Patent Application: US-201313898625-A

Abstract:
the invention relates to the provision of a novel cell population that can be used for tissue regeneration and the treatment of disease states associated with cell degeneration for age related tissue changes . the cell population are derived from adult stem / progenitor cells which are characterised by being positive or negative to the thy1 . 1 cell marker .

Description:
pancreatic ducts were isolated from 12 month old albino swiss ( glasgow ) rats by dissection and minced , prior to seeding in cmrl medium . the pdp cells emerged as a confluent monolayer after approximately 5 weeks in culture . these were then harvested and washed in pbs . pdpcs were maintained in culture in 20 mls cmrl 1066 medium ( invitrogen , paisley , uk .) supplemented with 10 % foetal bovine serum ( sigma , poole , uk ), 2 mm glutamax , 1 . 25 μg / ml amphotericin b , and 100 u / ml penicillin , 100 μg / ml streptomycin , ( all invitrogen , paisley , uk ) in t75 culture flasks with 0 . 2 μm filter caps ( corning , uk ) at 37 ° c . in a 5 % co2 atmosphere . sub - confluent cultures were passaged by the total removal of culture medium by pipette and the washing of the adherent cells by the addition of 10 mls calcium and magnesium - free hanks balanced salt solution ( hbss ), ( cambrex bio - science , wokingham , uk ) to the flask for 5 minutes at room temperature . after the removal of the hbss from the flask by pipette , 2 mls of trypsin - versene solution ( 200 mg / l versene , 500 mg / l trypsin ) was added to the flask . the flask was periodically examined microscopically until dissociation of the cell monolayer can be confirmed . cells were then removed by pipette and re - cultured as above at a density of ⅕ to 1 / 10 as desired , by the addition of 20 mls of fresh culture medium . pdpcs were maintained longterm in cmrl 1066 medium ( invitrogen , paisley , uk ) supplemented with 5 % foetal bovine serum ( sigma , poole , uk ), 2 mm glutamax , 1 . 25 ug / ml amphotericin b , and 100 u / ml penicinllin / streptomycin ( all invitrogen , paisley ) in t75 with 0 . 2 um filter caps at 37 ° c . in a 5 % co 2 atmosphere . pdpcs grow in a 37 ° c ., humidified 5 % co 2 atmosphere as a monolayer , and were passaged when 90 % confluent with trypsin - edta ( invitrogen ). cells were counted and replated at a density of 3300 cells / cm 2 . magnetic activated cell sorting ( macs ) was performed for isolation and depletion using 1 ug of primary antibody , mouse anti - rat thy1 . 1 ( cd90 ) ( serotec ) per 10 6 target cells for 20 minutes at 4 ° c . as per the manufacturers protocol ( dynabeads goat anti mouse igg ( dynal biotech )). sorted cell populations were resuspended cells in maintenance culture media and replated in tissue culture flasks . macs was performed on each positive and negative sorted population twice before use in experiments . all sorted population were checked with fluorescence activated flow cytometry before use in subsequent differentiation experiments . cells were resuspended in 0 . 5 % bsa in hbss . they are then centrifuged at 1000 × rpm for 10 minutes and the resulting cell pellet is resuspended in hbbs . after a viability count with trypan blue ( invitrogen , paisley , uk ), 1 × 10 6 cells / ml were labelled with 100 μl primary antibody . primary antibodies used were against cd90 ( serotec mca47r , 1 : 75 ), cd44 ( serotec mca643 , 1 : 10 ), cd49f ( serotec mca2034 , 1 : 50 ), cd147 ( serotec mca729 , 1 : 10 ), c - kit ( santa cruz sc - 19983 , 1 : 20 ), cd71 ( serotec mca 155ft , 1 : 10 ), cd24 ( bd biosciences 551133 , 1 : 50 ), cd45 ( bd biosciences 554875 , 1 : 50 ), cd31 ( serotec mca1334ga , 1 : 50 ) and cd34 ( santa cruz sc - 7324 , 1 : 50 ). secondary antibodies were added in 0 . 2 % bsa / pbs for 45 minutes at 4 ° c . in the dark . the cells were then washed and centrifuged three times at 1000 × rpm in 0 . 2 % bsa / pbs before labelling by the addition of 100 μl fitc - conjugated fab2 fragment of rabbit anti - mouse immunoglobulins ( dako cytomation , ely , uk 1 : 20 ) in 0 . 2 % bsa for 45 minutes at 4 ° c . in the dark . an isotype fitc control was also performed . after washing 3 × and centrifuging as before , the resulting cell pellet was resuspended in 1 ml of hbss and the cells analysed using a beckman coulter xl flow cytometer ( beckman coulter , high wycombe , uk ). for pancreatic differentiation thy1 . 1 positive and thy1 . 