the bone marrow stroma contains primitive and potentially self - renewing cells that can differentiate into various mesenchymal cells .
these mesenchymal stem cells ( msc ) are capable of supporting the expansion and differentiation of hematopoietic stem cells ( hsc ) in vitro and enhance hematopoietic engraftment in vivo ( 1 - 5 ) .
furthermore , msc express class i , but not class ii , histocompatibility antigens , and they are not immunogenic in in vitro assays and in preclinical or clinical models .
recently , it has been reported that there are mesenchymal precursors in cord blood and peripheral blood of normal individuals .
the results obtained demonstrate that after systemic infusion of cord blood msc into nude mice , human dna was found in the marrow and in ex vivo expanded stromal cells grown from the marrow of transplanted animals .
several groups also have reported bone and fat differentiation capacity from cord blood - derived nonhematopoietic cells ( 6 - 10 ) . despite the low initial frequency in cord blood , msc have a tremendous expansion potential in culture . due to the limited number of hsc in a given cord blood unit , the outcome of hsc transplantation ( hsct ) could be improved by coengraftment of the msc in addition to the hematopoietic precursors .
several lines of evidence suggest that msc produce some essential hematopoietic growth factors ( 1 - 5 ) .
based on these reports , we hypothesized that exogenously administered msc with cytokine gene - transfected would show additive or augmented effects on hsc expansion . in this study , mononuclear cells ( mnc ) were harvested from cord blood and seeded in long - term culture for ex vivo msc expansion .
we transfected culture - expanded msc with granulocyte macrophage - colony stimulating factor ( gm - csf ) and stem cell factor ( scf ) cytokine genes and then cotransplanted with cord blood mnc in nonobese diabetic / severe combined immunodeficiency ( nod / scid ) mice to further promote hsc engraftment .
although cd34 + cells can be selected for the preparation of hsc , many cord blood hsct practically use unselected mnc .
therefore , we chose unselected mnc samples instead of selected samples as the source of stem cells for transplantation .
our results show that human cells were significantly increased in cotransplanted mice , and the number was even higher with the cytokine gene - transfected msc than with transplantation of mnc alone .
these data not only provide direct evidence for the presence of msc in cord blood , but could also add to therapeutic profile of expanded msc for clinical use .
cord blood samples were collected from the umbilical vein after full - term vaginal delivery .
were suspended at a concentration of 110 cells / ml in mesencult medium ( stemcell technologies , inc . , vancouver , bc , canada ) and seeded in t-25 flasks . cultures were maintained at 37 in humidified air and 5% co2 with a regular change of medium .
after removal of non - adherent cells , the adherent layer was further cultured until 80 - 90% confluence . at this point ,
the cell population was expanded by successive passage and resulting adherent cells were used for experiments .
the adherent cells were detached with 0.05% trypsin - ethylenediamine tetraacetic acid and washed with phosphate - buffered saline ( pbs ) .
the cells were washed with pbs containing 2% bovine serum albumin , and incubated with fitc - conjugated cd45 ( bd biosciences , san jose , ca , u.s.a . ) , pe - conjugated cd73 ( bd biosciences ) , cd105 ( dinona , inc . , seoul , korea ) , and cd166 ( bd biosciences ) on ice for 30 min in the dark .
after fixation with 1% paraformaldehyde , flow cytometry was performed on the facsort instrument ( becton dickinson , san jose , ca , u.s.a . ) . for plasmid transfections , pegfp - n1 dna ( clontech , palo alto , ca , u.s.a . ) that encodes an enhanced green fluorescent protein ( egfp ) was used .
expanded msc were transfected with an egfp - expressing vectors after cloning with gm - csf ( pegfp - n1/gm - csf ) or scf ( pegfp - n1/scf ) . to create pegfp - n1/gm - csf or pegfp - n1/scf ,
after sequencing of gm - csf and scf genes , ecori was used for digestion to insert cdna3.1 and noti was treated for scf .
stable msc transfectants that expressed high levels of gfp were cloned by limiting dilution and selected using 2 mg / ml neomycin ( invitrogen corp . ,
the gfp - expressing cells were screened by a microplate spectrofluorometer ( shimadzu , kyoto , japan ) and confirmed by an enzyme - linked immunosorbent assay ( elisa ) ( r & d systems , minneapolis , mn , u.s.a . ) .
stable clones of msc with gfp and gm - csf or scf were maintained in dulbecco modified eagle medium ( gibco laboratories , grand island , ny , u.s.a . ) in the presence of neomycin until transplantation .
gfp - positive msc were visualized using a fluorescence microscope ( olympus , japan ) and then used for transplantation to nod / scid mice .
nod / scid mice were purchased from korea research institute of bioscience and biotechnology ( kribb , daejeon , korea ) and were maintained in sterile microisolator cages .
