Detection of haematopoietic stem cells and progeny and uses thereof

Abstract

The present invention relates to a method of identifying a haematopoietic stem cell (HSC) or progeny thereof comprising the steps of: obtaining a cell sample including HSC or progeny thereof; detecting the presence of at least one carbohydrate sequence having a sequence of at least one disaccharide repeat of glucuronic acid and N-acetylglucosamine or an equivalent thereof; and identifying a HSC or progeny thereof having the sequence or equivalent thereof. The invention also relates to methods of enriching cell populations for HSC or progeny thereof, for isolating HSC or progeny thereof and cell preparations obtained using the methods of their invention and their uses.

1 . A method of identifying a haematopoietic stem cell (HSC) or progeny thereof comprising the steps of: 
 obtaining a cell sample including HSC or progeny thereof;    detecting the presence of at least one carbohydrate sequence having a sequence of at least one disaccharide repeat of glucuronic acid and N-acetylglucosamine or an equivalent thereof; and    identifying a HSC or progeny thereof having the sequence or equivalent thereof.    
     
     
         2 . A method of identifying a haematopoietic stem cell (HSC) or progeny thereof comprising the steps of: 
 obtaining a cell sample including HSC or progeny thereof;    detecting the presence of hyaluronic acid (HA) or hyaluronic acid synthase (HAS) or a fragment thereof on a cell; and    identifying as a HSC or progeny thereof a cell having HA, HAS or a fragment thereof on the cell.    
     
     
         3 . The method according to  claim 2  wherein HA or HAS is detected.  
     
     
         4 . The method according to  claim 3  wherein a peptide sequence corresponding to a portion of HAS or an equivalent thereof is detected.  
     
     
         5 . The method according to  claim 2  wherein the sample is exposed or combined with a marker for hyaluronic acid (HA) or hyaluronic acid synthase (HAS) or a fragment thereof in a manner which facilitates interaction of the marker with the cells.  
     
     
         6 . The method according to  claim 5  wherein the marker includes a label to enhance identification of the marker.  
     
     
         7 . The method according to  claim 6  wherein a fluorescent, radioactive or enzymatic label is linked to the marker to enhance detection.  
     
     
         8 . The method according to  claim 5  wherein the marker identifies hyaluronic acid (HA) or hyaluronic acid synthase (HAS) or a fragment thereof on a cell surface.  
     
     
         9 . The method according to  claim 8  wherein the marker is selected from the group consisting of: antibodies to HA, HAS or a fragment thereof, binding proteins to HA, HAS or a fragment thereof; nucleic acid detection systems which can detect expression of HA or HAS by the presence of DNA, RNA, mRNA or HA or HAS protein; and enzymatic, fluorescent or colorimetric assays for HA, HAS or a fragment thereof.  
     
     
         10 . The method according to  claim 8  wherein the marker is hyaluronic acid binding protein (HABP) or a synthetic indicator based on HABP.  
     
     
         11 . A method according to  claim 2  wherein the sample is enriched for CD34 +  cells prior to detection of HA, HAS or a fragment thereof.  
     
     
         12 . A method for obtaining a cell population enriched in HSCs or progeny thereof comprising the steps of: 
 obtaining a cell sample containing HSC or progeny thereof;    combining the cell population with a binding protein for HA, HAS or fragment thereof detecting the presence of HA, HAS or a fragment thereof on a cell; and    selecting out those cells which are identified by the presence of HA, HAS or a fragment thereof.    
     
     
         13 . A method according to  claim 12  further comprising detecting the presence of HA, HAS or a fragment thereof on a cell, wherein the method of detection involves the use of an antibody to HA or HAS; or a binding protein (HABP) to HA.  
     
     
         14 . A method of removing HSCs or progeny thereof from a cell population comprising the steps of: 
 obtaining a cell sample comprising HSCs or progeny thereof;    detecting the presence of HA, HAS or a fragment thereof on a cell; and    selecting out those cells which are identified by the presence of HA, HAS or a fragment thereof on the cell.    
     
     
         15 . A method for isolating subpopulations within a HSC population comprising using a method according to  claim 2  and specific markers for specific cell lineages to identify and isolate the cell lineages.  
     
     
         16 . A method for distinguishing subpopulations within a HSC population comprising pre-enrichment for CD34 + , identifying HSCs or progeny thereof by detection of HA, HAS or a fragment thereof according to  claim 8  and using additional markers separately or in combination with a marker for HA, HAS or a fragment thereof to distinguish the subpopulations.  
     
     
         17 . A diagnostic assay for determining numbers of HSCs or progeny thereof in a sample wherein the cells and antibody or marker are combined under conditions sufficient to allow specific binding of the antibody or marker to HA or HAS and the HSCs or progeny thereof which are then quantitated.  
     
     
         18 . A method of isolating a HSC or progeny thereof comprising: obtaining a cell population comprising HSC or progeny thereof; detecting the presence of HA, HAS or a fragment thereof on a cell; selecting for those cells which are identified by the presence of HA, HAS or a fragment thereof on the cell; and isolating those cells identified by the presence of HA, HAS or a fragment thereof.  
     
     
         19 . A method of isolating a HSC or progeny thereof comprising: 
 obtaining a cell population comprising HSCs or progeny thereof; combining the cell population with a binding protein for HA, HAS or fragment thereof;    selecting for those cells which are identified by the binding protein for HA, HAS or fragment thereof; and    isolating cells identified by the binding protein.    
     
