Patent Application: US-10341993-A

Abstract:
a basal serum - free medium and a hematopoietic cell growth and differentiation - promoting serum - free medium based thereon are provided for the maintenance , cultivation , growth and differentiation of erythroid progenitor cells , other hematopoietic progenitor cells , and leukemia cells in which the effects of various growth factor compounds can be quantitatively evaluated . both media are wholly serum - free and contain no intrinsic growth factor compounds . the hematopoietic growth and differentiation medium consists essentially of the basal serum - free medium to which has been added at least one primarily but not exclusively growth - promoting agent selected from heme or hemin , interleukin - 3 and recombinant human stem cell factor , and at least one primarily but not exclusively cell differentiation - promoting agent selected from erythropoietin , insulin - like growth factor and a retinoid .

Description:
the basal serum - free medium and the sf growth and differentiation medium as defined above with their minimum number of components provide media in which useful results on cell viability , growth and differentiation , capable of meaningful scientific interpretation can be obtained . according to the preferred embodiments of the invention , however , additional ingredients are added as discussed herein , to produce an optimal sf medium . the present invention provides a novel improved sf medium for the growth and differentiation of human erythroid progenitor cells from peripheral blood . the activity in this medium of each of its major components has been systematically investigated . it has been found that in the presence of a &# 34 ; clean &# 34 ;, fatty acid - free and globulin - free crystallized albumin , rhu il - 3 and rhu epo were barely sufficient to support the production of erythroid bursts ; the bursts were small , developed in minimal numbers , and required benzidine staining for their confirmation . full maturation of the erythroid bursts necessitated the addition of hemin , which made possible the direct scoring of hemoglobinized bursts in situ . optimal growth was attained when igf - i and retinyl acetate were added . it is believed that this is the first sf medium which , using a bpa - free bsa and a defined source of bpa , fully supports the production of bursts in vitro by circulating erythroid progenitor cells from the normal human adult . it is very important to make the erythroid culture medium completely free of serum products inasmuch as these introduce not only undefined activities that promote growth , but also undefined inhibitory factors , as our results obtained with a &# 34 ; dirty &# 34 ; albumin and serum illustrate in the following examples . a medium containing fbs and cohn &# 39 ; s fr . v bsa , even after it had been improved by the addition of hemin , retinyl acetate and igf - 1 , had a 33 - 72 % lower day - 14 colony - forming efficiency than our own improved sf medium . this is demonstrated in example 2 below , and illustrated in accompanying fig2 . as described above , the first aspect of the invention described and claimed herein comprises basal serum - free medium in which hematopoietic cells will not proliferate . the first component thereof is minimum essential medium , a term well understood by those skilled in the art of cell culturing . specific preferred examples thereof include α medium , imdm , and iscove &# 39 ; s modified dulbecco &# 39 ; s medium . such minimum essential medium is then modified according to the invention to render it specifically suitable for the maintenance of hematopoietic cells and leukemia cells . for this purpose , there are added to it the four deoxyribonucleosides , the four ribonucleosides and the other additives referred to above . the phosphatidyl choline which is used is suitably l - α - phosphatidyl choline dipalmitoyl synthetic . the c 16 - c 24 unsaturated fatty acid is suitably linoleic acid or oleic acid . the cholesterol component is suitably porcine liver cholesterol . the antioxidant is suitably beta - mercaptoethanol or α - thioglycerol , or most preferably a mixture of both . one or more antibiotic substances are also included , to protect the medium against infection by airborne or other stray bacteria . suitable such antibiotics include sodium penicillin g and streptomycin sulphate . preferably a combination of these two antibiotics is used . the semi - solid matrix material is suitably a carbohydrate such as methylcellulose , agar or agarose , with methyl - cellulose being most preferred . suitable relative amounts of the various constituents of the basal serum - free medium are , in final molarity or in grams per milliliter of final volume : each deoxyribonucleoside : from 1 μg / ml to 100 mg / ml , and preferably from 5 to 10 mg / ml ; each ribonucleoside : from 1 μg / ml to 100 mg / ml , and preferably from 5 to 10 mg / ml ; l - glutamine : from 0 . 1 mm to 20 mm and preferably from 1 to 2 mm ; albumin : from 1 mg / ml to 100 mg / ml and preferably from 10 to 30 mg / ml ; transferrin : from 1 μg / ml to 1 mg / ml and preferably from 27 to 270 μg / ml ; phosphatidyl choline : from 0 . 1 μg / ml to 100 μg / ml and preferably from 5 to 10 μg / ml ; fatty acid : from 0 . 1 to 100 μg / ml and preferably from 2 to 10 μg / ml ; cholesterol : from 0 . 1 to 100 μg / ml and preferably from 1 to 10 μg / ml ; antioxidant : from 1 μm to 1 mm and preferably from 0 . 1 to 0 . 2 mm ; antibiotic : from 0 . 