Patent Application: US-79038910-A

Abstract:
an isolated mammalian progenitor cell or its progeny , isolated from articular cartilage , is disclosed . further is disclosed an equine progenitor cell isolated from the surface zone of equine articular cartilage tissue and uses thereof . also disclosed are a method of tissue repair , a medicament , and a method of treating mammalian cartilage damage using such progenitor cells or progeny .

Description:
in the following detailed description of the illustrated embodiments , reference is made to the accompanying drawings that form a part hereof , and in which is shown by way of illustration , specific embodiments in which the invention may be practiced . these embodiments are described in sufficient detail to enable those skilled in the art to practice the invention . also , it is to be understood that other embodiments may be utilized and that process , materials , reagent , and / or other changes may be made without departing from the scope of the present invention . equine bmsc were isolated from bone marrow stroma aspirates from the metacarpal bone ( mc iii ) using the classical plastic adhesion method . briefly , bone marrow was extracted under sterile conditions and blasted with serum free medium ( dmem with 4500 mg / l glucose and glutamax - 1 ™ [ dmem - hg / gm ] plus 100 μg ml − 1 gentamicin and 10 mm hepes buffer ) to liberate the cellular constituents . the cell solution was allowed to separate into a lipid and aqueous layer after which the aqueous layer ( containing the bmsc ) was filtered through a 40 μm nylon cell strainer . after centrifugation at 500 × g for 5 minutes , the supernatant was discarded and the cell pellet resuspended in serum free medium . cells and medium were transferred into culture flasks and incubated at 37 ° c . at 5 % co 2 . cells were allowed to settle for 4 days to allow for attachment . after 4 days flasks were washed thoroughly to ensure removal of all non - adherent cell types . cells were incubated at 37 ° c . at 5 % co 2 and received growth media changes every 48 hours . growth media consisted of serum free medium plus 10 % foetal bovine serum ( fbs ) and 10 ng ml − 1 fibroblastic growth factor - 2 ( fgf - 2 . peprotech , usa ). equine surface zone cartilage was harvested from the medial and lateral condyles of the distal end of mc iii . small , isolated cartilage defects typically seen in working horses were present in a minority of joints and avoided during tissue harvesting . chondrocytes were isolated by a sequential 700 i . u . ml − 1 pronase and 300 i . u . ml − 1 collagenase digestion as previously described ( archer et al ., 1990 ). following enzymatic digestion the cell suspension was filtered through a 40 μl cell strainer and centrifuged at 500 × g for 5 minutes . chondrocytes were re - suspended in 1 ml serum free medium ( dmem / f12 with glutamax - 1υ plus 100 μg ml − 1 gentamicin , 0 . 1 mm ascorbate - 2 - phosphate and 10 mm hepes buffer ) and counted using a haemocytometer . with the aim of isolating a progenitor population , a differential adhesion onto fibronectin was performed ( jones and watt , 1993 ; dowthwaite et al ., 2004 ). six - well culture plates were pre - treated with 1 % bovine fibronectin ( fn ) solution diluted in sterile pbs / ace ( 1 mm cacl 2 and 1 mm mgcl 2 distilled water + pbs , sterile filtered ) and incubated overnight at 4 ° c . chondrocytes were seeded at 4000 cells per ml − 1 with 1 ml added per well and incubated at 37 ° c . at 5 % co 2 for 20 minutes in serum free medium . after 20 minutes , the media plus non - adherent cells were carefully removed and replaced with growth medium ( serum free medium plus 10 % fbs ). on day 6 , colonies that consisted of greater than 32 cells were marked for cloning . a population of more than 32 cells was chosen as this number indicates that the colony derived from more than 5 population doublings of a single cell . the original cell can therefore be discounted as a transit amplifying cell and assumed to be a progenitor ( jones and watt , 1993 ). colonies were isolated using polystyrene cloning cylinders ( sigma - aldrich , uk ), trypsinised and transferred into 12 - well plates containing growth medium with the addition of 10 ng ml − 1 fgf - 2 . clones were cultured until confluent then passaged accordingly . in order to characterise the bmsc and acpc , cells were fixed and labelled with antibodies for cd90 ( bd biosciences , uk ) and cd166 ( serotec , uk ). additionally , notch - 1 expression of bmsc and acpc was determined using the notch - 1 c - 20 antibody ( santa cruz biotechnology inc . uk ). monolayer cultures of acpc and bmsc were washed with pbs , fixed in 4 % paraformaldehyde for 5 minutes and air dried . following a 30 - minute incubation with 0 . 