Patent Application: US-201213369245-A

Abstract:
this invention relates to the use of cells of a medullary or extra - medullary white adipose tissue , in particular of an extra - medullary stromal vascular fraction and / or mature dedifferentiated adipocytes of any origin for initiating the formation of a functional vascularisation .

Description:
it should be understood that these examples are given only by way of illustration of the subject of the invention , of which they in no way constitute a limitation . induction of a neovascularization , by means of mouse svf - cult cells , in a mouse muscle rendered ischemic seven - week - old male c57b1 / 6 or nu / nu mice ( harlan , france ) are raised in a controlled environment ( cycle of 12 hours of light and 12 hours of darkness at 21 ° c .) with free access to water and to the standard food ration . at the end of the experiments , the mice are sacrificed by cervical dislocation under anesthesia with co 2 . the inguinal adipose tissue and the muscle are rapidly removed and treated for the subsequent analyses . the animals are anesthetized by isoflurane inhalation . a ligature is applied to the right femoral artery . the mouse is subsequently injected with 10 6 svf - cult cells , intramuscularly in the limb rendered ischemic . 1 . 1 . 3 isolation of the cells of the adipose tissue stromal - vascular fraction and of the bone marrow cells the bone marrow cells are obtained by washing the tibias and femurs and then isolating the low - density mononuclear cells by centrifugation on a ficoll density gradient ( 34 ). the cells of the stromal - vascular fraction are isolated from adipose tissue according to the protocol of björntorp et al . ( 14 ) with minor modifications . briefly , the mouse inguinal adipose tissue is subjected to digestion with 2 mg / ml of collagenase ( sigma ) in pbs phosphate buffer containing 0 . 2 % of bsa at 37 ° c . for 45 minutes . after elimination of the nonhydrolyzed fragments by filtration through a 100 μm nylon membrane , the mature adipocytes are separated from the pallets of svf - ext cells by centrifugation ( 600 g , 10 minutes ). the svf - ext cells are seeded at a density of 30 000 cells / cm 2 in dmem f12 medium supplemented with 10 % of newborn calf serum ( ncs ). after 6 hours of culture , the nonadherent cells are removed by washing , and then the ( adherent ) cells are cultured for a few days ( 1 to 3 ) before being used ; svf - cult cells are thus obtained . the vessel density was evaluated by high - definition microangiography at the end of the treatment period ( 36 ). the angiographic score is expressed by the percentage of pixels per image that are occupied by vessels , in an area of quantification . the microangiographic analysis is supplemented by evaluation of the capillary density using an anti - body directed against total fibronectin ( 36 ). the capillary density is then calculated in random fields of a defined area , using the optilab / pro software . the functionality of the vascular network after the ischemia is analyzed by laser doppler perfusion imaging , carried out in the mouse as described in j s silvestre et al . ( 36 ). firstly , the angiogenic potential of the adipose tissue was evaluated with mouse svf - cult cells , by comparison with bone marrow mononuclear cells . these cells are prepared from inguinal adipose tissue and placed in cultures so as to obtain a limited expansion for 1 - 3 days ( number of successive passages limited to less than 10 ). the transplantation of 1 × 10 6 svf - cult cells clearly improves the neovascularization of the tissue in hind limbs rendered ischemic , as shown by the 2 . 6 - fold increase in the angiographic score ( fig1 a , p & lt ; 0 . 01 ), the 2 . 3 - fold increase in the doppler tissue perfusion score ( fig1 b , p & lt ; 0 . 001 ) and the 1 . 6 - fold increase in the capillary density ( fig1 c , p & lt ; 0 . 01 ). the degree of neovascularization observed after the injection of 1 × 10 6 svf - cult cells is comparable to that observed after the injection of 1 × 10 6 bone marrow mononuclear cells ( fig1 a - c ). the culture process according to the invention very significantly improves the angiogenic potential of the svf - cult cells , as shown by the very poor neovascularization observed after direct injection of svf - ext cells ( not placed in culture with limited expansion as in the invention ). furthermore , experiments with cells from the vascular stroma originating from brown adipose tissue , known to be more vascularized than white tissue , proved to be fruitless . the mouse svf - ext and svf - cult cells are prepared as specified in example 1 . the corresponding human cells are prepared in a similar manner , from samples of abdominal dermolipectomy or of nephrectomy containing human abdominal subcutaneous tissue , obtained with the patients &# 39 ; consent . the cells are labeled in phosphate buffered saline containing 0 . 