1 negative cell populations ( passage 30 ) were plated at 6600 cells / cm 2 cell density . after 24 hours maintenance media was removed and monolayers were washed thrice with hbss . cells were subsequently cultured in dmem : f12 ( lonza ) supplemented with 1 × its , 1 . 25 μg / ml amphotericin b , and 100μ / ml penicillin / streptomycin ( all invitrogen , uk ), nicotinamide 10 mm ( sigma ), kgf 10 ng / ml ( sigma ) and 0 . 2 % bsa ( sigma ) for hepatogenic differentiation cells were plated at 6600 cells / cm 2 in t75 and 6 well plates , and at 2500 cells / cm 2 in chamber slides ( nunc ) at 24 hours maintenance was replaced , after washing thrice with hbss , dmem : f12 ( lonza ) supplemented with fibroblast growth factor - 4 10 ng / ml ( sigma ), 1 × its , 100μ / ml penicinllin / streptomycin ( invitrogen ) and 0 . 2 % bovine serum albumin ( sigma ). medium changes were performed thrice weekly and cells were harvested for rna extraction from undifferentiated thy1 . 1 positive and thy1 . 1 negative cells at day 0 and day 28 for pancreatic differentiation and at day 0 , 7 , 14 , 21 and 28 for hepatic differentiation . cells undergoing hepatic differentiation in chamber slide were washed twice with pbs and fixed with 4 % paraformaldehyde for 15 minutes at room temperature between days 10 - 14 . undifferentiated thy1 . 1 positive and negative populations were also grown in chamber slides concurrently and were fixed as above at 90 % confluency . for staining of intracellular proteins cells were fixed as above . cells were thrice washed in pbs and permeabilized with 0 . 1 % triton x - 100 ( sigma - aldrich ) for 10 minutes . slides were incubated with donkey serum for twenty minutes and the incubated with previously optimised primary antibodies diluted in 0 . 5 % bsa / pbs against rat albumin ( abcam ab14255 , 1 : 100 ), ck19 ( biodesign int . m08029m , 1 : 100 ), ck7 ( chemicon mab3226 , 1 : 100 ), ck 18 ( sigma f - 4772 ) and vimentin ( abcam ab8979 , 1 : 50 ) for 1 hour . slides were washed thrice in pbs followed by the appropriate fitc labelled secondary antibody ( abcam ab6749 or dako cytomation f0313 , ely , uk ). omission of the primary antibody was performed as negative control . frozen sections of rat liver were used as positive control . slides were washed three times before mounting in vectashield and were visualised and photographed by fluorescence microscopy . total rna was extracted by using trizol ® according to the manufacturer &# 39 ; s instructions and quantified by genequant analyser . samples were dnase treated ( ambion ) and reverse transcription to cdna performed using superscript ii reverse transcriptase ( invitrogen ) according to manufacturer &# 39 ; s instructions . no rt negative controls were performed for all samples . rt - pcr was performed using taq polymerase ( invitrogen ). the housekeeping gene bactin was used to assess template quality . all pcr reactions were performed using a peltier thermal cycler - 200 . nested pcr was performed for pancreatic differentiation experiments . the following specific olignucleotide primers were used pdx 1 , insulin ii and glucagon ( pdx - 1 forward , 5 - cggccacacagctctacaagg - 3 ( seq id no : 1 ), reverse , 5 - ctccggttctgctgcgtatgc - 3 ( seq id no : 2 ), nested reverse 5 - ttccaggcccccagtctcgg - 3 ( seq id no : 3 ) ( 305 bp ), insulin , forward 5 - atggccctgtggatccgctt - 3 ( seq id no : 4 ); reverse , 5 - tgccaaggtctgaaggtcac - 3 ( seq id no : 5 ); nested forward , 5 - cctgctcatcctctgggagcc - 3 ( seq id no : 6 ) ( 209 bp ); glucagon , forward , 5 - gaccgtttacgtggctgg - 3 ( seq id no : 7 ); reverse , 5 - cggttcctcttggtgttcatcaag - 3 ( seq id no : 8 ); nested forward , 5 - acaaggcagctggcagcatgc - 3 ( seq id no : 9 ) ( 210 bp ). rat pancreatic total rna was reverse transcribed and used as positive control . the following specific oligonucleotide primers were used for hepatic differentiation experiments , albumin ( 141 bp ) forward5 - ctgggagtgtgcagatatcagagt - 3 ( seq id no : 10 ), reverse 5 - gagaaggtcaccaagtgctgtagt - 3 ( seq id no : 11 ), hnf3 beta ( 63 bp ) forward 5 - cctactcgtacatctcgctcatca - 3 ( seq id no : 12 ), reverse - cgctcagcgtcagcatctt ( seq id no : 13 ), hnf1 ( 138 bp ) alpha forward 5 - agctgctcctccatcatcaga - 3 ( seq id no : 14 ), reverse 5 - tgttccaagcattaagttttctattctaa - 3 ( seq id no : 15 ), gata4 ( 173 bp ) forward 5 - catgcttgcagttgtgctag - 3 ( seq id no : 16 ), reverse 5 - attctctgctacggccagta - 3 ( seq id no : 17 ), alpha - fetoprotein ( 124 bp ) forward 5 - gtcctttcttcctcctggagat - 3 ( seq id no : 18 ), reverse 5 - ctgtcactgctgatttctctgg - 3 ( seq id no : 19 ), cyp2b1 ( 549 bp ) forward 5 - gagttcttctctgggttcctg - 3 ( seq id no : 20 ), reverse 5 - actgtgggtcatggagagct - 3 ( seq id no : 21 ), ck19 ( 193 bp ) forward 5 - agtaacgtgcgtgctgacac - 3 ( seq id no : 22 ), reverse 5 - agtcgcactggtagcaaggt - 3 ( seq id no : 23 ), ck18 ( 70 bp ) forward 5 - ggacctcagcaagatcatggc - 3 ( seq id no : 24 ), reverse 5 ccacgatcttacgggtagttg - 3 ( seq id no : 25 ). the pcr products then underwent agarose gel electrophoresis and were visualised by ethidium bromide staining rat liver tissue was used as positive control . periodic acid schiff staining for glycogen storage was performed on undifferentiated thy1 . 