1 μg / ml to 250 μg / ml and preferably from 25 to 100 μg / ml ; matrix material : from 0 . 1 mg / ml to 10 mg / ml and preferably from 1 to 3 mg / ml . in making up the medium for optimal cell growth and differentiation , as determined by the maximum yield of colonies and number of cells per colony over a given period of time , from a given number of cells plated out , in an easily studiable condition , together with a maximized degree of hemoglobinization by the cells , the additional ingredients should be added to the basal serum - free medium , in the following approximate amounts : hemin or heme : from 50 μm to 1 mm and preferably from 200 μm to 500 μm ; retinoid : from 0 . 1 nm to 0 . 1 mm and preferably from 1 mm to 100 nm ; stem cell factor : from 0 . 5 ng / ml to 1 μg / ml and preferably from 25 to 100 ng / ml ; interleukin - 3 : from 0 . 1 ng / ml to 1 μg / ml and preferably from 5 to 25 ng / ml ; erythropoietin : from 0 . 1 ng / ml to 1 μg / ml and preferably from 10 to 30 ng / ml ; insulin - like growth factor : from 1 pg / ml to 1 μg / ml and preferably from 20 to 100 ng / ml . the problem of providing a defined bpa required for the growth of erythroid bursts has been a major stumbling block in the development of truly sf media . most recipes are in fact merely serum - deprived media , 5 , 10 - 13 as they call for the use of cohn &# 39 ; s fr . v bsa , which is known to be contaminated with bpa , as well as lipids , small proteins and other low molecular weight molecules . 13 delipidation with activated charcoal not only removes lipids but other small molecules as well , and has resulted in the loss of the erythropoietic activity associated with albumin . 6 , 7 . a source of bpa then had to be provided , either by the addition of leukocyte conditioned media 7 or serum lipoproteins of low to intermediate density . 14 investigators have reported bpa - like effects in their media , even though the albumin had been treated with activated charcoal ; the source of such bpa was traced to the semi - purified epo preparations utilized 10 and , when rhu epo was used , to the accessory cells of bone marrow ( monocytes , t - cells , fibroblasts ) 7 . it has been reported that accessory cells of bone marrow produce bpa . 7 , 15 , 17 when we did not remove accessory cells from our pb mnc preparations , as described in example 1 below , these cells did not appear to provide significant amounts of bpa in our system , judging from the fact that in the absence of added il - 3 and hemin , no bursts developed ( fig1 ). however , production of erythroid bursts by progenitors among pb mnc was completely abolished by removal of adherent cells in serum - free medium containing il - 3 and hemin but no scf ; addition of rhu scf partially restored burst formation . thus either non - adherent erythroid progenitor cells have a requirement for another growth factor ( s ) normally secreted by adherent cells , or else intimate cell - cell contact interactions between adherent cells and erythroid progenitors are required for burst production . in our novel culture system we have confirmed the well - documented bpa - like activity of il - 3 for human erythroid growth in vitro ; its introduction into our medium as a defined bpa made up for the loss of an undefined bpa resulting from our use of a &# 34 ; clean &# 34 ; bsa preparation and recombinant epo . in the absence of scf , the bpa in our sf culture medium appears in fact to be supplied by two defined entities , rhu il - 3 and purified hemin as demonstrated in example 1 and fig1 and example 2 and fig2 reported below . hemin ( iron protoporphyrin ix ) is a relatively small ( 652 dalton ) molecule , known to act in the process of normal erythropoiesis . in addition it has distinct proliferative effects in hematopoietic culture . hemin has been shown to enhance the proliferation of both the multipotential stem cells cfu - gemm and the early erythroid progenitor cells bfu - e in serum - containing or serumdeprived ( i . e . containing undefined bpa &# 39 ; s ), &# 34 ; sf &# 34 ; cultures of murine and human bone marrow . recently , a novel growth factor termed stem cell factor , scf , has been reported to stimulate primitive pluripotential hemopoietic progenitors in mice 18 , 19 , rats 20 and humans 18 - 21 . several groups have isolated scf , also termed mast cell growth factor ( mgf ) and have shown it to be the ligand for the c - kit receptor protein . the product of the c - kit proto - oncogene has tyrosine kinase activity and maps to the mouse w locus on chromosome 5 . these findings demonstrated that the genetic anemias of w / w v and sl / sl d mice were interconnected , with the former having defects in the c - kit receptor and the latter defective production of scf , the c - kit receptor ligand being the product of the steel ( sl ) locus . besides the soluble forms of scf , membrane - bound forms have also been reported 22 . serum - containing ( sc ) studies of the effects of scf on highly enriched , adherent cell - deprived , human bone marrow ( bm ) bfu - e preparations have shown that it can potentiate primitive hemopoietic colony - formation , and have suggested that it synergises with diverse cytokines , among which are epo and il - 3 20 - 21 . no comparable studies exist for human bfu - e from peripheral blood . the novel serum - free ( sf ) growth and differentiation medium of the present invention is capable of supporting the growth of circulating erythroid progenitors . however , even though the medium is capable of supporting burst formation at low cell density , removal of adherent cells from the peripheral blood mononuclear cell ( pb mnc ) suspensions prevents burst formation in sf medium . in accordance with another feature of the present invention , it has been found that recombinant human stem cell growth factor ( scf ) can replace the stimulatory activity of adherent cells and thus at least partially restore burst - formation . this is further described in example 9 below . the present invention provides strong indications that the effect of igf - i on burst formation is not limited simply to potentiation of the effect of any epo that might still be present in the medium . it has been shown that when defined bpas ( il - 3 and hemin ) are provided , the basal serum - free medium according to the invention is entirely free of any epo - like activity capable of inducing erythroid differentiation . moreover the present work shows that in the presence of an antibody to epo , igf - i is fully capable of supporting erythropoietic burst production . since experiments according to the present invention were unable to obtain growth of bfu - e with a combination of epo and igf - i in the absence of il - 3 , hemin , and retinyl acetate , it appears that igf - i does not have a bpa - like effect upon early bfu - e . this fits with the known incapacity of igf - i to act as a competence factor , i . e . it cannot recruit quiescent cells into the cell cycle . we have also been able to confirm in our culture system that rhu epo has no bpa - like activity either . 8 thus igf - i can function in vitro on its own as an erythroid differentiation factor for bfu - e from normal peripheral blood . igf - i must also function as a growth factor for the progenitors of burst - component colonies . we have found that the cellularity of these colonies was greatly increased by igf - i whether or not epo or retinyl acetate were present . this indicates that igf - i can stimulate cell proliferation within the developing burst , either by promoting self - renewal of the early bfu - e itself , or by targeting progenitor cells that are later in the differentiation sequence than the early bfu - e and promoting their proliferation . the work in support of the present invention appears to represent the first demonstration of epo - like activity of rhu igf - i on burst and colony formation from human pb mnc in a demonstrably sf medium . the sf medium of the present invention provides the opportunity to investigate the sensitivities of human circulating erythroid progenitors to the growth factors that are active in erythropoiesis in vitro . we found that these progenitors required a 100 - fold higher concentration of igf - i than of epo to reach maximal stimulation ( fig8 ). we further found that the result of the combined addition of epo and igf - i was only partially additive ( fig1 ). the combination of epo and igf - i in the presence of il - 3 and hemin gave a greater day - 14 colony - forming efficiency than either factor alone , but less than the sum of their separate effects ( fig . 1 ). this observation suggests that there may be two classes of bfu - e - derived erythroid progenitors which overlap with respect to their sensitivities to each of these factors . the observation that igf - i ( in the presence of il - 3 ) can completely replace epo constitutes strong evidence for the existence of an igf - i - dependent mechanism for proliferation and differentiation of normal circulating bfu - e , which can operate in vitro when epo levels are low or absent . that such an epo - independent mechanism might also function in vivo is suggested by the finding that plasma from a patient with chronic renal failure and low epo levels could nevertheless support erythropoiesis because of its igf - i content . 23 we have found that the addition of vitamin a ( retinyl ) acetate or all - trans retinoic acid at physiological concentration 24 , 25 ( in the absence of epo ) greatly enhances the effect of igf - i on erythropoiesis in vitro . even at ineffective concentrations of igf - i added to the medium , this vitamin is responsible for the expression of a background number of day - 14 erythroid colonies , and it synergizes with hemin in stimulating production of increased numbers of these colonies . retinyl acetate or all - trans retinoic acid thus appears to act as a potentiator of the functions of other growth factors in the sf medium . the invention is further described for illustrative purposes in the following specific examples , constituting the &# 34 ; most preferred embodiments &# 34 ;. after informed consent , peripheral blood was obtained by venipuncture from healthy donors and was immediately placed in α - minimal essential medium ( α - mem ) containing 2 % fetal bovine serum ( fbs ) (# sp80219 , gibco , grand island , n . y .) and 10 u / ml of preservative - free sodium heparin (# 820 5077 mf , gibco ). peripheral blood mononuclear cells ( pb mnc ) were separated by ficoll - hypaque ( pharmacia , montreal , p . q .) density - gradient centrifugation at 400 × g for 40 min . adherent cells were removed by 90 min exposure to the plastic of 50 ml falcon tissue culture flasks (# 3013 , becton dickinson , rutherford , n . j .) in the presence of 2 % fbs + α - mem , without agitating the flask at the time of removal of the cell suspension . cell suspensions were washed three times ( 400 × g , 10 min ), the first wash in the presence of 2 % fbs + α - mem , and the subsequent two washes in α - mem alone . cell counts were made with the trypan blue ( 0 . 