Patent Application: US-9414706-A

Abstract:
the present invention relates to immortal pluripotent stem cells derived from a human leukaemia cell line , preferably a human monocytoid cell line and more preferably the human monocytoid cell line , thp1 . the present invention further relates to cell lines derived from the immortal pluripotent stem cell line having the phenotype of cell strains characteristic of human tissues , particularly having a human hepatocyte phenotype , as well as the methods for preparing thereof . the present invention further relates to the use of the derived cell line with a human hepatocytic phenotype for the production of albumin and blood coagulation factors .

Description:
purely by way of non - limiting example , the present invention will now be described in detail with reference to some preferred embodiments . psc - thp1 cells have been directed to becoming specialised ( in the sense of maturing and differentiating ) and become cells with the typical functions of hepatocytes ( psc - thp1 liver like cells ). psc - thp1 cells have been induced to differentiate , assuming the morphology and functional characteristics of hepatocytes by subjecting them in culture to the continual stimulation generated by hgf ( hepatocyte growth factor ), glucose , a combination of interleukin 2 and interleukin 6 and aminoacids . for said purpose , various concentrations of hgf have been tested . at low hgf concentrations ( 25 and 50 ng / ml ), the differentiation of pscs towards hepatocytic cells is slow and the appearance of numerous oval cells is observed ( 80 % of the cells in culture ), some mature hepatocytes ( 10 % of the cells in culture ) which produce high concentrations of albumin and some immature hepatocytes or hepatoblasts ( 10 % of the cells in culture ) which produce alpha - feto - protein and a small activity , between 3 % and 5 %, of certain coagulation factors such as antithrombin iii , factor viii and von willebrand &# 39 ; s factor . at medium doses of hgf ( 75 and 100 ng / ml ), the differentiation of pscs accelerates as shown by the appearance of numerous mature hepatocytes ( 80 % of the cells in culture ) which produce a high concentration of albumin ( 40 - 120 g per litre of albumin / 5 million cells per ml in culture ). the number of oval cells is moderate ( 15 % of the cells in culture ), and the number of immature hepatocytes or hepatoblasts is also moderate ( 5 % of the cells in culture ) producing alpha - feto - protein . the coagulation factors , the activity of which is between 5 % and 80 %, become more abundant and are listed in detail in table 1 . at high hgf concentrations ( 150 and 200 ng / ml ), the differentiation of pscs slows down and oval cells represent the majority of the observable cells ( 70 % of the cells in culture ), the immature hepatocytes or hepatoblasts are reasonably abundant ( 25 % of the cells in culture ) and produce alpha - feto - protein , while mature hepatocytes producing a high concentration of albumin are rare ( 5 % of the cells in culture ). furthermore , at such high hgf concentrations modest activity ( between 0 % and 5 %) of the coagulation factors listed in table 1 is detected . the thp - 1 monocytoid cells [ 16 ] have been washed 3 times by centrifugation at 160 g for 10 minutes at room temperature in rpmi 1640 medium ( life technologies , grand island , n . y .) and resuspended in 15 cm plates ( lab - tek chamber slides , nunc , kamstrup , denmark ) at the final concentration of 1 × 10 6 cells / ml in rpmi 1640 medium supplemented with : 10 % fcs ( celbio , milan , italy ); 100 units / ml of penicillin ; 100 μg / ml streptomycin ; 160 mg / l gentamycin ( schering - plough , milan , italy ); 2 mm l - glutamine ( life technologies ; growth medium ); 50 ng / ml m - csf ( macrophage colony - stimulating growth factor , peprotech inc ., america , new jersey ); 1000 units / ml lif ( leukaemia inhibitory factor , santa cruz biotechnology , california , usa ); 1000 units / ml il - 2 ( human recombinant interleukin 2 ); 3 nm phorbol - 12 - myristate 13 - acetate ( pma , ( santa cruz biotechnology , california , usa ). two types of controls have been prepared : a negative control of untreated thp - 1 cells ( 1 ) and one control of thp - 1 cells treated with lif ( leukaemia inhibitory factor ) alone ( 2 ): ( 1 ) the control thp - 1 monocytoid cells [ 16 ] have been washed 3 times by centrifugation at 160 g for 10 minutes at room temperature in rpmi 1640 medium ( life technologies , grand island , n . y .) and resuspended in 15 cm plates ( lab - tek chamber slides , nunc , kamstrup , denmark ) at a final concentration of 1 × 10 6 cells / ml in rpmi 1640 medium supplemented with : 10 % fcs ( celbio , milan , italy ); 100 units / ml of penicillin ; 100 μg / ml streptomycin ; 160 mg / l gentamycin ( schering - plough , milan , italy ); 2 mm l - glutamine ( life technologies ; growth medium ). ( 2 ) the thp - 1 monocytoid cells [ 16 ] have been washed 3 times by centrifugation at 160 g for 10 minutes at room temperature in rpmi 1640 medium ( life technologies , grand island , n . y .) and resuspended in 15 cm plates ( lab - tek chamber slides , nunc , kamstrup , denmark ) at a final concentration of 1 × 10 6 cells / ml in rpmi 1640 medium supplemented with : 10 % fcs ( celbio , milan , italy ); 100 units / ml of penicillin ; 100 μg / ml streptomycin ; 160 mg / l gentamycin ( schering - plough , milan , italy ); 2 mm l - glutamine ( life technologies ; growth medium ); 1 . 