1 positive and thy1 . 1 negative cells and on thy1 . 1 positive and negative populations at day 21 of hepatic differentiation . human liver sections were used as positive control . cells were fixed in 4 % paraformaldehyde at room temperature for 10 minutes . cells were thrice washed in pbs and permeabilized with 0 . 1 % triton x - 100 ( sigma - aldrich ) for 10 minutes and washed with pbs x2 and ddh20 x1 . cells were immersed in periodic acid solution ( 1 g / dl ) for 5 minutes at room temperature . wells rinsed in distilled water three times . cells immersed in schiff &# 39 ; s reagent for 15 mins at room temperature . cells washed in running tap water for 5 minutes . cells counterstained in haematoxylin solutions for 90 seconds . rinse cells in running tap water for 15 - 30 seconds . macs sorting of pdpcs was used to isolate populations expressing thy1 . 1 positive cells at more than 98 . 5 % and thy1 . 1 negative cells at 98 . 7 % purity respectively . these populations were then cultured and reassessed by flow cytometry regularly every 10 - 12 days and prior to any differentiation or characterisation experiments . phenotypically , thy1 . 1 positive and negative populations demonstrated distinct differences in morphology : the thy1 . 1 positive population exhibited a fibroblast like morphology ( fig1 , panel a ), while thy1 . 1 negative populations exhibited a more epithelial like morphology ( fig1 , panel d ). several differences were also observed in the expression of cell surface markers between thy1 . 1 positive and thy1 . 1 negative cell populations . both cell lines expressed cd147 , cd44 and cd49f . both were cd71 low and did not express the haematopoetic markers cd31 , cd34 , cd45 and c - kit . in contrast to the thy1 . 1 positive sorted cell population , the thy1 . 1 negative population was positive for cd24 ( fig2 a and 2 b ). the sub populations were also assessed by immunocytochemistry with hepatic , biliary and mesenchymal markers albumin , vimentin , ck7 and ck19 ( fig3 ). both thy1 . 1 sorted populations were negative for albumin , ck 7 and vimentin ( fig3 — panels b , c , f , g , j and k ). the thy1 . 1 positive population was also negative for ck 19 ( fig3 , panel o ), whereas the thy1 . 1 negative cells were weakly positive ( fig3 , panel n ). both populations were positive for c - met and nestin by rt pcr ( data not shown ). in summary , the thy1 . 1 negative cell population expresses the following cell surface marker profile : the thy1 . 1 positive cells express the following cell surface marker profile : thy1 . 1 positive and thy1 . 1 negative populations exhibited markedly different morphological changes in pancreatic differentiation media . the thy1 . 1 positive population , initially fibroblast - like in morphology , formed matted cell clusters by days 14 - 21 , and formed into islet - like spherical clusters by day 28 , which eventually detached from the parent cell layer ( fig1 , panels d , h , i ). in contrast , the thy - 1 . 1 negative cells remained in a monolayer with a small epithelial like morphology , with no development of three dimensional structures ( fig1 , panels a and b ). rt - pcr analysis of undifferentiated thy1 . 1 positive cells demonstrated positive pdx - 1 expression , but no expression of insulin or glucagon ( fig4 - b ). differentiated cell clusters all positive for the transcriptional expression of all three markers ( fig4 panel b ). thy1 . 1 negative cells , however , expressed pdx - 1 when grown in either maintenance , or differentiation media . notably , when grown in differentiation medium , despite showing no morphological changes , insulin transcription was detected in the thy1 . 1 negative population . glucagon was not expressed in the undifferentiated thy - 1 . 1 negative cells nor was it induced in vitro after differentiation ( fig4 panel a ). thy - 1 . 