Pluripotent embryonic-like stem cells derived from corneal limbus, methods of isolation and uses thereof

Abstract

The present disclosure describes mammalian pluripotent embryonic-like stem cells (ELSCs) isolated from corneal limbal tissue, a non-embryonic tissue. The ELSCs of the present disclosure are capable of proliferating in an in vitro culture, maintain the potential to differentiate into cells of endoderm, mesoderm, and ectoderm lineage in culture, and are capable of forming embryoid-like bodies when placed in suspension culture. Thus, these cells possess multi-lineage differentiation potential and self-renewing capability. ELSCs may be a promising therapeutic tool, and may provide new therapeutic alternatives for various diseases, conditions, and injuries.

1 . Isolated mammalian pluripotent embryonic-like stem cells (ELSCs) which (i) are capable of proliferating in an in vitro culture, (ii) maintain the potential to differentiate into cells of endoderm, mesoderm or ectoderm lineage in culture, and (iii) are capable of forming embryoid-like bodies when placed in suspension culture.  
     
     
         2 . The isolated stem cells of  claim 1 , wherein the mammalian ELSCs are human ELSCs.  
     
     
         3 . The isolated stem cells of  claim 1 , wherein the ELSCs are derived from corneoscleral limbus tissue.  
     
     
         4 . The isolated stem cells of  claim 3 , wherein the corneoscleral limbus tissue is human tissue.  
     
     
         5 . The isolated stem cells of  claim 1 , wherein the ELSCs remain substantially undifferentiated after 20 passages in culture.  
     
     
         6 . The isolated stem cells of  claim 1 , wherein the ELSCs remain substantially undifferentiated after 100 passages in culture.  
     
     
         7 . Isolated mammalian pluripotent embryonic-like stem cells (ELSCs) which (i) are isolated from corneoscleral limbus, (ii) are capable of proliferating in an in vitro culture, and (iii) maintain the potential to differentiate into lineage-committed endodermal, ectodermal, or mesodermal cells.  
     
     
         8 . The isolated stem cells of  claim 7 , wherein the mammalian ELSCs are human ELSCs.  
     
     
         9 . The isolated stem cells of  claim 7 , wherein the ELSCs are capable of forming embryoid-like bodies when placed in suspension culture.  
     
     
         10 . The isolated stem cells of  claim 7 , wherein the ELSCs remain substantially undifferentiated after 20 passages in culture.  
     
     
         11 . The isolated stem cells of  claim 7 , wherein the ELSCs remain substantially undifferentiated after 100 passages in culture.  
     
     
         12 . A method of isolating a population of mammalian pluripotent embryonic-like stem cells (ELSCs), comprising the steps of: 
 (a) isolating corneal limbal tissue from a donor;    (b) culturing the corneal limbal tissue to expand corneal limbal cells in culture; and    (c) isolating a population of pluripotent ELSCs from the cultured corneal limbal cells by sorting the corneal limbal cells to select for one or more undifferentiated cell-specific surface markers.    
     
     
         13 . The method of  claim 12 , wherein the mammalian ELSCs are human ELSCs.  
     
     
         14 . The method of  claim 12 , wherein the donor is human.  
     
     
         15 . The method of  claim 12 , wherein the corneal limbal tissue is cultured on an extracellular matrix.  
     
     
         16 . The method of  claim 15 , wherein the extracellular matrix is mammalian amniotic membrane.  
     
     
         17 . The method of  claim 15 , further comprising the step of dissociating the cultured corneal limbal cells from the extracellular matrix prior to isolating the pluripotent ELSCs.  
     
     
         18 . The method of  claim 12 , wherein the corneal limbal tissue is cultured in culture media supplemented with one or more soluble factors selected from the group consisting of dimethyl sulphoxide, recombinant human epidermal growth factor, insulin, sodium selenite, transferrin, basic fibroblast growth factor, and leukemia inhibitory factor.  
     
     
         19 . The method of  claim 12 , wherein the corneal limbal tissue is cultured until the corneal limbal cells become confluent.  
     
     
         20 . The method of  claim 12 , wherein the corneal limbal cells are sorted using magnetic-affinity cell sorting (MACS).  
     
     
         21 . The method of  claim 12 , wherein the corneal limbal cells are sorted using fluorescence-activated cell sorting (FACS).  
     
     
         22 . The method of  claim 12 , wherein the undifferentiated cell-specific surface markers are selected from the group consisting of SSEA-4, SSEA-3, CD73, CD105, CD31, CD54, and CD117.  
     
     
         23 . The method of  claim 12 , wherein the undifferentiated cell-specific surface marker selected for is SSEA-4.  
     
     
         24 . The method of  claim 12 , further comprising culturing the isolated population of pluripotent ELSCs to produce an embryonic-like stem cell line.  
     
     
         25 . The method of  claim 24 , wherein the pluripotent ELSCs are cultured in culture media supplemented with one or more soluble factors selected from the group consisting of dimethyl sulphoxide, recombinant human epidermal growth factor, insulin, sodium selenite, transferrin, basic fibroblast growth factor, and leukemia inhibitory factor.  
     
     
         26 . The method of  claim 12 , wherein the isolated population of pluripotent ELSCs comprises at least about 70% ELSCs.  
     
     
         27 . The method of  claim 12 , wherein the isolated pluripotent ELSCs are capable of forming embryoid-like bodies when placed in suspension culture.  
     
     
         28 . The method of  claim 12 , wherein the pluripotent ELSCs remain substantially undifferentiated after 20 passages in culture.  
     
     
         29 . The method of  claim 12 , wherein the pluripotent ELSCs remain substantially undifferentiated after 100 passages in culture.  
     
     
         30 . The method of  claim 13 , further comprising the step of differentiating the human ELSCs into endodermal lineage-committed cells.  
     
     
         31 . The method of  claim 13 , further comprising the step of differentiating the human ELSCs into mesodermal lineage-committed cells.  
     
     
         32 . The method of  claim 13 , further comprising the step of differentiating the human ELSCs into ectodermal lineage-committed cells.