000 units / ml of lif ( leukemia inhibitory factor , santa cruz biotechnology , america , california ). all samples have been incubated for 15 days in a heraeus thermostatically controlled incubator at a temperature of 37 ° c . in an atmosphere containing a constant flow of 8 % co 2 ( v / v in air ). samples of all the cells forming the subject of the study , have been washed 3 times by centrifugation at 160 g for 10 min at 37 ° c . and have been subjected to cytofluorometric analysis ( epics profile ii , coulter , hialeath , fla .) after staining with , anti - human mouse monoclonal antibodies ( mabs ), conjugated to r - phycoerythrin , anti - human cd14 ( santa cruz biotechnology , california , usa ), anti - human cd34 ( santa cruz biotechnology , california , usa ), anti - cd45 ( santa cruz biotechnology , california , usa ), anti - c - kit ( santa cruz biotechnology , california , usa ) and anti - c - met ( santa cruz biotechnology , california , usa ). the cells tested by cell cytofluorimetry ( facs ), are largely positive for all the markers of stem cell expression ( cd14 +, cd29 +, cd34 +, cd44 +, cd45 −/+, cd71 +, cd90 +, cd105 +, cd117 +, c - met ). upon completion of the incubation period , all the cells show fibroblastoid morphology . the cells detached using 2 % lidocaine ( sigma aldrich , milan , italy ) in pbs [ 17 , 21 ] have been washed 3 times by centrifugation at 160 g for 10 minutes at 37 ° c . in rpmi 1640 ( life technologies , grand island , n . y .) and have been incubated for a second time in accordance with the following description . the treated controls , destined to remain undifferentiated pluripotent stem cells ( psc - thp1 ), have been resuspended at a final concentration of 1 × 10 5 cells per ml in 6 well plates ( lab - tek chamber slides , nunc , kamstrup , denmark ) in 0 . 5 ml per well of final solution composed of rpmi 1640 medium supplemented with : 10 % fcs ( celbio , milan , italy ); 100 units / ml of penicillin ; 100 μg / ml streptomycin ; 160 mg / l gentamycin ( schering - plough , milan , italy ); 2 mm l - glutamine ( life technologies ; growth medium ); 50 ng / ml m - csf ( macrophage colony - stimulating growth factor , peprotech inc ., america , new jersey ); 1000 units / ml lif ( leukaemia inhibitory factor , santa cruz biotechnology , california , usa ); 1000 units / ml il - 2 ( human recombinant interleukin 2 ); 3 nm phorbol - 12 - myristate 13 - acetate ( pma , santa cruz biotechnology , california , usa ). the cells have been incubated for 23 days in a heraeus thermostatically controlled incubator at a temperature of 37 ° c . in an atmosphere containing a constant flow of 8 % co 2 ( v / v in air ) with 0 . 25 ml of medium being replaced every 5 - 7 days . the samples destined for stimulation to become specialised hepatocytic cells ( psc - thp1 liver like cells ) have been resuspended at a final concentration of 1 × 10 5 cells per ml in 6 well plates ( lab - tek chamber slides , nunc , kamstrup , denmark ) in 0 . 5 ml per well of final solution composed of rpmi 1640 medium supplemented with : 300 ml / l nutrient ham mixture f - 12 ( gibco , grand island , n . y . ); 4 ml / l hank &# 39 ; s solution ( sigma aldrich , milan , italy ); 100 units / ml of penicillin ; 100 μg / ml streptomycin ; 160 mg / l gentamycin ( schering - plough , milan , italy ); 2 mm l - glutamine ( life technologies ; growth medium ); 50 ng / ml m - csf ( macrophage colony - stimulating growth factor , peprotech inc ., new jersey , usa ); 1000 units / ml lif ( leukaemia inhibitory factor , santa cruz biotechnology , california , usa ); 100 μg / l il - 2 ( recombinant human interleukin 2 ); 50 μg / l il - 6 ( recombinant human interleukin 6 ); 3 nm phorbol - 12 - myristate 13 - acetate ( pma , santa cruz biotechnology , california , usa ); hgf ( hepatocyte growth factor , peprotech inc ., new jersey , usa ), which has been tested at the following concentrations : 25 ng / ml , 50 ng / ml , 75 ng / ml , 100 ng / ml , 150 ng / ml and 200 ng / ml ; 5 ul / ml of non - essential aminoacid solution ( sigma aldrich , milan , italy ); 10 g / l glucose ( sigma aldrich , milan , italy ); 10 − 7 m dexamethazone phosphate ( soldesam 4 mg / ml solution for injections , ab . farmacologico