Patent Application: US-56870205-A

Abstract:
a method of producing stem / progenitor cells from human or animal origin . a population , from an embryonic , fetal or adult source , preferably from bone marrow , blood , fat , muscle , heart , intestine , kidney , liver , lung , pancreas , skin or neural tissues , that includes stem / progenitor cells , is treated with one or more first cytostatic or cytotoxic agents to which the stem / progenitor cells are less sensitive than the other cells of the population . preferably , the agent selectively deplete from the population cells that are negative with respect to expressing a transporter gene of the first agent while sparing cells that are positive with respect to expressing that gene . preferably , the population also is treated with one or more cytokines and / or growth factors .

Description:
the present invention is of a process for the preparation of enriched populations of stem / progenitor cells for various cell therapy applications , without the continuous use of xenogeneic antibodies directed against cell surface molecules , affinity columns or density so gradients . the invention includes a method for enrichment and expansion of stem / progenitor cells based on their relatively high expression levels of multidrug resistant − 1 ( mdr1 , also termed abcb1 ) gene and / or abcg2 gene and / or mrp gene , as compared to more differentiated progenitor and mature cells . the invention relates to expansion of stem / progenitor cells ex - vivo ( in the presence of cytokines and growth factors ) using selective killing of mdr1 and / or abcg2 and / or mrp low / negative cells by cytostatic and / or cytotoxic agents while avoiding the killing of mdr1 and / or abcg2 and / or mrp positive stem / progenitor cells . the principles and operation of stem / progenitor cell enrichment according to the present invention may be better understood with reference to the drawings and the accompanying description . cells were obtained from human umbilical cord blood ( ucb ) after normal fall - term delivery . the cells were layered on a ficoll - hypaque gradient ( 1 . 077 g / ml sigma ), and centrifuged at 500 g for 30 minutes . the mononuclear cells in the interface layer were collected and washed three times in phosphate - buffered saline ( pbs ; biological industries ) containing 0 . 5 % bovine serum albumin ( bsa ). to purify the cd133 + cells , the mononuclear cell fraction was subjected to two cycles of immunomagnetic bead separation using a minimacs cd133 progenitor cell isolation kit ( miltenyi biotec ,), according to the manufacturer &# 39 ; s recommendations . the purity of the cd133 + population thus obtained was evaluated by flow cytometry ( see section 3 . 1 . 3 ). purified cd133 + cells were cultured in 24 - well culture plate at densities of 2 - 4 × 10 4 cells in alpha - mem medium ( biological industries ) supplemented with 10 % fetal calf serum ( biological industries ) and the following human recombinant cytokines ( pepro tech , inc ., rocky hill , n . j ., usa ): trombopoietin , interleukin - 6 , flt - 3 ligand and stem cell factor , at final concentration of 50 ng / ml each as well as interleukin - 3 and il2 at 20 ng / ml , in the absence or presence of colchicine in a concentration scale as indicated . cells were expanded at 37 ° c . in a humidified atmosphere of 5 % co 2 in air . cultures were depopulated twice a week and a fresh medium in which growth factors and colchicine were added . at various time points , cells were counted after staining with trypan blue and harvested cells were used for enumeration of cd133 + cells following immunophenotype analysis . the cells were washed with a pbs solution containing 1 % bsa and stained ( at 4 ° c . for 30 min ) with fluorescein isothiocyanate ( fitc )- or phycoerythrin ( pe )- conjugated antibodies that are specifically described in the next sections . cells were washed in the abovementioned buffer and analyzed by flow cytometer ( becman coulter ). cells were passed at a rate of up to 1000 cells / second , using a 488 - nm argon laser beam as the light source for excitation . cells stained with fitc - and pe - conjugated isotype control antibodies were used to determine background fluorescence . the percentage of cd133 + cell fraction from total cells was determined by flow cytometry using . pe - anti - cd133 + antibody ( miltenyi biotec ). fold enrichment was calculated by dividing the cd133 + cell percentage of culture growing with cytostatic agent by the cd133 + cell percentage of culture growing without cytostatic agent . since cultures were depopulated twice a week , the culture volume was calculated by multiplying the actual volume with the number of passages . fold expansion was calculated by dividing the cd133 + cell content of the culture by the initial number of cultured cd133 + cells . the percentages of early stem cell subsets were determined from the purified fresh and / or cultured cd133 + cell fraction . cells were dually stained with pe anti - cd34 and fitc anti - cd38 ( dako ) for determination of cd34 + cd38 − cells by flow cytometry analyses with pe anti - cd133 + antibody ( miltenyi biotec ). 3 . 1 . 