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    <TD>Child's Nervous System</TD></TR>
  <TR>
    <TD>=A9&nbsp;Springer-Verlag&nbsp;2007</TD></TR>
  <TR>
    <TD>10.1007/s00381-007-0515-2</TD></TR></TBODY></TABLE><!--Begin =
Abstract-->
<H2 class=3Drubric>Original Paper</H2>
<DIV class=3DHeading1><A name=3Dtitle></A>Cytotoxicity of human =
umbilical cord=20
blood-derived mesenchymal stem cells against human malignant glioma =
cells </DIV>
<P class=3DAuthorGroup>Seok-Gu&nbsp;Kang<SUP>1</SUP>,=20
Sin&nbsp;Soo&nbsp;Jeun<SUP>2&nbsp;<A=20
href=3D"http://www.springerlink.com/content/l0182768h43vj125/fulltext.htm=
l#ContactOfAuthor2"><IMG=20
alt=3D"Contact Information"=20
src=3D"http://www.springerlink.com/content/l0182768h43vj125/contact.gif" =

border=3D0></A></SUP>, Jung&nbsp;Yeon&nbsp;Lim<SUP>2</SUP>,=20
Seong&nbsp;Muk&nbsp;Kim<SUP>3</SUP>, =
Yoon&nbsp;Sun&nbsp;Yang<SUP>4</SUP>,=20
Won&nbsp;IL&nbsp;Oh<SUP>4</SUP>, Pil-Woo&nbsp;Huh<SUP>1</SUP> and=20
Chun&nbsp;Kun&nbsp;Park<SUP>2</SUP></P>
<TABLE>
  <TBODY>
  <TR vAlign=3Dtop>
    <TD><SPAN class=3DAffiliation><A =
name=3DAff1></A>(1)&nbsp;</SPAN></TD>
    <TD><SPAN class=3DAffiliation>Department of Neurosurgery, The =
Catholic=20
      University of Korea College of Medicine, Uijeongbu St. Mary=92s =
Hospital,=20
      65-1, Kumoh-dong, Uijeongbu, 480-130, South=20
Korea</SPAN></TD></TR></TBODY></TABLE>
<TABLE>
  <TBODY>
  <TR vAlign=3Dtop>
    <TD><SPAN class=3DAffiliation><A =
name=3DAff2></A>(2)&nbsp;</SPAN></TD>
    <TD><SPAN class=3DAffiliation>Department of Neurosurgery, College of =

      Medicine, The Catholic University of Korea, Kangnam St. Mary=92s =
Hospital,=20
      505 Banpo-dong, Seocho-gu, Seoul, 137-701, South=20
Korea</SPAN></TD></TR></TBODY></TABLE>
<TABLE>
  <TBODY>
  <TR vAlign=3Dtop>
    <TD><SPAN class=3DAffiliation><A =
name=3DAff3></A>(3)&nbsp;</SPAN></TD>
    <TD><SPAN class=3DAffiliation>Department of Biomedical Science, The =
Catholic=20
      University of Korea College of Medicine, Kangnam St. Mary=92s =
Hospital, 505=20
      Banpo-dong, Seocho-gu, Seoul, 137-701, South=20
Korea</SPAN></TD></TR></TBODY></TABLE>
<TABLE>
  <TBODY>
  <TR vAlign=3Dtop>
    <TD><SPAN class=3DAffiliation><A =
name=3DAff4></A>(4)&nbsp;</SPAN></TD>
    <TD><SPAN class=3DAffiliation>MEDIPOST Biomedical Research =
Institute,=20
      MEDIPOST, Pungnap-2 dong, Songpa-gu, Seoul, 138-736, South=20
  Korea</SPAN></TD></TR></TBODY></TABLE>
<P><A name=3DContactOfAuthor2></A></P>
<TABLE class=3DContact>
  <TBODY>
  <TR>
    <TD vAlign=3Dtop><IMG alt=3D"Contact Information"=20
      =
src=3D"http://www.springerlink.com/content/l0182768h43vj125/contact.gif" =

      border=3D0></TD>
    =
<TD><STRONG>Sin&nbsp;</STRONG><STRONG>Soo&nbsp;</STRONG><STRONG>Jeun</STR=
ONG><STRONG></STRONG><BR><STRONG>Email:=20
      </STRONG><A=20
    =
href=3D"mailto:ssjeun@catholic.ac.kr">ssjeun@catholic.ac.kr</A></TD></TR>=
</TBODY></TABLE>
<P class=3DAffiliation><STRONG>Received:=20
</STRONG>3&nbsp;September&nbsp;2007&nbsp;&nbsp;<STRONG>Published online: =

