the term “ cells ” in the present invention refers to isolated mammalian tissue cells including , but not limited to , stem cells , progenitor cells , bone marrow - derived cells ( bmc ), and bone marrow - derived mononuclear cells ( bm - mnc ]. other cell types may also be used in the methods of the invention , including endothelial progenitor cells ( epc ), cord blood - derived cells , adipose derived cells , spleen - derived cells , hematopoietic stem cells , hematopoietic progenitor cells , mesenchymal stem cells ( msc ), stromal cells , myocardial stem cells , myocardial cells , neuronal progenitor cells , precursor cells , mature cells , somatic cells , smooth muscle cells , fibroblasts , dendritic cells , astrocytes , and islet cells . cells can be treated as described in the invention so as to enhance the surface cxcr4 expression and improve the efficiency of engraftment . cells which are treated with the invented method are referred to as treated cells . cells can be obtained from the same individual human or animal into which the treated cells will be injected , or can be obtained from different individual . the treated cells will be used for therapy for diseases or for research in animals . the term “ invented solution ” refers to any buffered isotonic solution , containing calcium ions in a concentration that increases the concentration of cxcr4 surface receptors on target cells , in comparison to an equivalent solution that does not contain calcium . additional components , in addition to saline , buffer and calcium ion , can include glucose , salt , amino acids , vitamins and similar well - known additives to cell culture solutions . a preferred ph range is a ph of about 7 - 7 . 5 . added glucose in the range of about 1 to about 30 mm is a preferred additive . the term “ invented treatment ” refers to incubation of target cells in the invented solution (“ treatment ”) for certain times at certain temperature , for example , 2 hrs at 37 ° c . the incubation duration can be 0 . 5 hr to 15 hrs , preferably about 1 to about 4 hrs . the incubation temperature can be from 18 ° c . to 37 ° c . the treatment can be carried out prior to or during cell harvesting , cell processing , or cell injection . the term “ cxcr4 ” or “ cxc chemokine receptor 4 ” refers to a protein with the property of binding with chemokine stromal cell - derived factor - 1 ( sdf - 1 ). in particular , cxcr4 usually refers to the surface receptor for sdf - 1 . 1 . cxcr4 surface expression on bmc was enhanced by the invented treatment mouse bm ( bone marrow ) was collected by flushing the femur with phosphate buffered saline ( pbs ). after centrifugation , the cells were suspended in 1 . 2 ml red blood cell lysis solution ( sigma ) and incubated at 37 ° c . for 5 minutes followed by addition of 10 ml pbs and centrifugation to remove the lysed red blood cells . the bmc ( bone marrow cells ) numbering about 1 × 10 6 cells were then incubated in pbs (“ untreated ”) or the invented solution (“ treated ”) for 4 hrs at 37 ° c . after a wash in phosphate - buffered saline ( pbs ), the bmc were re - suspended in 100 μl protein blocking solution with 20 fluorescent fitc - conjugated rat anti -( mouse cxcr4 ) monoclonal antibody ( mab ) ( bd - pharmingen ), and incubated at 4 ° c . for 1 hr . the fluorescence on the cell surface was measured by flow cytometry ( facs ) using isotype igg stained bmc as a control ( control , grey line ) to set the gate ( fig1 a ). the results are shown in fig1 . there were significantly more cells expressing surface cxcr4 in the treated bmc ( 43 ± 5 %) than the untreated bmc ( 10 ± 1 %) ( fig1 b ). there was also significantly more surface cxcr4 on each treated cell than the untreated cell . the mean fluorescence intensity ( mfi ) increased from 11 ± 2 % untreated to 26 ± 3 % after the treatment ( fig1 c ). bmc migration towards sdf - 1 was determined using a modified boyden chamber assay . bmc were pre - incubated with either pbs or the invented solution for 4 hours at 37 deg . c . and transferred to the upper chamber of inserts in a 24 - well plate containing dmem and 100 ng / ml sdf - 1 . migrated cells were counted after 6 hrs incubation at 37 ° c . inhibitors amd3100 , ly294002 , l - nmma , and anti - cxcr4 antibody were added to the upper chamber along with bmc . as shown in fig2 ( legend : *, p & lt ; 0 . 05 versus treated group ; #, p & lt ; 0 . 05 versus other groups ), cell mobility increased 2 - fold after the invented treatment relative to untreated control ( 32 . 9 ± 8 . 4 vs . 15 . 6 ± 2 . 9 ; p & lt ; 0 . 05 , n = 4 , fig2 ). the mobility of the treated bmc was significantly reduced by addition of pi - 3k inhibitor ly294002 , or nitric oxide synthase inhibitor l - nnma , cxcr4 antagonist amd3100 , or anti - cxcr4 antibody ( fig2 ; all p & lt ; 0 . 05 ). these results indicate that cell mobility is enhanced by the treatment , and that the enhancement is through sdf - 1 / cxcr4 interaction . as shown in fig3 , bmc from gfp mice were incubated with the invented solution ( treated ) or with pbs ( untreated ), and injected into the tail veins of mice after surgical implementation of hindlimb ischemia . t sdf1 protein was also injected into the ischemic hindlimb muscle to enhance bmc homing . sdf - 1 protein ( 100 ng ) was injected into the ischemic hindlimb muscle of wt bl6 mice twice at consecutive days after the surgery . the hindlimb muscles were recovered 7 days after the cell injection , stained with dapi for nucleus ( light white ), and examined for gfp cells ( bright white , arrow pointed ) incorporation under fluorescent microscopy . ( a ) untreated bmc , b ) treated bmc , c ) untreated bmc + sdf - 1 , d ) treated bmc + sdf - 1 . e ) the incorporated gfp cells were quantified as cells per high power field ( hpf ). * p & lt ; 0 . 05 , and ** p & lt ; 0 . 