2 % triton - x ( sigma - aldrich ) diluted in pbs , cells were rehydrated in pbs / t ( pbs + 1 % tween 20 ; polyethylenesorbitan monolaurate ; sigma - aldrich ) for 5 minutes then blocked for one hour with ( a ) 5 % normal goat serum for cd90 and cd166 ( b ) 5 % normal rabbit serum for notch - 1 at room temperature . primary antibodies diluted in pbs / t ( cd90 5 μg ml − 1 , cd166 and notch - 1 c20 10 μg ml − 1 ) were incubated with the cells at 4 ° c . overnight . appropriate species specific igg immunoglubulins diluted in pbs / t ( 5 μg ml − 1 ) and pbs served as negative controls . after washing in pbs / t , relevant fitc conjugated secondary antibodies diluted in pbs / t ( 10 μg ml − 1 ) were incubated for 1 hour at room temperature . goat anti - mouse fitc conjugated secondary antibody was applied to cd90 and cd166 treated cells ( including controls ). rabbit anti - goat fitc conjugated secondary antibody was applied to notch - 1 treated cells ( including controls ). dishes were washed thoroughly with pbs and mounted onto slides with vectorshield ® mounting media containing 4 ′, 6 - diamidino - 2 - phenylindole ( dapi ) ( vector laboratories , uk ). fluorescent labelling was observed and photographed using an olympus bx61 microscope . to compare the chondrogenic ability of bmsc and acpc , cells were cultured in chondrogenic medium in pellet mass culture . briefly , cells were harvested and resuspended in chondrogenic medium consisting of dmem - hg / gm , plus 2 % fbs , 100 μg ml − 1 gentamicin , 10 mm hepes buffer , 10 μg ml − 1 its , 0 . 1 mm ascorbate - 2 - phosphate , 10 nm dexamethasone and 10 ng ml − 1 transforming growth factor beta - 1 ( tgf - β1 , peprotech ). cells were placed in 1 . 5 ml tubes and centrifuged at 500 × g for 5 minutes to form a pellet consisting of 0 . 5 × 10 6 cells . pellets were incubated at 37 ° c . and 5 % co 2 and media changed every three days . after 21 days , chondrogenic pellets were fixed in neutral buffered formalin solution , photographed and embedded into paraffin wax , immunohistochemistry on the chondrogenic pellets was performed for aggrecan and collagen types i , ii and x . briefly , pellet sections were blocked with the appropriate serum for 30 minutes at room temperature . excess serum was removed and the sections were incubated with the primary antibodies , aggrecan - igd ; 6b4 ( kind gift from professor bruce cater son , cardiff university ), collagen type i ( 10 μg ml − 1 ; abcam , uk ), collagen type ii ( 10 μg ml − 1 ; dshb , usa ) and collagen type x ( 10 μg ml − 1 ; abcam ) in pbs / t overnight at 4 ° c ., sections were then washed in pbs / t and incubated with appropriate fitc - conjugated secondary antibodies ( 10 μg ml − 1 ) for 1 hour at room temperature . after 3 washes in pbs , sections were mounted under a coverslip using either vectorshield ® mounting medium containing propidium iodide ( pi ) or dapi . images were viewed and recorded using either the spe1000 confocal microscope system ( leica microsystems , uk ) or an inverted fluorescence microscope ( olympus bx61 ). at each experimental run , the appropriate immunoglobulin ( 10 μg ml − 1 ) replaced the primary antibody , as a negative control . for 6b4 +, collagen type ii and collagen type x , sections were subjected to a chondroitinase ( 0 . 25 u ml − 1 ; sigma - aldrich ) and hyaluronidase ( 2 u ml − 1 ; sigma - aldrich ) pre - treatment for 1 hour at 37 ° c . for collagen type i , cells were subjected to proteinase k ( 2 . 0 μg ml − 1 ; sigma - aldrich ) digest for 15 minutes . pellet cultures were established as described above . osteogenic differentiation medium comprised of dmem - hg / gm , 10 % fbs , 10 mm β - glycerophosphate , 10 nm dexamethasone and 0 . 1 mm l - ascorbic - acid - 2 - phosphate . pellets were incubated at 37 ° c . and 5 % co 2 and media changed every three days . after 21 days , osteogenic pellets were fixed in neutral buffered formalin solution , photographed and processed into technovit 9100 new ® ( taab laboratories , uk ) using the chemical catalytic method fully described by yang et al ., ( 2003 ) with fully destabilised resin at all steps as recommended by singhrao et al ., ( 2009 ). pellets were sectioned using glass knives to a thickness of 1 μm as previously described ( singhrao et al ., 2009 ). sections were de - acrylated in 2 - methoxyethyl acetate ( sigma - aldrich ) for 3 - 4 h followed by rehydration through xylene , a series