycans, entactin, and nidogen.
Dependencies are inferred from the claim text.