4 %, # 630 - 5250 , gibco ) dye - exclusion method . serum - free culture of pb mnc was performed with a modification of the technique previously described , 6 in flat bottomed 1 . 5 × 1 . 0 cm plastic wells (# 76 - 000 - 04 , flow laboratories , maclean , va ., now discontinued ; nunclon delta , nunc , roskilde , denmark , sl - 24 well multidishes -# 1 - 43982 can also be used ). between 5 × 10 4 and 1 × 10 5 pb mnc were plated in 0 . 5 ml of final culture medium containing α - mem , 0 . 8 % of 1 , 500 centipoise methylcellulose ( methocel a4m , premium grade , dow chemical co ., midland , mich . ), 1 % fatty acid - and globulin - free crystallized bsa i ( sigma ) which was subsequently deionized with analytical grade ion exchange resin ( ag 501 - x8 ( d ), biorad labs , richmond , calif . ), 2 × 10 - 4 m β - mercaptoethanol ( bdh biochemicals , poole , england ), 270 μg / ml fully iron - saturated bovine transferrin ( sigma ), 7 × 10 - 7 m d - α - tocopherol ( clinic products , windsor , ont . ), 8 μg / ml l - α - phosphatidyl choline dipalmitoyl synthetic ( sigma ), 5 . 6 μg / ml oleic acid ( sigma ), 7 . 8 μg / ml porcine liver cholesterol , grade 1 ( sigma ), 10 μg / ml of each of the four deoxy - and ribonucleosides ( sigma ), 2 mm l - glutamine ( sigma ), 100 u / ml penicillin g and 50 μg / ml streptomycin sulfate ( gibco ) ( this combination of ingredients constituting a specific example of a basal serum - free medium according to the invention ). e . coli - derived recombinant human somatomedin - c ( referred to as rhu igf - i ) having the natural amino acid sequence was purchased from amersham , oakville , ont ., or from amgen , thousand oaks , calif . the recombinant human preparations of erythropoietin ( rhu epo ) and interleukin - 3 ( rhu il - 3 ) were from amgen . bovine type 1 hemin ( ferric chloride protoporphyrin ix ) was purchased from sigma (# h - 2250 ), with ˜ 97 % purity by spectrophotometric assay . retinyl acetate was from nutritional biochemicals corp ., cleveland , ohio . recombinant human scf was a gift from dr . a . bernstein ( s . linenbeld research inst ., ont .). the final concentrations of these growth and differentiation factors used in the optimal medium were as follow : rhu igf - i 0 . 26 μg / ml to 2 . 6 μg / ml ( 3 × 10 - 8 to 3 × 10 - 7 m ), rhu epo 3 . 0 u / ml ( 27 ng / ml , 9 × 10 - 10 m ), rhu il - 3 5 . 5 ng / ml ( 2 × 10 - 10 m ), rhu scf 100 ng / ml ( 3 nm ), retinyl acetate or all - trans retinoic acid 3 × 10 - 8 m , and hemin 65 . 2 or 163 . 0 μg / ml ( 1 . 0 or 2 . 5 × 10 - 4 m ). petri dishes containing the wells were incubated at 37 ° c . in a humidified atmosphere and 5 % co 2 for 7 to 9 days for erythroid colonies and 14 to 16 days for erythroid bursts . all erythroid colonies and bursts were scored by in situ observation with an inverted microscope . polyclonal rabbit anti - epo antibody hcc - 1400 was obtained from terry fox laboratory , vancouver , b . c ., and used at a final dilution of 1 : 50 , at which concentration it neutralizes 6 u / ml of human epo . an erythroid burst is defined as either a single colony or a cluster of &# 34 ; burst - component &# 34 ; or &# 34 ; day - 14 &# 34 ; colonies , each having at least 50 hemoglobinized cells , scored at days 14 to 16 of growth . hemoglobinized colonies with & lt ; 50 cells and separated from one another could also be observed ; even when they appeared to belong to a single burst , their counts were not included in the counts of bursts . contiguous colonies which collectively comprised at least 50 cells were scored as a burst . burst - component colonies , also referred to as &# 34 ; day - 14 colonies &# 34 ; or &# 34 ; subcolonies &# 34 ;, were easy to count and their numbers could be considered reliable even when crowding of the cultures made the distinction between individual bursts questionable . comparison of the effects of rhu epo and rhu igf - i in the presence or absence of rhu il - 3 and hemin in the initial experiments , we examined the effects growth of several growth factor permutations upon the of erythroid day - 14 burst - component colonies from pb mnc . accompanying fig1 presents the results graphically . on fig1 triplicate or sextuplicate determinations from one or several experiments are expressed as means ± s . e . faf , gf bsa ( 1 %) present together with the entirety of the basal serum - free medium components as described under &# 34 ; clonal cell culture &# 34 ; above , was unable , by itself , to support the development of burst - component colonies ( fig1 bar 1 ) and the same negative result was obtained when epo was added ( fig1 bar 2 ), confirming that the bsa preparation employed was operationally devoid of a bpa - like activity and that the basal serum - free medium by itself does not support growth of primary hematopoietic cells . hence , in order to obtain burst - formation we would need to add an exogenous source of bpa to the medium . the same results showed that the recombinant epo preparation was equally devoid of a bpa contaminant , which is often present in semi - purified epo preparations . 10 this contrasted with the results obtained when rhu epo alone was added to cohn &# 39 ; s fr . v bsa ( 1 %). under these conditions 18 ± 2 . 