milan . srl ); 2 mg / l scopolamine ( n - butylbromide hyoscine , buscopan 20 mg / ml solution for injections , boehringer ingelheim italy s . p . a . ); 10 % fcs ( celbio , milan , italy ), or the fcs can be replaced with the substances listed in table 2 : after 23 days in culture , the surnatants of the test cultures are collected and stored at − 80 ° c . for the following laboratory analyses : the concentrations of microalbumin , calcium , alpha - feto - protein , glucose , glycogen and urea . after 23 days in culture , cell suspensions of all the test samples have been obtained following incubation for 5 - 8 minutes with 2 % lidocaine ( sigma aldrich , milan , italy ) in pbs , by pipetting the cell suspension up and down in the well and then harvesting the solution obtained , as described in the references [ 17 , 21 ]. the cells have been washed three times by centrifugation at 160 g for 10 minutes at 37 ° c . with unsupplemented rpmi 1640 ( life technologies , grand island , n . y .). from the cell pellet obtained , a small portion of the cells have been resuspended in 15 ml tubes ( lab - tek chamber slides , nunc , kamstrup , denmark ) at a final concentration of 5 × 10 5 cells / ml for the subsequent phenotypic analyses ( western blotting , direct immunofluorescence and facs ) and a portion of the cells have been centrifuged at 160 g for 10 minutes at 4 ° c . in pbs and the cell pellet obtained has been dried completely and immediately frozen at − 80 ° c . for subsequent rna analysis ( pcr ). the samples have been subjected to phenotypic analysis by western blotting for markers using anti - cd 14 ( santa cruz biotechnology , california , usa ), anti - cd 29 ( santa cruz biotechnology , california , usa ), anti - cd 34 ( santa cruz biotechnology , california , usa ), anti - cd 44 ( santa cruz biotechnology , california , usa ), anti - cd 45 ( santa cruz biotechnology , california , usa ), anti - cd 71 ( santa cruz biotechnology , california , usa ), anti - cd 90 ( santa cruz biotechnology , california , usa ), anti - cd 105 ( santa cruz biotechnology , california , usa ), anti cd 117 / c - kit ( santa cruz biotechnology , california , usa ), anti c - met ( santa cruz biotechnology , california , usa ), anti cytokeratin 7 ( santa cruz biotechnology , california , usa ), anti cytokeratin 8 ( santa cruz biotechnology , california , usa ), anti cytokeratin 18 ( santa cruz biotechnology , california , usa ), anti cytokeratin 19 ( santa cruz biotechnology , california , usa ), anti cytokeratins 7 / 17 ( santa cruz biotechnology , california , usa ), anti albumin ( rockland immunochemicals , pennsylvania , usa ), anti alpha - feto - protein ( monosan europa , netherlands ). after five washes , the membranes have been incubated with the corresponding secondary antibodies ( 1 : 1000 ) conjugated to horseradish peroxidase ( hrp , santacruz biotechnologies inc ., santa cruz , calif ., usa ) for 1 hour at room temperature , as reported in the following tables . cells in suspension have been incubated with 0 . 2 mm mitotracker red for 10 minutes at 37 ° c . after three washes by centrifugation at 160 g for 10 minutes at room temperature in pbs ( ph 7 . 4 ), the cell pellets have been resuspended in a fixing solution of 4 % paraformaldehyde in rpmi 1640 at ph 7 . 4 , 1 hour at room temperature . after three washes in pbs , the cells have been resuspended in a solution of pbs and 0 . 1 % triton for 1 hour at 4 ° c . after three washes in pbs , the cells have been seeded onto slide covers and the liquid allowed to evaporate - off in air . the cells have been blocked with 20 % normal goat serum for one hour , and then incubated for 30 minutes with the following anti - human monoclonal antibodies : anti - cd 14 ( santa cruz biotechnology , california , usa ), anti - cd 29 ( santa cruz biotechnology , california , usa ), anti - cd 34 ( santa cruz biotechnology , california , usa ), anti - cd 44 ( santa cruz biotechnology , california , usa ), anti - cd 45 ( santa cruz biotechnology , california , usa ), anti - cd 71 ( santa cruz biotechnology , california , usa ), anti - cd 90 ( santa cruz biotechnology , california , usa ), anti - cd 105 ( santa cruz biotechnology , california , usa ), anti cd 117 / c - kit ( santa cruz biotechnology , california , usa ), anti c - met ( santa cruz biotechnology , california , usa ), anti cytokeratin 7 ( santa cruz biotechnology , california , usa ), anti cytokeratin 8 ( santa cruz biotechnology , california , usa ), anti cytokeratin 18 ( santa cruz biotechnology , california , usa ), anti cytokeratin 19 ( santa cruz biotechnology , california , usa ), anti cytokeratins 7 / 17 ( santa cruz biotechnology , california , usa ), anti albumin ( rockland immunochemicals , pennsylvania , usa ), anti alpha - feto - protein ( monosan europa , netherlands ) conjugated to r - phycoerythrin ( pe ) or fluorescein isothiocyanate ( fitc ). specific controls with the corresponding isotypes have been devised for each monoclonal antibody ( santa cruz biotechnology , california , usa ). the nuclei have been stained using hoechst solution ( dilution 1 : 1000 ). the cover slips , mounted onto slides using moviol have been examined by light microscopy [ 16 - 17 ]. the cells have been transferred into 50 ml tubes ( lab - tek chamber slides , nunc , kamstrup , denmark ) and washed three times by centrifugation at 160 g for 10 minutes at room temperature in pbs ( ph 7 . 