of graded ethanol to distilled water . resin autofluorescence was quenched by immersing tissue sections in 1 % sodium borohydride and equilibrated in pbs as previously described ( singhrao et al ., 2009 ). rehydrated , de - acrylated pellet sections were stained to assess the mineral content using the von kossa silver impregnation technique ( 5 % aqueous silver nitrate for 30 minutes under bright light , 5 % sodium carbonate in 25 % formalin for 5 minutes and 5 % sodium thiosulphate for 2 minutes with water washes in between ). examination and images were recorded using the leitz dmrb light microscope ( leica microsystems , uk ). for immunolabelling with osteocalcin , the rehydrated pellet sections were digested with 5 mg / ml bovine testes hyaluronidase ( sigma - aldrich )/ pbs for 1 h at 37 ° c . sections were treated to blocking reagent for 1 hr in pbs / t + 0 . 5 % normal rabbit serum ( x0902 , dakocytomation ). the tissue sections were incubated with goat anti - osteocalcin ( santa cruz biotechnology inc ) diluted 20 μg ml − 1 in the blocking reagent and incubated at 4 ° c . overnight . the appropriate immunoglobulin ( 10 μg ml − 1 ) replaced the primary antibody , as a negative control . following overnight incubation , the sections were incubated for 1 h at room temperature in secondary detection antibody conjugated to rabbit anti - goat - fitc ( f02016 , sigma - aldrich ) at 5 mg / ml diluted in blocking buffer . sections were mounted under a coverslip using vectorshield ® containing pi . examination and images were recorded using the spe1000 confocal microscope system ( leica microsystems ) adipogenic differentiation was induced in 2 - d monolayer cultures using a modified protocol described by koch et al . ( 2007 ). briefly , cells were seeded in 6 well plates at 5 × 10 4 cells per well and cultured in growth medium until sub - confluent . cells were then treated with dmem - hg / gm plus 10 μg ml − 1 insulin , 1 μm dexamethasone , 100 μm indomethacin , 500 μm 3 - isobutyl - 1 - methyl xanthine ( ibmx ) and 15 % normal rabbit serum with medium changes taking place every 2 days . wells treated with growth medium were also set up as negative controls . after 6 days exposure to adipogenic induction medium , the cells were fixed with neutral buffered formalin solution and stained with oil red o for the presence of lipid droplets ( barbero et al ., 2003 ). cells were counterstained with haematoxylin for 1 minute . acpc were isolated by differential adhesion onto fibronectin . adherent cells formed a large number of colonies from a low seeding density . colonies were observed 6 days after initial seeding and were subsequently expanded in monolayer culture ( fig1 a & amp ; 1 b ). bmsc formed colonies on adherence to plastic - bound serum proteins ( fig1 c ). bmsc exhibited a higher rate of proliferation indicated by an average of 21 population doublings in 14 days , whilst acpc undertook on average 22 population doublings in 21 days . there was no indication of senescence in either cell type at this stage . immunolabelling of monolayer cultures demonstrated that both acpc and bmsc expressed the putative stem cell markers cd90 ( fig2 a & amp ; 2 b ) and cd166 ( fig2 c & amp ; 2 d ) in addition to the cell fate selector gene , notch - 1 ( fig2 e & amp ; 2 f ). the majority of cells positively labeled in all acpc colonies in addition to expanded bmsc . differences in labelling intensity between cells were observed . chondrogenic induction of both cell types produced pellets that were smooth and iridescent in appearance ( fig3 a & amp ; 3 b ) and stained positively for toluidine blue and safranin o ( images not shown ). bmsc pellets were spherical and generally larger than acpc pellets which were also more varied in size and shape . there was some evidence of zonal organisation in pellets from both cell types indicated by flattened cells at the surface and rounder cells towards the centre ( images not shown ). immunohistochemistry revealed positive labelling for type ii collagen ( fig3 c & amp ; 3 d ), aggrecan ( fig3 e & amp ; 3 f ) and type i collagen ( fig3 g & amp ; 3 h ), type x collagen was not detected in acpc pellets but was detected in all bmsc pellets representing a terminal cartilage phenotype ( fig3 i & amp ; 3 j ). osteogenic induction of acpc and bmsc produced pellets that were irregular and matt in appearance ( fig4 a & amp ; 4 b ) and were positive upon von kossa staining ( fig4 c & amp ; 4 d ). immunohistochemistry revealed positive labelling for osteocalcin suggesting bone formation ( fig4 e & amp ; 4 f ). adipogenic induction in both