     
         20 . A method of isolating a HSC or progeny thereof comprising 
 obtaining a cell population comprising HSCs or progeny thereof;    combining the cell population with an antibody for HA, HAS or fragment thereof;    selecting for those cells which are identified by the antibody for HA, HAS or fragment thereof; and    isolating cells identified by the antibody.    
     
     
         21 . A method according to  claim 20  wherein the antibody is specific for HA or HAS.  
     
     
         22 . A method according to  claim 21  wherein the antibody is a pan-species αHA polyclonal sheep antibody.  
     
     
         23 . A composition of enriched HSCs and progeny thereof comprising an enriched population of cells including CD34 + , CD38 − , thy1 + , and HA +  cells.  
     
     
         24 . Use of a composition according to  claim 23  in autologous engraftment.  
     
     
         25 . Use of a composition according to  claim 23  modified by gene transfer, to correct genetic defects or provide genetic capabilities naturally lacking in the HSCs or progeny thereof or their progeny. I would cancel this—these types of claims are not favored by the USPTO  
     
     
         26 . Use of a composition according to  claim 23  to isolate and define factors associated with regeneration and differentiation of HSCs or their progeny.  
     
     
         27 . A method of measuring the content of HSC or their progeny said method comprising: 
 obtaining a cell population comprising HSC or progeny thereof;    combining the cell population with a binding protein for HA, HAS or fragment thereof;    selecting for those cells which are identified by the binding protein for HA, HAS or fragment thereof; and    quantifying the amount of selected cells relative to the quantity of cells in the cell population prior to selection with the binding protein.    
     
     
         28 . A method of measuring the content of HSC or progeny thereof said method comprising: 
 obtaining a cell population comprising HSC or progeny thereof; combining the cell population with an antibody for HA, HAS or fragment thereof;    selecting for those cells which are identified by the antibody for HA, HAS or fragment thereof; and    quantifying the amount of selected cells relative to the quantity of cells in the cell population prior to selection with HA or HAS antibody.    
     
     
         29 . Use of the method according to  claim 27  or  28  for diagnosis of a HSC associated condition.  
     
     
         30 . A composition for detecting HSC or progeny thereof in a population, said composition comprising an indicator of HA or HAS or a fragment thereof and a carrier.  
     
     
         31 . The composition according to  claim 30  wherein the indicator of HA, HAS or a fragment thereof includes a detection means which can identify HA or HAS or a fragment thereof on a HSC or progeny thereof.  
     
     
         32 . The composition of  claim 31  wherein the indicator is an antibody or binding protein to HA, HAS or a fragment thereof.  
     
     
         33 . The composition of  claim 32  wherein the binding protein is HABP.  
     
     
         34 . The composition according to  claim 31  comprising additional markers to distinguish specific cell lineages.  
     
     
         35 . A method of diagnosing a condition associated with HSCs or progeny thereof by identifying the presence of HSC or progeny thereof in a cell population by a method according to  claim 2 .  
     
     
         36 . A method of controlling proliferation and/or differentiation in a HSC or progeny thereof, said method comprising modulating expression and/or activity of HA, HAS or a fragment thereof in a HSC or progeny thereof.  
     
     
         37 . A method according to  claim 36  wherein modulation of HA or HAS expression and/or activity in HSC is achieved using antagonists, inhibitors, mimetics or derivatives of the HA or HAS.  
     
     
         38 . A method of treating a HSC associated condition comprising administering an effective amount of a composition comprising an enriched population of HSC or progeny thereof and wherein said enriched population of HSC or progeny thereof is prepared by a method according to  claim 12 .  
     
     
         39 . Use of cells prepared by a method according to  claim 12  to reconstitute an immunocompromised host or as a source of cells for specific lineages, by providing for their maturation, proliferation and differentiation into one or more selected lineages with factors selected from erythropoietin, colony stimulating factors, interleukins, and stromal cells associated with the HSCs or progeny thereof.  
     
     
         40 . Use of HSCs and progeny thereof prepared by a method according to  claim 12  in isolation and evaluation of factors associated with differentiation and maturation of hematopoietic cells.  
     
     
         41 . Use of HSCs prepared by a method according to  claim 20  for the treatment of genetic diseases.  
     
     
         42 . A method of treating a HSC associated condition wherein said condition results from uncontrolled proliferation of HSCs or progeny thereof, said method comprising controlling expression of HA, HAS or a fragment thereof in a HSC or progeny thereof.  
     
     
         43 . A method of treating a HSC associated condition wherein said condition results from uncontrolled proliferation of HSCs or progeny thereof, said method comprising reducing expression and/or activity of HA, HAS or a fragment thereof in a HSC or progeny thereof.  
     
     
         44 . A method of treating a HSC associated condition wherein said condition results from differentiation of HSCs or progeny thereof, said method comprising controlling expression and/or activity of HA, HAS or a fragment thereof in a HSC or progeny thereof.  
     
     
         45 . A method of treating a HSC associated condition resulting from differentiation of HSCs or progeny thereof, comprising reducing expression and/or activity of HA, HAS or a fragment thereof in a HSC or progeny thereof.  
     
     
         46 . Use of a marker for hyaluronic acid (HA) or hyaluronic acid synthase (HAS) or a fragment thereof in the preparation of a diagnostic format for identifying or isolating HSCs or progeny thereof.