Patent Application: US-76531597-A

Abstract:
haematopoietic stem cells are immobilized on a substrate coated with a fibrin matrix and including a substance capable of both binding to the fibrin matrix and also having an rgd amino acid sequence for binding to the stem cells . the substance may be fibronectin or thrombospondin . the substrate is generally in the form of a closed bag formed of a carbon dioxide - permeable and oxygen - permeable plastics material which allows culturing of the stem cells . the cultured stem cells may re - engraft a patient following chemotherapy or to correct haemotological deficiencies . stem cells may be harvested from peripheral blood onto the coated substrate . the stem cells in contact with the coated substrate are good candidates for gene therapy to introduce a heterologous gene e . g . employing a transfection vector .

Description:
embodiments of the present invention will now be described by way of example only . in order to validate the use of cell line kgla as a model system for hpc cells ( including hpc stem cells ), we characterised the pattern of cell adhesion molecule expression on hpc cells derived from normal bone marrow and peripheral blood following mobilisation with high - dosage cyclophosphamide and recombinant human granulocyte colony stimulating factor , and compared it with expression from kgla . samples were separated by ficoll discontinuous density - gradient centrifugation . the mononuclear cell layer was removed , and the cells washed twice in a handling medium ( phosphate buffered saline pbs ! containing 1 % bovine serum albumin , 0 . 1 % sodium azide and 0 . 02 % ethylene diaminetetra - acetic acid edta !). the cells were incubated for 15 min in 0 . 1 % human gamma globulin ( sigma ) in pbs to effect fc receptor blockade . aliquots of 5 × 10 5 cells per test were incubated with purified monoclonal antibody to one of a panel of cell adhesion molecules ( table i ) for 15 min . the cells were washed twice in handling medium , and incubated with the second step reagent -- a sheep anti - mouse r - phycoerythrin conjugate ( sigma ), again for 15 mins . the cells were washed again , incubated for 15 min with mouse serum , to blockade the second - step reagent , and then incubated with the third step reagent -- an anti - cd34 monoclonal antibody directly conjugated to fluorescein isothiocyanate ( bg12 - fitc , becton dickinson ). the cd34 cell surface antigen identifies the hpc cells ( which typically constitute only about 1 % of the total cells ). the cells were washed a final time , and fixed in 2 % paraformaldehyde ( sigma ) in pbs , prior to analysis . appropriate control samples were established using monoclonal antibodies to cd45 as a positive control , and an isotype - specific antibody of irrelevant specificity as negative control , appropriately stained with each fluorochrome . 30 - 50 , 000 cells were acquired through a lymphoblastoid acquisition gate using a facscan flow cytometer ( becton dickinson ), with prior standardisation of the instrument settings and two - colour compensation using control samples . data was acquired and analysed using lysis ii software ( becton dickinson ). we demonstrated expression of icam - 1 , pecam - 1 , lfa - 3 , lfa - 1 , vla - 4 , vla - 5 , l - selectin and hcam ( table ii ). peripheral blood hpc demonstrated a similar pattern of expression , except in that there was less expression of lfa - 3 , and vla - 5 . vla - 4 and vla - 5 are members of the b 1 integrin family , and recognise and adhere with high - avidity to fibronectin . l - selectin recognises carbohydrate moieties such as sialyl lewis x , with low avidity binding . hcam recognises collagen type 1 and hyaluronic acid . the lower levels of vla - 5 expression by circulating progenitors is suggestive that the vla - 5 : fibronectin adhesion pathway may be instrumental in mediating hpc - stromal adhesion . in view of the problems associated with obtaining highly purified human hpc populations , we therefore utilised the primitive human haematopoietic cell line kgla as a model early progenitor during development of a binding assay . dual immunocytometry revealed kgla to be cd34 positive , and to express a similar cell adhesion molecule profile to normal haematopoietic progenitors ( table ii ). development of a functional binding assay to assess adhesion of haematopoietic progenitors to extracellular matrix components we explored the functional binding of kgla to a panel of bone extracellular matrix components using a 51 chromium radiolabelling assay adapted from the work of cheryl hardy and jose minguell ( 1993 ). 