Patent Application: US-49427504-A

Abstract:
the enrichment of a population of keratinocyte stem cells from a preparation of keratinocytes and kscs , includes pre - enrichment by contacting a sample of keratinocytes and kscs with a collagen - coated culture plate for a sufficient time for kscs to adhere , followed by washing away the non - adherent cells and recovering the adherent cells . the recovered adherent cells are sorted by their expression level of egfr so that cells are recovered that have an expression level of egfr of less than about 50 % of the maximum level of egfr expression , and the recovered cells have an expansion potential of at least 10 9 after about 100 days in culture .

Description:
i / pre - selection of a cell population of “ adherent ” cells , which represents 10 % of the total keratinocytes and essentially includes the kscs . 1 — selection of cells from the population of “ adherent ” cells : the selection method is based on the adhesion ability of keratinocytes . the adhesion substrate chosen is a plastic support on which type i collagen is adsorbed . the preparations of keratinocytes originating from cutaneous samples is applied to the adhesion substrate , followed by incubation at 37 ° c . for a short time . the cells which have not adhered to the support are sampled , then the adherent cells are detached by mild trypsinization and recovered separately . it has been determined that an incubation duration of 12 minutes is suitable for the selection of a population enriched with cells capable of initiating a cell expansion in vitro . the cells having adhered are designated “ adherent ”. 2 — phenotypical characteristics of cells of the population of “ adherent ” cell : analyses of the expression of surface molecules involved in cell adhesion were carried out by flow cytometry on the non - fractionated population , as well as on the population of adherent cells and the population of non - adherent cells ( fig9 ). it was observed that the population of adherent cells is significantly enriched with cells expressing the α6 chain of the integrins ( cd49f ) ( approximately 70 % of the cells have the α6 integrin at their surface ), a criterion associated with the most primitive keratinocytes , whilst the population of non - adherent cells is less rich in this ( approximately 30 % of the cells have the α6 integrin at their surface ). an expression of the α2 and β1 chains of the integrins is detected in a less specific manner at the surface of the cells originating from the total ( non - fractionated ), adherent , and non - adherent populations . 3 — functional characteristics of the cells of the population of “ adherent ” cells : statistical analysis carried out on several independent samples ( n & gt ; 5 experiments ) shows that the population of adherent cells represents only 10 . 4 % of the total keratinocytes , but has a considerable enrichment with clonogenic cells ( fig1 a ). in fact , the realization of short - term clonogenic tests ( 6 days ) on these two cell populations indicates that 2 . 3 % of the keratinocytes originating from the population of non - adherent cells over 12 minutes have the ability to generate clones on a plastic support whilst only 0 . 2 % of the cells of the population of non - adherent cells are clonogenic ( fig1 b ). the content of each one of the populations of cells selected by the method of rapid adhesion on type i collagen in keratinocytes having a strong expansion potential , a property more representative of the physiological function of the kscs , was evaluated . the results obtained from several independent samples ( n & gt ; 5 experiments ) show that the most primitive cells , which express the strongest long - term proliferation potential , are enriched in the population of adherent cells ( fig2 ). in fact , the cells contained in this population make it possible to obtain a cumulative expansion greater than that of the total population . on the other hand , the population of non - adherent cells appears to be mostly constituted by later keratinocytes , and expresses only a lower proliferation potential . ii / screening of a fraction of cells called “ adherent egf - r low ” which represents 20 % to 45 % of the cells of the population of “ adherent ” cells , therefore 2 % to 4 . 5 % of the total keratinocytes , and essentially includes kscs . 