Patent Application: US-52978908-A

Abstract:
abstract markers , antibodies and recombinant scfvs for mesenchymal stem cell sub - populations and osteoclasts . the present invention relates to specific epitopes of surface membrane bound glycoproteins expressed by mesenchymal stem cells and pre - osteoclasts and relates to antibodies such as monoclonal antibodies and recombinant scfv or fragments thereof , raised to the particular epitope and their use in identifying , isolating , and characterization of mesenchymal stem cell sub - populations such as that termed “ stromal progenitor cells ” in bone marrow and identifying , isolating , and characterization of pre - osteoclasts in peripheral blood . by binding to a specific epitope on the cell surface , limbin / evc - 2 detection and separation by conventional cell sorting methodologies are facilitated .

Description:
standard molecular biology and recombinant biotechnology methods were used to create the scfv phage library from spleens and bone marrow of chickens immunised with cultured human mesenchymal stem cells , which were isolated from adult human bone marrow . the scfvs were expressed on the surface of phages and screened for their specificity against cultured human mscs . chickens were selected for development of the phage display scfv library since immunisation of chickens with human stem cells gives rise to a substantial and specific immune response . human , chicken evolutionary separation and lack of tolerance of human antigens by chickens makes chickens an excellent choice for production of an scfv phage display library . a better immune response to highly conserved surface membrane proteins results from use of chickens , the libraries are easier to create due the lower number of immunoglobulin genes in chickens and the libraries are cheaper and more effective than buying a large commercial library . sa brown chickens were obtained from agro - bio ™ ( la ferte st . aubin , france ); tr1 reagent and superscript ™ first strand synthesis system from invitrogen ™ ( carlsbad , calif . ); chloroform , 2 - propanol , ethanol , agarose and molecular biology grade water from sigma ™ ( st . louis , mo . ); perfectprep ™ gel cleanup from eppendorf ™ ( hamburg , germany ); xl i blue and quick - pik ™ electroelution capsules from stratagene ™ ( la jolla , calif . ); anti igy and rabbit anti - chicken fitc conjugate from pierce ™ ( rockford , ill . ); rnase removal solution “ mercury ” from clp direct ( san diego , calif . ); enzyme free cell dissociation solution from specialty media ™ ( phillipsburg , n . j .) and molecular biology consumables from new england biolabs ™ ( ipswich , mass .). electrocompetent xl1 blue ™ ( stratagene ™; la jolla , calif . ); trypsin , bovine serum albumin , sodium chloride , peg 8000 , glycine , triethylamine , superbroth medium and pbs tablets from sigma ™ ( st . louis , mo . ); antibiotics from roche ™ ( base1 ; switzerland ); ebm - 2 from cambrex ™ ( berkshire , uk ); micropulser ™ ( biorad ™; hercules , calif . ); sequencing primers were obtained from mwg ™ ( martinsried , germany ) bone marrow aspirates were obtained from the iliac crest of normal donors after informed consent was given . the mscs were isolated and expanded in culture as described previously in after percoll fractionation or by direct plating [ 7 , 48 ]. the aspirates were washed with dulbecco &# 39 ; s phosphate - buffered saline ( d - pbs ). the cell - containing fraction was gently layered onto a percoll cushion ( 1 . 073 g / ml ), at a density of 1 − 3 × 10 8 nucleated cells / 25 ml and centrifuged at 1 , 100 × g for 30 min at 20 ° c . the nucleated cell fraction at the interface with density 1 . 073 g / ml was collected , washed once with d - pbs and resuspended in msc culture medium ( 10 % fetal bovine serum ( fbs ) in dulbecco &# 39 ; s modified eagle &# 39 ; s medium containing 1 . 0 g / l glucose ( dmem - lg ) with antibiotic / antimycotic supplements ). cells were plated at 1 . 