1 positive and negative pdpcs were culture in serum free , fgf4 containing media to assess hepatic potential . thy - 1 . 1 positive cells demonstrated a morphological change from fibroblastoid like to epithelial / cuboidal morphology . furthermore by day 28 luminal structures were evident throughout the culture with flattened epithelium . occasional three dimensional islet like structure similar to those seen in the pancreatic differentiation plates were also observed in the hepatic differentiation plates . the thy - 1 . 1 negative population remained in a monolayer with no evidence of three dimensional structures or of lumen like structures and no marked change in morphology ( fig1 a , c ). the inventor further examined the differentiation of the two populations by rt - pcr over a 28 day period . rt - pcr was performed for endodermal specific genes hnf3 beta and gata 4 , early liver maker alpha feto protein and ck18 , mature liver markers hnf1alpha , albumin and the cytochrome p450 enzyme cyp2b1 . undifferentiated thy1 . 1 negative cells expressed hnf3 — beta and ck19 by rt - pcr , but did not express albumin , ck18 , hnf1alpha , cy2b 1 , gata4 or alpha fetoprotein ( fig5 , panel b ) none of the other early , or mature liver markers , or cy2b1 were induced in the thy1 . 1 negative population . interestingly , undifferentiated thy1 . 1 positive pdpcs expressed the early endodermal markers hnf3 - beta , gata 4 and alpha fetoprotein , but did not express later markers of hepatocyte differentiation , hnf1 - alpha and albumin , until day 14 of hepatic differentiation ( fig5 panel a ). ck 19 , normally expressed by biliary cells , was also induced during days 14 - 28 consistent with the appearance of the lumenal structures in culture . the induction of albumin expression was confirmed by immunocytochemistry . undifferentiated cells , stained negatively for albumin content ( fig3 , panel a ), while day 14 differentiated cells were strongly positive for albumin staining ( fig3 , panel d ). interestingly , differentiated cells at the day 10 and 14 time points , stained negatively for the biliary markers ck19 and ck7 ( fig3 , panels n and g ), but did stain positively for vimentin in the lumen like structures only ( fig3 , panel l ). ck 18 was also expressed in undifferentiated thy1 . 1 positive cells and throughout the 28 day differentiation period . cyp2b 1 was present in undifferentiated thy1 . 1 positive cells and throughout the differentiation period . undifferentiated thy1 . 1 negative cells were negative for ck7 , vimentin and albumin expression and weakly positive for ck19 by immunocytochemistry . the thy1 . 1 positive population was negative for ck 19 the presence of stored glycogen , as determined by pas staining , was not observed in thy - 1 . 1 positive or negative pdpcs nor in day 21 differentiated thy - 1 . 1 negative cells . however positive staining with pas indicative of glycogen storage was observed in the thy1 . 1 positive differentiated cells by day 21 ( fig6 ). a cell subpopulation described herein ( e . g ., a thy1 . 1 positive or thy1 . 1 negative cell population ) can be employed in an animal model of disease such as diabetes . in one embodiment , a subpopulation of cells is used in a rodent concordant xenograft model of streptozotocin ( stz ) induced diabetes . c57bl / 6 mice are made diabetic by injection of stz on day 0 , while 750 , 000 cells ( e . g ., thy1 . 1 positive cells ) are injected into the tail vein of treated animals on day 3 . control animals are given an injection of saline or an equivalent number of c57bl / 6 bone marrow cells . blood glucose is monitored every 3 days . stabilization of blood glucose and / or increased survival relative to controls indicate that the administered cells give rise to insulin production in the animals . provided herein are details on the in vitro culture , selection and characterisation of thy1 . 1 positive and thy1 . 1 negative pdpc sub - populations . furthermore , there is disclosed details as to their potency with respect to differentiation to pancreatic and hepatic lineages and the provision of a cell population sorted using the marker thy1 . 1 , which displays lineage bipotentiality in vitro . thy1 . 1 is a cell surface protein whose function is not clearly understood . however , it has been suggested to be involved in cellular recognition ( gunter et al ., 1984 ; williams , 1985 ), cellular adhesion ( he et al ., 1991 ; hueber et al ., 1992 ) and signal transduction ( kroczek et al ., 1986 ). thy1 . 