1 × 10 7 kgla were incubated with 200 kbq 51 chromium ( amersham international ) in 100 μl of foetal calf serum fcs ! for 1 hr . and washed twice in large volumes of iscove &# 39 ; s modified dulbecco &# 39 ; s medium imdm !, with 10 % fcs . the radiolabelled cells were aliquoted at a concentration of 2 × 10 5 / 200 μl , and dispensed into 2cm 2 wells of a 24 well flat - bottomed tissue culture dish ( costar ). the wells were prior coated with extracellular matrix components known to be present in bone marrow stroma ( gordon , 1988 ; clark , gallagher & amp ; dexter , 1992 ) ( table iii ). 100 μl of a solution containing 100 μg / ml ( collagens ) in 0 . 1 % acetic acid in distilled water , or 50 μg / ml ( fibronectin and proteoglycans ) in distilled water , was added to each well and allowed to dry in a 37 ° c . oven for 1 hr . 1 % denatured bovine serum albumin ( sigma ) was used to coat negative control wells . the cells were incubated with the substrate for 2 hrs at 37 ° c ., and the supernatant removed . the wells were washed twice with imdm 10 % fcs , and the washes and supernatant added to a scintillation vial . 0 . 5 ml of 0 . 1 % non - idet in distilled water was added to each well for 15 min to lyse the adherent cells . this was also removed into a scintillation vial . both adherent and non - adherent fractions were counted for 30 min in a hewlett - packard gamma counter , and the percentage binding calculated . each assay was carried out in triplicate within each plate , and the mean percentage binding calculated . this assay system was standardised , and its reproducibity demonstrated . kgla were found to bind to tissue culture grade plastics ( for comparison purposes ), but not to tuta clx blood - bag plastic . the former was blocked by coating the well with denatured bsa or other proteins . kgla bound significantly to plasma and tissue fibronectin , but not to various collagens or proteoglycans ( table iv ). percentage adherence to fibronectin was remarkably constant over a range of cell concentrations between 1 × 10 4 to 2 × 10 6 cells per 2cm 2 well , but fell sharply at 1 × 10 7 cells / well , consistent with a maximal binding capacity of 430 , 000 cells / cm 2 . whether the maximal binding capacity is a reflection of saturation of fibronectin binding sites or of steric hinderance is currently unclear . binding was abrogated at 4 ° c ., but stable at room temperature and 37 ° c . the use of various incubating media ( imdm or rpmi , with and without fcs ) made no difference to binding . in addition , binding reached a plateau after 1 hr , with no benefit to prolonged incubation periods . fibronectin binding could be abrogated by carrying out the substrate incubation in a medium from which divalent cations had been removed ( hanks balanced salts solution ( northumbria biologicals ) with 10 mmol edta ( p , 0 . 01 ), or in a 3 mmol solution of an arginine - glycine - asparatate - serine peptide ( sigma ) in imdm ( p , 0 . 01 ), though not in a solution of imdm with 3 mmol of a control peptide ( arginine - glycine - glutamate - serine ) ( sigma ). finally , it was found that it made no difference to the % kgla binding whether the fibronectin was dried onto the well surface , or simply incubated at room temperature for 1 hr . the possibility of coating a flexible plastics material was investigated . a tuta clx blood bag was cut open along the side seams , and a section of one side wag clamped in a bio - dot ( bio - rad ) dot blot apparatus so that the inner bag surface was exposed at the bottom of the 96 wells of the apparatus ( 96 well plate layout ). human plasma fibronectin pfn ! ( sigma ) and tissue fibronectin tfn ! derived from foreskin fibroblasts ( sigma ) were dissolved in pyrogen - free water to give a stock solution at 500 μg / ml . 50 μl of doubling dilutions of the pfn and tfn solutions were added across well columns 1 - 11 . column 12 contained water only ( control ). after 1 hr at room temperature , the well contents were removed , and the wells were washed 4 times with pyrogen - free water . rabbit polyclonal anti - plasma fibronectin rppfn ! ( sigma ) and mouse monoclonal anti - tissue fibronectin mmtfn ! ( sigma ) were diluted 1 : 500 in buffer 1 ( phosphate buffered saline ( ph 7 . 2 ) containing 0 . 1 % tween 20 , and 0 . 