6 determination of pgp ( mdr1 ) surface expression in stem cell subsets cells were dually stained with pe anti - cd133 + antibody ( miltenyi biotec ) and with mrk16 antibody against pgp and fitc secondary goat anti mouse antibody ( jackson ). stained cells were subjected to facs analyses . total rna was isolated using rneasy mini kit ( quiagen sciences , germantown , md ., usa ), according to the manufacturer instructions . rna expression was measured by rt - pcr cdna was synthesized by reverse transcription system ( promega , madison , wis ., usa ), according to manufacturer instructions . the cdna was used as a template for subsequent amplification of the human mdr1 gene transcript . amplification of the β - actin gene was used as internal control . r : 5 ′- aactgaagtgaacatttctg - 3 ′ for human mdr1 , ( product size 487 bp ) and the primers reaction mixtures contained the following : 0 . 5 μg cdna , 25 pm of each primers and 2 × reddymix pcr master mix ( abgene , surrey , uk ), which contains 1 . 25 u thermoprime plus dna polymerase , 75 mm tris - hcl ( ph 8 . 8 ), 20 mm ( nh 4 ) 2 so 2 , 1 . 5 mm mgcl 2 , 0 . 01 % tween 20 , 0 . 2 mm of each dntp , precipitant and red dye for electrophoresis . cycling parameters were : denaturation in 94 ° c . for 1 min , annealing in 61 ° c . for 1 min , and extension in 72 ° c . for 1 min . samples were cycled using a paltier thermal cycler , ptc - 200 , ( mj research , watertown , mass ., usa ). the rt - pcr , products were separated by 2 % agarose gel electrophoresis and visualized under uv light using ethidium bromide staining . gels were scanned and analyzed by edas 290 electrophoresis documentation and analysis system kodak ). enriched cd133 + stem cell population was prepared from human , ucb - derived mnc by cd133 immunomagnetic beads isolation kit . the mdr1 expression level of ucb - derived cd133 + cells in comparison to ucb - derived mnc and cd133 − cells was studied using rt - pcr and flow cytometry ( fig1 and 2 , respectively ). as shown in fig1 a , mdr1 rna expression level in ucb - derived cd133 + cells is significantly higher than its expression in both ucb - derived mnc and cd133 − cells . the expression level of the house - keeping gene , β - actin , was similar in all the tested sub - populations ( fig1 b ). the purity of cd133 + cells was measured by flow cytometry ( fig2 ). results indicated that approximately 80 % of the cd133 enriched cells were positive for cd133 and for pgp ( mdr1 ) antigens . pgp expression in ucb derived cd133 + cells is significantly higher than its expression in mnc ( 81 . 9 % v . s . 2 . 2 %, respectively ). moreover , most of the ucb - derived cd133 + cells also express pgp . these results indicate that cd133 + stem cells express higher level of mdr1 relatively to the other cells and , therefore , may be further selected by cytostatic agents to enrich their fraction . 3 . 2 . 2 effect of cytostatic agent treatment on enrichment and expansion of cd133 + , stem cell population purified ucb - derived cd133 + cells were cultured in cytokine - supplemented liquid medium in the continuous presence or absence of cytostatic agent ( colchicine ) in a concentration scale ( fig3 ). flow cytometry analysis after 2 weeks expansion ( of three independent experiments ) demonstrated a dose - dependent enrichment of ucb - derived cd133 + cell - fraction by the cytostatic agent colchicine and revealed the optimal concentration of 2 . 5 ng / ml in which the highest cd133 + cells enrichment was achieved ( fig3 ). although intrinsic variability was demonstrated in the ex vivo expansion potential of cd133 + cells derived from different cord blood donors , the use of cytostatic agent substantially increased the renewal potential , resulting in prolongation of cd133 + cell enrichment and expansion ( table 1 and table 3 ). cd34 + hematopoietic progenitors comprise a heterogeneous population . the minority , cd34 + cd38 − are lineage uncommitted progenitors , whereas the majority , cd34 + cd38 + cells , are lineage committed cells . to determine the effect of cytostatic agent on cd34 + cd38 − early stem cell sub - population , purified ucb derived cd133 + cells after 1 week in culture in the absence or presence of colchicine were analyzed by flow cytometry for cd34 and cd38 surface antigen expression . the results ( summarized in table 2 ) indicated an increase in the percentage of cd34 + cd38 − sub - population in cultures treated with cytostatic agent comparing to un - treated cultures . these results demonstrated that colchicine enabled preferential proliferation of early progenitor cells with cd133 + and cd34 + 38 − phenotype , resulting in the observed increased ex vivo expansion . results obtained after expansion of cd133 positive cells for two weeks in the absence or presnece of cytostatic agent ( colchicine ) at 2 . 5 ng / ml . results from three independent experiments of different ucb donors are shown . based on the abovementioned results , the invention provides a process for the selection , enrichment and expansion of stem / progenitor cells subsets that are commonly a constituent of umbilical cord blood , peripheral blood , and bone marrow . the process could be also suitable for selective enrichment and expansion of other types of stem / progenitor cells that predominantly express pgp ( mdr1 ). while the invention has been described with respect to a limited number of embodiments , it will be appreciated that many variations , modifications and other applications of the invention may be made . abbott b l . abcg2 ( bcrp ) expression in normal and malignant hematopoietic cells . hematol oncol . 2003 , 21 : 115 - 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