1 expression observed in various stem cell populations , notably the oval cell population in adult rat liver , has led to the supposition that thy1 . 1 may allow cells to recognize and adhere to stromal tissue , potentially as repair cells after injury . ( masson et al ., 2006 ; petersen et al ., 1998 ; terrace et al ., 2007 ). thy1 . 1 is also expressed on stem cells of the fetal liver , umbilical cord blood and mesenchymal stem cells in humans , mouse and rat . the present findings of greater in vitro potency within the thy1 . 1 positive population would be consistent with these observations . they also demonstrate a method of isolation and purification with which to enable further use of such cells . previous studies have demonstrated hepatic differentiation of a number of different cell types , including bone marrow derived mscs , mapcs , endometrial and pancreatic derived mscs ( jiang et al ., 2002 ; meng et al ., 2007 ; schwartz et al ., 2002 ; seeberger et al ., 2006 ). thy1 . 1 positive subpopulations of pdpcs share the morphological phenotype and express a number of cell surface markers with these populations , including cd44 +, cd24 -, cd45 -, cd31 - and cd34 . in contrast to this however , thy1 . 1 positive pdpcs appear to be a distinct cell type , expressing gata4 , hnf3 - beta and alpha feto protein , which have not been described as expressed for any of these other cell types . hnf3 - beta is a marker of definitive endoderm believed to play an important role in endoderm competency ( gualdi et al 1996 ) while gata4 is a transcription factor required for ventral foregut endoderm development and for early liver gene expression ( gualdi et al ., 1996 ; rossi et al ., 2001 ). hnf3 - beta has been demonstrated to direct nucleosome positioning within the context of the albumin enhancer ( mcpherson et al ., 1996 ; cirillo and zaret , 1999 ) with the subsequent facilitation of binding of gata4 to the albumin enhancer . both gata4 −/− and hnf3 - beta −/− embryos show defects in foregut morphogenesis ( duncan et al ., 1997 ). therefore the expression of hnf3 - beta and gata4 in undifferentiated pdpcs and the subsequent fgf stimulated induction of expression of liver specific genes such as albumin and hnf1 - alpha , is consistent with the proposal that hnf 3 - beta and gata 4 co - operate to control the potential of these cells to commit to a hepatic fate . moreover , the presence of pas staining in the thy1 . 1 positive population after 21 days of differentiation , demonstrated a functional characteristic of more mature hepatocytes , which is consistent with the expression of hnf1 - alpha . hnf1 - alpha is known to bind to genes whose products are related to mature hepatic functions , including carbohydrate storage and synthesis and lipid metabolism ( odom et al ., 2004 ). undifferentiated thy1 . 1 positive pdpcs express afp . this observation is consistent with reports describing afp expression in nestin positive islet derived progenitor cells and low level afp and ttr expression , prior to hepatogenesis in the early ventral foregut endoderm . this expression is subsequently lost in endoderm isolated from cardiac mesodermal signalling ( gualdi et al ., 1996 ; jung et al ., 1999 ; zulewski et al ., 2001 ). it has also been suggested that this is a feature of the default pancreatic fate of ventral foregut endoderm , ( deusch et als ). expression of afp in the thy1 . 1 positive pdpc population , which demonstrates capacity to both pancreatic and hepatic lineages , would not be inconsistent with this finding . ( deutsch et al ., 2001 ) significantly , the undifferentiated thy1 . 1 negative population , while expressing hnf3beta , did not express gata 4 , or alpha feto - protein , nor were they induced during the differentiation experiment . no evidence of hepatic competency was observed in the thy1 . 1 negative population . this is congruent with pdx - 1 expression and the absence of gata4 expression within this undifferentiated population . vimentin was not expressed in either undifferentiated thy1 . 1 positive or negative populations but was expressed in the cells forming the ductal - like structures during hepatic differentiation . vimentin is considered to represent a mesenchymal marker . however , masson et al observed coexpression of thy1 . 