6 × 10 5 cells / cm 2 in t - 175 flasks . cultures were maintained at 37 ° c . in a humidified atmosphere and 5 % co 2 . at the end of p0 , adherent colonies were detached by cell scrapping and the cells were cryopreserved in 10 % dmso / 90 % fbs until used . the cryo - preserved stem cells were thawed in a 37 ° c . water bath and spun down in an eppendorf ™ desktop centrifuge at 1000 rpm for 5 minutes . the freezing medium was aspirated and the cells resuspended in a total of 10 ml of fully supplemented cell culturing medium and left in a 37 ° c . water bath for 45 minutes . the stem cells were spun down again and washed with pbs solution . the washing step was repeated once . 50 , 000 cells of each cell line were resuspended in a total volume of 200 μl pbs and injected intradermally under the wing of the chickens . all chickens were pre - bled prior to immunizations . two chickens ( no . 261 and 262 ) were injected with mesenchymal stem cells , the birds received a second boost injection of 50 , 000 cells three weeks later to enhance the immune response . a final boost was performed another three weeks later . the chickens were bled on day 32 and the eggs were collected starting three days after the final immunization step . all birds were sacrificed on day 49 , spleen and bone marrow was harvested , homogenized and stored in tr1 reagent ( invitrogen ™) method for preparation of mrna from spleen and bone marrow homogenates all the equipment used was cleaned with rnase removal solution ( clp direct ) prior to the experiments . homogenized spleen and bone marrow samples from chickens were thawed in a 30 ° c . water bath ; 10 ml of tr1 reagent was added and mixed thoroughly . the tubes were spun for 10 minutes at 2500 g and 10 ml of each supernatant was transferred into fresh polypropylene centrifugation tubes . 6 ml of chloroform was added under a laminar flow hood and the tubes were inverted several times . after 5 minutes , the samples were mixed again , incubated at room temperature for 5 minutes to allow nuclear proteins to dissociate from the rna and spun at 4 ° c ., 17 , 000 g for 15 minutes . the aqueous phase was transferred into fresh tubes , 7 ml of isopropanol added and the rna was precipitated by centrifugation at room temperature , 15 , 000 rpm for 20 minutes . the supernatant was discarded , the pellet washed with 70 % ethanol and precipitated again by centrifugation at 15 , 000 rpm for 10 minutes . the purified rna was dissolved in water and examined for yield and purity in a shimadzu ™ spectrophotometer . first - strand cdna synthesis was carried out according to the instructions in the invitrogen ™ instruction manual included with the superscript ™ first strand ™ synthesis system for rt - pcr . briefly , 25 μg of chicken mrna was mixed in depc water with dntp &# 39 ; s , oligo ( dt ) and incubated at 65 ° c . for 5 minutes . the samples were chilled on ice for 1 minute , mixed with rt buffer , mgcl 2 , dtt ( dithiothreitol ), rnase out ™ rnase inhibitor and incubated at 42 ° c . for 2 minutes . 5 μl of superscript ii ™ reverse transcriptase was added to the tubes and incubated for 50 minutes at 42 ° c . all reactions were terminated at 70 ° c . for 15 minutes , chilled on ice and incubated with rnase h for 20 minutes at 37 ° c . amplification of the target cdna using pcr was carried out according to the instructions in given in “ phage display — a laboratory manual ” by carlos barbas et . al ( 6 ). briefly , short linker scfv libraries were generated for all samples obtained from chickens 261 & amp ; 262 ( samples were pooled ; msc library ), 263 ( 7 day epc library ), 264 ( out - growth library ) and 284 ( control library ; pbs library ) using primers cscvho - f ( sense ) ( seq id no 11 ) and cscg - b ( seq id no 12 ) to amplify the chicken v h domains . similarly , primers cscvk ( sense ) ( seq id no 13 ) and ckjo - b ( reverse ) ( seq id no 14 ) were utilized to amplify the v λ domains . cscvho - f : chicken v h domain sense : seq id no : 11 cscg - b : chicken v h domain antisense : seq id no : 12 cscvk : chicken v λ domain sense : seq id no : 13 ckjo - b : chicken v λ domain antisense : seq id no : 14 the first - round pcr amplification of chicken v h sequences for the construction of scfv libraries . the primers cscvho - f ( short linker ) are paired with the cscg - b reverse primer to amplify v h segments from chicken cdna . the sense primers have a sequence tail that corresponds to the linker sequence that is used in the overlap extension pcr . the reverse primer has a sequence tail containing an sfi i site ; this tail is recognized by the reverse extension primer used in the second round pcr . 0 . 5 μg of cdna was amplified in 30 pcr cycles using 60 pmole of each primer according to the following protocol : an initial cycle at temperature of 94 ° c . for 5 minutes that resulted in the initial denaturation of the cdna . this was followed by 30 cycles of temperatures 94 ° c . for 15 seconds ; 56 ° c . for 15 seconds and 72 ° c . for 90 seconds . a final , one time extension step for 10 minutes at a temperature of 72 ° c ., was included at the end of the pcr protocol . the pcr products were then precipitated with ethanol and sodium acetate , stored at − 20 ° c . for 60 minutes before being spun down at 17 , 500 g at 4 ° c ., dissolved in water and analysed on a 2 % agarose gel . the pcr products were excised from the gel and eluted using eppendorf perfectprep ™ gel cleanup kit . the amplification of chicken v λ sequences for the construction of scfv libraries . the cscvk sense primer is combined with the ckjo - b reverse primer to amplify v λ gene segments from chicken cdna . cscvk has a 5 ′ sequence tail that contains an sfi i site and is recognized by the sense extension primer in the second round pcr . the reverse primer has a linker sequence tail that is used in the overlap extension . briefly , 0 . 5 μg of cdna was amplified in 34 pcr cycles using 60 pmole of each primer according to the following protocol : an initial cycle , at temperature of 94 ° c ., for a duration of 4 minutes , that resulted in the initial denaturation of the cdna . this was followed by 34 cycles of temperatures of 94 ° c . for 45 seconds ; 50 ° c . for 1 min and 72 ° c . for 90 sec . a final , one time extension step for 10 minutes at a temperature of 72 ° c . was included at the end of the pcr protocol . the pcr products were precipitated with ethanol and sodium acetate , stored at − 20 ° c . for 60 minutes , before being spun down at 17 , 500 g , 4 ° c ., dissolved in water and analysed on a 2 % agarose gel . the pcr products were excised from the gel , eluted using eppendorf &# 39 ; s perfectprep ™ gel cleanup kit and analysed in a shimadzu ™ spectrophotometer . an “ overlap extension pcr ” was performed to generate full length , short linker single chain antibodies . this pcr combines the chicken v h and v λ fragments for the construction of scfv libraries . the sense and reverse extension primers used in this second round of pcr ( csc - f ; seq id no : 5 and csc - b ; seq id no : 6 ) recognize the sequence tails that were generated in the first round of pcr . briefly , 100 ng of both v h and v λ pcr products were overlapped and amplified in 25 pcr cycles using 60 pmole of csc - f and csc - b primers according to the following protocol : an initial cycle , at temperature of 94 ° c ., for a duration of 5 minutes , that resulted in the initial denaturation of the cdna . this was followed by 25 cycles of temperatures of 94 ° c . for 15 seconds ; 56 ° c . for 15 seconds and 72 ° c . for 2 minutes . a final , one time extension step for 10 minutes at a temperature of 72 ° c . was included at the end of the pcr protocol . the pcr products were precipitated with ethanol and sodium acetate , stored at − 20 ° c . for 60 minutes , before being spun down at 17 , 500 g , 4 ° c ., dissolved in water and analysed on a 2 % agarose gel . the pcr products were excised from the gel , eluted using eppendorf &# 39 ; s perfectprep ™ gel cleanup kit and analysed in a shimadzu ™ spectrophotometer . the pcr products containing the msc library were digested with sfi i for 5 hours at 50 ° c . and subcloned into phagemid vector pcomb3xss targeting repertoire of immune scfv libraries to adult human mesenchymal stem cells electrocompetent xl1 blue bacteria were thawed on ice and mixed with the recombinant scfv libraries in a cuvette on ice . electroporation was performed with a micropulser ( biorad ™) at 2 . 5 kv , 25 μf , 200ω . the bacteria were transferred from the cuvette into glass tubes and incubated at 37 ° c ., 220 rpm for 1 hour . ampicillin ( 25 μg / ml ) and tetracycline ( 10 μg / ml ) were added to the transformed bacteria , incubated another hour at 37 ° c ., 220 rpm and transferred to a 500 ml flask containing 183 superbroth medium , ampicillin and tetracycline . 2 ml of vcsm13 helper phage was added , the samples incubated for 2 h , 37 ° c ., 220 rpm . kanamycin was added at 25 μg / ml and all cultures were incubated for 6 h , 37 ° c ., 220 rpm . the bacteria were spun down at 3 , 000 rpm for 15 minutes and the bacterial pellet stored at − 80 ° c . for future plasmid preparation purposes . the phages were precipitated from the supernatant by addition of 8 g peg , 6 g nacl followed by an incubation period of 30 minutes on ice and centrifugation at 15 , 000 g for 15 minutes at 4 ° c . the phage pellet was washed once with 1 % bsa / pbs and passed through a 0 . 2μ on filter . aliquots of the original input library and the screened library were electroporated into xl1 blue as described above . the number of transformants was 5 × 10 8 / library . twenty clones were randomly picked from a superbroth / carbenicillin plate and the scfv amplified by pcr using primers ompseq ( seq id no 7 ) and gback ( seq id no 8 ) the pcr products were digested with alul for 4 hours at 37 ° c . and analyzed on 4 % gels for unique restriction digest patterns . the isolation of the scfvs with reactivity against antigens on the mscs surface was achieved by iterative cycles of biopanning . biopanning is typically performed by incubating the library of phage - displayed scfvs with the targets , immobilized either on a plastic plate or on paramagnetic beads . the phages are allowed to bind to the immobilized target on the mscs , after which the unbound phage is washed away and the bound material is eluted . the eluted phages are then re - amplified and several additional cycles of binding and amplification are performed in order to enrich for phage clones , which have the ability to bind to the desired antigen target . this msc - specific phagemid library was transformed into e . coli , and rescued by the addition of vcsm13 helper phage . the resulting phage expressing scfv were added to cultured human mscs to select for msc - specific binders in a process known as panning . in total three rounds of panning were performed . in the first round of panning , the phage library was incubated with approximately 2 . 5 × 10 5 cultured human mscs with the addition of 7 × 10 6 pbmcs ( to remove non - specific scfv ). cells and phage were then incubated at 4 ° c . for 30 minutes at 150 rpm . unbound phage and pbmcs were washed away and phage bound to mscs were rescued for the next round of panning . approximately 5 × 10 4 human mscs and 3 × 10 6 pbmcs were used in the second round of panning and in the final most stringent round of panning , 5 × 10 3 mscs and 8 × 10 6 pbmcs were used . human mscs from the final round of panning were harvested and the phage used to infect e . coli which were subsequently plated and grown overnight at 37 ° c . colonies from these plates were picked and analysed for scfv insert using pcr . each pcr reaction was also digested with alui to identify clones producing unique scfv