Patent Application: US-201313742699-A

Abstract:
the invention concerns a homogenous population of human stem cells isolated from the full depth of human cartilage tissue and / or isolated from aged human cartilage ; and uses thereof .

Description:
12 well plates were coated with 10 μg ml − 1 plasma fibronectin ( sigma , uk ) in pbs containing 1 mm mgcl 2 and 1 mm cacl 2 ( pbs +) overnight at 4 ° c . dishes were blocked with 1 % bsa ( sigma ) in pbs + before chondrocytes were added . control dishes were treated with pbs + overnight at 4 ° c . tissue was obtained from patients undergoing hemiarthrotomy with full institutional ethical approval . full depth cartilage was removed from the grossly normal femoral condyle and incubated in 1 : 1 dmem / f12 ( gibco ) containing 10 % fcs ( gibco ) overnight . chondrocytes were then isolated by sequential pronase ( roche )/ collagenase ( sigma ) digestion as previously described ( dowthwaite et al 2004 ). briefly cartilage chips were incubated with pronase ( 70 units ml − 1 in dmem / f12 containing 5 % fcs ) for 3 hours at 37 ° c . pronase was removed and cartilage incubated with collagenase ( 300 units ml − 1 in dmem / f12 containing 5 % fcs ) overnight at 37 ° c . chondrocytes were centrifuged at 2000 rpm for 5 minutes , supernatant removed and resuspended in serum free dmem / f12 and counted . after isolation , chondrocytes ( 1 , 000 ml − 1 ) were seeded into individual wells of 12 well plates and incubated at 37 ° c . for 20 minutes in 1 : 1 dmem / f12 containing 0 . 1 % gentamycin ( dmem / f12 −). after 20 minutes , media ( and non - adherent cells ) was removed and placed in a second dish for 40 minutes at 37 ° c . before this media ( and non - adherent cells ) was removed and placed in a third dish . after removal of media at 20 and 40 minutes , fresh 1 : 1 dmem / f12 containing 0 . 1 % gentamycin and 10 % fcs ( dmem / f12 +) was added to the remaining adherent cells which were maintained in culture for up to 17 days . controls comprised cells subjected to differential adhesion on dishes coated with 1 % bsa in pbs +. within 3 hours of plating , initial chondrocyte adhesion was assayed by counting the total number of cells adhering to the bottom of the dish using an inverted microscope equipped with phase contrast optics and expressed as a percentage of the initial seeding density . colonies of chondrocytes consisting of more than 32 cells were counted using the same microscope at 8 , 12 , 14 and 17 days . colony forming efficiency ( cfe ) was calculated by dividing the number of colonies by the initial number of adherent cells . once colonies consisting of more than 32 cells had formed , they were identified under a light microscope . clones were trypsinised ( 0 . 25 %; gibco ) and extensively subcultured in dmem / f12 + 10 % fcs . cell numbers were calculated at each passage and population dynamics plotted . clonal cell lines were immunolabelled with antibodies to notch 1 ( c20 , 5 ug ml − 1 ; santa cruz biotechnology ), stro1 ( neat tc supernatant , gift from r oreffo , southampton university ) and msx1 ( n20 , 5 ug ml − 1 ; santa cruz biotechnology ) after fixing for 5 minutes in either 95 % etoh ( notch 1 , stro1 ) or 10 % nbfs ( msx1 ). primary antibodies were localised using relevant fluorescently conjugated antibodies and observed under a fluorescent microscope . clonal cell lines were labelled with 10 um cell tracker green ( invitrogen ) following the manufacturers instructions and injected into the wing bud of 3 day old ( hh st 12 - 14 ) chick embryos which had been previously windowed . embryos were resealed with sellotape and incubated for various times up to day 10 ( hh st 36 - 37 ) and wings were fixed in 10 % nbfs and processed for wax embedding . samples were sectioned at 10 μm , dewaxed and mounted in dpx before being examined under a fluorescent microscope . additional sections were immunolabelled with antibody 5d8 ( anti human type i collagen ; abcam ) and observed under a fluorescence microscope . as shown in fig5 , wax sections of aged human tissue that have been labelled with antibody to the transcription factor msx 1 , a marker for stem cells , showed that stem cells were present in both the surface and middle zones of cartilage tissue . this goes against conventional wisdom which presumed that stem cells were only present in the surface of cartilage tissue . archer c and francis - west p ( 2003 ) the chondrocyte . int . j biochem cell biol . 35 , 401 - 404 . brittberg , m , lindahl , a , nilsson , a , ohlsson , c , isakssin , o , peterson , l . ( 1994 ) treatment of deep cartilage defects in the knee with autologous chondrocyte transplantation . n engl j med . 331 , 889 - 895 . dowthwaite , g p , flannery , c r , lewthwaite , j , flannelly , j , archer , c w and pitsillides a a . ( 2003 ) a mechanism underlying the movement requirement for synovial joint cavitation . matrix biol . 22 , 311 - 322 . dowthwaite , g p , bishop j c , redman s n , khan i m , rooney p , evans d j , haughton l , bayram z , boyer s , thomson b , wolfe m s , archer c w . ( 2004 ). the surface of articular cartilage contains a progenitor cell population . j cell sci . 117 , 889 - 897 flannery c r , hughes c e , schumacher b l , tudor d , aydelotte m b , kuettner k e , caterson b . 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