Isolation and growth of stem cells from hemangiomas

Abstract

The present invention describes stem cells and progenitor cells derived from hemangiomas, including testing of angiogenic inhibitors using these cells. The invention as described is useful in providing a process to culture and propagate hemangioma stem cells and generate xenograft models to develop treatments for infantile hemangiomas and other types of vascular lesions.

1 . A composition comprising an enriched population of stem cells, wherein the stem cells form tumor spheres in vitro. 
   
   
       2 . The composition of  claim 1 , wherein the stem cells are derived from a human, heterologous vascular tumor. 
   
   
       3 . The composition of  claim 2 , wherein the vascular tumor is a hemangioma. 
   
   
       4 . The composition of  claim 1 , wherein the stem cells express CD133 or SALL4, or a combination thereof. 
   
   
       5 . The composition of  claim 4 , wherein the stem cells express SALL4. 
   
   
       6 . The composition of  claim 1 , wherein the stem cells grow in vitro in the absence of serum or serum replacement. 
   
   
       7 . The composition of  claim 1 , wherein the stem cells are capable of differentiating into endothelial-like tissue when contacted with VEGF. 
   
   
       8 . A method of screening agents that modulate cell proliferation comprising:
 a) contacting a progenitor cell or stem cell from hemangioma tissue with a test agent;   b) comparing a characteristic property associated with the cells in the presence of the test agent with characteristic property of the cells in the absence of the test agent; and   c) determining the affect of the test agent on the property,   wherein a test agent modulates cell proliferation when a change in a property associated with the cell is affected.   
   
   
       9 . The method of  claim 8 , wherein the property is selected from the group consisting of growth characteristics; tumor sphere formation; karyotype; immunohistochemical profile; gene expression profile; self-renewal capacity; and/or differentiation capacity. 
   
   
       10 . The method of  claim 9 , wherein the growth characteristics are determined by contacting the progenitor or stem cell contained within a xenograft transplanted in a non-human animal. 
   
   
       11 . The method of  claim 8 , wherein the immunohistochemical profile comprises a tissue specific marker selected from the group consisting of GLUT-1, merosin, Fc-gamma-RII, Lewis Y antigens, β4-integrin, CD133, and SALL4, or a combination thereof. 
   
   
       12 . The method of  claim 8 , wherein the determining step comprises generating global maps for SALL4 binding to promoter regions in the presence and absence of the test agent. 
   
   
       13 . The method of  claim 8 , further comprising determining the effect of the test agent in the presence and absence of an angiogenesis inhibitor. 
   
   
       14 . The method of  claim 8 , wherein the test agent is an angiogenesis inhibitor. 
   
   
       15 . The method of  claim 13  or  14 , wherein the angiogenesis inhibitor is selected from the group consisting of VEGFR-1, NRP-1; angiopoietin 2, TSP-1, TSP-2, angiostatin, endostatin, vasostatin, calreticulin, platelet factor 4, IMP, CDAI, Meth-1, Meth-2, INF-α, INF-β, INF-γ, CXCL10, IL-4, IL-12, IL-18, prothrombin (kringle domain-2) antithrombin III fragment, prolactin, VEGI, SPARC, osteopontin, maspin, canstatin, proliferin-related protein, restin, bevacizumab, carboxyamidotriazol, TNP-470, CM101, suramin, thrombospondin, angiostatic steroids/heparin, cartilage-derived angiogenesis inhibitory factor, matrix metalloproteinase inhibitors, 2-methoxyestradiol, Tecogalan, α v β 3  inhibitors, and linomide. 
   
   
       16 . A non-human animal containing a xenograft transplant comprising an hemangioma derived progenitor cell, stem cell, or differentiated cell derived from human infantile hemangioma tissue, wherein the stem cell or progenitor cell self renews in culture, and forms a tumor sphere from a single cell in suspension. 
   
   
       17 . The non-human animal of  claim 16 , wherein the differentiated cell is an endothelial-like cell. 
   
   
       18 . The non-human animal of  claim 16 , wherein the non-human animal is murine. 
   
   
       19 . A tumor sphere derived from an immortalized hemangioma cell comprising a progenitor cell or stem cell from human infantile hemangioma tissue. 
   
   
       20 . The tumor sphere of  claim 19 , wherein the tumor sphere comprises cells which express CD133 or SALL4, or a combination thereof. 
   
   
       21 . The tumor sphere of  claim 20 , wherein the cells express SALL4. 
   
   
       22 . The tumor sphere of  claim 20 , wherein the tumor sphere grows in vitro in the absence of serum or serum replacement. 
   
   
       23 . The tumor sphere of  claim 20 , wherein cells isolated from the tumor sphere are capable of forming endothelial-like cells when contacted with VEGF. 
   
   
       24 . An immortalized hemangioma cell line comprising a progenitor cell or stem cell from human infantile hemangioma tissue, wherein the stem cell or progenitor cell self renews in culture, and forms a tumor sphere from a single cell in suspension in vitro. 
   
   
       25 . The immortalized hemangioma cell line of  claim 24 , wherein the cell line is isolated from a human, heterologous vascular tumor. 
   
