Patent Application: US-34334608-A

Abstract:
disclosed is a growth factor supplement for stem cell culture media , stem cell culture media supplemented with the growth factor supplement , and methods for growing and maintaining stem cells in culture . the invention particularly relates to human stem cells , more particularly human embryonic stem cells , neonatal stem cells , adult stem cells , and ips cells .

Description:
substantially similar results were obtained in the presence of media supplemented with a combination of growth factors each at a concentration of 10 ng / ml comprising human interleukin 5 , human interleukin 6 and human interleukin 7 ; or with a combination of growth factors comprising human interleukin 5 , human interleukin 6 , human interleukin 7 and human granulocyte macrophage colony stimulating factor ( gm - csf ), or with a combination of growth factors comprising human interleukin 5 , human interleukin 6 , human interleukin 7 , human gm - csf and human gro gamma / cxcl3 , or with a combination of growth factors comprising human interleukin 5 , human interleukin 6 , human interleukin 7 , human gm - csf , human gro gamma / cxcl3 and ccl2 mcp1 . although the present invention has been described with reference to particular embodiments , it is to be appreciated that various adaptations and modifications may be made without departing from the spirit and scope of the invention . the invention is only to be limited by the appended claims . shiloh laboratories engineered human ht1080 fibrosarcoma cells with an inducible system that forces cell cycle arrest and terminal differentiation of the cells ( called fibrosen ™), as disclosed in co - owned and co - pending u . s . patent application ser . no . 12 / 115 , 451 , filed may 5 , 2008 and incorporated by reference herein . fragments of the senescence triggering protein p21 under regulation of the lactose operon promoter are repressed by constitutive expression of the lac repressor protein . upon addition of iptg , the repression is removed p21 factors trigger differentiation of the fibrosen ™ cells . these cells provided a confluent feeder layer upon which hescs may be plated . not only did hescs grow well on the fibrosen ™ feeder system , but cell numbers increased dramatically , to as much as 7 - fold . an intuitive hypothesis from these results is that some factors secreted from the fibrosen cells are assisting the enhanced proliferation of the hescs . shiloh laboratories undertook a proteomics approach to identifying what factors are present in the medium in differentiated fibrosen ™ cultures , with respect to media from non - differentiated fibrosen ™ cells ( fig1 ). media lacking serum was added to the cells and incubated for either 3 or 5 days . supernatants from each culture were removed , centrifuged , filtered with 0 . 2 um pes filter . supernatant proteins from either non - differentiated ( lacking iptg ) or differentiated ( with iptg ) cells were then incubated on triplicate cytokine antibody array membranes ( ray biotech ) overnight at 37 ° c . the next day , biotinylated antibodies to all of the spotted cytokines on the array was incubated with the membrane , washed then incubated with 35 s methionine labeled steptavidin . labeled proteins were the membranes were washed and exposed to a phosphorimaging plate . spots were visualized using a fuji phosphorimager . the visualized spots were quantitated with the phosphorimager software and triplicate data were averaged . statistically significant results were used to identify 6 growth factors that had at least 2 - fold higher concentrations in media from differentiated cells than control non - differentiated cells . these factors were identified as il5 , il6 , il7 , gm - csf , groγ , and mcp1 . all of these growth factors are commercially available . the next logical experiment was to add these factors in a feeder free system to determine if they could increase the growth rates of hescs . h9 cells were plated at a density of 50 , 000 cells per well in a 12 well plate coated with matrigel . prosen growth factors . il - 5 ( gf1 ) stimulates b cell growth and is associated with janus kinase ( jak ) 2 [ 66 ]. il - 6 ( gf2 ) acts as a pro - inflammatory cytokine that triggers signal transduction through jak and stat tyrosine kinases [ 67 ]. il7 ( gf3 ) is a hematopoietic growth factor secreted by the stromal cells capable of stimulating the proliferation of lymphoid progenitors by signal transduction through the jak / stat pathway [ 68 ] gm - csf ( gf4 ) is secreted by macrophages , t cells , mast cells , endothelial cells and fibroblasts and functions as a white blood cell growth factor by inducing protein tyrosine phosphorylation , ras , raf - 1 and map kinase [ 69 ], groγ ( gf5 ) works through the chemokine receptor cxcr2 [ 70 ] and plays a role in inflammation . mcp1 ( gf6 ) is a chemokine that recruits monocytes , memory t cells , and dendritic cells to sites of tissue injury and infection and