4 ). the pellets have been resuspended at a final concentration of 5 × 10 5 cells / ml in pbs . the cells in suspension have been incubated with 0 . 2 mm mitotracker red for 10 minutes at 37 ° c . after washing three times with pbs the cells have been fixed for 1 hour in 4 % paraformaldehyde in pbs ( ph 7 . 4 ) at room temperature . after washing three times in pbs , the cells have been resuspended in a solution of pbs and 0 . 1 % triton for 1 hour at 4 ° c . after washing three times in pbs , the cells have been resuspended in a blocking solution of 20 % normal goat serum for 1 hour . after washing three times in pbs , the samples have been incubated for 30 minutes with the following anti - human monoclonal antibodies : anti - cd 14 ( santa cruz biotechnology , california , usa ), anti - cd 29 ( santa cruz biotechnology , california , usa ), anti - cd 34 ( santa cruz biotechnology , california , usa ), anti - cd 44 ( santa cruz biotechnology , california , usa ), anti - cd 45 ( santa cruz biotechnology , california , usa ), anti - cd 71 ( santa cruz biotechnology , california , usa ), anti - cd 90 ( santa cruz biotechnology , california , usa ), anti - cd 105 ( santa cruz biotechnology , california , usa ), anti cd 117 / c - kit ( santa cruz biotechnology , california , usa ), anti c - met ( santa cruz biotechnology , california , usa ), anti cytokeratin 7 ( santa cruz biotechnology , california , usa ), anti cytokeratin 8 ( santa cruz biotechnology , california , usa ), anti cytokeratin 18 ( santa cruz biotechnology , california , usa ), anti cytokeratin 19 ( santa cruz biotechnology , california , usa ), anti cytokeratins 7 / 17 ( santa cruz biotechnology , california , usa ), anti albumin ( rockland immunochemicals , pennsylvania , usa ), anti alpha - feto - protein ( monosan europa , netherlands ). all the antibodies used are monoclonal , conjugated to r - phycoerythrin ( pe ) or fluorescein - isothiocyanate ( fitc ). specific controls with the corresponding isotypes have been devised for each monoclonal antibody ( santa cruz biotechnology , california , usa ). the samples have been subjected to quantitative analysis using a laser cytofluorimeter ( epics profile ii , coulter , hialeath , f l ) at 488 nm and referred to the percentage of fluorescent cells ( pfc ) as the geometric mean . threshold values ( gates ) have been established using control samples labelled with the corresponding isotypes . all values have been analysed with a minimum threshold of 15 , 000 cells . rna extraction has been performed using tri reagent solution ( molecular research center , inc .) in accordance with the manufacturers instructions . the cell pellets have been resuspended in 1 ml of tri reagent solution per sample and incubated for 5 minutes at room temperature . following incubation , 100 μl of bromochloropropane have been added and the samples incubated again for 15 minutes at room temperature . after centrifugation at 12000 g for 15 minutes at 4 ° c ., the aqueous phases have been recovered and transferred into eppendorf tubes ( approx . 500 μl ), with the addition of 500 μl of isopropanol . the samples have been incubated for 10 minutes at room temperature , then centrifuged at 12000 g for 8 minutes at 4 ° c . and washed with 1 ml of 75 % ethanol , then centrifuged at 7500 g for 5 minutes an finally the supernatant has been removed using a syringe and the pellets have been carefully dried under a fume hood . finally , the rna samples have been resuspended in 50 μl of dnase - free water . resuspend in 50 μl of freshly opened , autoclaved milliq h 2 o , read 5 μl using the spectrophotometer ( dilution : 1 : 100 ). messenger rnas have been retro - transcribed using the applied biosystems “ cdna archive kit ” ( applied biosystems ). reactions have been prepared in a total volume of 50 μl , with 10 μl of sample solution + h 2 o in the presence of 0 . 