1 — fraction of cells composed of 45 % of the cells of the “ adherent ” cell ponulation most weakly expressing the egf - r : the population composed of the adherent cells over 12 minutes on type i collagen was firstly separated into 2 fractions of equivalent size on the basis of the level of expression of the egf - r detectable at their surface . the fraction qualified as “ egf - r low ” contains 45 % of the adherent keratinocytes most weakly expressing the egf - r and the fraction qualified as “ egf - r high ” contains 45 % of the adherent keratinocytes most strongly expressing this receptor ( fig3 ). these experiments were carried out on several independent samples ( n & gt ; 5 ). the results obtained demonstrate in a reproducible manner that the most primitive keratinocytes which have the greatest expansion potential are significantly enriched in the egf - r low fraction ( fig4 ). this correlates with the microscopic observation of the cells at different culture times , which reveals a production of keratinocytes which are small in size , non - differentiated in the longer term in the cultures initiated from the egf - r low fraction ( fig5 ), a criterion associated with the primitive keratinocytes ( 9 ). 2 — fraction composed of 20 % of the cells of the population of “ adherent ” cells most weakly expressing the egf - r : the population of adherent cells was finally separated into 4 fractions of equivalent size on the basis of the level of expression of the egf - r detectable at the surface of the cells . four fractions of equal representativity and each comprising 20 % of the cells of this population were defined as “ egf - r −/+ ”, “ egf - r ++ ”, “ egf - r +++ ”, and “ egf - r ++++ ” fractions ( fig6 ): the fractions called “ egf - r −/+ ” and “ egf - r ++ ” are included in the egf - r low fraction and each contain 20 % of the cells of the adherent population ; the fractions called “ egf - r +++ ” and “ egf - r ++++ ” are included in the egf - r high fraction and each contain 20 % of the cells of the adherent population . an inverse proportionality was observed between the level of expression of the egf - r at the surface of the cells thus selected , and their long - term proliferation potential ( fig7 ). in fact , the greatest cumulative expansion is obtained from the egf - r −/+ fraction whilst the value obtained from the egf - r ++++ fraction proves to be the lowest . the egf - r ++ and egf - r +++ fractions behave in intermediate manner ( n & gt ; 3 independent experiments carried out ). these experimental results validate the possibility of using a mitogenic growth factor receptor ( egf tyrosine kinase receptor ) as a phenotypical marker in order to select a fraction of keratinocytes significantly enriched in kscs . 1 — controlled trypsinization of the cells during successive subcultures : the objective is to limit the stress caused to the cells during the successive detachments and subcultures of the long - term cultures , a problem which could lead to an underestimation of their potential . firstly a controlled proteolytic treatment is carried out ( 0 . 05 % trypsin - 0 . 02 % edta ) which allows the detachment of some of the cells whilst avoiding subjecting them to too drastic a proteolytic treatment which could alter their viability . the cells most difficult to detach for which mild proteolysis has proved ineffective are then subjected to a stronger proteolytic treatment ( 0 . 25 % trypsin - 0 . 02 % edta ), which finally makes it possible to recover all of the population . a comparative study of the impact of different detachment methods on the viability of the cells was carried out . 2 — seeding density of the culture flasks suitable for optimal expansion of the keratinocytes : in order to evaluate which seeding densities are compatible with a good demonstration of the expansion potential of the cells tested , during the initiation of the long - term cultures as well as at each successive subculture , experiments were carried out over a 7 - day period . these made it possible to determine that seeding densities comprised between 2500 and 7000 cells / cm 2 are the most suitable since they make it possible to obtain a cell expansion by a factor of 15 to 20 , whilst lower and higher seeding densities produce poorer results . fig1 a and 1b : representativity and clonogenicity of the cell populations of “ adherent ” cells and “ non - adherent ” cells . fig1 a and 1b show the enrichment in clonogenic cells obtained after selection by the method of adhesion on type i collagen . the total keratinocytes isolated from a skin sample were separated into a cell population called “ adherent ” and a population called “ non - adherent ” by a 12 - minute stage of adhesion on type i collagen . the representativity of each fraction relative to the total cell population