twelve to 24 hr after irradiation , cells in a final volume of 200 l were injected through the tail vein .
we designed 4 experimental groups , and each group received 110 cells as a total : group 1 ( n=5 ) received mnc only , group 2 ( n=7 ) received a mixture of mnc plus msc , 0.510 respectively , group 3 ( n=7 ) received a mixture of mnc plus gm - csf msc , 0.510 respectively , and group 4 ( n=6 ) received a mixture of mnc plus scf msc , 0.510 respectively , per mouse .
six to 8 weeks later , blood samples were collected from the retroorbital venous plexus of the nod / scid mice .
an aliquot of the blood was used for complete blood cell counts by sysmex xe-2100 ( sysmex corp .
human cell engraftment was detected by flow cytometry , using fitc - conjugated cd45 ( bd biosciences ) and pe - conjugated cd34 ( bd biosciences ) antibodies .
a part of venous blood was also exposed to a hypotonic solution , and fixed in methanol / acetic acid fixative .
nuclei were then stained with cep x ( dxz1 ) alpha satellite probe ( vysis , inc . , downers grove , il , u.s.a . ) and
cep y ( dyz1 ) satellite iii probe ( vysis ) for fluorescence in situ hybridization ( fish ) identification of human cells .
a total of at least 500 - 700 nuclei were examined in each case under a fluorescent microscope .
differences in engraftment percentages between experimental conditions were analyzed by student - t test . a p value less than 0.05 was considered statistically significant .
cord blood samples were collected from the umbilical vein after full - term vaginal delivery .
were suspended at a concentration of 110 cells / ml in mesencult medium ( stemcell technologies , inc . , vancouver , bc , canada ) and seeded in t-25 flasks . cultures were maintained at 37 in humidified air and 5% co2 with a regular change of medium .
after removal of non - adherent cells , the adherent layer was further cultured until 80 - 90% confluence . at this point ,
the cell population was expanded by successive passage and resulting adherent cells were used for experiments .
the adherent cells were detached with 0.05% trypsin - ethylenediamine tetraacetic acid and washed with phosphate - buffered saline ( pbs ) .
the cells were washed with pbs containing 2% bovine serum albumin , and incubated with fitc - conjugated cd45 ( bd biosciences , san jose , ca , u.s.a . ) , pe - conjugated cd73 ( bd biosciences ) , cd105 ( dinona , inc . , seoul , korea ) , and cd166 ( bd biosciences ) on ice for 30 min in the dark .
after fixation with 1% paraformaldehyde , flow cytometry was performed on the facsort instrument ( becton dickinson , san jose , ca , u.s.a . ) .
for plasmid transfections , pegfp - n1 dna ( clontech , palo alto , ca , u.s.a . ) that encodes an enhanced green fluorescent protein ( egfp ) was used .
expanded msc were transfected with an egfp - expressing vectors after cloning with gm - csf ( pegfp - n1/gm - csf ) or scf ( pegfp - n1/scf ) . to create pegfp - n1/gm - csf or pegfp - n1/scf ,
after sequencing of gm - csf and scf genes , ecori was used for digestion to insert cdna3.1 and noti was treated for scf .
stable msc transfectants that expressed high levels of gfp were cloned by limiting dilution and selected using 2 mg / ml neomycin ( invitrogen corp . ,
the gfp - expressing cells were screened by a microplate spectrofluorometer ( shimadzu , kyoto , japan ) and confirmed by an enzyme - linked immunosorbent assay ( elisa ) ( r & d systems , minneapolis , mn , u.s.a . ) .
stable clones of msc with gfp and gm - csf or scf were maintained in dulbecco modified eagle medium ( gibco laboratories , grand island , ny , u.s.a . ) in the presence of neomycin until transplantation .
gfp - positive msc were visualized using a fluorescence microscope ( olympus , japan ) and then used for transplantation to nod / scid mice .
nod / scid mice were purchased from korea research institute of bioscience and biotechnology ( kribb , daejeon , korea ) and were maintained in sterile microisolator cages .
twelve to 24 hr after irradiation , cells in a final volume of 200 l were injected through the tail vein .
we designed 4 experimental groups , and each group received 110 cells as a total : group 1 ( n=5 ) received mnc only , group 2 ( n=7 ) received a mixture of mnc plus msc , 0.510 respectively , group 3 ( n=7 ) received a mixture of mnc plus gm - csf msc , 0.510 respectively , and group 4 ( n=6 ) received a mixture of mnc plus scf msc , 0.510 respectively , per mouse .
six to 8 weeks later , blood samples were collected from the retroorbital venous plexus of the nod / scid mice .
