Screening assays for identifying differentiation-inducing agents and production of differentiated cells for cell therapy

Abstract

The invention relates to assays for screening growth factors, adhesion molecules, immunostimulatory molecules, extracellular matrix components and other materials, alone or in combination, simultaneously or temporally, for the ability to induce directed differentiation of pluripotent and multipotent stem cells.

1 . A method for evaluating the differentiation of totipotent, nearly totipotent, or pluripotent stem cells, or cells therefrom, in response to one or more chemical or biological agents or physical conditions, comprising:
 (a) separating individual totipotent, nearly totipotent, or pluripotent stem cells, or cells therefrom, or groups of such cells, in culture medium into one or a plurality of separate wells which may be open or closed, which wells may be in the same or different apparatus;   (b) exposing said separate wells of cells to one or more putative differentiation-inducing conditions simultaneously or sequentially; and   (c) screening said individual cells or groups of cells to detect markers of differentiation of said individual cells or groups of cells,   wherein the markers are indicative of differentiation to myocardial cells.   
     
     
         2 . (canceled) 
     
     
         3 . The method of  claim 1 , wherein said nearly totipotent, or pluripotent stem cells, or cells therefrom, are selected from the group consisting of human cells, primate cells, bovine cells, porcine cells, murine cells, rat cells, sheep cells, canine and feline cells. 
     
     
         4 . The method of  claim 1 , wherein said one or more putative differentiation-inducing conditions are selected from the group consisting of growth factors, cytokines, tissue extracts, nucleic acids, factors involved in cell-to-cell interactions, adhesion molecules and extracellular matrix components, extracts of extracellular components from tissue, media components, environmental conditions, and living cells that induce differentiation by cell-cell interactions. 
     