was evaluated , as well as their clonogenic keratinocytes content . fig1 a shows the percentage of cells constituting the population of adherent cells relative to the total cells ( hatched column ) and the percentage of cells constituting the population of non - adherent cells relative to the total cells ( filled - in column ). fig1 b shows the percentage of clonogenic cells respectively in the population of adherent cells ( hatched column ) and in the population of adherent cells ( filled - in column ). the results obtained show that the cell population called “ adherent ” is significantly enriched in clonogenic cells , since it represents only 10 % of the total keratinocytes but contains a percentage of clonogenic cells 10 times higher than that of the population called “ non - adherent ”. fig2 : comparison of the expansion potential of the keratinocytes contained in the non - fractionated population , with the “ adherent ” and “ non - adherent ” cell populations . the total keratinocytes isolated from a skin sample were separated into a cell population called “ adherent ” and a population called “ non - adherent ” by a 12 - minute stage of adhesion on type i collagen . the cells contained in the 2 populations , as well as those of the total non - fractionated population , were subjected to a long - term culture test ( cumulative total number of cells produced ), in order to compare their expansion potential . the x - axis represents time ( expressed in number of days ) and the y - axis the cumulative expansion ( number of cells produced on day x / number of cells on day 1 ). the curve with black squares corresponds to the results obtained from the cells of the total population , those with triangles from the cells of the adherent population ( 12 minutes on collagen i ) and those with circles from the cells of the non - adherent population ( 12 minutes on collagen i ). the results obtained show that the cell population called “ adherent ” essentially contains primitive cells with a strong expansion potential , whilst the cell population called “ non - adherent ” is mostly composed of later keratinocytes which only express a reduced potential expansion . fig3 a and 3b : separation of the population of keratinocytes of “ adherent ” cells into 2 fractions on the basis of the level of expression of the egf - r . keratinocytes of the “ adherent ” fraction , pre - enriched in primitive cells by a stage of adhesion on type i collagen , were marked by a fluorescent antibody directed against the egf - r ( antibody directed against the egf - r extracellular domain ). the signal was analyzed in semi - quantitative manner by flow cytometry . fig3 a shows a marking profile representative of the egf - r obtained from keratinocytes of the adherent fraction ( darker surface ), as well as the corresponding control profile ( lighter surface ). the x - axis represents the intensity of fluorescence measured by flow cytometry and expressed on a logarithmic scale . the y - axis represents the distribution of the cells analyzed as a function of the intensity of the fluorescent signal measured . fig3 b makes it possible to visualize the gaussian distribution of the level of expression of the egf - r at the surface of the keratinocytes of the adherent fraction ( x and y - axes identical to those of fig3 a ). the results obtained show a distribution of the signal sufficiently spread - out to allow the screening of cells either expressing the egf - r weakly , or expressing this receptor strongly . the profile presented illustrates a separation of the adherent population into 2 fractions : the one , called “ egf - r low ”, contains 45 % of the adherent cells expressing the lower level of egf - r at their surface ; the other , called “ egf - r high ”, contains 45 % of the adherent cells which express the stronger level of egf - r at their surface . fig4 : comparison of the long - term expansion potential of the keratinocytes contained in the “ egf - r low ” and “ egf - r high ” phenotype fractions . the total keratinocytes isolated from a skin sample were pre - enriched in primitive cells by the method of adhesion on type i collagen , then separated into 2 fractions : the one , called “ egf - r low ”, contains 45 % of the adherent cells expressing the lowest level of egf - r at their surface ; the other , called “ egf - r high ”, contains 45 % of the adherent cells which express the highest level of egf - r at their surface . the long - term expansion potential ( cumulative total number of cells produced ) of these 2 fractions was compared . the x - axis