sequences . the recombinant scfvs raised and characterised in this fashion have been denoted tmsc1 , tmsc2 , tmsc3 and tmsc4 and are characterised by seq id nos : 5 , 7 , 3 and 9 respectively . the short chain variable fragment is understood to mean any fragment , which retains the antigen binding specificity of the antibody . these sequences are primers for library region flanking sequences . ompseq recognizes a sequence upstream of the scfv in the phagemid pcomb3xss , whilest gback binds to a sequence downstream of the scfv in pcomb3xss . these primers are used to verify that the subcloning into the phagemid has successfully been accomplished ( vs . primers 1 - 6 that were used to amplify chicken antibodies prior to subcloning ). the unique clones identified from the library of the invention secrete functional scfv into the supernatant for routine purification and analysis by flow cytometry . frozen glycerol stocks of unique scfv were used to inoculate a super broth ( sb ) agar plate containing carbenicillin ( carb ) and incubated at 37 ° c . overnight . a single colony from this plate was used to inoculate 2 ml of pre - warmed sb + carb and incubated at 37 ° c . for 8 hours . the 2 ml culture was then used to inoculate 250 ml of sb + carb and incubated for 3 hours at 37 ° c . at 250 rpm . expression of scfv was induced by addition of 0 . 4 ml of 0 . 5 m iptg to the culture . after 11 hours the culture was placed on ice and centrifuged at 11 , 000 rpm for 20 minutes at 4 ° c . the supernatant was filter sterilised using a 0 . 2 μm filter . a 6 × histidine ( his ) residue is tagged to the scfv to allow protein purification , and the presence of a haemaglutinin ( ha ) decapeptide tag allows for detection of scfv using anti - ha antibody . proteins that are engineered to express six histidine residues in tandem can be purified using a resin that contains ni 2 + ions that are immobilised by covalent linkage to nitrilotriacetic acid ( nta ). immidazole and nacl were added to the culture supernatant to a final concentration of 1 mm and 0 . 5m respectfully and was then added to a ni - nta agarose column ( qiagen ™) and allowed to drip through overnight at 4 ° c . the column was washed with 5 mm imidazole and bound scfv was eluted with 0 . 25m imidazole , concentrated using centricon filtration system and buffer exchanged with pbs . the quality of the scfv preparation was analyzed by immunoblotting . a 10 % sds - page was transferred onto a nitrocellulose membrane ( 30 v , 1 hour ). the membrane was blocked over night in 3 % bsa / pbs solution and then incubated in a 1 : 1000 dilution of anti - ha antibody ( roche ™) for 1 hour at room temperature . the scfv tmsc3 was detected by addition of a 1 : 5000 dilution of anti - rat hrp antibody ( roche ™) and detected using ecl ™ reagents . silver staining confirmed that the his - tag purification was successful . the scfv clones tmsc1 , 2 , 3 , and 4 were submitted to mwg ( germany ) for sequencing analysis using primers ompseq ( seq id no : 17 ) and gback ( seq id no : 18 ). scfv tmsc3 was submitted to rzpd ™ ( heidelberg ) for identification of the cell surface antigen . 50 μg of the scfv was used to detect potential binding domains in human fetal brain cdna expression library . this library contains 38 , 000 different proteins . rabbit anti - ha antibody was used as a secondary antibody . limbin is known to be the expression product of the evc2 ( ellis van creveld syndrome 2 ) gene [ 50 ]. techniques of the present invention have , for the fist time , determined that limbin is expressed on the cellular surface of mesenchymal stem cells . the invention provides the unexpected result that limbin can be used to selectively isolate and characterise mesenchymal stem cells . the current literature suggests a role of limbin in ellis von crefeld syndrome and dwarfism in cattle [ 51 ]. although the exact function of this protein is unknown , it appears to be important for