     
         5 . The method of  claim 4 , wherein said growth factors and cytokines are selected from the group consisting of the Fibroblast Growth Factor family of proteins (FGF1-23), the Tumor necrosis factor (TNF) superfamily (TNFSF), the insulin-like growth factor family IGF-1 and their binding proteins, the matrix metalloproteinases PDGF, Flt-3 ligand, Fas Ligand, B7-1(CD80), B7-2(CD86), DR6, IL-13 R alpha, IL-15 R alpha, GRO beta/CXCL2 (aa 39-107), IL 1-18, II-8/CXCL8, GDNF, G-CSF, GM-CSF, M-GSF, PDGF-BB, PDGF-AA, PDGF-AB, IL-2 sR alpha, IL-2 sR beta, Soluble TNF RII, IL-6 sR, Soluble gp130, PD-ECGF, IL-4 sR, beta-ECGF, TGF-alpha, TGF-beta sRII, TGF-beta 5, LAP (TGF-beta 1), BDNF, LIF sR alpha, LIF, KGF/FGF-7, Pleiotrophin, ENA-78/CXCL5, SCF, beta-NGF, CNTF, Midkine, HB-EGF, SLPI, Betacellulin, Amphiregulin, PIGF, Angiogenin, IP-10/CXCL10, NT-3, NT-4, MIP-1 alpha/CCL3, MIP-1 beta/CCL4, I-309/CCL1, GRO alpha/CXCL1, GRO beta/CXCL2, GRO gamma/CXCL3, Rantes/CCL5, MCP-1/CCL2, MCP-2/CCL8, MCP-3/CCL7, IFN-gamma, Erythropoietin, Thrombopoietin, MIF, IGF-I, IGF-II, VEGF, HGF, Oncostatin M, HRG-alpha (EGF Domain), TGF-beta 2, CNTF R alpha, Tie-2/Fc Chimera, BMP-4, BMPR-IA, Eotaxin/CCL11, VEGF R1 (Fit-1), PDGF sR alpha, HCC-1/CCL14, CTLA-4, MCP-4/CCL13, GCP-2/CXCL6, TECK/CCL25, MDC/CCL22, Activin A, Eotaxin-2/MPIF-2/CCL24, Eotaxin-3/CCL-26 (aa 24-94), TRAIL R1 (DR4), VEGF R3 (Fit-4)/SDF-1 alpha(PBSF)/CXCL12, MSP, BMP-2, HVEM/VEGF R2 (KDR), Ephrin-A3, MIP-3 alpha/CCL20, MIP-3 beta/CCL19, Fractalkine/CX3CL1 (Chemokine Domain), TARC/CCL17, 6Ckine/CCL21, p75 Neurotrophin R (NGF R), SMDF, Neurturin, Leptin R/Fc Chimera, MIG/CXCL9, NAP-2/CXCL7, PARC/CCL18, Cardiotrophin-1 (CT-1), GFR alpha-2, BMP-5, IL-8/CXCL8 (Endothelial Cell Derived), Tie-1, Viral CMV UL146, VEGF-D, Angiopoietin-2, Inhibin A, TRANCE/RANK L, CD6/Fc Chimera, CF, dMIP-1 delta/LKN-1/CCL15(68 aa), TRAIL R3/Fc Chimera, Soluble TNF RI, Activin RIA, EphA1, E-Cadherin, ENA-70, ENA-74, Eotaxin-3/CCL26, ALCAM, FGFR1 alpha (IIIc), Activin B, FGFT1 beta (IIIc), LIGHT, FGFR2 beta (IIIb), DNAM-1, Follistatin, GFR alpha-3, gp 130, I-TAC/CXCL11, IFN-gamma RI, IGFBP-2, IGFBP-3, Inhibin B, Prolactin CF, RANK, FGFR2 beta (IIIc), FGFR4, TrkB, GITR, MSP R, GITR Ligand, Lymphotactin/XCL1, FGFR2 alpha (IIIc), Activin AB, ICAM-3 (CD50), ICAM-1 (CD54), TNF RII, L-Selectin (CD62L, BLC/BCA-1/CXCL13, HCC-4/CCL16, ICAM-2 (CD102), IGFBP-4, Osteoprotegerin (OPG), uPAR, Activin RIB, VCAM-1 (CD106), CF, BMPR-II, IL-18 R, IL-12 R beta 1, Dtk, LBP, SDF-1 alpha (PBSF)/CXCL12 (synthetic), E-Selectin (CD62E), L-Selectin (CD62L), P-Selectin (CD62P), ICAM-1 (CD54), VCAM-1 (CD106), CD31 (PECAM-1), hedgehog family of proteins, Interleukin-10, Epidermal Growth Factor, Heregulin, HER4, Heparin Binding Epidermal Growth Factor, bFGF, MIP-18, MIP-2, MCP-1, MCP-5, NGF, NGF-B, leptin, Interferon A, Interferon A/D, Interferon B, Interferon Inducible Protein-10, Insulin Like Growth Factor-II, IGBFBP/IGF-1 Complex, C10, Cytokine Induced Neutrophil Chemoattractant 2, Cytokine Induced Neutrophil Chemoattractant 2B, Cytokine Induced Neutrophil Chemoattractant 1, Cytokine Responsive Gene-2, and any fragment thereof and their neutralizing antibodies. 
     
     
         6 . The method of  claim 4 , wherein said factors involved in cell-cell interactions are selected from the group consisting of the ADAM (A Disintegrin and Metalloproteinase) family of proteins including ADAM 1,2,3A, 3B, 4-31 and TS1-9, ADAMTSs (ADAMs with thrombospondin motifs), Reprolysins, metzincins, zincins, and zinc metalloproteinases and their neutralizing antibodies. 
     
     
         7 . The method of  claim 4 , wherein said adhesion molecules are selected from the group consisting of Ig superfamily CAM's, Integrins, Cadherins and Selectins and their neutralizing antibodies. 
     
     
         8 . The method of  claim 4 , wherein said nucleic acids that may be tested are those that encode or block by antisense, ribozyme activity, or RNA interference with transcription factors that are involved in regulating gene expression during differentiation, genes for growth factors, cytokines, and extracellular matrix components, or other molecular activities that regulate differentiation. 
     
     
         9 . The method of  claim 4  wherein said cell-cell interactions include placing the cells being assayed in cell-cell contact with cells of another differentiated cell type, or in the presence of media conditioned by cells of another differentiated cell type. 
     
     
         10 . The method of  claim 4  wherein said tissue extracts comprise materials derived from early stage embryos, fetuses, or adult tissues. 
     
     
         11 - 86 . (canceled) 
     
     
         87 . The method of  claim 4 , comprising exposing the stem cells to endothelial cells.