5 single - colony bursts developed per 2 × 10 5 cells . however , the results thus obtained with fr . v bsa were erratic , suggesting that this source of albumin has variable quantities of contaminating bpa and may , at times , have none , at least operationally . the addition of a defined , exogenous bpa - like activity in the form of 5 . 5 ng / ml of rhu il - 3 was also unable to promote erythroid burst - formation in the absence of any added epo ( fig1 bar 3 ), demonstrating that the bsa employed is also devoid of an epo - like activity . in fact , examination of these cultures showed the presence of morphologically erythroid - like colonies that failed to mature , along with granulocytic and monocytic colonies . the addition of 0 . 1 mm hemin to this basal level of il - 3 did not elicit erythroid differentiation of these colonies either ( fig1 bar 4 ), and addition of rhu epo and rhu il - 3 showed the presence of a few day - 14 erythroid colonies ( or single - colony bursts ) scorable only with acid benzidine ( fig1 bar 5 ). addition of 0 . 1 mm hemin to the combination of epo and il - 3 dramatically increased the colony - forming efficiency ( 7 - fold , fig1 bar 6 ). under these conditions , colonies could now be scored directly in the microscope by their orange colour alone . a comparison of bars 6 and 7 in fig1 shows that , in the presence of hemin and il - 3 , addition of igf - i could effectively replace epo with respect to burst formation . however , when epo and igf - i were added together ( at the previously established concentrations ), their combined effect was greater than that obtained with either factor alone , but less than the sum of their separate effects ( fig1 bar 8 ). it was reported in 1982 that some retinoids can significantly increase erythroid burst formation . 26 hence , in order to improve the efficiency of the medium , we tested the effects of combining hemin and retinyl acetate with the full complement of recombinant factors , epo , il - 3 , and igf - i , except for scf . we had in this medium previously found that , by themselves , retinyl acetate and all - trans - retinoic acid also had an epo - like activity , in that they appeared to function as differentiation factors . 27 , 28 fig2 also shows that with respect to numbers of burst - component colonies , the effect of a combination of hemin and retinyl acetate ( bar 4 - bar 1 ) was significantly greater than the sum of their separate effects [( bar 2 - bar 1 )+( bar 3 - bar 1 )], suggesting a synergism , but this effect was not detectable for bursts ( bar 4 - bar 1 ) vs [( bar 2 - bar 1 )+( bar 3 - bar 1 ) ]. together , hemin and retinyl acetate induced a 4 . 8 - fold increase in the number of burst - component colonies and a 3 . 5 - fold increase in the number of bursts . this contrasts with a 2 . 8 - fold increase in burst - component colonies and a 2 . 6 - fold increase in bursts obtained by stimulation with retinyl acetate only . however , in the presence of serum ( 10 % fbs ) and a &# 34 ; dirty &# 34 ; bsa ( cohn &# 39 ; s fr . v ), no synergism was apparent ( fig2 bars 6a and 7a for bursts , and 6b and 7b for colonies ). the highest number of burst - component colonies in serum was significantly lower than that in sf medium ( bar 6 vs bar 4 ), suggesting the presence of inhibitory factors in serum . similar results have been obtained with hemin together with all - trans retinoic acid atra ( fig3 ). to find out whether atra behaves in the same way as retinyl acetate , we substituted atra for ra ( at 30 nm ) in the same sf medium . we found that in the absence of any added bpa - like activity ( il - 3 or hemin ) and of any epo - like activity ( epo or igf - i ), atra was not capable by itself of supporting early colony growth and differentiation ( fig3 bar 1 ). in the presence of epo , the normal regulator of erythireporetic differentiation , atra did not provide a bpa - like activity in that no day - 16 colony formation was detected ( fig3 bar 2 ). in the presence of rhu - il - 3 , erythroid colonies could be morphologically recognized but no hemoglobinization could be detected ( fig3 bar 3 ). substituting optimal concentrations of epo and igf - 1 for atra ( on an il - 3 background ) yielded a comparable number of day - 16 colonies ( not significantly different from bar 5 , fig3 ; t = 2 . 46 for 2 degrees of freedom and p & gt ; 0 . 05 ) and a much stronger degree of hemoglobinization ( fig3 bar 6 ); still , other erythroid - like colonies could be observed which had not matured . however , colony maturation was dramatically increased when atra was added to complete the sf medium ( fig3 bar 7 ), as it induced a 4 - fold increase in the number of hemoglobinized d16 colonies . these findings show that atra functions in a manner analogous to ra , and strongly suggest that these retinoids act as essential co - factors of erythroid differentiation . the relation between the number of bursts produced and the number of pb mnc plated was investigated . the results in fig4 show that , in the presence of 5 . 5 ng / ml of rhu il - 3 , 3 . 0 u / ml rhu epo , 3 × 10 - 8 m igf - i , 0 . 1 mm hemin and 3 × 10 - 8 m retinyl acetate , the number of bursts varied linearly with the cell concentration between 1 and 10 × 10 4 cells / 0 . 