normal growth and development [ 50 , 52 - 55 ]. researchers have determined that the evc2 gene is active in several organs and tissues before birth , including the heart , lungs , liver , kidneys , pancreas , and in muscles used for movement ( skeletal muscles ) [ 50 , 51 ]. changes in the evc2 gene are thought to also cause a skeletal disorder called weyers acrodental dysostosis [ 54 ]. people with this condition can have mild short stature , but often are of average height . other characteristic features include extra fingers and toes ( polydactyl ), unusually formed nails , and dental abnormalities [ 54 ]. only one evc2 mutation has been associated with weyers acrodental dysostosis . one skilled in the art will appreciate that limbin protein , can be used to generate scfvs for mesenchymal stem cells . an additional advantage of the present invention using scfvs over conventional methods concerns the fact that when mouse and rat mabs are used , it is essential to use fc - blocking agents to prevent non - specific binding of mab to cells . in the present invention , this is not necessary since chicken derived scfvs are used . cultured mscs were harvested by cell scrapping and washed with facs buffer ( dmem media + 1 % bovine serum albumin ( bsa )+ 0 . 02 % sodium azide ). a total of 50 μl of 10 μg / ml of column purified scfv was added to 1 × 10 6 cells per sample and incubated for 30 minutes on ice . cells were washed twice with media + 1 % bsa and binding of scfv to cells was detected by the addition of 50 μl of a 1 : 50 dilution of anti - ha fitc antibody for 30 minutes on ice . samples were washed twice in facs buffer . resuspended in 200 μl dmem + dapi ( 200 nm ) to gate out dead cells and debris . flow cytometry analysis was performed on the facs aria with diva software ( bd ). the ability of tmsc1 , 2 , 3 , and 4 scfvs to bind adult stem cells in human bone marrow was analysed as follows : human bone marrow was supplied by cambrex . 1 ml of human bone marrow was centrifuged at 350 g for 5 minutes at 4 ° c . and resuspended 1 ml of red blood cell lysing buffer and incubated at room temperature for 90 seconds . cells were centrifuged and washed with facs buffer ( dmem media + 1 % bsa + 0 . 02 % sodium azide ) and incubated with 20 % human serum in facs buffer and incubated on ice for 30 minutes ( this is fc blocking step when using cd markers ). the cells were then incubated with 50 μl of purified scfv at 10 μg / ml on ice for 30 minutes . cells were washed twice with facs buffer and incubated with 50 μl of a 1 : 50 dilution of rat anti - ha fitc ( miltenyi ) plus 10 μl of either cd3 apc , cd14 apc , cd19 apc cy7 , cd34 apc , cd45 pe cy7 , cd56 apc ( bd biosciences ™), and cd235a apc cy 7 incubated on ice for 30 minutes in the dark . cells were washed and analysed on a bd facsaria sorter . for mouse bone marrow , mice were sacrificed and bone marrow harvested . red blood cells were lysed using rbc lysing buffer ( sigma ) and cells resuspended in facs buffer . the cells ( 1 × 10 6 cells ) were then incubated with 50 μl of purified scfv at 10 μg / ml on ice for 30 minutes . cells were washed twice with facs buffer and incubated with 50 μl of a 1 : 50 dilution of rat anti - ha fitc ( miltenyi ) plus 10 μl of cd44 apc , incubated on ice for 30 minutes in the dark . cells were washed and analysed on a bd facsaria sorter . horse bone marrow was treated with red blood cell lysing buffer ( bd pharmlyse ) and resuspended in facs buffer . the cells ( 1 × 10 6 cells ) were then incubated with 50 μl of purified scfv at 10 μg / ml on ice for 30 minutes . cells were washed twice with facs buffer and incubated with 50 μl of a 1 : 50 dilution of rat anti - ha fitc ( miltenyi ) incubated on ice for 30 minutes in the dark . cells were washed and analysed on a bd facsaria sorter . human mesenchymal stem cells ( hmscs ) in monolayer culture will undergo adipogenic differentiation in the presence of adipogenic media containing dexamethasone , insulin , 3 - methyl - isobutylxanthine ( mix ) and indomethacin [ 45 ]. adipogenic differentiation is determined by the formation of lipid vacuoles . method : for each assay 2 wells ( one &# 39 ; treated &# 39 ; and one ‘ control ’) of hmscs are prepared at 2 × 10 5 cells per well of a 6 well plate ( in a volume of 2 - 3 mls ) and incubated at 37 ° c . and 5 % co 2 . cells are fed three times per week with hmsc medium until they become confluent . upon confluency 2 . 