Three dimensional heterogeneously differentiated tissue culture

Abstract

The present invention provides an artificial tissue culture comprising a heterogeneous population of cells of at least two different tissue sections, wherein said tissue sections are in a three dimensional structure, method of generating such a tissue and kits suitable for said method or maintain a three dimensional tissue culture.

1 . An in vitro grown artificial Three-dimensional neuronal tissue culture comprising a heterogeneous population of cells of at least two different progenitor and neuronal differentiation layers, wherein at least on progenitor layer comprises outer radial glia cell. 
     
     
         2 . Tissue culture according to  claim 1 , wherein said tissue sections form at least two layers, preferably at least one layer being shaped around a globular tissue body. 
     
     
         3 . Tissue culture according to  claim 1  or  2 , wherein said tissue develops apical and dorsal tissue sections. 
     
     
         4 . Tissue culture according to any one of  claims 1  to  3 , wherein said tissue is cerebral tissue. 
     
     
         5 . Tissue culture according to any one of  claims 1  to  4 , wherein cells of said culture express one or more gene expression markers selected from forebrain markers BF1 and Six3, hindbrain markers Krox20 and Ils1, preferably wherein forebrain markers are expressed in increased amounts as compared to hindbrain markers. 
     
     
         5 . Tissue culture according to any one of  claims 1  to  5 , wherein cells of said culture express one or more gene expression markers selected from Otx1, Otx2, FoxG1, Auts2, Tuj1, Brn2, Satb2, Ctip2, calretinin. 
     
     
         7 . A method of generating an artificial tissue culture comprising providing a multicellular aggregation of pluripotent stem cells culturing said multicellular aggregation in a three dimensional matrix, preferably a gel, thereby expanding said cells in a multicellular aggregation, wherein said cells are allowed to differentiate, and culturing said expanded and optionally differentiated multicellular aggregation of cells in a suspension culture. 
     
     
         8 . The method of  claim 7 , wherein said pluripotent cell an induced pluripotent cell, especially an induced pluripotent cell that has been isolated from a patient. 
     
     
         9 . The method of  claim 7  or  8 , wherein said expanded cells differentiate into unipotent stem cells. 
     
     
         10 . The method of any one of  claims 7  to  8 , wherein the three dimensional matrix comprises collagen or an extracellular matrix from the Engelbreth-Holm-Swarm tumor, or any component thereof selected from laminin, collagen, entactin, and heparan-sulfated proteoglycan or any combination thereof. 
     
     
         11 . The method of investigating a developmental neurological tissue effect comprising decreasing or increasing the expression in a gene of interest in a cell at any stage during the method of any one of  claims 7  to  10 . 
     
     
         12 . The method of screening a candidate therapeutic agent suitable for treating a developmental neurological tissue detect of interest, comprising performing the method of  claim 11  and administering the candidate agent to said cells at any stage during the method, preferably at all stages. 
     
     
         13 . The method of testing a candidate drug for neurological effects, comprising administering a candidate drug to an artificial culture according to any one of  claims 1  to  6  and determining an activity of interest of the cells of said culture and comparing said activity to an activity of cells to the culture without administering said candidate drug, wherein a differential activity indicates a neurological effect. 
     
     
         14 . The method of obtaining a differentiated neural cell comprising the step of providing an artificial culture according to any one of  claims 1  to  6  and isolating a differentiated neural cell of interest, or comprising the step of generating an artificial tissue culture according to any one of  claims 7  to  10  further comprising the step of isolating a differentiated neural cell of interest. 
     
     
         15 . A kit for generating an artificial three-dimensional neuronal tissue culture according to any one of  claims 1  to  6  or for performing the method of any one of  claims 7  to  14  comprising a medium comprising a three dimensional matrix and nutrients and a medium comprising retinoic acid and nutrients, optionally further comprising a medium comprising nutrients and a ROCK inhibitor and/or optionally comprising a medium comprising nutrients but lacking growth factors that would differentiate neural tissue to a particular fate.