TISSUE MODELING IN EMBRYONIC STEM (ES) CELL SYSTEM
Provided are embryonic stem (ES) cell-derived tissue modeling systems. In particular, systems for the de novo generation of tissue by parallel drug selection of cell types constituting the tissue of interest in one culture of differentiating ES cells is described as well as the use of such systems in transplantation and drug development.
1 - 81 . (canceled)
82 . A method of modeling or obtaining neuronal tissue, comprising:
(a) obtaining neuronal cells, wherein a first population of pluripotent cells is differentiated into neuronal cells;
(b) obtaining astrocytes, wherein a second population of pluripotent cells is differentiated into astrocytes;
(c) co-culturing the neuronal cells obtained in step (a) with astrocytes obtained in step (b); and
(d) allowing integration and alignment of the neuronal cells and astrocytes into viable neuronal tissue;
wherein the neuronal cells upon integration and alignment with the astrocytes acquire morphology of neuronal tissue.
83 . The method of claim 82 , wherein the first population of pluripotent cells comprises a selectable marker gene operably linked to a neuronal cell specific regulatory sequence.
84 . The method of claim 82 , wherein said selectable marker confers resistance to puromycin.
85 . The method of claim 82 , wherein the first population of pluripotent cells comprises a reporter gene operably linked to a neuronal cell-specific regulatory sequence.
86 . The method of claim 85 , wherein the first population of pluripotent cells further comprises a selectable marker gene operably linked to a neuronal cell-specific regulatory sequence, and wherein said neuronal cell-specific regulatory sequence operably linked to the reporter gene is the same as said neuronal cell-specific regulatory sequence operably linked to the selective marker gene.
87 . The method of claim 86 , wherein said reporter gene is selected from different color versions of enhanced green fluorescent protein (EGFP).
88 . The method of claim 86 , wherein said marker gene and said reporter gene are contained in the same recombinant nucleic acid molecule.
89 . The method of claim 88 , wherein said marker gene and said reporter gene are contained in the same cistron.
90 . The method of claim 89 , wherein said regulatory sequence is a promoter sequence and wherein the promoter sequence is nestin promoter sequence.
91 . The method of claim 82 , further comprising analyzing the physiological or developmental status or both of the neuronal cells and astrocytes.
92 . The method of claim 91 , wherein said status is analyzed by recording the extracellular field potentials with a microelectrode array.
93 . The method of claim 82 , wherein said first and/or second population of pluripotent cells is genetically engineered to overexpress or inhibit the expression of a target gene.
94 . The method of claim 82 , wherein the neuronal tissue is produced and contained in a container.
95 . The method of claim 94 , comprising taking three or more measurements, optionally at different positions within the container.
96 . The method of claim 94 , wherein said container is a well in a microtiter plate.
97 . The method of claim 96 , wherein said microtiter plate is a 24-, 96-, 384- or 1586-well plate.
98 . The method of claim 82 , further comprising culturing the neuronal cells and astrocytes in the presence of oligodendrocytes.
99 . A neuronal tissue obtained by the method of claim 98 .
100 . The method of claim 99 , further comprising analyzing the physiological or development status or both of the neuronal cells, astrocytes, and oligodendrocytes.
101 . The method of claim 99 , wherein the status is analyzed by monitoring the differentiation of electrical activity of the neuronal cells, astrocytes, and oligodendrocytes on an array.