5 ml plated . the regression line relating these variables extrapolated through a point not significantly different from the origin , suggesting that the medium is performing satisfactorily for burst production . the efficiency of burst - production was approximately 1 burst / 1 , 300 / pb / mnc plated . bfu - e required for rhu il - 3 and hemin in the improved sf medium a titration of rhu il - 3 ( fig5 ) in the sf medium showed that the number of day - 14 burst - component colonies varied linearly with the log of rhu il - 3 concentration . in the absence of added il - 3 , no bursts could be detected ; the erythroid colonies that were present had a soft orange colour and had & lt ; 50 cells . at il - 3 concentrations of up to 0 . 28 ng / ml , hemoglobinized bursts were practically absent , but burst - component colonies could still be recognized by their morphology and a faint orange colour , even though their number and the cellularity of each colony were very low . between 0 . 28 and 5 . 5 ng / ml , colony numbers increased , they had a mean cellularity of about 100 cells / colony , and a few , much larger colonies could also regularly be observed . a plateau of activity was reached at 5 . 5 ng / ml of rhu il - 3 , without any toxic effects . from these data , we chose to use 5 . 5 ng / ml of rhu il - 3 as our standard concentration of added bpa - like activity for the improved sf medium . in the improved sf medium , hemin facilitated visualization of hemoglobinized colonies by increasing the intensity of their colour : at 10 μm , hemin did not increase the number of bursts or their colonies ( fig6 ), but conferred on them a stronger hemoglobinization , their colour now being a definite orange instead of the soft , pale orange colour of the colonies present in the absence of hemin ; at 100 μm hemin , the colonies became redder , and at 250 μm they seemed to attain a maximal degree of redness . besides this qualitative effect , which greatly facilitated in situ scoring , both the number of day - 14 bursts and the number of day - 14 burst - component colonies increased linearly with increasing hemin concentration between 10 and 250 μm ( fig6 ). over this range , the number of day - 14 burst - component colonies increased 4 - fold and the number of day - 14 bursts increased 2 - fold . the optimal concentration of hemin for both maximal hemoglobinization and number of day - 14 bursts and their component colonies derived from 10 5 pb mnc was 250 μ m . at this cell density , the 500 μm concentration of hemin was too high for purposes of colony and burst enumeration ; colony growth became practically confluent as early as day - 12 , and by day - 14 some lysis was detectable . still higher concentrations tested ( 1 mm ) were toxic and resulted in widespread lysis of cells in the colonies . titration of rhu igf - i in the absence of epo , with and without retinyl acetate next , we titrated the number of burst - component colonies against rhu igf - i concentration , in the absence of epo or retinyl acetate ( fig7 ). at low concentrations (& lt ; 10 - 10 m ), the number of erythroid burst - component colonies was significantly lower than would have been expected if we extrapolated back the linear component of the response curve between 10 - 10 and 3 × 10 - 10 m igf - i ( fig7 lower curve , closed squares ). this suggests that a threshold concentration of igf - i may be necessary for erythroid burst - component colony formation to be detected . titration of burst - component colonies against rhu igf - i concentration , using the same pb mnc but performed in the presence of retinyl acetate ( also without epo ), showed that the effect of igf - i was proportional to the log of its molarity , between 3 × 10 - 11 and 3 × 10 - 8 m , without having reached a plateau of activity at the highest concentration tested ( fig7 upper curve , closed circles ). accordingly , the half - maximal stimulation must be & gt ; 3 × 10 - 10 m (& gt ; 2 . 6 ng / ml ). at 3 × 10 - 12 m , rhu igf - i appeared to be ineffective against a background of early erythroid colonies which were apparently promoted by the presence of the retinoid at a concentration of 3 × 10 - 8 m . once again , retinyl acetate at the highest effective concentration greatly enhanced (˜ 5 - fold ) the number of burst - component colonies scored . comparison of the titration of rhu epo and rhu igf - i under optimal sf conditions for burst - production the effects of either epo or igf - i on the production of burst - component colonies , each titrated in the improved sf medium , but in the absence of the other gf , were compared . the results are expressed as raw data ( fig8 a and 8b ) and as percentage of the maximal stimulation observed with each gf ( fig8 c ). the epo dose - response curve ( continuous line joining the closed circles ) is the mean of 3 separate experiments from different donors . epo in our medium showed the familiar type of dose - response curve , plateauing at 3 - 6 u / ml or 9 - 18 × 10 - 10 m ( epo mol . wt . 34 , 000 ). below 2 × 10 - 11 m rhu epo , the number of burst - component colonies lay within the background level induced by retinyl acetate alone , in the absence of any epo or igf - i . from this normalized epo curve , the half - maximal stimulation of the hormone upon burst - component colony formation was 2 . 7 × 10 - 10 m ( 0 . 89 u / ml ), which corresponds to an epo protein concentration of 8 . 1 ng / ml . a rhu igf - i titration from 2 experiments with different donors is also shown in fig8 b ( one in closed circles , the other in open squares ). each is expressed both as raw data ( fig8 b ) and as a percentage of its own maximal effect ( fig8 c ). it is apparent that the concentration of rhu igf - i needed to reach maximal activity ( at 10 - 7 m ) is nearly two logs of molarity higher than what we have observed with epo in the same medium . between 3 × 10 - 11 and 3 × 10 - 7 m rhu igf - i , the number of burst - component colonies varied proportionately with the log of igf - i concentration , its half - maximal stimulation lying around 6 . 