corresponds to the culture time expressed in days and the y - axis to the cumulative expansion already defined . the curve with black squares corresponds to the results obtained from the cells of the egf - r low fraction and that with diamonds to those obtained from the cells of the egf - r high fraction . the results obtained show that the cell fraction called “ egf - r low ” essentially contains primitive cells with a high expansion potential , whilst the cells of the cell fraction called “ egf - r high ” have a lower expansion potential . fig5 a , 5 b , 5 c and 5 d : cultures originating from the “ egf - r low ” and “ egf - r high ” fractions . fig5 a corresponds to a culture originating from the egf - r low fraction ( day 34 of culture ) fig5 b corresponds to a culture originating from the egf - r high fraction ( day 34 of culture ) fig5 c corresponds to a culture originating from the egf - r low fraction ( day 70 of culture ) fig5 d corresponds to a culture originating from the egf - r high fraction ( day 70 of culture ) the total keratinocytes isolated from a skin sample were pre - enriched in primitive cells by the method of adhesion on type i collagen , then separated into two fractions : the one , called “ egf - r low ”, contains 45 % of the adherent cells expressing the lowest level of egf - r at their surface ; the other , called “ egf - r high ”, contains 45 % of the adherent cells which express the highest level of egf - r at their surface . the production of cells which are small in size , a criterion associated with the kscs , was monitored over time by microscopic observation in the cultures originating from each of these 2 fractions . the results obtained show that the cell fraction called “ egf - r low ” ensures a more prolonged production of primitive cells which are small in size than the cell fraction called “ egf - r high ”. fig6 : separation of the population of “ adherent ” keratinocytes into 4 fractions on the basis of the level of expression of the egf - r . keratinocytes of the population called “ adherent ”, pre - enriched in primitive cells by a stage of adhesion on type i collagen , were marked by a fluorescent antibody directed against the egf - r ( antibody directed against the extracellular domain of the egf - r ). the signal was analyzed in a semi - quantitative manner by flow cytometry . the x - axis represents the intensity of fluorescence measured by flow cytometry and expressed on a logarithmic scale . the y - axis represents the distribution of the cells analyzed as a function of the intensity of the fluorescent signal measured . the flow cytometry profile presented illustrates a separation of the adherent population into 4 fractions : the fractions called “ egf - r −/+ ” and “ egf - r ++ ” are included in the egf - r low fraction and each contain 20 % of the cells of the adherent population ; the fractions called “ egf - r +++ ” and “ egf - r ++++ ” are included in the fraction egf - r high and each contain 20 % of the cells of the population of adherent cells . fig7 : comparison of the long - term expansion potential of the keratinocytes contained in the fractions of “ egf - r −/+ ”, “ egf - r ++ ”, “ egf - r +++ ”, and “ egf - r ++++ ” phenotype . the total keratinocytes isolated from a skin sample were pre - enriched in primitive cells by the method of adhesion on type i collagen , then separated into 4 fractions : 2 extreme fractions containing respectively 20 % of the cells expressing the egf - r , “ egf - r −/+ ” and “ egf - r ++++ ” most weakly and strongly , as well as 2 fractions having intermediate “ egf - r ++ ” and “ egf - r +++ ” markings . the long - term expansion potential of these 4 fractions was compared ( cumulative total number of cells produced ). the curve with black triangles corresponds to the results obtained from the cells of the egf - r −/+ fraction , that with diamonds from the cells of the egf - r ++ fraction , that with circles from the cells of the egf - r +++ fraction , that with squares from the cells of the egf - r ++++ fraction . the results obtained show that the cell fraction called “ egf - r −/+ ” is the most enriched in primitive cells having a high long - term expansion potential . fig8 a , 8 b , 8 c and 8 d : ability of the adh +++ egf - r low primitive cells to generate in vitro a pluristratifled epidermis . the culture substrate used is a devitalized human dermis free from epidermis , prepared according to the method described by regnier et al ., ( 1981 ) ( 10 ). the keratinocytes originating from the different adh +++ egf - r high ( 4 or 7 successive culture passages ) or adh +++ egf - r low ( 4 or 7 successive culture passages ) sub - populations , were applied to this dermal substrate and cultured for 6 days in dmem / ham f12 medium ( invitrogen ), containing 10 % of foetal calf serum ( invitrogen ); 10 ng / ml of egf ( bd biosciences , usa ); 0 . 