2 % of fetal calf serum ; they are incubated with anti - mouse or anti - human monoclonal antibodies ( mabs ) coupled to fluorescein isothiocyanate ( fitc ), to phycoerythrin ( pe ) or to peridinin chlorophyll protein ( percp ), for 30 minutes at 4 ° c . after washing , the cells are analyzed by flow cytometry ( facs calibur , becton dickinson ). the data obtained are then analyzed using the cell quest software ( becton dickinson ). all the antibodies come from bd biosciences , with the exception of cd144 , which comes from serotec . all the statistical analyses are carried out by means of the non - paired t - test using the prisme ™ software ( graphpad software ). the comparative analysis of the phenotype of the svf - ext and svf - cult ( human or murine ) cells was carried out by flow cytometry . since the results obtained with the human and murine cells are comparable , only the results relating to the human cells are presented . the svf - ext cells obtained from subcutaneous human adipose tissue are heterogeneous , as shown by the dispersion diagram in fig2 a . the culturing of these cells for 1 - 3 days under the conditions of the invention results in homogenization of the cell population , as shown by the obtaining of a single cell population , called svf - cult ( fig2 b ). the antigenic phenotype confirms the dispersion diagram . the svf - ext cells are heterogeneous and comprise various populations , in particular hematopoietic cells ( cells positive for the cd45 marker ) and a population of nonhematopoietic cells ( negative for the cd45 marker ) expressing the markers cd34 , cd13 and hla abc ( fig2 c ). the stromal - vascular fraction does not contain a significant proportion of mature endothelial cells , as shown by the absence of labeling with the antibodies directed against ve - cadherin ( cd144 ) and the cd31 marker ( fig2 c ). in the svf - cult population ( conditions of the invention ), the population is composed predominantly of undifferentiated cells , with 90 + 3 % of cells expressing the cd34 marker and 99 + 0 . 2 % of cells being positive for the cd13 and hla abc markers . on the other hand , these svf - cult cells express neither the markers characteristic of hematopoietic cells ( cd45 ) or of monocytes / macrophages ( cd14 ), nor the cd144 and cd31 markers , which are characteristic of differentiated endothelial cells ( fig2 c ). these results show that the cellular expansion for 1 - 3 days in vitro ( or ex vivo ) promotes the proliferation of a homogeneous population of cells ( svf - cult cells ) that possess some surface antigens characteristic of cells with proangiogenic potential , but no surface marker characteristic of differentiated cells . induction of neovascularization , with svf - cult cells , in mouse muscle rendered ischemic , and differentiation of this population into endothelial cells seven - week - old male nu / nu mice ( harlan , france ) are raised under the same conditions as those disclosed in example 1 . the samples of human adipose tissue are identical to those used in example 2 . the human and mouse svf - cult cells are isolated as specified in examples 1 and 2 . the quantification of the neovascularization and the phenotypic analysis are carried out as specified , respectively , in examples 1 and 2 . the effect of the injection ( or transplantation ) of the human svf - cult cells on revascularization is evaluated in immunodeficient nude mice . as for the mouse svf - cult cells , the injection of 1 × 10 6 human svf - cult cells after 15 days of ischemia of the hind limbs makes it possible to obtain a significant increase in the angiographic score and in the cutaneous blood flow ( by a factor , respectively , of 1 . 6 and 1 . 5 when compared with the nude mice rendered ischemic and not treated , p & lt ; 0 . 