an aliquot of the blood was used for complete blood cell counts by sysmex xe-2100 ( sysmex corp . ,
human cell engraftment was detected by flow cytometry , using fitc - conjugated cd45 ( bd biosciences ) and pe - conjugated cd34 ( bd biosciences ) antibodies .
a part of venous blood was also exposed to a hypotonic solution , and fixed in methanol / acetic acid fixative .
nuclei were then stained with cep x ( dxz1 ) alpha satellite probe ( vysis , inc . ,
downers grove , il , u.s.a . ) and cep y ( dyz1 ) satellite iii probe ( vysis ) for fluorescence in situ hybridization ( fish ) identification of human cells
. a total of at least 500 - 700 nuclei were examined in each case under a fluorescent microscope .
primary cultures of msc mainly consisted of colonies of spindle - shaped fibroblastoid cells ( fig .
wright - giemsa staining of detached msc showed a large cell population characterized by abundant cytoplasms with occasional vacuoles and projections , fine nuclear chromatins , and one or more nucleoli ( fig .
flow cytometry analyses demonstrated that msc expressed cd73 , cd105 , and cd166 in all cases , but were negative for cd45 ( fig .
the purity of expanded msc was 85 - 97% . at 24 hr after transfection , increased gfp expression was identified both by spectrofluorometry and elisa .
the highest efficiency of the transfection was obtained after 5 - 7 days of culture both with pegfp - n1/gm - csf and pegfp - n1/scf ( fig . 3 , 4 ) .
elisa detected increased gm - csf levels from msc cultures after transfection ( 273.78148.33 pg / ml ) compared to those before transfection ( 34.3517.06 pg / ml ) ( p<0.05 , n=4 ) .
scf also increased from 1.301.05 pg / ml to 24.1020.21 pg / ml post transfection , however , there were no statistical differences ( p>0.05 , n=4 ) . in mnc transplantation alone , the absolute numbers of total nucleated cell ( tnc ) count was 0.080.04 ( 10/l ) ( n=5 ) comparable to 0.130.05 ( 10/l ) ( n=7 ) in mice transplanted with mnc plus msc ( table 1 ) . however , higher levels of human xx or xy cell by fish ( fig .
5 ) were found in transplantation of mnc plus msc , 0.410.41 ( 10/l ) vs. 1.520.81 ( 10/l ) ( p<0.05 ) .
the tnc count , 4.630.96 ( 10/l ) ( n=7 ) , and the percentage of xx or xy cells , 5.680.58 ( 10/l ) , were significantly higher ( p<0.05 ) than those after transplantation of mnc alone . also , higher levels of tnc count , 2.930.33 ( 10/l ) ( n=6 ) , and xx or xy cells , 3.430.49 ( 10/l ) , were obtained following cotransplantation of mnc plus scf - transfected msc ( p<0.05 ) .
engraftment study with immunophenotypic analysis of cd45+/cd34 + cells by flow cytometry showed similar results ( table 1 , fig .
genetic modification of stem cells is an attractive target for gene therapy because of their higher proliferative capacity and long - term survival compared with other somatic cells .
msc have been demonstrated to be able to express exogenous protein for an extended period of time and to maintain this ability after transplantation in vitro ( 1 , 11 - 14 ) .
msc with forced expression of interferon beta were shown to inhibit the growth of malignant cells in vivo ( 11 , 12 ) .
gene - modification of msc with therapeutic cytokines also clearly augmented the antitumor effect and prolonged the survival of tumor - bearing animals ( 13 ) .
recently more efficient non - viral dna transfection methods have been introduced to obtain stably transfected human cells ( 14 , 15 ) .
it is possible that a therapeutic benefit from msc could be obtained by local production of growth factors and the provision of temporary immunosuppression .
human msc have a high expansion potential , genetic stability , and reproducible characteristics proven in widely dispersed laboratories ( 1 - 3 ) .
no adverse events during or after msc infusion have been reported , and no ectopic tissue formation has been observed .
based on these results , we hypothesized that exogenously administered msc with cytokine gene - transfected would show additive or augmented effects on hsc expansion . among the various cytokines
, we have previously demonstrated that the combination of scf , thrombopoietin , and flt3 ligand is sufficient for ex vivo expansion of cord blood ( 16 ) . with the addition of granulocyte ( g)-csf ,
the clonogenic and homing potentials of cord blood were even higher , although statistically significant differences were not observed .