4 μg / ml of hydrocortisone ( sigma chemical co ); 10 − 6 m of isoproterenol ( sigma chemical co . ); 5 μg / ml of transferrin ( sigma chemical co . ); 2 × 10 − 9 m of triiodothyronine ( sigma chemical co . ); 1 . 8 × 10 4 m of adenine ( sigma chemical co . ), and 5 μg / ml of insulin ( sigma chemical co .). the cultures were then placed at the air - liquid interface and continued in the absence of isoproterenol , transferrin , triiothyronine and adenine . the histological analysis of the reconstructed epidermes was carried out 7 days later . ( n . b . adh +++ indicates keratinocytes originating from the adherent population following pre - enrichment ). fig8 a shows a microscopic cross - section corresponding to an epidermis generated from adh +++ egf - r low keratinocytes at passage 4 . the epidermis obtained is correctly formed and differentiated . the scale represented , of maximum size 100 μm , is graduated every 10 μm . fig8 b shows a microscopic cross - section corresponding to an epidermis generated from adh +++ egf - r high keratinocytes at passage 4 . the epidermis obtained is correctly formed and differentiated . the scale represented , of maximum size 100 μm , is graduated every 10 μm . fig8 c shows a microscopic cross - section corresponding to an epidermis generated from adh +++ egf - r low keratinocytes at passage 7 . the epidermis obtained is correctly formed and differentiated . the scale represented , of maximum size 100 μm , is graduated every 10 μm . fig8 d shows a microscopic cross - section corresponding to an epidermis generated from adh +++ egf - r high keratinocytes at passage 7 . the epidermis obtained is degenerative . the scale represented , of maximum size 100 μm , is graduated every 10 μm . the results show that the organogenic potential of the adh +++ egf - r low sub - population is more durable than that of the keratinocytes of the adh +++ egf - r high sub - population . fig9 a , 9 b and 9 c : phenotyping by flow cytometry of the non - fractionated population of keratinocytes and of the adherent and non - adherent fractions obtained after pre - enrichment . immuno - phenotypical markings are carried out on samples of 50 , 000 cells in suspension in pbs / bsa at 0 . 2 %. in order to limit the non - specific binding of antibodies , the cells are firstly incubated in the presence of “ gamma globulins ” originating from the same species as the antibodies used for the markings ( rat γ globulin or mouse γ globulin , jackson immunoresearch laboratories inc ., immunotech , marseille , france ) for 10 minutes at 4 ° c . the cells are then incubated for 30 minutes at 4 ° c . in the presence of an anti - cd49f - fitc antibody ( α6 chain integrin ) produced in rats ( marking ) ( anti - human cd49f - fitc rat igg 2a ; goh3 clone , pharmingen , san diego , calif ., usa ) or a control non - specific antibody of the same isotype ( isotypic control ) ( rat igg 2a - fitc isotypic control , dako , glostrup , denmark ); an anti - cd49b - pe antibody ( α2 chain integrin ) produced in mice ( marking ) ( anti - human cd49b - pe mouse igg 2a ; 12f1 - h6 clone , pharmingen , san diego , calif ., usa ) or a control non - specific antibody of the same isotype ( isotypic control ) ( mouse igg 2a - pe isotypic control ; g155 - 178 clone , pharmingen , san diego , calif ., usa ); an anti - cd29 - pe antibody ( β1 chain integrin ) produced in mice ( marking ) ( anti - human cd29 - fitc mouse igg 1 ; 4b4 clone , coulter corp ., miami , fla ., usa ) or a control non - specific antibody of the same isotype ( isotypic control ) ( mouse igg 1 - fitc isotypic control ; becton dickinson , san jose , calif ., usa ). the cells are then washed in pbs / bsa before being analyzed by flow cytometry . for each sample , the statistics are based on at least 10 , 000 events taken into account . fig9 represents the percentage ( y - axis ) of cells of the non - fractionated population ( before passage on collagen , white column ), the adherent population ( 12 minutes on type i collagen , black column ) and the non - adherent population ( 12 minutes on type i collagen , grey column ) positive to the immunological marking of the α6 , α2 and β1 integrins . these results are the averages and standard deviations obtained for 3 independent experiments . fig9 a