25 iu of rnase ( promega ). the samples have been incubated at 25 ° c . for 10 minutes and then at 37 ° c . for 2 hours . genomic dna has been removed using the “ turbo dna - free ” system ( ambion ), by incubation for 30 minutes at 37 ° c . the dnas has been removed by incubating each sample with 5 μl dnas removing agent , for 2 minutes a room temperature . the samples have then been centrifuged at 10000 × g for 1 minute , and finally , 1 μl of each reverse transcribed sample used for real time pcr amplification . the real time pcr amplification has been performed using the iqsybr green supermix 1000 × 50 ml system ( 1708884 - biorad ) with primers for albumin , alpha - feto - protein , c - met and gadph from sigma . amplification reactions have been performed using an “ abi prism 7700 sequence detector ” ( perkin elmer ). the data have been analysed using the sequence detection 1 . 9 . 1 software ( perkin elmer ). alpha - feto - protein , urea , albumin and microalbumin assays in the cell culture supernatants tested all measurements have been performed using a modular analytics p automated system ( roche - hitachi ). all measurements have been performed using a maldi - tof - ms ( micro mx , waters , manchester , uk ) automated system . the peptides identified have been processed using the profound - peptide mapping on - line search engine for the reconstruction of the sequences and the identification of the corresponding proteins using the pmf , peptide mass fingerprinting technique ( embl - ebi , european bioinformatic institute ; ncbi , national center for biotechnology information ). electrolyte ( na + , k + , cl − ) assay in the cell culture supernatants tested electrolyte assay have been performed using ise ( ion - sensitive ) electrodes , fitted with a membrane with such physico - chemical characteristics as to make them selective for a given ion . the magnitude of the electromotive force or voltage at the membrane of the ise electrode with respect to a reference electrode , is proportional to the concentration of the selected ion in the test sample . the supernatant is reacted with reactive r1 ( buffer / atp / nadp ) and reactive r2 ( hk / g - 6 - pdh ). in the presence of such reagents , the reaction of phosphorylation of glucose to g - 6 - p due to the hexokinase ( hk ) and in the presence of atp occur ; the g - 6 - p formed is oxidised by the enzyme g - 6 - pdh with the consumption of nadp . measurement of the rate of formation of nadph , determined photometrically , is proportional to the concentration of glucose contained in the tested sample . after seven days of incubation with rpmi 1640 containing 1000 units / ml of lif , 50 ng / ml m - csf and 3 nm phorbol - 12 - myristate 13 - acetate ( pma ), the samples revealed the morphological transformation of the thp1 cells from rounded to an elongated fibroblastoid shape adhering to the culture plates . the controls showed a purely rounded shape and were completely in suspension . after 23 days of incubation with rpmi 1640 , supplemented as described previously , containing 1000 units / ml of lif , 50 ng / ml of m - csf , 50 , 75 , 100 , 150 , 200 ng / ml hgf and 3 nm phorbol - 12 - myristate 13 - acetate ( pma ), the samples revealed the morphological transformation of the thp1 cells from rounded to tetrahedral shape adhering to the culture plates , positive for the production of : albumin , alpha - feto - protein , glycogen , cytokeratins 7 , 8 , 17 , 18 and 19 , ov - 6 , c - met receptor ( facs , immunohistochemistry , wb , pcr ) and some coagulation factors ( western blot , maldi - tof ms , 2d electrophoresis ), as listed in table 1 . the controls showed only a rounded shape and were all in suspension and negative for albumin , alpha - feto - protein , glycogen , cytokeratins 7 , 8 , 17 , 18 and 19 , ov - 6 . characterisation of the thp - 1 cells vs . pscs - thp cells and pscs - thp1 liver like cells the results pertaining to the expression of the surface antigen cd14 +, cd29 +, cd34 +, cd44 +, cd45 −/+, cd71 +, cd90 +, cd105 +, cd117 +, c - met , cytokeratin 7 , cytokeratin 8 , cytokeratin 18 , cytokeratin 19 , cytokeratins 7 / 17 have been expressed on a quantitative scale , as shown in table 5 . 1 . albumin ( rockland immunochemicals , pennsylvania , usa ) the results ( table 6 ) show negativity for human albumin in the sample of rpmi medium , slight positivity for the production of human albumin in the thp - 1 control cells , thin positivity in the psc - thp1 cells , in contrast to marked positivity for the production of human albumin in the hepg2 cells and abundant positivity in the psc - thp1 liver like cells and in the human albumin . after 48 hours ( day 2 ) incubation after stimulation , the following samples have been tested : rpmi medium , thp - 1 controls , psc - thp1 liver like cells , i . e . sample as ( hgf , hepatocyte growth factor , 100 ng / ml , since this is the optimal dose ) and hepg2 cells ( positive control hepatoblastoid cell line ). in particular , the results show that the production of human alpha - feto - protein in the sample of rpmi medium and in the thp - 1 control cells is negative , with marked positivity in the psc - thp1 liver like cells and abundant positivity in the hepg2 cells ( positive control ) as shown in table 7 . the following