0 ml of the appropriate media is added to each of the wells . on day 1 ( post confluency ), treated &# 39 ; wells are fed with filter sterilized adipogenic induction medium ( 0 . 2 ml of 1 mm dexamethasone solution , 0 . 4 ml of 100 mm indomethacin solution , 2 ml of antibiotic - antimycotic solution , 20 ml fetal bovine serum , 2 ml 1 mg / ml insulin , 0 . 2 ml of 500 mm mix , 175 . 2 ml of hg - dmem ) and ‘ control ’ wells fed with hmsc growth ( 20 ml fetal bovine serum , 2 ml antibiotic - antimycotic solution , 178 ml low glucose dmem ). on days 5 , and 9 ‘ treated ’ wells are fed with adipogenic induction medium and control wells with hmsc growth medium . while on days 4 , 8 , 12 , 15 , and 17 treated wells are fed with adipogenic maintenance medium ( 2 ml of antibiotic - antimycotic solution 20 ml fetal bovine serum , 2 ml 1 mg / ml insulin , 176 ml of hg - dmem ) and control wells with hmsc growth medium . on day 19 the cells are fixed by rinsing each well with 2 ml of sterile dpbs , followed by 10 % formalin and incubated for 30 minutes at room temperature , cells are rinsed with 1 ml of dpbs and resuspended in 2 ml of dpbs . adipogenesis can be quantified using oil red o staining , briefly , pipet working solution of oil red o ( mix 6 parts of stock oil red o ( 0 . 3 g / 100 ml isopropanol 99 %) with 4 parts of distilled water ) until the layer of cells is covered and let stand for 5 minutes . aspirate off and rinse with tap water . pipet hematoxylin onto the plate and stain for 1 minute , wash in warm tap water for 4 min and observe staining on a microscope . stained structures are indicative of the lipid vacuoles which are characteristic of adipogenic cells . extract the oil red o using isopropanol . quantify the extracted stain using a spectrophotometer or 96 - well plate reader capable of reading absorbance at 490 nm - 520 nm method : cultured mscs are trypsinized , counted and resuspended in 0 . 5 ml complete chondrogenic medium ( dmem ( high glucose )+ 6 . 25 μg / ml bovine insulin , transferring , selenous acid , 5 . 33 μg / ml linoleic acid , 1 . 25 mg / ml bsa , 100 mm dexamethasone , 50 μg / ml ascorbic acid - 2 - phosphate , 40 μg / ml proline , 1 mm sodium pyruvate , 100 u / ml penicillin , 10 , 000 μg / ml streptomycin , 250 ng / ml amphotericin b + tgf - β3 @ 10 ng / ml ) such that each chondrogenic pellet contains 2 × 10 5 cells in a 15 ml polypropylene conical screw - top tube and incubated at 37 ° c ., 5 % co 2 [ 46 ]. after 24 hours the cells at the bottom of each tube contract into a ball or disc with a diameter of ½ to 1 mm . medium is changed 3 times a week and chondrogenic differentiation begins in 1 to 2 weeks . quadruplicate pellets are set up for each assay time point for day 0 , 14 , and 21 . two pellets at each time point are used for histological evaluation , and the remaining two are used for biochemical analysis . chondrogenesis can be quantified . briefly , pellets are harvested by paraffin embedding or frozen sectioned or placed in the appropriate solution for biochemical analysis ( papain digestion of pellets for analysis of sulfated glycosaminoglycans ( s - gag )). for high - quality thin histological sections , harvested pellets are immediately fixed for 30 to 60 min with an isotonic solution of 4 % paraformaldehyde or with 10 % buffered formalin . pellets are then transferred from the fixation solution to a 70 % ethanol solution in preparation for dehydration , paraffin