shows the percentage ( y - axis ) of cells of the non - fractionated population ( white column ), the adherent population ( 12 minutes on type i collagen , black column ) and the non - adherent population ( 12 minutes on type i collagen , grey column ) positive to the immunological marking of the α6 integrin . fig9 b shows the percentage ( y - axis ) of cells of the non - fractionated population ( white column ), the adherent population ( 12 minutes on type i collagen , black column ) and the non - adherent population ( 12 minutes on type i collagen , grey column ) positive to the immunological marking of the α2 integrin . fig9 c shows the percentage ( y - axis ) of cells of the non - fractionated population ( white column ), the adherent population ( 12 minutes on type i collagen , column noire ) and the non - adherent population ( 12 minutes on type i collagen , grey column ) positive to the immunological marking of the β1 integrin . the results show that approximately 70 % of the cells of the population of adherent cells express the α6 integrin whilst approximately 40 % of the cells of the non - fractionated population express this integrin and only approximately 30 % of the cells of the non - adherent population express it . after elimination of the sub - cutaneous tissue using a scalpel , the skin sample is cut up into fragments of approximately 5 mm × 5 mm , then decontaminated by an antibiotic treatment ( gentamycin ( life technologies ltd , paisley , scotland ), 3 successive 10 - minute baths in dmem culture medium ( life technologies ltd , scotland )). in order to allow the separation of the dermis from the epidermis , the sample is then subjected to a proteolytic treatment ( dispase ( boehringer , roche diagnostics gmbh , mannheim , germany ) overnight at 4 ° c ., then 30 minutes at 37 ° c .). the fragments of epidermis separated from the dermal tissue are placed in a 0 . 05 % trypsin - 0 . 02 % edta solution ( biological industries , kibbutz beit haemek , israel ) ( 15 minutes at 37 ° c .). the preparation is stirred periodically in order to promote the dissociation of the cells . the effect of the trypsin is then neutralized by the addition of a culture medium containing 10 % foetal calf serum ( fcs ) ( sigma - aldrich , st quentin fallavier , france ) ( dmem + 10 % fcs ). the cell suspension is homogenized , then washed in keratinocyte culture medium ( keratinocyte growth medium , kgm ) ( kgm bullet kit , biowhittaker , clonetics corp ., san diego , calif ., usa ). the cells in suspension are counted with a microscope using a malassez cell . the viability of the samples is estimated by the method of exclusion with trypan blue ( life technology , cergy pontoise cedex , france ). ii / enrichment of a preparation of keratinocytes in primitive cells from the basal layer of the epidermis the basal layer of the epidermis contains the most primitive keratinocytes called “ stem cells ”. these cells can be separated from the more mature keratinocytes on the basis of their rapid adhesion property . this stage allows a pre - enrichment of the preparation in primitive cells . the cell suspension is placed in culture flasks “ covered ” with type i collagen ( solution of collagen i ( sigma chemical co ltd , irvine , uk ) diluted by a factor of 2 in pbs , applied to the flasks over 45 minutes , followed by drying after elimination of the surplus ), at a density of 200 , 000 cells / cm 2 . after 12 minutes , the keratinocytes not having adhered are eliminated by washing in pbs buffer . the adherent cells thus selected are detached from the support by a mild trypsinization ( 0 . 05 % trypsin - 0 . 02 % edta ( biological industries , kibbutz beit haemek , israel ) for 3 to 5 minutes at 37 ° c .). after neutralization of the trypsin ( dmem + 10 % fcs ), the cells are recovered , washed , then resuspended in kgm medium . the fraction of the adherent cells selected by this method represents approximately 10 % of the total keratinocytes of the epidermis . the keratinocytes are cultured in kgm medium at a low density ( 1600 , 3200 and / or 4800 cells / cm 2 ) in order to obtain well individualized clones which are easily observable with a microscope . the culture medium is renewed 3 times per week until the experiments are stopped ( 6 to 12 days ). the clones are then fixed and stained , then counted and classified according to the number of cells which compose them . for staining , the preparations are firstly fixed for 30 minutes at ambient temperature with formalin ( formaldehyde in 35 % solution ) ( merck , darmstadt , germany ) diluted to 