</STRONG>30&nbsp;October&nbsp;2007 </P>
<DIV class=3DAbstract><A name=3DAbs1></A><SPAN =
class=3DAbstractHeading>Abstract</SPAN>
<DIV class=3DAbstractSection>
<DIV class=3D""><SPAN class=3DAbstractSectionHeading><A=20
name=3DASec1></A>Background&nbsp;&nbsp;</SPAN>Mesenchymal stem cells =
(MSCs)=20
represent a potential useful source for cell-based glioma therapies =
because=20
these cells evidence both orthodox and unorthodox plasticity and also =
show=20
tropism for cancer. In this study, the authors attempted to access the=20
cytotoxicity of human umbilical cord blood (hUCB)-derived MSCs, with or =
without=20
cytokine activations against malignant glioma cells. </DIV></DIV>
<DIV class=3DAbstractSection>
<DIV class=3D""><SPAN class=3DAbstractSectionHeading><A =
name=3DASec2></A>Materials and=20
methods&nbsp;&nbsp;</SPAN>hUCB-derived MSCs were activated by =
interleukin-2,=20
interleukin-15, granulocyte macrophage colony-stimulating factor, and=20
combinations. The hUCB-derived MSCs and activated hUCB-derived MSCs were =

effector cells. The cytotoxicity of the unactivated hUCB-derived MSCs =
and=20
activated hUCB-derived MSCs against the target cells (human malignant =
glioma=20
cells) was estimated via visual survival cell assays and transwell =
inserts.=20
Phenotypic changes occurring in these hUCB-derived MSCs before and after =

cytokine activation were determined via flow cytometry. The secreted =
proteins=20
from these effector cells were estimated via enzyme-linked immunosorbent =
assays.=20
</DIV></DIV>
<DIV class=3DAbstractSection>
<DIV class=3D""><SPAN class=3DAbstractSectionHeading><A=20
name=3DASec3></A>Results&nbsp;&nbsp;</SPAN>We noted a significant =
cytotoxicity of=20
hUCB-derived MSCs against malignant glioma cells. In addition, the =
hUCB-derived=20
MSCs activated with cytokines evidenced significantly higher =
cytotoxicity than=20
that observed with unactivated hUCB-derived MSCs. Differentiated immune=20
effectors cells from the hUCB-derived MSCs after cytokine activation =
were not=20
shown to have increased in number. However, the activated hUCB-derived =
MSCs=20
secreted more immune response-related proteins (interleukin 4, =
interferon-&#947;)=20
than did the unactivated hUCB-derived MSCs. </DIV></DIV>
<DIV class=3DAbstractSection>
<DIV class=3D""><SPAN class=3DAbstractSectionHeading><A=20
name=3DASec4></A>Conclusion&nbsp;&nbsp;</SPAN>The data collected herein =
confirm=20
for the first time that hUCB-derived MSCs, with or without activation, =
evidence=20
significant cytotoxicity against human malignant glioma cells, and the =
immune=20
response-related proteins secreted in this process may perform relevant=20
functions. </DIV></DIV></DIV>
<P class=3DKeyword><SPAN=20
class=3DKeywordHeading>Keywords&nbsp;&nbsp;</SPAN>Cytokine&nbsp;-&nbsp;Cy=
totoxicity&nbsp;-&nbsp;Malignant=20
glioma&nbsp;-&nbsp;Mesenchymal stem cell&nbsp;-&nbsp;Umbilical cord =
blood </P>
<DIV class=3D""><A name=3DSec1></A>
<HR>