cell types revealed a positive result via oil red o staining ( fig5 ). in the negative controls kept in growth medium , no lipid staining was detectable in either cell type . articular chondrocytes and mesenchymal stromal cells are the two main cell sources used in cell - based cartilage repair therapies ; however , it is unclear if one is more suitable than the other . here , equine acpc and bmsc were isolated , partially characterised and their differentiation potential compared and contrasted . as yet , there is no unique equine mesenchymal stem cell marker . here , bmsc expressed cd90 , a putatative mesenchymal stem cell marker associated with bone marrow - derived . adipose - derived and umbilical cord - derived equine stem cells ( stephan et al ., 2007 ; pascucci at al ., 2007 ; hoynowski et al ., 2007 ). additionally , bmsc were further characterised by the expression of cd166 , a putatative human stem cell marker , thus demonstrating cross - reactivity between the two species . in comparison to bmsc , acpc demonstrated similar expression of the chosen cell membrane markers . they expressed both cd90 and cd166 , thus providing further evidence for their progenitor / stem cell status . the cell - fate signalling receptor , notch - 1 was also found to be expressed by both bmsc and acpc . although not a unique marker of acpc , notch - 1 signalling has been previously implicated in the maintenance of clonality and proliferation of bovine acpc ( dowthwaite et al ., 2004 ). in situ , it has been suggested that notch 1 signalling may play one of two roles in the surface zone of articular cartilage ; it may function to maintain cells in a proliferative state , or it may promote chondrocyte differentiation and cartilage growth ( dowthwaite at al ., 2004 ). notch 1 has also been implicated in tissue boundary specification and its expression at the articular surface is consistent with this role ( williams et al ., 2009 ). furthermore , notch 1 has also been implicated during the initial stages of chondrogenesis in human bone marrow stromal cells ( oldershaw at al ., 2007 ). it is , therefore , suggested that notch 1 is a possible candidate marker for cell selection in cartilage repair procedures . acpc were found to have the capacity to proliferate extensively in vitro in the presence of fgf - 2 and , like bmsc , maintain their morphological and growth characteristics over the study period . previous reports have demonstrated a population doubling time of 6 days for normal equine chondrocytes ( nixon et al ., 1992 ). here , we show that acpc have a population time more akin to bmsc with the former having a population doubling time of 1 day as , on average , they undertook 22 population doublings in 21 days . bmsc exhibited a slightly higher rate of proliferation indicated by an average of 21 population doublings in 14 days , and hence a population doubling time of less than 1 day . both acpc and bmsc have demonstrated pluripotency via differentiation into chondrogenic , osteogenic and adipogenic lineages . under chondrogenic differentiation , acpc pellets demonstrated a proteoglycan rich matrix , type ii collagen expression and a lack of type x collagen , confirming a hyaline cartilage phenotype . bmsc also demonstrated a proteoglycan rich matrix , with type ii collagen but type x collagen was also detected which suggested differentiation into terminally differentiated chondrocytes and thus the formation of a hypertrophic cartilage . hypertrophic cartilage is formed during the late stages of endochondral ossification , whereby mesenchymal cells differentiate through a series of cellular phenotypes into type x collagen synthesising hypertrophic chondrocytes before undergoing cell death . subsequent vascular in - growth from the perichondrium allows the infiltration of osteoblasts which replace the tissue with a mineralised bone matrix . ( lefebvre and smits ., 2005 ). hypertrophic cartilage not only lacks the specialised functional properties of hyaline cartilage but could also lead to the formation of mineralised bone and would , therefore , be detrimental to any cartilage repair procedure . under chondrogenic differentiation , both cell types expressed type i collagen . given the occurrence of significant amounts of type ii collagen in pellets from both cell types , it has been suggested that the matrix remodels from a fibrocartilage to a hyaline type cartilage with time in culture which is also seen in normal cartilage differentiation in the embryo ( craig et al ., 1987 ). it has also been questioned whether the presence of type ii collagen alone is a valid marker of cartilage repair quality ( roberts et al ., 2001 ). a more recent cartilage repair study revealed 78 % of cartilage biopsies had a fibrocartilaginous repair tissue ( roberts et al ., 2003 ). notably , the average time interval between graft and biopsy was greatest for biopsies of hyaline compared to fibrocartilage tissue which suggests that the initial repair tissue may be fibrocartilaginous , but may remodel over time to become more ‘ hyaline - like .’ all the same , in this study , both bmsc and acpc expressed type i and type ii collagen in this study with no apparent differences observed between the two cell types . under osteogenic differentiation both cell types demonstrated evidence of mineralisation via von kossa staining and positive osteocalcin immunolabelling . osteogenic differentiation has previously been reported in equine bone marrow - derived , adipose - derived umbilical cord - derived stem cells and equine peripheral blood derived progenitors ( stephan et al ., 2007 ; pascucci et al ., 2007 ; hoynowski et al ., 2007 , koerner et al ., 2006 ). we are , however , the first to report osteogenic differentiation of equine cells in 3d culture . marrow stromal cells have been previously described to enhance bone repair in human studies ( quarto et al ., 2001 ; marcacci et al ., 2007 ; morishita et al ., 2006 ) and although further molecular characterisation is required , acpc have clinical implications to augment equine bone repair and regeneration . our findings for adipogenic differentiation are comparable to other recently published studies . we found that the addition of 15 % rabbit serum significantly enhanced adipogenic differentiation in both cell types . our work confirmed that of janderova et al . ( 2003 ), who also demonstrated that by using 15 % rabbit serum instead of foetal calf serum , over 90 % of all human marrow stromal cells were oil red o positive within 6 days of adipogenic induction . we can conclude that there were no apparent differences observed between the two cell types for adipogenic differentiation . in addition to presenting a novel cell source for equine cartilage repair , acpc may be of great use in research targeting cartilage repair in humans . the horse represents a superior animal model for human cartilage repair for many reasons including similarity of joint anatomy and size and articular cartilage thickness . an ability to create multiple defects , as demonstrated in the trochlear ridge model , allows the comparison of different cell - based repair therapies and enhanced experimental throughput ( frisbie et al ., 2007 ). unlike smaller animal models , horses are athletic and , therefore , demonstrate comparable functional needs to humans . in addition , the ability to control exercise and monitor clinical recovery post - operatively improves the long term assessment of cartilage repair techniques ( frisbie at al ., 2007 ). we have demonstrated the first isolation of equine chondroprogenitors . equine acpc and bmsc demonstrated functional equivalence in their multipotent differentiation capacity . however , chondrogenic induction of equine acpc did not result in a hypertrophic cartilage phenotype , therefore , equine acpc are considered desirable in producing cartilage capable of functional repair . additionally , acpc hold an advantage over normal chondrocytes as they can divide to produce more cells at a faster rate and maintain their phenotype when cultured extensively . the foregoing description is presented for purposes of illustration and description of the various aspects of the invention . one of ordinary skill in the art will recognize that additional embodiments of the invention are possible without departing from the teachings herein . this detailed description , and particularly the specific details of the exemplary embodiments , is given primarily for clarity of understanding , and no unnecessary limitations are to be imported , for modifications will become obvious to those skilled in the art upon reading this disclosure and may be made without departing from the spirit or scope of the invention . relatively apparent modifications , of course , include combining the various features of one or more figures with the features of one or more of other figures . all such modifications and variations are within the scope of the invention as determined by the appended claims when interpreted in accordance with the breadth to which they are fairly , legally and equitably entitled . 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 . 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