samples have been tested : rpmi medium , hepg2 cells ( positive control hepatoblastoid cell line ), thp - 1 controls , psc - thp1 , i . e . a sample as per example 1 , psc - thp1 liver like cells , i . e . a sample at a concentration of 25 ng / ml hgf ( hepatocyte growth factor ) after 4 , 18 , 24 , 72 , 96 , 120 and 196 hours from stimulation . in particular , the results are negative for the production of human alpha - feto - protein in the sample of rpmi medium and in the thp - 1 control cells , abundantly positive in the hepg2 cells ( positive control ) below , and markedly positive in the psc - thp1 liver like cells with a peak after 18 hours from incubation with the hgf growth factor at a concentration of 25 ng / ml , as summarised in tables 8 and 9 . the following samples have been tested : rpmi medium , hepg2 cells ( positive control hepatoblastoid cell line ), thp - 1 controls , psc - thp1 , i . e . a sample as per example 1 , psc - thp1 liver like cells , i . e . a sample at a concentration of 50 ng / ml hgf ( hepatocyte growth factor ) after 4 , 18 , 24 , 72 , 96 , 120 and 196 hours from stimulation . in particular , the results are negative for the production of human alpha - feto - protein in the sample of rpmi medium and in the thp - 1 control cells , abundantly positive in the hepg2 cells ( positive control ) below , and markedly positive in the psc - thp1 liver like cells with a peak between 24 and 72 hours from incubation with the hgf growth factor at a concentration of 50 ng / ml , as summarised in tables 10 and 11 . the following samples have been tested : rpmi medium , hepg2 cells ( positive control hepatoblastoid cell line ), thp - 1 controls , psc - thp1 , i . e . a sample as per example 1 , psc - thp1 liver like cells , i . e . a sample at a concentration of 100 ng / ml hgf ( hepatocyte growth factor ) after 4 , 18 , 24 , 72 , 96 , 120 and 196 hours from stimulation . in particular , the results are negative for the production of human alpha - feto - protein in the sample of rpmi medium and in the thp - 1 control cells , abundantly positive in the hepg2 cells ( positive control ) below , and markedly positive in the psc - thp1 liver like cells with a peak after 24 hours from incubation with hgf at a concentration of 100 ng / ml , as summarised in tables 12 and 13 . the following samples have been tested : rpmi medium , hepg2 cells ( positive control hepatoblastoid cell line ), thp - 1 controls , psc - thp1 , i . e . a sample as per example 1 , psc - thp1 liver like cells , i . e . a sample at a concentration of 200 ng / ml hgf ( hepatocyte growth factor ) after 4 , 18 , 24 , 72 , 96 , 120 and 196 hours from stimulation . in particular , the results are negative for the production of human alpha - feto - protein in the sample of rpmi medium and in the thp - 1 control cells , and abundantly positive in the hepg2 cells ( positive control ) below , and markedly positive in the psc - thp1 liver like cells with a peak between 24 and 72 hours from incubation with hgf at a concentration of 200 ng / ml , as summarised in tables 14 and 15 . the growth factor hgf binds to the met receptor ; the hgf receptor binding or activating monoclonal antibodies , or the hgf receptor binding or activating peptides , are agonists of the met receptor too , and as such can induce the same effects as the hgf natural ligand . this concept is supported by the international patent application pct / ep2004 / 05097 entitled “ anti - hgf - r antibodies and the use thereof ”. hence , various concentrations of hgf have been tested . at low hgf concentrations ( 25 and 50 ng / ml ), the differentiation of pscs - thp1 towards hepatocytic cells is slow and the appearance of numerous oval cells is observed ( 80 % of the cells present in culture ) some mature hepatocytes ( 10 % of the dells in culture ) which produce a high concentration of albumin and some immature hepatocytes or hepatoblasts ( 10 % of the cells in culture ) which produce alpha - feto - protein . at medium doses of hgf ( 75 and 100 ng / ml ), the differentiation of pscs accelerates as shown by the appearance of numerous mature hepatocytes ( 80 % of the cells in culture ) which produce a high concentration of albumin ( 90 g per litre of albumin / 5 million cells per ml in culture ). the number of oval cells is moderate ( 15 % of the cells in culture ), and the number of immature hepatocytes or hepatoblasts producing alpha - feto - protein is also moderate ( 5 % of the cells in culture ). at high hgf concentrations ( 150 and 200 ng / ml ), the differentiation of pscs slows down and oval cells represent the vast majority of the observable cells ( 70 % of the cells in culture ), the immature hepatocytes or hepatoblasts are reasonably abundant ( 25 % of the cells in culture ) and produce alpha - feto - protein , while mature hepatocytes producing a high concentration