Methods of isolating distinct pancreatic cell types

Abstract

Methods of isolating distinct specific cell types within mixed populations of cells. Methods of isolating specific cell types among pancreatic cells, particularly from human islets of Langerhans. Markers and combinations thereof for use in methods of isolating insulin producing islet beta cells for treatment of diabetes.

What is claimed is: 
     
         1 . A method of prevention or treatment of diabetes comprising transplanting of a population of human beta cells to a human subject in need thereof, wherein at least 80% of said human beta cells express CD9+/CD56+ cell surface markers, wherein said population of beta cells comprises mature, functional insulin-producing beta cells. 
     
     
         2 . The method of  claim 1 , wherein said beta cells express at least one pair of markers selected from the group consisting of: (i) CD9 and CD73, (ii) CD9 and CD221, (iii) CD9 and CD81, and (iv) CD9 and CD147. 
     
     
         3 . The method of  claim 1 , wherein said beta cells exhibit the CD9+/CD56+/CD73+/CD221+/CD87+/CCR4+/CD165+/CD85J+/CD153+/CD68+/WNT16+/CD6+/CD77+/CD61+/CD32+ expression signature. 
     
     
         4 . The method of  claim 1 , wherein said population of human beta cells is obtained by sorting cells of human adult islets of Langerhans using a combination of at least two cell-surface markers relevant to the cell type to be isolated, wherein the at least one distinct type of cells is selected from the group consisting of: beta cells and delta cells, and wherein one of the at least two cell-surface markers is CD9 and the cell isolation is for an enriched population of cells expressing high levels of the cell-surface marker CD9. 
     
     
         5 . The method of  claim 4 , wherein said sorting using said combination of at least two cell-surface markers is effected sequentially and the cell-surface marker CD9 is the first marker used for isolation. 
     
     
         6 . The method of  claim 4 , further comprising the steps of:
 i. obtaining said cells of human adult islets of Langerhans from recovered or extracted pancreatic tissue;   ii. exposing the cells obtained in (i) to a probe capable of identifying CD9+ cells and to at least one additional probe; and   iii. isolating cells expressing CD9 and the additional probe by sorting of cells, thereby isolating an enriched population of at least one distinct cell type selected from the group consisting of: beta cells and delta cells.   
     
     
         7 . The method of  claim 4 , wherein the cells are isolated using a combination of CD9 and at least one cell-surface marker selected from the group consisting of: CD56, EGFR, CD4, CD73, CD87, CCR4, CD165, CD85J, CD221, CD153 (CD30L), CD142, CD134, ITGB7, CD68, WNT16, CD18, CD6, CD77, CD61, and CD32. 
     
     
         8 . The method of  claim 4 , wherein said sorting is performed with anti CD9 and anti CD56 antibodies, wherein said anti-CD9 and said anti-CD56 are used for positive selection. 
     
     
         9 . The method of  claim 4 , wherein said sorting is performed with anti-CD9 and anti-EGFR antibodies, wherein said anti-CD9 is used for positive selection and said anti-EGFR is used for negative selection. 
     
     
         10 . The method of  claim 9 , wherein the at least one additional probe is capable of identifying a cell-surface marker is selected from the group consisting of: CD4 (NP_000607.1), CD73 (NP_001191742.1, CD87 (NP_002650.1), CCR4 (NP_005499.1), CD165 (Gene ID 23449), CD85J (NP_001075106.1), CD221(NP_000866.1), CD153 (CD30L) (NP_001235.1), CD142 (NP_001171567.1), CD134 (NP_003318.1), ITGB7 (NP_000880.1), CD68 (NP_001035148.1), WNT16 (NP_057171.2), CD18 (NP_000202.2), CD6 (NP_001241679.1), CD77 (NP_059132.1), CD61 (NP_000203.2), and CD32 (NP_001002273.1). 
     
     
         11 . The method of  claim 6 , wherein the enriched cells of the population of at least one distinct cell type selected from the group consisting of: beta cells and delta cells are subject to additional iterations of steps (ii)-(iii). 
     
     
         12 . The method of  claim 6 , wherein the sorting of the enriched cells of the population of at least one distinct cell type selected from the group consisting of: beta cells and delta cells (iii) is performed via fluorescence activated cell sorting (FACS). 
     
     
         13 . The method of  claim 1 , wherein said population of human beta cells is obtained by sorting cells from a heterogeneous population of cells recovered or extracted from pancreatic tissue, the method comprising sorting the cells using a combination of cell-surface markers CD9 and EGFR. 
     
     
         14 . The method of  claim 13 , wherein said sorting is performed with anti CD9 and anti EGFR antibodies, wherein said anti-CD9 is used for a positive selection and wherein said anti EGFR is used for a negative selection. 
     
     
         15 . The method of  claim 13 , wherein the heterogeneous population of cells is selected from the group consisting of cells recovered or extracted from pancreatic tissue, committed lineages of stem cells and cultures of differentiated stem cells. 
     
     
         16 . The method of  claim 1 , wherein said population of human beta cells is obtained by sorting cells from a heterogeneous population of cells recovered or extracted from pancreatic tissue, the method comprising sorting the cells using a combination of at least the following cell-surface markers selected from the group consisting of:
 (i) CD9 and CD73,   (ii) CD9 and CD221,   (iii) CD9 and CD81,   (iv) CD9 and CD147,   (v) CD9 and CD49B,   (vi) CD9 and CD44,   (vii) CD9 and CD142,   (viii) CD9 and CD18,   (ix) CD9 and CD134,   (x) CD9 and CD4, and   (xi) CD9 and ITGB7.   
     
     
         17 . The method of  claim 16 , wherein said CD9 is for positive selection. 
     
     
         18 . The method of  claim 16 , further comprising sorting the cells using the CD56 cell surface marker. 
     
     
         19 . The method of  claim 8 , wherein said CD56 is for positive selection. 
     
     
         20 . The method of  claim 16 , wherein each of said CD9, said CD73, said CD221, said CD81, and said CD147 is for positive selection. 
     
     
         21 . The method of  claim 16 , wherein each of said CD49B, said CD44, said CD142, said CD18, said CD134, said CD4 and said ITGB7 is for negative selection. 
     
     
         22 . The method of  claim 15 , wherein said stem cells adult stem cells, induced pluripotent stem cells or embryonic pluripotent stem cells.