1 and vimentin in portal structures , as well as demonstrating vimentin expression in epithelial cells within tissue sections and in culture of fetal liver epithelial cells . ( masson et al ., 2006 ) the data pertaining to pancreatic differentiation are intriguing . no morphological evidence of islet like clusters was observed in the thy1 . 1 negative population . in contrast , thy1 . 1 positive pdpcs could readily be induced to a pancreatic lineage with characteristic morphological changes resulting in three dimensional islet like structures and the transcriptional expression of pdx - 1 , insulin and glucagon . the detection of pdx - 1 transcriptional expression in both populations indicates their potential to become insulin producing cells . notably , however , thy1 . 1 negative cells when grown in differentiation medium , despite showing no morphological changes , expressed insulin . glucagon was not expressed in the undifferentiated thy - 1 . 1 negative cells , nor was it induced in vitro after differentiation ( fig4 panel ). various different candidate populations of pancreatic progenitor / stem cell have been described previously , including islet progenitor cells expressing nestin or other neuronal stem cell markers , ( abraham et al ., 2004 ; cornelius et al ., 1997 ; lechner et al ., 2002 ; ramiya et al ., 2000 ). another population have been shown to express pdx - 1 , a known marker for insulin producing cells and these cells can stimulate both ductal and endocrine differentiation in vitro under appropriate conditions ( bonner - weir et al ., 2000 ; otonkoski et al ., 1993 ). moreover , there is evidence that pancreatic ductal epithelial cells have the potential to dedifferentiate to a progenitor cell capable of proliferation and formation of new islets and acini ( bonner - weir et al ., 2004 ) and most recently , ck19 + non - endocrine pancreatic epithelial cells ( nepcs ) were reported to be partially induced to differentiate into insulin producing cells in vivo , when in the presence of fetal pancreatic tissue ( hao et al ., 2006 ). the precise physiological role played by these cells has been questioned . dor et al . have challenged the view that neogenesis from ductal or progenitor cells occurs , instead arguing that beta cell replication , rather than new islet generation is the predominant mechanism by which pancreatic endocrine tissue regenerates after near - total pancreatectomy ( dor et al ., 2004 ) although this interpretation still remains controversial ( bonner - weir and weir , 2005 ). the present findings are consistent with a role for a non beta cell that can produce insulin as potentially facilitating such a process . it is clear that these cells offer an alternative insulin producing cell source for transplantation therapies . the inventor observed a time course of both morphologic and gene expression changes indicative of hepatic lineage differentiation by use of a serum free fgf - 4 containing differentiation protocol . the potential bipotentiality of embryonic ventral endoderm for pancreas and liver differentiation has been investigated in explant experiments where ventral endoderm differentiated to hepatic lineage by proximity to the cardiac mesoderm . the absence of inductive factors , such as fgf - 1 , fgf - 2 and fgf - 4 , secreted by cardiac mesoderm allow the default pancreatic pathway of ventral endoderm to continue ( deutsch et al ., 2001 ) and general fgf signalling antagonist inhibits heptogenesis in vitro ( jung et al ., 1999 ) and fgf - 4 ( zhu et al ., 1999 ). the present data are entirely congruent with this concept . the inventor has demonstrated isolation and characterisation of pdpcs , which in vitro , demonstrate potency and transcriptional responses to signalling consistent with a population of bipotential endodermal progenitors . previously , administration of unsorted pdpc populations in a murine streptozocin induced diabetes model have demonstrated differentiation and production of rat insulin with concurrent stimulation of mouse pancreatic regeneration ( shiels 2005 and wo 2006 / 120476 , both incorporated herein by reference ). 1 . abraham , e . j ., s . kodama , j . c . lin , m . ubeda , d . l . faustman and j . f . habener : human pancreatic islet - derived progenitor cell engraftment in immunocompetent mice . am . j . pathol . dev . 164 , 817 - 830 ( 2004 ). 2 . bonner - weir , s ., e . toschi , a . inada , p . reitz , s . fonseca , t . aye and a . sharma : the pancreatic ductal epithelium serves as a potential pool of progenitor cells . paediatric diabetes dev . 5 , 16 - 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