CD34(+) cells and their methods of use
View Patent ↗The present invention provides isolated cells that are selectively enriched for hematopoietic progenitor cells that are precursors for natural killer (NK) cells. These cells are both CD34 and CD45RA positive and express C-integrin β 7 , and are referred to as CD34 dim CD45RA (+) C-integrin β 7 bright . The invention provides methods for isolating these cells, for inducing formation of CD56 bright NK cells, and for treating diseases associated with immunodeficiency and cancer.
1. A method of isolating CD34 dim CD45RA (+) integrinβ 7 bright cells from a biological sample comprising the steps of:
separating cells expressing at least one first marker protein by using an antibody to the at least one first marker protein;
isolating mononuclear cells using Ficoll-centrifugation;
isolating CD34(+)-cell positive cells using at least one antibody to CD34(+); and
sorting cells in a cell sorter using at least one second marker protein.
2. The method of claim 1 , wherein said first marker protein is chosen from CD3, CD4, CD19, CD36, and glycophorin A.
3. The method of claim 1 , wherein said second marker protein is chosen from CD34, CD45RA, and integrin β 7 .
4. The method of claim 1 , wherein said second marker protein is integrin β 7 .
5. The method of claim 1 , wherein the biological sample is obtained from lymph nodes.
6. The method of claim 4 , wherein the isolated cells are selected using cytoadhesion.
7. The method of claim 4 , wherein the isolated cells are selected using immunomagnetic separation.
8. The method of claim 6 wherein said cytoadhesion uses binding to galactose.
9. The method of claim 8 wherein said cytoadhesion uses binding to N-acetyl galactosamine.
10. The method of claim 9 , wherein said N-acetyl galactosamine cytoadhesion is affinity chromatography.
11. The method of claim 9 , wherein the N-acetyl galactosamine is conjugated to BSA.
12. The method of claim 4 , wherein the isolated cells are purified by equilibrium density centrifugation.
13. The method of claim 1 , further comprising fractionating said purified population of hematopoietic precursor cells by fluorescence activated flow cytometry, velocity sedimentation or counter flow centrifugal elutriation.
14. Isolated NK precursor cells that are CD34(+), constitutively express CD45RA and high surface density integrin α 4 β 7 .
15. The isolated NK precursor cells according to claim 14 that express heterotrimeric high affinity IL-2Rαβγ.
16. A method for producing a population of functional hematopoietic precursor cells enriched for NK precursor cells, comprising:
(a) obtaining a population of hematopoietic precursor cells;
(b) selecting CD34+, CD45RA(+) cells from said population of precursor cells; and
(c) further selecting cells expressing integrin β 7 bright on their surfaces.
17. A method for isolating CD34(+) hematopoietic precursor cells, comprising
(i) at least one negative selection step in which cells expressing one or more marker proteins selected from the group of CD3, CD4, CD19, CD36, and glycophorin A are separated and removed from the desired cell set;
(ii) a Ficoll-centrifugation step in which mononuclear cells are separated from other cell types;
(iii) a CD34(+)-cell positive selection step in which antibodies to CD34(+) are used to select CD34(+) cells; and
(iv) a cell-sorting step in which cells that are CD34 dim , CD45RA(+), and integrinβ 7 bright are separated from other cells.