05 % sodium azide ), and 50 μl was added to each well of the microplate . after 90 min . at room temperature the well contents were removed , and wells were washed with buffer 2 ( phosphate buffered saline ( ph 7 . 2 ) containing 0 . 1 % tween 20 , 0 . 05 % sodium azide , 1 % bovine serum albumin , and 4 % polyethylene glycol ). alkaline phosphatase conjugated goat anti - rabbit 1gg apar ! ( zymed ) and alkaline phosphatase conjugated rabbit anti - mouse ig apam ! were diluted 1 : 1200 in buffer 2 . 50 μl was added to the appropriate well series , and after 120 min at room temperature the well contents were removed , and the wells washed 5 times with buffer 2 and a further thrice with pyrogen - free water . the top manifold of the bio - dot apparatus was unclamped , and washed with sodium hydroxide , detergent ( decon ) and distilled water to remove / destroy any of the above reagents which may have attached to the manifold . the manifold was clamped back into position over the sheet of blood bag plastic , and alkaline phosphatase substrate solution ( sigma ) added at 140 μl per well . colour could be seen to be developing at the well bottom , at the blood bag surface . the apparatus was left on an orbital mixer for 40 min at room temperature , 100 μl was transferred from each well to a half area microplate ( costar ), and the optical density was read at 405 nm ( minus 650 nm reference ). the results are illustrated graphically in fig1 . there was some background binding or the alkaline phosphatase labelled anti ig antibodies ( apar and apam ) to fibronectin alone ( 3 , 6 ). the anti - tissue fibronectin antibody ( mmtfn ) showed relatively weak selective binding to pfn ( 2 ), and only slightly better binding to tfn ( 4 ). this may be a reflection of the particular concentrations of the anti - tfn antibody ( mmtfn ) and the anti - mouse ig antibody ( apam ) used in this experiment . the anti - pfn antibody ( rppfn ) showed strong selective binding to both pfn ( 1 ) and tfn ( 5 ), and showed that binding of tfn to the plastic surface was superior to that of pfn at lower concentration ranges ( 0 . 5 to 8 μg / ml ). both pfn and tfn achieved saturation binding at approximately 20 μg / ml , but showed some evidence of &# 34 ; prozone &# 34 ; above approximately 50 μg / ml , i . e . there was some decrease in binding suggesting less firm anchorage of fibronectin to plastic at higher coating concentrations . this usually suggests multivalency of binding , such that more valencies are used at lower concentrations to establish firm binding while at higher concentrations there is competition for binding sites , less valencies are occupied , and the reagent is more readily detached . it proved necessary to adapt the 51 chromium adhesion assay in order to examine kgla ( used as a model for hpc cell binding ) adhesion to tuta clx blood - bag plastic . the base of a 24 well plate was removed , a blood - bag cut open , and the internal surface adhered to the base of the plate . care was taken to ensure that adhesive did not spread onto the test surface , and that a water - seal was achieved around each well individually . wells were tested for continence with 1 ml of distilled water . in an initial series of experiments , kgla adhesion was examined to the plastic itself , following coating with plasma or tissue fibronectin , or following coating with bsa . the results are summarised in table v . kgla showed no significant binding to any of wells under these conditions , and we suspect that the negligable charge on the surface of the blood - bag plastic leads to only a very loose coating with fibronectin . we considered a number of options to increase the extent and avidity of fironectin binding , including pre - coating the surface with a fibrin glue . fibrin glue consists of two proteinaceous compounds , which form an adhesive matrix on reconstitution and mixing . one component is prepared from cryoprecipitate obtained from a large pool of voluntary uk blood donations . the material is lyophilised and subjected to heat treatment ( 80 ° c . for 72 hrs ) to minimise the risk of viral transmission . the lyophilised croprecipitate consists mainly of fibrinogen ( 225 mg / vial ) and factor xiii ( 50 units / vial ), but probably also small amounts of other plasma proteins such as fibronectin , thrombospondin and von willebrand &# 39 ; s factor . the solvent for this component is water containing 20 mm tris buffer ( ph 7 . 