<DIV class=3Dheading2>Introduction</DIV>
<P class=3D"">Although many advances have recently been made in the =
conventional=20
treatment of malignant gliomas via surgery, radiotherapy, and =
chemotherapy, this=20
disease continues to have a very generally poor prognosis [<CITE><A=20
href=3D"http://www.springerlink.com/content/l0182768h43vj125/fulltext.htm=
l#CR9">9</A></CITE>,=20
<CITE><A=20
href=3D"http://www.springerlink.com/content/l0182768h43vj125/fulltext.htm=
l#CR14">14</A></CITE>,=20
<CITE><A=20
href=3D"http://www.springerlink.com/content/l0182768h43vj125/fulltext.htm=
l#CR34">34</A></CITE>].=20
New trials regarding the combination of chemotherapeutic agents in an =
orthotopic=20
model or new target agents in clinical field have been attempted; =
however, the=20
results of these trials have not proven satisfactory [<CITE><A=20
href=3D"http://www.springerlink.com/content/l0182768h43vj125/fulltext.htm=
l#CR7">7</A></CITE>,=20
<CITE><A=20
href=3D"http://www.springerlink.com/content/l0182768h43vj125/fulltext.htm=
l#CR15">15</A></CITE>,=20
<CITE><A=20
href=3D"http://www.springerlink.com/content/l0182768h43vj125/fulltext.htm=
l#CR29">29</A></CITE>,=20
<CITE><A=20
href=3D"http://www.springerlink.com/content/l0182768h43vj125/fulltext.htm=
l#CR31">31</A></CITE>,=20
<CITE><A=20
href=3D"http://www.springerlink.com/content/l0182768h43vj125/fulltext.htm=
l#CR36">36</A></CITE>].=20
Therefore, there is a definite need to develop innovative and effective=20
treatments for this condition. Stem cells (SCs) may perform crucial =
functions=20
with regard to the treatment of malignant glioma, a condition that is =
deeply=20
troubling and disruptive to both patients and their families [<CITE><A=20
href=3D"http://www.springerlink.com/content/l0182768h43vj125/fulltext.htm=
l#CR6">6</A></CITE>].=20
</P>
<P class=3D"">Mesenchymal SCs (MSCs), which reside within the stromal =
compartment=20
of the bone marrow, have the capacity to differentiate into cells of =
connective=20
tissue lineages [<CITE><A=20
href=3D"http://www.springerlink.com/content/l0182768h43vj125/fulltext.htm=
l#CR3">3</A></CITE>,=20
<CITE><A=20
href=3D"http://www.springerlink.com/content/l0182768h43vj125/fulltext.htm=
l#CR32">32</A></CITE>]=20
and cells that are not a part of their normal repertoire [<CITE><A=20
href=3D"http://www.springerlink.com/content/l0182768h43vj125/fulltext.htm=
l#CR26">26</A></CITE>,=20
<CITE><A=20
href=3D"http://www.springerlink.com/content/l0182768h43vj125/fulltext.htm=
l#CR28">28</A></CITE>,=20
<CITE><A=20
href=3D"http://www.springerlink.com/content/l0182768h43vj125/fulltext.htm=
l#CR38">38</A></CITE>].=20
Recent studies have indicated that MSCs have the capacity to target =
therapeutic=20
genes to malignant glioma [<CITE><A=20
href=3D"http://www.springerlink.com/content/l0182768h43vj125/fulltext.htm=
l#CR11">11</A></CITE>,=20
<CITE><A=20
href=3D"http://www.springerlink.com/content/l0182768h43vj125/fulltext.htm=
l#CR23">23</A></CITE>=96<CITE><A=20
href=3D"http://www.springerlink.com/content/l0182768h43vj125/fulltext.htm=
l#CR25">25</A></CITE>].=20
Thus, gene therapies involving such as interleukin (IL) 2 [<CITE><A=20
href=3D"http://www.springerlink.com/content/l0182768h43vj125/fulltext.htm=
l#CR25">25</A></CITE>]=20
and interferon-&#946; [<CITE><A=20
href=3D"http://www.springerlink.com/content/l0182768h43vj125/fulltext.htm=
l#CR24">24</A></CITE>],=20
which employ MSCs as a targeting vehicle, may potentially allow for the=20
development of a novel therapeutic approach for malignant glioma. As the =
notion=20
of unorthodox plasticity in MSCs remains somewhat interesting [<CITE><A=20
href=3D"http://www.springerlink.com/content/l0182768h43vj125/fulltext.htm=
l#CR2">2</A></CITE>,=20
<CITE><A=20
href=3D"http://www.springerlink.com/content/l0182768h43vj125/fulltext.htm=
l#CR5">5</A></CITE>,=20
<CITE><A=20
href=3D"http://www.springerlink.com/content/l0182768h43vj125/fulltext.htm=
l#CR20">20</A></CITE>,=20
<CITE><A=20
href=3D"http://www.springerlink.com/content/l0182768h43vj125/fulltext.htm=
l#CR28">28</A></CITE>,=20
<CITE><A=20
href=3D"http://www.springerlink.com/content/l0182768h43vj125/fulltext.htm=
l#CR38">38</A></CITE>],=20
the authors utilized rat MSCs as direct immune effector cells, rather =
than=20
simple gene vehicles, and presented the results regarding the direct =
cytotoxic=20
effect of rat MSCs against rat malignant glioma cells [<CITE><A=20
href=3D"http://www.springerlink.com/content/l0182768h43vj125/fulltext.htm=
l#CR13">13</A></CITE>].=20
</P>
<P class=3D"">Human umbilical cord blood (hUCB) is a rich source of both =