   
       26 . The immortalized hemangioma cell line of  claim 25 , wherein the tumor sphere comprises cells which express CD133 or SALL4, or a combination thereof. 
   
   
       27 . The immortalized hemangioma cell line of  claim 26 , wherein the cells express SALL4. 
   
   
       28 . The immortalized hemangioma cell line of  claim 27 , wherein the cells grow in vitro in the absence of serum or serum replacement. 
   
   
       29 . The immortalized hemangioma cell line of  claim 24 , wherein the stem cell is capable of differentiating into endothelial-like tissue when contacted with VEGF. 
   
   
       30 . A method of producing stem cells comprising:
 a) isolating cells from hemangioma tissue;   b) culturing the cells of step (a) in serum free media until tumor spheres are formed;   c) isolating cells from the tumor spheres; and   d) culturing the isolated cells of step (c) in serum free media.   
   
   
       31 . The method of  claim 30 , wherein the stem cell expresses SALL4. 
   
   
       32 . The method of  claim 30 , wherein the stem cells are capable of differentiating into endothelial-like tissue when contacted with VEGF. 
   
   
       33 . A method of treating hemangiomas comprising administering an agent screened in  claim 8 . 
   
   
       34 . The method of  claim 33 , further comprising co-administration of one or more of the angiogenesis inhibitors selected from the group consisting of VEGFR-1, NRP-1; angiopoietin 2, TSP-1, TSP-2, angiostatin, endostatin, vasostatin, calreticulin, platelet factor 4, IMP, CDAI, Meth-1, Meth-2, INF-α, INF-β, INF-γ, CXCL10, IL-4, IL-12, IL-18, prothrombin (kringle domain-2) antithrombin III fragment, prolactin, VEGI, SPARC, osteopontin, maspin, canstatin, proliferin-related protein, restin, bevacizumab, carboxyamidotriazol, TNP-470, CM101, suramin, thrombospondin, angiostatic steroids/heparin, cartilage-derived angiogenesis inhibitory factor, matrix metalloproteinase inhibitors, 2-methoxyestradiol, Tecogalan, α v β 3  inhibitors, and linomide. 
   
   
       35 . The method of  claim 33 , wherein the hemangioma is infantile hemangioma. 
   
   
       36 . A method of screening combinations of agents that modulate cell proliferation comprising:
 a) contacting hemangioma derived stem cells with one or more first agents that induce differentiation of the stem cells;   b) allowing the stem cells to differentiate;   c) contacting the differentiated cells with a second agent; and   d) determining the affect of the second agent on the differentiated cells,   wherein combining the first and second agents modulates proliferation of the differentiated cell.   
   
   
       37 . The method of  claim 36 , wherein the hemangioma derived stem cells express CD133 or SALL4, or a combination thereof 
   
   
       38 . The method of  claim 36 , wherein the first agent is selected from the group consisting of retinoic acid, BMP, VEGF, GATA factors, EGF, FGF-2, PDGF, HGF, laminin, nicotinamide, GM-CSF, IL-3, collagen IV, dexamethasone, TGF-β, fibronectin, erythropoietin, thrombopoietin, G-CSF, TNF-α, and insulin. 
   
   
       39 . The method of  claim 38 , wherein the first agent is VEGF. 
   
   
       40 . The method of  claim 36 , wherein differentiation is determined by detecting the presence or absence of one or more cell-type differentiation markers. 
   
   
       41 . The method of  claim 36 , wherein the second agent is selected from the group consisting of small molecules, nucleic acids, proteins, polypeptides, peptides, and peptidomimetics. 
   
   
       42 . The method of  claim 41 , wherein the second agent modulates the expression of SALL4. 
   
   
       43 . The method of  claim 42 , wherein the second agent is selected from the group consisting of 5′ azacytidine, 5′ aza-2-deoxycytidine, 1-B-D-arabinofuranosyl-5-azacytosine, dihydroxy-5-azacytidine, MG132, PSI, lactacystin, expoxomicin, and bortezomib. 
   
   
       44 . The method of  claim 42 , wherein the second agent is an RNAi which decreases the expression of SALL4. 
   
   
       45 . The method of  claim 36 , wherein the second agent is selected from the group consisting of VEGFR-1, NRP-1; angiopoietin 2, TSP-1, TSP-2, angiostatin, endostatin, vasostatin, calreticulin, platelet factor 4, IMP, CDAI, Meth-1, Meth-2, INF-α, INF-β, INF-γ, CXCL10, IL-4, IL-12, IL-18, prothrombin (kringle domain-2) antithrombin III fragment, prolactin, VEGI, SPARC, osteopontin, maspin, canstatin, proliferin-related protein, restin, bevacizumab, carboxyamidotriazol, TNP-470, CM101, suramin, thrombospondin, angiostatic steroids/heparin, cartilage-derived angiogenesis inhibitory factor, matrix metalloproteinase inhibitors, 2-methoxyestradiol, Tecogalan, α v β 3  inhibitors, and linomide. 
   
   
       46 . The method of  claim 36 , wherein combining the first and second agents inhibits proliferation of the differentiated cell. 
   
   
       47 . A method of treating hemangiomas comprising administering a combination of agents screened in  claim 36 .