of albumin are rare ( 5 % of the cells in culture ). characterisation of the thp - 1 cells in comparison to pscs - thp cells and pscs - thp1 liver like cells by cytofluorimetry ( facs ) adult blood contains hematopoietic progenitor cells . among the hematopoietic cells , the so - called “ hsc ” cells ( cd14 + and cd34 +) are pluripotent cells ( zhao , 2003 ) which , when suitably stimulated using growth factors , can differentiate and specialise into cells typical of a number of body parts , including the liver [ 1 - 2 ]. thp - 1 cells in culture incubated with 50 ng / ml mcsf show minimal expression at the seventh day and stabilise on the fifteenth day , when they show widespread and marked stable fluorescence for cd14 +, cd29 +, cd34 +, cd44 +, cd45 −/+, cd71 +, cd90 +, cd105 +, cd117 +, c - met ( pscs - thp1 ); after the fifteenth day said fluorescence is maintained stably in culture . untreated controls show fluorescence for cd29 +, cd34 +, cd44 +, cd90 +, cd105 +. from the fifteenth day thp - 1 cells in culture incubated with 25 , 50 , 100 , 200 ng / ml hgf show no fluorescence for cd14 +, cd29 +, cd34 +, cd44 +, cd45 −/+, cd90 +, cd105 + ( pscs - thp1 ), but are slightly positive for c - met and cd117 and show high fluorescence for albumin , alpha - feto - protein and cytokeratins ( 7 , 17 , 8 , 18 and 19 ) and cd71 . untreated controls show fluorescence for cd14 and cd45 . the results are reported in tables 16 and 17 as the geometric mean +/− the standard deviation ( sd ). at low hgf concentrations ( 25 and 50 ng / ml ), the differentiation of pscs - thp1 towards hepatocytic cells is slow and the appearance of several mature hepatocytes is observed ( 10 % of the cells present in culture ) which produce a high concentration of albumin , increasing progressively from day 7 to day 23 , and some immature hepatocytes or hepatoblasts ( 10 % of the cells in culture ) which produce alpha - feto - protein . at medium doses of hgf ( 100 ng / ml ), differentiation of the pscs accelerates as demonstrated by the appearance of numerous mature hepatocytes ( 80 % of the cells in culture ) which produce a high concentration of albumin ( 90 g / l of albumin / 5 million cells per ml in culture ), again with progressive increase from day 7 to day 23 . the number of immature hepatocytes or hepatoblasts producing alpha - feto - protein is moderate ( 5 % of the cells in culture ). at high concentrations of hgf ( 200 ng / ml ), the differentiation of pscs slows down , the immature hepatocytes or hepatoblasts are reasonably represented ( 25 % of the cells in culture ) and produce alpha - feto - protein , while mature hepatocytes producing a high concentration of albumin are rare ( 5 % of the cells in culture ). concentrations of albumin above the minimum detectable limits are observed in only 2 samples treated with hgf ( psc - thp1 liver like cells ) sampled at day 15 to 30 of the experiment . in accordance with the previous analysis , said data indicate the production of albumin by psc - thp1 liver like cells derived from the pluripotent psc - thp1 stem cell line . the data relating to microalbumin assays in the samples tested confirm the previous data on albumin , both numerically and temporally . infact , concentrations of microalbumin above the minimum detectable limits are observed in only 3 samples treated with hgf ( psc - thp1 liver like cells ) sampled at day 7 , 15 and 30 of the experiment . at all incubation times , the thp - 1 control cells show no significant presence of albumin by cytofluorimetry . by way of example , the data relating to the controls at day 23 are shown . at all incubation times , the thp - 1 cells treated with 50 ng / ml mcsf ( psc - thp1 ) show no detectable albumin by cytofluorimetry . by way of example , the data relating to the controls at day 23 are shown . pscs - thp1 cells incubated with hgf at 25 nanograms / ml ( 7 , 15 , 23 days ): at low concentrations of hgf ( 25 ng / ml and 50 ng / ml ), the differentiation of the pscs - thp1 cells towards hepatocytic cells is slow and the appearance of mature hepatocytic cells is observed ( 10 % of the cells in culture ) which produce a high concentration of albumin . pscs - thp1 cells incubated with hgf at 50 ng / ml ( 7 , 15 , 23 days ): as in the previous example , at reduced concentrations of hgf ( 25 and 50 ng / ml ), the differentiation of the pscs - thp1 towards hepatocytic cells appears moderate . pscs - thp1 cells incubated with hgf at 100 ng / ml ( 7 , 15 , 23 days ): at medium doses of hgf ( 100 ng / ml ), the differentiation of pscs accelerates as shown by the appearance of numerous mature hepatocytes ( 80 % of the cells in culture ) which produce a high concentration of albumin ( 90 g per litre of albumin / 5 million cells per ml in culture ) again with progressive increase from day 7 to day 23 . pscs - thp1 cells incubated with hgf at 200 