embedding , sectioning , and staining export of s - gag to the extracellular matrix is a hallmark of the chondrogenic phenotype . determination of s - gag accumulation depends on the metachromatic change demonstrated by dimethylmethylene blue when complexed to s - gag , and the consequent shift in the absorption spectrum of the dye . briefly , pellets are digested with papain , and a solution of dmmb is added to the digest . a positive reaction results in a decrease in absorbance at 595 nm the values obtained are compared against a standard curve prepared using known quantities of chondroitin sulfate . method : human mscs plated at a density of 3 × 10 4 cells per well in a 6 - well plate are grown in the absence and presence of osteogenic supplements ( 0 . 1 ml of 1 mm dex solution , 10 ml of 1m ( 3 - glycerophosphate solution and 5 ml of 10 mm asap ) in hmsc culture media ( 100 ml fetal bovine serum , 10 ml antibiotic - antimycotic solution , 890 ml low glucose dmem ) for 16 days , with media changes performed twice weekly and a media volume of 2 ml per well [ 47 ]. osteogenesis can be quantified by measuring calcium deposition on day 16 . 29 . walsh , s ., et al ., expression of the developmental markers stro - 1 and alkaline phosphatase in cultures of human marrow stromal cells : regulation by fibroblast growth factor ( fgf )- 2 and relationship to the expression of fgf receptors 1 - 4 . bone , 2000 . 27 ( 2 ): p . 185 - 95 . 30 . stewart , k ., et al ., further characterization of cells expressing stro - 1 in cultures of adult human bone marrow stromal cells . j bone miner res , 1999 . 14 ( 8 ): p . 1345 - 56 . 31 . gronthos , s , and p j simmons , the growth factor requirements of stro - 1 - positive human bone marrow stromal precursors under serum - deprived conditions in vitro . blood , 1995 . 85 ( 4 ): p . 929 - 40 . 32 . gronthos , s ., et al ., the stro - 1 + fraction of adult human bone marrow contains the osteogenic precursors . blood , 1994 . 84 ( 12 ): p . 4164 - 73 . 33 . sigurjonsson , o . e ., k . o . guethmundsson , and s . guethmundsson , [ mesenchymal stem cells . a review .] . laeknabladid , 2001 . 87 ( 7 / 8 ): p . 627 - 632 . 34 . caplan , a . i ., review : mesenchymal stem cells : cell - based reconstructive therapy in orthopedics . tissue eng , 2005 . 11 ( 7 - 8 ): p . 1198 - 211 . 35 . hui , j . h ., et al ., mesenchymal stem cells in musculoskeletal tissue engineering : a review of recent advances in national university of singapore . ann acad med singapore , 2005 . 34 ( 2 ): p . 206 - 12 . 36 . gindraux , f ., et al ., human and rodent bone marrow mesenchymal stem cells that express primitive stem cell markers can be directly enriched by using the cd 49 a molecule . cell tissue res , 2006 . 37 . heckmann , l ., et al ., mesenchymal progenitor cells communicate via alpha and beta integrins with a three - dimensional collagen type i matrix . cells tissues organs , 2006 . 182 ( 3 - 4 ): p . 143 - 54 . 38 . letchford , j ., et al ., isolation of c 15 : a novel antibody generated by phage display against mesenchymal stem cell - enriched fractions of adult human marrow . j immunol methods , 2006 . 308 ( 1 - 2 ): p . 124 - 37 . 39 . boiret , n ., et al ., characterization of nonexpanded mesenchymal progenitor cells from normal adult human bone marrow . exp hematol , 2005 . 33 ( 2 ): p . 219 - 25 . 40 . deschaseaux , f ., et al ., direct selection of human bone marrow mesenchymal stem cells using an anti - cd 49 a antibody reveals their cd 45 med , low phenotype . br j haematol , 2003 . 122 ( 3 ): p . 506 - 17 . 41 . jones , e . a ., et al ., optimization of a flow cytometry - based protocol for detection and phenotypic characterization of multipotent mesenchymal stromal cells from human bone marrow . cytometry b clin cytom , 2006 . 70 ( 6 ): p . 391 - 9 . 42 . knight , r . l ., et al ., tissue engineering of cardiac valves : re - 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