5 × 10 - 10 m igf - i , which corresponds to a protein concentration of 5 . 6 ng / ml . it is worth remarking that the overall numbers of burst - component colonies obtained by stimulation with each growth factor alone , as seen at plateau concentrations , were not significantly different from one another ( a range of 462 to 503 colonies / 10 5 mnc for all 5 experiments ). titration of rhu epo and rhu igf - i expressed in terms of the number of bursts produced ( fig9 a ) and as a percentage of their maximal effect ( fig9 b ) showed that the differential sensitivities to the respective growth factors were approximately the same as those observed for burst - component colonies , both for the proportional and the plateau components of the curves . from the actual numbers obtained in all these experiments we calculated the number of burst - component colonies per burst both at the background level of retinoids ( i . e . in the absence of both igf - i and epo , or at their ineffective concentrations ), and at their plateau levels . the value of background averaged 1 . 7 colonies ( with ≧ 50 cells each ) per burst ( 77 colonies per 42 bursts / 10 5 mnc ); the values at plateau averaged 4 colonies per burst ( 467 colonies per 116 bursts ) at maximal igf - i stimulation and 5 . 4 colonies per burst ( 488 colonies per 91 bursts ) at maximal epo stimulation . this shows that one of the actions common to both growth factors is to enhance the proliferation of bfu - e - derived progenitors within each burst . these experiments showed , under optimal conditions , that igf - i can substitute for epo in the production of erythroid bursts . an investigation was conducted to determine whether erythroid bursts would develop under the influence of igf - i in the presence of antibody directed against epo . in fig1 , bar 1 shows the number of bursts obtained in the full sf medium but lacking igf - i , with 6 . 0 u / ml of epo . comparison with bar 4 , in which anti - epo antibody was added to the same medium containing epo , shows that the antibody was effective in inhibiting burst formation that was dependent on epo . bar 3 shows that burst formation under the influence of igf - i was not affected by the same anti - epo antibody . similar results were obtained when the day - 14 cultures were scored for burst - component colonies . this conclusively demonstrates that the production of erythroid bursts under the influence of igf - i does not occur through an epo - dependent mechanism . bars 4 and 5 show that in this sf medium a limited number of bursts form which do not require either epo or igf - i , and the anti - epo antibody has no effect on their development . evidently , the presence of il - 3 , hemin and retinyl acetate or all - trans - retinoic acid is sufficient to support the production of these &# 34 ; epo - and igf - i - independent bursts &# 34 ;. role of accessory cells in burst production in an improved sf medium the experiments described thus far were done with ficoll - hypaque density gradient - separated cells , and they provided information on the growth factor requirements of human erythroid progenitors among pb mnc , against a sf background . to find out whether or not accessory cells play a role under these conditions , we removed plastic adherent cells . fig1 shows that production of erythroid bursts could be completely eliminated by this procedure , despite the presence of il - 3 , hemin , retinyl acetate , epo and igf - i at what would otherwise be optimal concentrations . thus accessory cells clearly still play a crucial role in the production of erythroid bursts by pb mnc in the improved sf medium . effect of recombinant human stem cell growth factor as a replacement for adherent cells recombinant human ( rhu ) scf was added to the sf cultures of ficoll - hypaque density gradient separated mnc which had been exposed to 1 . 5 hours &# 39 ; adherence to plastic . when the rhu scf was added to cultures of untreated pb mnc under sf conditions at concentrations ranging from 30 to 1500 pm ( 1 to 50 ng / ml ) in the absence of il - 3 , they showed virtually no change in the number of bursts ( fig1 a closed circles ) or of burst - component colonies ( bcc ) ( fig1 b , closed circles ). in the presence of 0 . 8 nm rhu il - 3 , no significant increase ( t = 2 , with 2 df and p & gt ; 0 . 1 ) in burst - formation was observed at 1 . 5 nm rhu scf ( fig1 a open circles ). in contrast , adherent celldepleted pb mnc showed a significant increase of burst - and bcc - formation upon the addition of as little as 30 pm rhu scf ( fig1 a and 12b , closed squares ), but no increase upon the addition of il - 3 ( in the presence of hemin and 1 . 5 or 3 . 