17 ) also have shown that scf , interleukin ( il)-3 , il-6 , and gm - csf could induce the up - regulation of several adhesion molecules involved in the mobilization and homing of hsc .
currently gm - csf is one of the most widely used cytokines for the purpose of stem cell mobilization and for the treatment of neutropenic patients .
therefore , we transfected culture - expanded cord blood msc with gm - csf and scf cytokine genes , and then cotransplanted with cord blood mnc in nod / scid mice to further promote hsc engraftment .
we demonstrated that human cells were significantly increased in cotransplanted mice and even higher with the cytokine gene - transfected msc than in transplantation of mnc alone .
cotransplantation studies in animal models as well as in humans showed that primary or culture - expanded msc promote the engraftment of hsc ( 18 - 22 ) .
cotransplantation of msc and cord blood ( 18 , 19 , 21 ) or mobilized peripheral blood ( 20 ) cd34 + cells resulted in a significantly higher level of engraftment than with transplantation of cd34 + cells only .
cotransplantation of human leukocyte antigen ( hla)-identical sibling culture - expanded msc with an hla - identical sibling hsc was also feasible and safe without immediate infusional or late msc - associated toxicities ( 22 ) .
several lines of evidence suggest that msc produce several essential hematopoietic growth factors including il-6 , il-11 , scf , leukemia inhibitory factor , and flt3 ligand and express adhesion molecules and extracellular matrix proteins that are known to play an important role in hsc homing such as vcam1 , e - selectin , collagen i , and fibronectin ( 1 - 5 , 20 , 23 ) .
different studies report that msc have an immunomodulatory effect and are able to suppress proliferation of t lymphocytes both by cell - cell interaction and via soluble factors ( 22 - 25 ) .
it has been claimed that there are mesenchymal precursors in cord blood and peripheral blood of normal individuals ( 6 - 8 , 26 , 27 ) . despite the low initial frequency in cord blood , msc have a tremendous expansion potential in culture .
compared to bone marrow msc , cord blood cultures were slower to establish , but once established , the growth was maintained over multiple passages .
using optimized isolation and culture conditions , cells showing a characteristic mesenchymal morphology and immune phenotype were isolated .
the frequency of msc ranged from 0 - 2.3 clones per 110 mnc . due to the limited number of hsc in a given cord blood unit
, the outcome of hsc transplantation could be improved by coengraftment of the msc in addition to the hematopoietic precursors .
( 8) previously investigated the capability of transplanted cord blood msc to home and survive in the marrow of nude mice .
they have detected human dna after transplantation in the marrow of recipients as well as in ex vivo - expanded stromal cells prepared from the marrow of transplants . in this study we found that cord blood msc cotransplantation enhances human cell engraftment .
this enhancement was greater after cotransplantation of gm - csf- and scf - transfected msc . according to wagner et al .
( 26 ) several genes involved in mesodermal differentiation were differently expressed on msc from human bone marrow , adipose tissue , or umbilical cord blood .
these results implied that potentials of mesodermal development might be different depending on msc preparations . by upregulating genes involved in proliferation and homing of cord blood msc
msc are multipotent and believed to facilitate the engraftment of hsc when transplanted simultaneously in animal studies , and recently , even in human trials .
in this study , we transfected culture - expanded cord blood msc with gm - csf and scf cytokine genes and then cotransplanted with cord blood hsc to further promote hsc engraftment . the tnc count and the engraftment of cd45+/cd34 + cells and xx or xy positive human cells
were significantly increased in cotransplanted mice and even higher with the cytokine gene - transfected msc ( gm - csf > scf , p<0.05 ) than in transplantation of hsc alone .
our results indicate that msc may serve as a platform to deliver hematopoietic growth factors in clinical stem cell transplantation .
delivering genes involved in homing and cell adhesions , cxcr4 or vla , would further increase the efficiency of stem cell transplantation in the future .
 mesenchymal stem cells ( msc ) are multipotent in nature and believed to facilitate the engraftment of hematopoietic stem cells ( hsc ) when transplanted simultaneously in animal studies and even in human trials . in this study , we transfected culture - expanded msc with granulocyte macrophage - colony stimulating factor ( gm - csf ) and stem cell factor ( scf ) cytokine genes and then cotransplanted with mononuclear cells ( mnc ) to further promote hsc engraftment . 
 mnc were harvested from cord blood and seeded in long - term culture for ex vivo msc expansion . 
 a total of 1107 mnc plus msc/l were introduced to the tail vein of nonobese diabetic / severe combined immunodeficiency mice . 
 after 6 - 8 weeks later , homing and engraftment of human cells were determined by flow cytometry and fluorescence in situ hybridization studies . the total nucleated cell count and the engraftment of cd45+/cd34 + cells and xx or xy positive human cells were significantly increased in cotransplanted mice and even higher with the cytokine gene - transfected msc ( gm - csf > scf , p<0.05 ) than in transplantation of mnc alone . 
 these results suggest that msc transfected with hematopoietic growth factor genes are capable of enhancing the hematopoietic engraftment . delivering genes involved in homing and cell adhesions , cxcr4 or vla , would further increase the efficiency of stem cell transplantation in the future . 