10 % in pbs buffer ( biowhittaker inc ., walkersville , mass ., usa ), then washed twice with water . the cells are then incubated for 2 minutes with filtered hematoxylin ( staining of the nuclei ) ( sigma diagnostics , st louis , mo ., usa ). after rinsing with water , the preparations are dried in air , then incubated for 5 minutes in 1 % eosin ( sigma diagnostics , st louis , mo ., usa ) in pbs buffer . they are again washed with water , then dried . iv / phenotyping by flow cytometry of the non - fractionated population of keratinocytes , of the adherent and non - adherent fractions obtained after the pre - enrichment immuno - phenotype markings are carried out on samples of 50 , 000 cells in suspension in pbs / bsa at 0 . 2 %. in order to limit the non - specific binding of antibodies , the cells are firstly incubated in the presence of “ gamma globulins ” originating from the same species as the antibodies used for the markings ( rat γ globulin or mouse γ globulin , jackson immunoresearch laboratories inc ., immunotech , marseille , france ) for 10 minutes at 4 ° c . the cells are then incubated for 30 minutes at 4 ° c . in the presence of an anti - cd49f - fitc antibody ( α6 chain integrin ) produced in rats ( marking ) ( anti - human cd49f - fitc rat igg 2a ; goh3 clone , pharmingen , san diego , calif ., usa ) or a control non - specific antibody of the same isotype ( isotypic control ) ( rat igg 2a - fitc isotypic control , dako , glostrup , denmark ); an anti - cd49b - pe antibody ( α2 chain integrin ) produced in mice ( marking ) ( anti - human cd49b - pe mouse igg 2a ; 12f1 - h6 clone , pharmingen , san diego , calif ., usa ) or a control non - specific antibody of the same isotype ( isotypic control ) ( mouse igg 2a - pe isotypic control ; g155 - 178 clone , pharmingen , san diego , calif ., usa ); an anti - cd29 - pe antibody ( β1 chain integrin ) produced in mice ( marking ) ( anti - human cd29 - fitc mouse igg 1 ; 4b4 clone , coulter corp ., miami , fla ., usa ) or a control non - specific antibody of the same isotype ( isotypic control ) ( mouse igg 1 - fitc isotypic control ; becton dickinson , san jose , calif ., usa ). the cells are then washed in pbs / bsa before being analyzed by flow cytometry . for each sample , the statistics are based on at least 10 , 000 events taken into account . the results presented in fig9 indicate that the population of adherent cells on the type i collagen frequently express the α6 integrin ( approximately 70 % of the positive cells ) relative to the non - fractionated population ( approximately 40 % of the positive cells ) and the non - adherent population ( approximately 30 % of the positive cells ). the population of adherent cells therefore has a significant enrichment in α6 - type integrins relative to the non - fractionated population . by comparison , the α2 and β1 integrins are not markers suitable for the evaluation of the enrichment obtained as the majority of the cells of the three populations ( non - fractionated , adherent , non - adherent ) express these integrins . v / marking of the egf receptor ( egf - r ) for analysis and screening by flow cytometry the cells in suspension are centrifuged and taken up in pbs buffer containing 0 . 2 % of bovine serum albumin ( bsa ) ( sigma chemical co ., st louis , mo ., usa ) ( pbs / 0 . 2 % bsa ). in order to limit the non - specific binding of antibodies , the cells are firstly incubated in the presence of “ rat gamma globulins ” ( rat γ globulin , jackson immunoresearch laboratories inc ., immunotech , marseille , france ) for 10 minutes at 4 ° c . the cells are then incubated for 30 minutes at 4 ° c . in the presence of a non - conjugated anti - egf - r antibody ( extracellular domain ) produced in mice ( marking ) ( anti - human egf - r mouse igg 2b ; egfr1 clone , dako , glostrup , denmark ) or a control non - specific antibody of the same isotype as the anti - egf - r antibody ( isotypic control ) ( mouse igg 2b isotypic control , immunotech , marseille , france ). after 2 successive washings in pbs / 0 . 