hematopoietic SCs [<CITE><A=20
href=3D"http://www.springerlink.com/content/l0182768h43vj125/fulltext.htm=
l#CR33">33</A></CITE>]=20
and MSCs [<CITE><A=20
href=3D"http://www.springerlink.com/content/l0182768h43vj125/fulltext.htm=
l#CR39">39</A></CITE>].=20
In addition, hUCB-derived SCs evidence higher proliferation and =
expansion=20
potential than has been observed in their adult bone marrow counterparts =

[<CITE><A=20
href=3D"http://www.springerlink.com/content/l0182768h43vj125/fulltext.htm=
l#CR35">35</A></CITE>,=20
<CITE><A=20
href=3D"http://www.springerlink.com/content/l0182768h43vj125/fulltext.htm=
l#CR37">37</A></CITE>].=20
Furthermore, the authors have detected more cytotoxic capability of =
immune=20
effector cells form hUCB than with cells derived from peripheral blood =
against=20
malignant glioma cells and medulloblastoma cells [<CITE><A=20
href=3D"http://www.springerlink.com/content/l0182768h43vj125/fulltext.htm=
l#CR12">12</A></CITE>].=20
hUCB may, therefore, be considered to be an alternative source of MSCs =
[<CITE><A=20
href=3D"http://www.springerlink.com/content/l0182768h43vj125/fulltext.htm=
l#CR39">39</A></CITE>].=20
</P>
<P class=3D"">In the current study, we attempted, for the first time, to =
access=20
the cytotoxicity of hUCB-derived MSCs and activated hUCB-derived MSCs =
against=20
malignant glioma cells. Moreover, we reviewed the changes in cellular =
phenotype=20
and immune response-related proteins from hUCB-derived MSCs before and =
after a=20
variety of cytokines activation. </P></DIV>
<DIV class=3D""><A name=3DSec2></A>
<HR>