5 ), and aprotinin ( trasylol , bayer ) at 3 , 000 kallikrein inactivator units / ml . the latter acts as a proteolytic enzyme inhibitor . the second component is lyophilised human thrombin , at 1000 iu / vial , reconstituted with a solution of 4 mm calcium chloride . when the two components are mixed , the thrombin activates the fibrinogen to fibrin , and also activates factor xiii to factor xiiia , which stabilises the fibrin though cross - linking . other proteins which may be present ( such as fibronectin ) will also be cross - linked into the matrix by factor xiiia . we coated tuta clx blood - bag plastic with fibrin matrix alone , fibrin matrix onto the surface of which plasma fibronectin had been incubated using the standard protocol ( fibronectin contains fibrinogen binding domains ), and fibrin matrix into which fibronectin had been added ( to the fibrinogen component at 0 . 05 mg / ml ) such that the fibronectin was cross - linked into the matrix by factor xiiia . the results are tabulated in table v . table i______________________________________cell adhesion molecules studied . cytoadhesion monoclonalmolecule ( cd ) ligand antibody ( source ) ______________________________________immunoglobulin gene superfamily . icam - 1 ( cd54 ) lfa - 1 84hio ( immunotech ) pecam - 1 ( cd31 ) 5 . 6e ( immunotech ) lfa - 3 ( cd58 ) lfa - 2 aicd58 ( immunotech ) integrin family . β . sub . 1 vla subfamily . vla - 4 ( cdw49d / cd29 ) fibronectin hp2 / 1 ( immunotech ) vla - 5 ( cdw49e / cd29 ) fibronectin sam1 ( immunotech ) β . sub . 2 leukocyte adhesion subfamilylfa - 1 ( cd11a / cd18 ) icam1 / 2 iot16 ( immunotech ) β . sub . 3 cytoadhesion subfamily . vitronectin vitronectin amf7 ( immunotech ) receptor ( cd51 / cd61 ) selectin family . l - selectin carbohydrate dreg56 ( immunotech ) proteoglycan analogues . hcam ( cd44 ) collagen f10 - 44 - 2 ( serotech ) hyaluronic acidcd36 / limp ii family . thrombospondin collagen fa - 152 ( immunotech ) receptor ( cd36 ) thrombospondin______________________________________ table ii______________________________________cell adhesion molecule expression . adhesionmolecule bone marrow peripheral blood kg1a . ______________________________________icam - 1 ( cd54 ): 91 ± 11 % 93 ± 11 % 98 . 6 ± 3 % pecam - 1 ( cd31 ): 91 ± 7 % 94 ± 6 % 30 ± 13 % lfa - 3 ( cd58 ): 65 ± 25 % 27 ± 18 % 98 ± 1 % lfa - 1 ( cd11a ): 84 ± 12 % 78 ± 15 % 99 ± 1 % vla - 4 ( cdw49d ): 67 ± 25 % 71 ± 23 % 99 ± 3 % vla5 -( cdw49e ): 62 ± 19 % 32 ± 19 % 99 ± 3 % vnr ( cd51 ): 9 ± 7 % 5 ± 6 % 7 ± 3 % l - selectin : 64 ± 22 % 60 ± 28 % 5 ± 4 % hcam ( cd44 ): 98 ± 2 % 97 ± 5 % 99 ± 0 . 5 % cd36 : 20 ± 11 % 14 ± 14 % 35 ± 13 % ______________________________________ table iii______________________________________extracellular matrix components studied . ______________________________________collagen type i , acid soluble from human placenta . ( sigma , type viii ) collagen type iii , acid soluble from human placenta . ( sigma , type x ). collagen type iv , acid soluble from human placenta . ( sigma , type vi ) fibronectin , from human plasma . ( sigma ). fibronectin , from human foreskin fibroblasts . ( sigma ). heparan sulphate , sodium salt from bovine kidney . ( sigma ). chondroitin sulphate a . sodium salt from bovinetrachea . ( sigma ). hyaluronic acid , from human umbilical cord . ( sigma ). ( gordon , 1988 ; clark , gallagher & amp ; dexter , 1992 ) thrombospondin . ______________________________________ table iv______________________________________kg1a binding to extracellular matrix components . ______________________________________denatured albumin 8 . 3 ± 5 . 1 % collagen type i 5 . 2 ± 1 . 7 % collagen type iii 10 . 1 ± 7 . 9 % collagen type iv 4 . 3 ± 2 . 1 % heparan sulphate 4 . 5 ± 1 . 2 % chondroitin sulphate 5 . 2 ± 1 . 7 % hyaluronic acid 6 . 9 ± 1 . 3 % plasma fibronectin 28 . 3 ± 5 . 2 % thrombospondin 76 . 2 ± 12 . 8 % ______________________________________ table v______________________________________kg1a binding to tuta clx blood - bag plastic . mean % adherence ± ( number ofsubstrate 1 standard error experiments ) ______________________________________plastic 7 . 5 ± 2 . 3 % ( 3 ) plasma fibronectin 7 . 4 ± 0 . 5 % ( 3 ) tissue fibronectin 7 . 5 ± 1 . 9 % ( 2 ) bovine serum albumin 5 . 0 ± 1 . 2 % ( 2 ) fibrin matrix 22 . 9 % ( 1 ) fibrin matrix + 38 . 9 % ( 1 ) pfn ( coated ) fibrin matrix + 32 . 5 ± 16 . 1 % ( 3 ) pfn ( inclusive ) ______________________________________ clark b . r ., gallagher j . t ., dexter t . m . 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