0 nmrhu scf ). the control point shown in parentheses ( closed squares at 0 rhu scf in fig1 b ) represents erythroid colonies with & lt ; 50 cells ( an average of 25 ) which are promoted by the bpa - like action of hemin alone and were tentatively grouped as clusters , possibly equivalent to undeveloped bursts ( fig1 a , closed square in parentheses ). some of these small hemin - dependent colonies approached the 50 - cell size , and the effect of rhu scf , under these conditions ( in the absence of il - 3 ), was to increase the cellularity ( up to 200 cells ) of the bcc ; it also rendered the colonies rounder and more compact . it thus allowed full burst development . a comparison of the effects of rhu scf and of the two defined bpa - like activities used in our sf medium ( 28 ), hemin and rhu il - 3 , upon burst - formation shows that optimal conditions for burst - formation appeared to require the presence of all three factors , rhu il - 3 , rhu scf and hemin ( fig1 c , open bar 3 ), even though the addition of rhu il - 3 to both hemin and rhu scf did not significantly increase the number of bcc observed ( cf open bars 2 and 3 , fig1 d ). removal of adherent cells appeared to prevent the capacity of hemin to promote erythroid colonies large enough for them to be classified as bcc ; the number of erythroid colonies with 20 to 50 hemoglobinized cells ( fig1 d , striped bar 1 ) and of &# 34 ; undeveloped bursts &# 34 ; ( fig1 c , striped bar 1 ) were fewer than the number of bcc and bursts formed in the absence of hemin ( fig1 c and 12d , closed bars 4 ). finally , addition of rhu il - 3 to a combination of hemin and rhu scf did not significantly increase the number of bursts obtained with hemin and rhu scf alone ( cf closed bars 2 and 3 , fig1 c ) and only marginally increased the number of bcc obtained with hemin and rhu scf ( cf closed bars 2 and 3 , fig1 d ). effect of proliferation - promoting growth factors ( hemin , rhu - il - 3 and rhu scf ) upon multi - lineage hematopoietic colonies from human bone - marrow normal bone - marrow mononuclear cells ( bm mnc ) separated and washed as described in materials and methods for pb mnc were grown in the basal serum - free medium , together with 3 . 0 u / ml of rhu epo , 3 × 10 - 8 m rhu igf - 1 and 3 × 10 - 8 retinyl acetate , with different combinations of proliferation - stimulatory activities ( 250 μm hemin , 10 ng / ml rhu il - 3 and 50 ng / ml rhu scf ). hemin is necessary to detect both erythroid ( cfu - e - and bfu - e - derived ) and non - erythroid ( cfu - gm - derived ) colonies ( fig1 bars 1 ), but the numbers of these colonies are significantly increased by the addition of rhu il - 3 ( bars 2 ). the addition of rhu scf in the absence of rhu il - 3 further increased the levels of these colonies over those obtained with rhu il - 3 in the absence of rhu scf ( cp . bars 3 and 2 ), and expression of multi - lineage cfu - gemm - derived colonies could be observed under these conditions ( cp . open bar 3 ). lastly , optimal levels of all four types of colonies were obtained when hemin , rhu il - 3 and rhu scf were all added together , making the medium complete ( bars 4 ). results shown in fig1 were obtained using human bone - marrow mononuclear progenitor cells from patients with diamond blackfan anemia ( dba ). bars 1 of fig1 are comparable to bars 1 of fig1 as obtained under the same culture conditions ( complete medium minus rhu il - 3 and rhu scf ). bars 2 of fig1 , however , show that dba patients have dramatically fewer cfu - e ( late erythroid progenitors ) and bfu - e ( early erythroid progenitors ) than normals do ( cp bars 3 of fig1 ) when rhu scf is added to hemin in the absence of rhu il - 3 . finally , the addition of rhu il - 3 to hemin in the absence of rhu scf ( fig1 , bars 3 ) exhibited the same greatly diminished expression of erythroid bone - marrow progenitors in dba but it also indicates a greater expression of non - erythroid ( cfu - gm ) progenitors in dba than in normals ( cp . open bar 3 from fig1 with the more densely stippled bar 2 of fig1 ). erythroid colony formation by polycythemia vera primary and continuous cell populations in an improved sf medium fig1 shows the cell - dose response curve for burst - formation by pb mnc from a polycythemia vera ( pv ) patient in our novel sf medium , without rhu scf ( closed circles ) and without both rhu scf and rhu epo ( stippled circles ). in both instances , the medium contained 10 ng / ml rhu il - 3 , 0 . 25 mm hemin , 3 × 10 - 8 m retinyl acetate and 3 × 10 - 8 m rhu igf - 1 . where indicated rhu epo was employed at 3 u / ml . either curve of fig1 extrapolates through points not significantly different from the origin , indicating that , with or without rhu epo , our novel sf medium is performing satisfactorily for burst - formation by pv pb mnc . the improved sf medium performs equally well for an analysis of &# 34 ; cfu - e - like &# 34 ; and &# 34 ; bfu - e - like &# 34 ; erythroid colonies obtained from an immortalized cell line isolated from the peripheral blood of another pv patient , as is shown by the two curves of fig1 , one for day 7 erythrocytic - like colonies ( open circles ) and the other for day - 14 burst - like colonies ( closed circles ). presumed autocrine growth of a bi - phenotypic leukemia cell line in sf medium a bi - phenotypic leukemia cell line ( b - 1 ) from a patient with all ( acute lymphoblastic leukemia ) was studied for its growth characteristics in our novel sf medium . growth could be detected with the basal sf medium alone ( in the absence of all peptide growth factors , namely rhu il - 3 , rhu scf , rhu epo and rhu igf - 1 , as well as in the absence of hemin and retinoids ), as shown in bar 2 of fig1 a . subtraction of d - α - tocopherol from the basal sf medium significantly diminished the viability of those biphenotypic leukemic cell day - 9 colonies ( bar 1 , fig1 a ) whereas addition of retinyl acetate further increased their growth ( bar 3 , fig1 a ). these results indicate that b - 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