2 % bsa , the cells are incubated in the presence of a secondary antibody produced in rats and coupled with phycoerythrin ( pe ), capable of recognizing the mouse antibodies ( rat anti - mouse igg 2a + b - pe , becton dickinson , san jose , calif ., usa ) ( 30 minutes at 4 ° c .). the cells are again washed twice , before being used for screening by flow cytometry ( facs vantage , becton dickinson , san jose , calf ., usa ). the population of adherent keratinocytes was separated into fractions having different levels of expression of the egf - r and the cells that they contain screened in order to characterize them from a functional point of view . vi / evaluation of the long - term expansion potential of the fractions of keratinocytes screened by flow cytometry the cells of each sub - population , screened by flow cytometry , are subjected to a functional test making it possible to evaluate their long - term expansion ability ( property associated with the most primitive cells called “ stem cells ”). after estimation of the viability by the method of exclusion with trypan blue , 60 , 000 cells of each sub - population are seeded in non -“ covered ” culture flasks , with a surface area of 25 cm 2 . the culture medium ( kgm ) is changed 3 times per week . the cells are detached from their support , counted and reseeded at a rate of 60 , 000 viable cells per 25 cm 2 flask when the cultures reach 70 - 80 % confluence , in order to avoid the cells becoming committed to differentiation following the contact inhibition phenomena . the detachment of the cells is carried out in 2 successive stages . the first is mild trypsinization ( 0 . 05 % trypsin - 0 . 02 % edta ( biological industries , kibbutz beit haemek , israel ) for 3 to 5 minutes at 37 ° c .) which makes it possible to detach approximately 80 % of the cells . the cells which are more difficult to detach for which mild proteolysis has proved ineffective are then subjected to stronger trypsinization ( 0 . 25 % trypsin - 0 . 02 % edta ( biological industries , kibbutz beit haemek , israel ) for 5 minutes at 37 ° c .) which makes it possible to recover all of the cell population . at each passage , the cumulative expansion obtained is calculated for each culture ( number of cells produced on day x / number of cells seeded on day 1 ). the experiments are continued until the cell proliferation potential is exhausted . the results are shown in the form of a cumulative expansion curve which makes it possible to visualize the total proliferation potential of each of the cell sub - populations tested . vii / ability of the epidermal stem cells selected to generate a reconstructed epidermis in vitro another characteristic of the adh +++ egf - r low keratinocytes which was evaluated is their organogenic potential , i . e . their ability to generate in vitro a pluristratified epidermis ( n . b . adh +++ means keratinocytes originating from the adherent population following pre - enrichment ). keratinocytes of the adh +++ egf - r high and adh +++ egf - r low sub - populations were cultured during 7 successive passages . at each passage , some of the amplified cells were sampled in order to be seeded on a dermal substrate . in the early passages ( passage 4 ), the keratinocytes originating from the cultures initiated with adh +++ egf - r high cells and with adh +++ egf - r low cells have showed a good ability to produce a good - quality epidermis ( fig8 a , fig8 b ). in both cases , the histology is characteristic of a correctly formed and differentiated epidermis : i ) a basal layer containing polygonal cells orientated perpendicularly to the dermal substrate , ii ) 3 to 4 layers of spiny cells , iii ) 4 to 6 layers of granular cells , and iv ) a compact stratum comeum comprising anucleated horn cells . at a higher number of passages ( passage 7 ), it was observed that only the keratinocytes originating from the adh +++ egf - r low sub - population remain capable of generating an epidermis ( fig8 c ), the cultures originating from the adh +++ egf - r high sub - population producing no more than a degenerative epidermis ( fig8 d ). these results demonstrate the organogenic potential of the adh +++ egf - r low sub - population which is more durable than that of the keratinocytes of the adh +++ egf - r high sub - population . 1 . aumailley m & amp ; t krieg ( 1996 ). laminins : a family of diverse multifunctional molecules of basement membranes . j invest dermatol . 106 : 209 - 214 . 2 . clark r a ( 1990 ). fibronectin matrix deposition and fibronectin receptor expression in healing and normal skin . j invest dermatol . 94 : 128 - 134 . 3 . couchman j r , austria m r & amp ; a wood ( 1990 ). fibronectin - cell interactions . j invest dermatol . 94 : 7 - 14 . 4 . couchman j r ( 1993 ). hair follicle proteoglycans . j invest dermatol . 101 : 60 - 64 . 5 . mc grath j a & amp ; r a eady ( 1997 ). heparan sulphate proteoglycan and wound healing in skin . j pathol . 183 : 264 - 271 . 6 . sage h ( 1982 ). collagens of basements membranes . j invest dermatol . 79 : 51 - 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