01 ) ( fig3 a and 3 b ). two possible mechanisms , which are not incompatible , may explain the proangiogenic effects : the release of angiogenic growth factors by the svf - cult cells or a direct contribution of the injected cells by incorporation ( or transplantation ) of the latter into the regenerated vessels . in fact , vegf is detected as being a potential angiogenic factor ( 31 + 8 ng / ml ). thus , in order to evaluate the ability of the svf - cult cells to be incorporated into new blood vessels , immunochemistry experiments were carried out using an antibody specific for the human cd31 marker , which does not react with mouse tissue . numerous cells positive for the cd31 marker forming a layer on the regenerated vessels are demonstrated in the treated hind limb ( fig3 c ). no cell positive for the cd31 marker is detected in the other hind limb which is not treated . the detection of human cd31 + cells strongly suggests that , under in vivo conditions , the svf - cult cells differentiate into endothelial cells and contribute directly to the vessel regeneration . spontaneous differentiation of human svf - cult cells into adipocytes or into endothelial cells , in vitro or in vivo in the matrigel ® matrix the human cells of the extramedullary stromal - vascular fraction ( svf ) are prepared and placed in culture as in example 2 . to test their potential for differentiation in vitro at the clonal level while preserving cellular function , the svf - cult cells are placed in culture in semi - solid medium ( methylcellulose ; 15 ). a primary culture of svf - cult cells is trypsinized , and then seeded at a concentration of 7 × 10 3 cells / ml into 1 . 5 ml of methocult mg3534 , mg , h4534 ( stemcell technologies ) or any other equivalent medium . the cells are cultured for 10 days in order to stimulate their development in terms of cells having an endothelial - type morphology , and then analyzed by immunolabeling . the colonies of the cultures in the presence of methylcellulose are washed with pbs buffer and fixed in a methanol / acetone mixture for 20 minutes at − 20 ° c . the preparations are then blocked in pbs containing 1 % bsa , and incubated for 1 hour with either anti - human cd31 antibodies ( dako , reference m0823 ) or anti - human vwf factor or anti - mouse vwf factor antibodies . the angiogenesis assay , in vivo , using the matrigel ® matrix , is carried out in the following way : the mice are given a subcutaneous injection of a volume of 0 . 5 ml of matrigel ® matrix containing 10 6 svf - cult cells isolated from mouse tissue or from human tissue . on the 14th day , the mice are sacrificed and the angiogenesis is analyzed as described in r . tamarat et al . ( 37 ). for the immunolabeling , the matrigel ® matrices are treated as described in n . nibbelink et al . ( 35 ). sections 5 μm thick are stained with alkaline phosphatase ( bcip / nbt ) after having been incubated with an alkaline phosphatase - coupled antibody from jackson , or else they are stained with diaminobenzidine ( dab ) after having been incubated with a primary antibody and then with a biotinylated secondary antibody ( dako carpinteria , ca ); the anti - human 0 × phos complex iv antibody comes from molecular probes ( eugene , oreg ., usa ). by way of comparison , svf - cult cells are cultured in an adipogenic medium ( björntorp et al ., mentioned above ). the differentiation of the svf - cult cells was analyzed in vitro , in a semi - solid medium that makes it possible to study cell differentiation at the clonal level while preserving cell function ( methylcellulose ), and in vivo after injection of cells associated with a solid matrix ( matrigel ®) under these conditions , the svf - cult cells form a network having a structure in the form of hollow tubes ( fig4 b ). antibodies directed , respectively , against the cd31 marker and against the von willebrand ( vwf ) factor strongly label the svf - cult cells ( fig4 c and 4 d ). when the svf - cult cells are injected in combination with a matrigel ® matrix , the cells form numerous tubular - type structures within the matrigel ® matrix . the presence of erythrocytes in the lumen of these tubular - type structures demonstrates the existence of a functional vascular structure ( fig4 e and f ). the antibodies directed against the cd31 marker and against the vwf marker positively label these structures resembling vessels ( fig4 g and h ). by comparison , the svf - cult cells cultured in an adipogenic medium differentiate into adipocytes ( fig4 a ). all these results show that the svf - cult cells spontaneously exhibit the phenotypic and functional properties of endothelial progenitor cells . the mature human adipocyte fraction , isolated from a sample of adipose tissue as described in example 1 , is washed carefully in dmem - f12 medium supplemented with 10 % of ncs and prepared in the form of a suspension at a concentration of 10 6 cells / ml . a sample of 100 μl of the cell suspension is transferred onto a 25 mm thermanox coverslip and placed in a 35 mm culture dish . the first coverslip is covered with a second , and , after incubation for 15 minutes at ambient temperature , 1 . 