01 versus all other groups ( n = 6 ). significantly more injected gfp + cells were detected in the ischemic muscles of the mice that received treated bmcs ( fig3 b ) than in mice that received untreated cells ( fig3 a ) in the absence of sdf - 1 . with exogenous sdf - 1 , both cells homed more efficiently ( fig3 e ). however , treated bmc ( fig3 d ) still homed more efficiently to the ischemic site than the control bmc ( fig3 c ). bl6 mice with an ischemic hindlimb were injected intravenously with bmc that had been incubated in pbs ( untreated ) or the invented solution ( treated ) at 37 ° c . for 4 hours . sdf - 1 protein was injected into the ischemic hindlimb muscle after the surgery . blood flow of the lower limbs was measured using a laser doppler perfusion image ( ldpi ) analyzer . fig4 shows the results . a ). representative laser doppler perfusion images at day 0 and day 21 after ischemia and cell injection . b ) capillaries in the ischemic muscle from the mice at day 21 were identified by alkaline phosphatase cryosectional staining . c ). cd34 immunostaining of paraffin sections obtained from the ischemic muscle of mice at day 7 . arrow points cd34 + cells . dark dots represents cells counter - stained with hemotoxylin . d ). quantitative measurement of perfusion ratio of ischemic limbs to that of normal limbs at day 21 ( n = 6 ). e ) quantification of capillary density on the tissue section , which is presented as the ratio of the number of capillaries to the number of muscle fibers . f ), quantification of cd34 + cells around each muscle fiber . * p & lt ; 0 . 05 versus treated group . # p & lt ; 0 . 05 versus other groups . it can thus be seen that injection of the treated bmc promoted significantly improved reperfusion ( fig4 a and d ) and higher capillary density ( fig4 b and e ) in ischemic limbs at 3 weeks after surgery both in the presence and absence , of sdf - 1 injections . the improved therapy was paralleled by significantly more cd34 + cells in the ischemic muscles following injections of the treated bmc ( fig4 c and f ). 5 . the invented treatment enhanced bmc homing to the bm of irradiated mice bmc ( 1 × 10 exp 7 ) isolated from mice were treated with a ) the invented method or b ) pbs ( untreated ), and then were intravenously injected into recipient mice that had received total body irradiation ( 550 cgy ) one day before cell injection . the mice were sacrificed 3 days later , and sections of the recovered bones were observed under fluorescence microscopy . as shown in fig5 , ( a ) shows cells injected with the treated bmc , and ( b ) shows cells injected with untreated bmc . the homed cells with red fluorescence were quantified as cells per view under 20 × objective lens ( c ). significantly more bmcs treated with the invented method homed into bone marrow than the untreated cells . hence , it has been discovered that treatment of bmcs with the inventive solution improves the ability of the bmcs to repair damage . the treated bmc , relative to control , have increased cxcr4 expression , improved cell mobility , improved homing to the tissue , and improved angiogenesis induction . the treatment of the invention is an ideal treatment in terms of minimization of adverse effect potential . the treatment is accomplished by adding an effective level of calcium to bmc cells in a pbs solution and incubating for 1 - 4 hours before injection of the cells into a target site , without necessarily removing the incubation solution . the total quantity of calcium injected during treatment is very small and unlikely to affect any other physiological process . addition of low levels of glucose can further improve the response . while treatment of animals with this procedure is of interest , the important aspect of this invention is the treatment of humans . it is likely that humans — and for that matter mammals other than mice — may have optimal response to this treatment system at varying levels of calcium concentration , and perhaps of other variables . accordingly , the invention is in an important aspect the use of optimal levels of calcium in the treatment of bone marrow cells to make them clinically useful in the restoration of one or more functions . determination of the optimum concentration is straightforward for any species once the utility is known . in particular , it is important for use in humans to determine experimentally . for the particular cells involved in the treatment , the optimal value of one or more of the concentration of calcium ions , the time of incubation , and the temperature of incubation , to allow for standardization of clinical procedures . 1 . tateishi - yuyama e , matsubara h , murohara t , et al . therapeutic angiogenesis for patients with limb ischaemia by autologous transplantation of bone - marrow cells : a pilot study and a randomized controlled trial . the lancet . 2002 ; 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( note that reference 43 is a publication by the inventor ). unless defined otherwise , all technical and scientific terms used herein have the same meanings as commonly understood by one of skill in the art to which the disclosed invention belongs . although any methods and materials similar or equivalent to those described herein can be used in the practice or testing of the present invention , the preferred methods , devices , and materials are as described . publications cited herein and the material for which they are cited are specifically incorporated by reference , where such incorporation is permitted . nothing herein is to be construed as an admission that the invention is not entitled to antedate such disclosure by virtue of prior invention , where relevant . those skilled in the art will recognize , or be able to ascertain using no more than routine experimentation , many equivalents to the specific embodiments of the invention described herein . such equivalents are intended to be encompassed by the following claims .
this invention relates to a method and compositions for activation of precursor cells via upregulation of cxcr4 surface receptor expression , to give the activated cells a better homing capability and higher viability . the method for activation involves incubation of the target cells with an isotonic activation solution containing an activation - effective amount of calcium . such activated cells have a better ability to engraft into target tissues to execute the therapeutic function .