ng / ml ( 7 , 15 , 23 days ): at high concentrations of hgf ( 200 , ng / ml ), the differentiation of the pscs slows the mature hepatocytes ( 5 % of the cells in culture ) producing a high concentration of albumin . samples of untreated thp - 1 cells in culture ( control ), treated with 50 ng / ml mcsf ( psc - thp1 ) and treated with various concentrations of hgf ( 25 , 50 , 100 , 200 ng / ml ) ( psc - thp1 liver like cells ) have been analysed at various incubation times ( days 7 , 15 , 23 ). at all incubation times , the thp - 1 control cells show no production of alpha - feto - protein by cytofluorimetry . by way of example , the data relating to the controls at day 23 are shown . at all incubation times , the thp - 1 cells treated with 50 ng / ml mcsf ( psc - thp1 ) show no production of alpha - feto - protein by cytofluorimetry . by way of example , the data relating to the controls at day 23 are shown . thp - 1 cells treated with hgf ( 25 , 50 , 100 , 200 ng / ml ) ( psc - thp1 liver like cells ): samples of cells in culture treated with various concentrations of hgf ( 25 , 50 , 100 , 200 ng / ml ) incubated for various times ( 7 , 15 , 23 days ) ( psc - thp1 liver like cells ) have been analysed . at low concentrations of hgf ( 25 and 50 ng / ml ), the differentiation of the pscs - thp1 cells towards hepatocytic cells is slow and the appearance of immature hepatocytes or hepatoblasts is observed ( 10 % of the cells in culture ) which produce alpha - feto - protein , with maximal expression at day 15 of incubation . pscs - thp1 cells incubated with hgf at 100 ng / ml ( 7 , 15 , 23 days ): at medium doses of hgf ( 100 ng / ml ), differentiation of the pscs accelerates as shown by the appearance of a moderate number of immature hepatocytes or hepatoblasts ( 5 % of the cells in culture ) producing alpha - feto - protein with progressive increase from day 7 to day 15 of incubation . pscs - thp1 cells incubated with hgf at 200 ng / ml ( 7 , 15 , 23 days ): at high concentrations of hgf ( 200 ng / ml ), the differentiation of pscs slows down , the immature hepatocytes or hepatoblasts are reasonably represented ( 25 % of the cells in culture ) and produce alpha - feto - protein , with a progressive increase from day 7 to day 15 , and a progressive decrease from day 15 to day 23 of incubation . the thp - 1 cells in culture incubated with 25 , 50 , 100 , 200 ng / ml hgf from the fifteenth day show no strong fluorescence for cytokeratins ( 7 , 17 , 8 , 18 and 19 ). all controls and psc - thp - 1 cells show no significant positive signal for alpha - feto - protein and the cytokeratins ( 7 , 17 , 8 , 18 e 19 ). the data relating to alpha - feto - protein assay in the supernatants show no detectable variation in concentration in the tested samples . the data relating to glucose assays in the supernatants show high concentrations from day 1 to day 7 of the second incubation of the experiment , with an increase in the samples treated with growth factors ( mcsf and hgf ). within the time range from day 7 to day 15 , a rapid decrease in the concentrations of glucose , first of all in the samples treated with hgf ( psc - thp1 liver like cells ) is detected . the data relating to the samples and controls at day 30 of the second incubation show concentrations below the detection limit . the data show that the mrna for gapdh is uniformly expressed in all tested samples , ensuring the correct storage of the mrna extracted , and the uniformity in the initial concentrations of the samples examined . the data show that the presence of the mrna for albumin is only clear in the hepg2 positive control and in the thp1 - psc liver like cells following stimulation with hgf ( optimal treatment range : 50 - 100 ng / ml hgf ) for at least 30 days ; in all other controls there appears to be no albumin mrna . the data show that the presence of the mrna for alpha - feto - protein is only clear in the hepg2 positive control , in the 2 thp1 - lif controls , in the thp1 - psc liver like cells following stimulation with hgf ( optimal treatment range : 50 - 100 ng / ml hgf ) for at least 10 days ( weak expression of the alpha - feto - protein mrna ), at day 15 ( maximum peak of expression of the alpha - feto - protein mrna ) and at day 30 ( slight expression of the alpha - feto - protein mrna ); in all other controls there appears to be no alpha - feto - protein mrna . the data show the presence of c - met mrna is rather well expressed in all the tested cells . the c - met mrna does not appear to be modulated by any treatment . the psc - thp1 liver like cells culture supernatants have been treated by western blotting , and the bands obtained have been cut out and digested . numerous peptides have been identified from the digestion . the peptides obtained have been processed using the profound - 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