5 ml of dmem f12 supplemented with 10 % of ncs are added . after 4 or 5 days of incubation , the adherent cells containing small lipid droplets ( cells of preadipocyte type ) appear ; they become modified into a fibroblast - type morphology devoid of lipid droplets ( hddac cells for human dedifferentiated adipose cells ). these fibroblastic - type cells then begin to actively divide and can undergo several passages without major modification of their characteristics . the dedifferentiated human adipocytes are placed in culture in methylcellulose and analyzed by immunolabeling as described in example 4 . furthermore , their angiogenic potential is analyzed in vivo , after injection in a matrigel ® matrix , as described in example 4 . the angiogenic potential of the svf - cult cells prepared as described in example 3 is analyzed in parallel . alternatively , the dedifferentiated human adipocytes are placed in culture in adipogenic medium ( björntorp et al ., mentioned above ). in order to obtain a homogeneous population of adipocyte precursor cells from adipose tissue and to confirm the existence of a precursor common to adipocytes and to endothelial cells derived from the svf - cult cells , mature adipocytes were dedifferentiated , according to previously described protocols ( 16 ; 17 ; 18 ; 19 ). the mature adipocytes isolated from adipose tissue represent 99 % of a population of floating cells . the only cellular contamination comes from macrophages rich in lipid droplets , with a ratio of a few contaminating cells per 1000 cells . when the adipocytes are placed in culture under the above - mentioned conditions ( 17 ), they initially lose their fatty acids and change their morphology to preadipocyte - type cells and then to fibroblast - type cells which can attach to the coverslip . this morphological change is associated with functional changes , given that the adipocytes also lose their enzymatic content for lipolysis and lipogenesis and also the molecular markers ( 17 ). the homogeneous population of human dedifferentiated adipocytes ( hddacs ) have the ability to proliferate and to differentiate again into adipocytes , when it is cultured in an adipogenic medium ( fig5 a ). the same homogeneous population of human dedifferentiated adipocytes ( hddacs ) cultured in a medium containing methylcellulose forms branchy alignments and structures in the form of a tube ( fig5 b ) and coexpresses , at more than 99 %, the same markers as the svf - cult cells ( cd13 , cd34 and hla abc ), including the vwf marker ( fig5 c ). as is the case for the svf - cult cells , when the hddac cells are injected in association with the matrigel ® matrix , they form numerous tubular - type structures , which contain erythrocytes in their lumen , demonstrating the existence of a functional vascular structure . these results are illustrated in fig6 , which illustrates the plasticity of the cells of the adipocyte line , for obtaining endothelial cells . the adipocyte progenitor cells have the ability to differentiate into adipocytes and to acquire a functional endothelial phenotype . the mature adipocytes can dedifferentiate into progenitor cells with a double proliferative potential . stimulation of neovascularization , with human dedifferentiated adipocytes , in mouse muscle rendered ischemic , and differentiation of these adipocytes into endothelial cells the angiogenic potential of the hddac cells was analyzed in nude mice as for the sv - cult cells ( example 3 ), which serve as comparison . the hddac cells are as effective as the svf - cult cells in restoring the vascularization of the hind limbs rendered ischemic ( fig5 d and 5 e ). as is the case for the svf - cult cells , numerous cells positive for the cd31 marker are identified , which form a layer on the newly formed vessels of the hind limb , into which the hddac cells were injected ( fig5 f ). use of the svf - cult cells to induce neovascularization in an atheromatous context ( murine apoe −/− model ) the angiogenic potential of the svf - cult cells was analyzed in 14 - week - old apoe deficient mice ( apoe knock - out ( apoe ko or apoe −/−); iffa - credo ), as in the c57b1 / 6 mouse ( example 1 ). the angiogenic potential of bone marrow mononuclear cells , in apoe ko mice , is analyzed in parallel , by way of comparison . the control group is given an injection of pbs , under the same conditions . more specifically , the neovascularization process was analyzed by laser doppler microangiography , 4 weeks after femoral occlusion . the statistical analysis was carried out by means of an anova - type variance test for comparing each parameter ( n = 6 for each group ). a bonferroni t test subsequently made it possible to identify the groups causing these differences . a value of p & lt ; 0 . 05 is considered to be significant . the administration of svf - cult cells increases the angiographic score by a factor of 2 ( p & lt ; 0 . 01 ) and the blood flow by a factor of 1 . 5 ( p & lt ; 0 . 01 ), in the hind limb rendered ischemic of the treated apoe ko mice , compared with the nontreated apoe ko mice ( table i ). the angiogenic potential of the svf - cult adipose cells is similar to that of the bone marrow mononuclear cells ( table i ). * the values represent the mean ± standard deviation on a group of 6 animals the treatment of the limb rendered ischemic of the apoe (−/−) mice is effective and promotes angiogenesis / neovascularization . this effect is as effective as the injection of bone marrow mononuclear cells . the svf - cult cells can serve their proangiogenic potential in an atheromatous context . improvement in the angiogenic potential of the svf - cult cells by modification of their redox state the angiogenic potential of the svf - cult cells treated , in vitro , with antimycin ( 40 nm ) and / or pyrrolidine dithiocarbamate ( pdtc ; 0 . 5 mm ) two days before the injection , was analyzed in the model of the mouse with a hind limb rendered ischemic , as described in example 1 . furthermore , after the injection of the svf - cult cells treated with antimycin alone , by adding antimycin to the culture medium , or not treated , the mice were or were not given a daily i . p . injection of antimycin ( 50 μl at 40 nm ). the mice treated similarly with pdtc alone or in combination with antimycin receive no treatment after the injection of cells . the angiogenic potential of the nontreated svf - cult cells , in mice not treated after the injection of the cells , was analyzed in parallel , by way of comparison . the control group was given an injection of ethanol , under the same conditions . the neovascularization process was analyzed by microangiography and , optionally , by laser doppler , 8 days after femoral occlusion . the statistical analysis was carried out by means of an anova - type variance test for comparing each parameter ( n = 5 for each group ). a bonferroni t test subsequently made it possible to identify the groups causing these differences . a value of p & lt ; 0 . 05 is considered to be significant . the effect of the modification of the redox state of the svf - cult cells on their proangiogenic potential was tested with a mitochondrial respiratory chain complex iii inhibitor which induces the production of active oxygen species and a modification of the redox state of cells ( antimycin ), and an antioxidant which limits the production of active oxygen species and the cellular redox state ( pdtc : pyrrolidine dithiocarbamate ). the results are given in tables ii and iii below . effect of the treatment in vitro or in vivo , of the svf - cult cells with the treatment of the svf - cult cells with antimycin alone , before injection into the limb rendered ischemic , has a significantly positive and substantial effect on revascularization , as shown by the 1 . 4 - fold increase in blood flow ( p & lt ; 0 . 05 ; tables ii and iii ) and the 1 . 3 - fold increase in the angiographic score ( p = 0 . 06 ; table iii ). this effect is prevented by an antioxidant ( table iii ), which indicates the involvement of active oxygen species and / or a modification of the redox state in the effects favorable to angiogenesis . on the other hand , antimycin has no effect when it is administered directly to the animal , after the injection of the svf - cult cells into the muscle rendered ischemic ( table ii ). 1 . ailhaud g . et al ., annu . rev . nutr ., 1992 , 12 , 207 - 33 . 2 . castellot j . j . et al ., proc . natl . acad . sci . usa , 1982 , 79 , 5597 - 601 . 3 . bouloumie a . et al ., ann . endocrinol . 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