is under the control of nuclear factor κb ( nfκb ) [ 71 ]. the concentration of cells was the lowest that could be used and have the h9 cells grow properly . using a low concentration to start the experiments allows for longer experiments to be performed in the same dish . in an attempt to mimic the fibrosen ™ cultures , the initial concentrations of each of the growth factors were 10 ng / ml and using all six factors at equal concentrations . for ease of discussion , the grouping of gf1 - gf6 ( il5 , il6 , il7 , gm - csf , groγ , and mcp1 ) was termed the prosen ™ supplement . please note that simply adding each of the growth factors alone ( i . e ., individually ) at 10 ng / ml concentration in mtesr1 did not yield an increase in viable cell density of h9 cells after 6 days . these concentrations were chosen based on the effective concentration of each factor as published by the manufacturer . using 10 ng / ml concentrations yielded more than 2 - fold increase in the number of h9 cells after 6 days of culture in mtesr1 media ( stem cell technologies ; results not shown ). the experiment was repeated with 1 and 0 . 1 ng / ml concentrations of the combined six growth factors . as can be seen in fig2 , the combination gf - 1 - gf6 at 1 ng / ml each was capable of increasing the number of h9 cells by more than 2 - fold after 6 days in culture . using 0 . 1 ng / ml of each growth factor had no different effect on h9 cell growth as mtesr1 media alone . media is changed every day in feeder - free culture to prevent differentiation of the cells . many laboratories have unsuccessfully attempted to use growth factor supplements to extend the media life of feeder free stem cell media . to determine if prosen ™ at 1 ng / ml is capable of extending the usable lifetime of mtesr media , cells were plated at 50 , 000 cells per well in 1 ml mtesr . the next day , prosen ™ supplement was added to begin the experiment ( day 0 ). viable cell density ( the number of viable cells / ml ) was determined at days 2 , 3 and 4 . media was not changed at all during this experiment . the results of these experiments are shown in fig3 . under these conditions , h9 cells in log phase were expected to double in about 30 hours . for h9 in mtes1r , viable cell density did not double each day . in fact , the cells began to die by day 3 and the culture was filled with mostly dead cells by day 4 . in contrast , using 1 ng / ml prosen ™ supplement allowed cells to continue to double for as long as 4 days without changing the media . it is suggested that using prosen ™ can lower the cost of using mtesr1 about 6 - fold by increasing the number of cells 2 - fold and using 3 times less media . the effects of the prosen ™ supplement on pluripotency of h9 cells after 6 days in culture was determined for cells receiving mtesr1 daily or every third day . results of oct 4 expression in h9 are shown in fig4 . more than 97 % of the cells stained for oct 4 transcription factor , a known marker of pluripotency , in both cells receiving fresh media daily or mtesr + prosen ™ supplement every 3 rd day . these results indicate that under short - term exposure to prosen ™ supplement does not adversely impact pluripotency of h9 cells . another effort put forth by a number of laboratories using feeder - free systems for growing hescs is to increase the plating efficiency of the cells . plating efficiency is defined as the number of colonies that grow per cell seeded into the culture . the plating efficiency drops dramatically when lower seeding cell numbers are used . to date , it has not been possible to plate a single hesc cell in a single well of a microtiter plate and get a colony to form in a feeder - free system . plating cells at a density lower than 3000 cells / cm 2 ( 30 , 000 cells / well in a 6 well plate ) do not form colonies . in an attempt to determine if prosen ™ supplement can enhance the plating efficiency of h9 cells on matrigel in mtesr1 media , h9 cells were digested with accutase to single cells , a process that normally does not allow for colony formation . digested h9 cells were plated at 30 , 000 per well in a 6 - well plate . one set of cells received the prosen ™ supplement , control cells did not . the mtesr1 medium with or without prosen ™ was changed every day for this experiment . colonies were counted after 6 days in culture . the results of these experiments are shown in fig5 . very few and very small colonies with mostly dead cells were observed in cultures with mtesr alone . adding prosen ™ lead to normal colony formation with plating efficiencies of greater than 50 %. using prosen ™ and accutase treatment may allow for true subcloning and possible automation of hesc cloning using facs sorting , limiting dilution , or other cloning techniques that isolate single cells . having cells of true clonal origin may aid percent conversion of hescs to differentiation committed embryoid cells or type - 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