Methods and devices for high throughput screening of conditions affecting stem cell differentiation

Abstract

Disclosed are methods and systems for testing the effects of various morphogens and/or feeder cells on the differentiation of pluripotent cells. The assays described herein can be used for determining the optimum conditions that lead to differentiation of stem cells. Once the optimum conditions for stem cell differentiation are determined, such cells may be used it a variety of therapies.

1 - 48 . (canceled) 
     
     
         49 . A method for identifying a pluripotency profile of stem cells comprising:
 culturing one or more stem cells in a cell culturing system, wherein the cell culturing system comprises a cell culturing plate comprising a layer of cell culturing substrate, a cell culture site comprising the one or more stem cells, and one or more morphogen sites comprising one or more morphogens, wherein the one or more morphogen sites are each positioned peripheral to the cell culture site, wherein each of the one or more morphogen sites physically overlap one another;   incubating the cell culturing system under conditions suitable to allow the one or more stem cells to differentiate into one or more differentiated cells and migrate on the cell culturing plate in response to the one or more morphogens; and   determining the migration pattern of the one or more differentiated cells on the cell culturing plate, wherein the migration pattern of the one or more differentiated cells is indicative of the pluripotency profile of the population of stem cells.   
     
     
         50 . The method of  claim 49 , further comprising isolating the one or more differentiated cells that have migrated towards the one or more morphogen sites. 
     
     
         51 . The method of  claim 49 , wherein the migration of a desired differentiated cell on the cell culturing plate towards the one or more morphogen sites is indicative of a target morphogen condition to induce differentiation. 
     
     
         52 . The method of  claim 49 , wherein the migration of the one or more differentiated cells towards a morphogen site is indicative of a stem cell morphogen present in the morphogen site. 
     
     
         53 . The method of  claim 49 , wherein the one or more morphogen sites comprise different concentrations of the same morphogen. 
     
     
         54 . The method of  claim 49 , wherein each of the one or more morphogen sites comprise a different morphogen. 
     
     
         55 . The method of  claim 49 , wherein the cell culture site is surrounded by at least one physical barrier. 
     
     
         56 . The method of  claim 49 , wherein each of the one or more morphogen sites are an equal distance away from the cell culture site. 
     
     
         57 . The method of  claim 49 , wherein each of the one or more morphogen sites comprise a chemokine or growth factor. 
     
     
         58 . The method of  claim 57 , wherein the chemokine or growth factor comprises a molecule selected from the group consisting of: decapentaplegic (Dpp), transforming growth factor beta (TGF-β), hedgehog, sonic hedgehog, wingless/wnt, epidermal growth factor, fibroblastic growth factor, insulin-like growth factor, retinoic acid, and mixtures thereof. 
     
     
         59 . The method of  claim 49 , wherein the cell culturing plate is physically divided into one or more compartments. 
     
     
         60 . A cell culturing system comprising a cell culturing plate, wherein the cell culturing plate comprises a layer of cell culturing substrate; a cell culture site comprising one or more stem cells; and two or more morphogen sites, wherein each of the two or more morphogen sites is positioned peripheral to the cell culture site, and wherein each of the morphogen sites is separated by a physical barrier. 
     
     
         61 . The cell culturing system of  claim 60 , wherein the two or more morphogen sites comprise different concentrations of the same morphogen. 
     
     
         62 . The cell culturing system of  claim 60 , wherein each of the two or more morphogen sites comprise a different morphogen. 
     
     
         63 . The cell culturing system of  claim 60 , wherein the two or more morphogen sites comprise one or more feeder cells. 
     
     
         64 . The cell culturing system of  claim 63 , wherein the two or more morphogen sites comprise a heterogeneous population of feeder cells or a homogenous population of feeder cells. 
     
     
         65 . The cell culturing system of  claim 60 , wherein each of the two or more morphogen sites are an equal distance away from the cell culture site. 
     
     
         66 . The cell culturing system of  claim 60 , wherein each of the two or more morphogen sites comprise a chemokine or growth factor. 
     
     
         67 . The cell culturing system of  claim 66 , wherein the chemokine or growth factor comprises a molecule selected from the group consisting of: decapentaplegic (Dpp), transforming growth factor beta (TGF-β), hedgehog, sonic hedgehog, wingless/wnt, epidermal growth factor, fibroblastic growth factor, insulin-like growth factor, retinoic acid, and mixtures thereof. 
     
     
         68 . The cell culturing system of  claim 60 , wherein the cell culturing substrate comprises an ingredient selected from the group consisting of gelatin, agarose and agar. 
     
     
         69 . The cell culturing system of  claim 68 , wherein the cell culturing substrate is solid or semi-solid. 
     
     
         70 . The cell culturing system of  claim 60 , wherein the cell culturing plate is at least one of visually or physically divided into one or more compartments. 
     
     
         71 . A method for identifying a stem cell morphogen comprising:
 culturing one or more stem cells in a cell culturing system, wherein the cell culturing system comprises a cell culturing plate comprising a layer of cell culturing substrate, a cell culture site comprising the one or more stem cells, and two or more morphogen sites comprising one or more test agents, wherein each of the two or more morphogen sites is positioned peripheral to the cell culture site, and wherein each of the morphogen sites is separated by a physical barrier;   incubating the cell culturing system under conditions suitable to allow the one or more stem cells to differentiate into one or more differentiated cells and migrate on the cell culturing plate in response to the one or more test agents; and   determining the migration pattern of the one or more differentiated cells on the cell culturing plate, wherein the migration pattern of the one or more differentiated cells is indicative of test agent's efficacy as a morphogen.