<DIV class=3Dheading2>Materials and methods</DIV>
<DIV class=3D""><A name=3DSec3></A>
<DIV class=3DHeading3>Collection and processing of hUCB</DIV>
<P class=3D"">The hUCB was obtained postpartum from the umbilical cords =
of=20
full-term health infants delivered via normal vaginal delivery. The hUCB =
were=20
extracted via the cannulation of the umbilical vein (with informed =
consent from=20
the mother) and were collected in bags containing 23&nbsp;ml of citrate=20
phosphate dextrose adenine 1 anticoagulant (Greencross, Yongin, =
Gyeonggi, South=20
Korea). hUCB was subjected to gravity flow and processed within =
24&nbsp;h of=20
collection. </P></DIV>
<DIV class=3D""><A name=3DSec4></A>
<DIV class=3DHeading3>Isolation of hUCB-derived MSCs</DIV>
<P class=3D"">The hUCB MSCs were isolated and cultivated as reported =
previously=20
[<CITE><A=20
href=3D"http://www.springerlink.com/content/l0182768h43vj125/fulltext.htm=
l#CR39">39</A></CITE>].=20
In brief, mononuclear cells were isolated by centrifugation in a =
Ficoll-Hypaque=20
gradient (density 1.077&nbsp;g/cm<SUP>3</SUP>, Sigma, St Louis, MO, =
USA). The=20
separated mononuclear cells were washed, suspended in &#945;-minimum =
essential medium=20
(&#945;-MEM, Gibco BRL, Carlsbad, CA, USA), supplemented with 10% fetal =
bovine serum=20
(FBS; HyClone, Logan, UT, USA), and seeded at a concentration of=20
5=D710<SUP>6</SUP> cells/cm<SUP>2</SUP>. Cultures were maintained at =
37=B0C in a=20
humidified atmosphere containing 5% CO<SUB>2</SUB> with a change of =
culture=20
medium twice a week. One to 3 weeks later, when the monolayer of =
mesenchymal=20
stem cells colonies reached 80% confluence, cells were trypsinized =
(0.25%=20
trypsin, HyClone), washed, resuspended in culture medium (&#945;-MEM =
supplemented=20
with 10% FBS) and subcultured at a concentration of 5=D710<SUP>4</SUP>=20
cells/cm<SUP>2</SUP>. </P></DIV>
<DIV class=3D""><A name=3DSec5></A>
<DIV class=3DHeading3>Malignant glioma cell line cultures</DIV>
<P class=3D"">The human malignant glioma cell line (U87MG) was obtained =
from the=20
American Type Culture Collection, and all experiments were conducted =
using the=20
cells of less than 15 passages from the original stock. The U87MG cells =
were=20
maintained in Dulbecco=92s Modified Eagles Media (DMEM; Gibco BRL, =
Rockville, MD)=20
supplemented with 10% fetal bovine serum (FBS), 2&nbsp;mM <SPAN=20
style=3D"FONT-VARIANT: small-caps"><SMALL>l</SMALL></SPAN>-glutamine =
(Gibco BRL),=20
100&nbsp;U/ml penicillin, and 100&nbsp;&#956;g of streptomycin (Gibco =
BRL). This cell=20
line was grown as a monolayer culture. </P></DIV>
<DIV class=3D""><A name=3DSec6></A>
<DIV class=3DHeading3>Establishment of green fluorescence protein stable =
malignant=20
glioma cell line</DIV>
<P class=3D"">The green fluorescence protein (GFP) stable cell line was =
prepared=20
as described previously [<CITE><A=20
href=3D"http://www.springerlink.com/content/l0182768h43vj125/fulltext.htm=
l#CR12">12</A></CITE>,=20
<CITE><A=20
href=3D"http://www.springerlink.com/content/l0182768h43vj125/fulltext.htm=
l#CR13">13</A></CITE>,=20
<CITE><A=20
href=3D"http://www.springerlink.com/content/l0182768h43vj125/fulltext.htm=
l#CR17">17</A></CITE>].=20
The U87MG cells were cultured in DMEM containing 10% FBS, then =
transfected with=20
the pEGFPN1 plasmid via lipofection. Forty-eight hours after =
transfection, the=20
cells were split at a 10:1 ratio and cultured in the presence of =
400&#956;g/ml of=20
neomycin analogue-G418 (Gibco BRL). After 21&nbsp;days in the selective =
medium,=20
individual G418-resistant colonies were isolated. Subsequently, a stable =
U87MG=20
cell line that constantly expresses GFP was established. Therefore, if =
the=20
target cells (U87MG) were alive, they would constantly express green=20
fluorescence. This method allowed for the direct observation of live =
target=20
cells via fluorescence microscopy. </P></DIV>
<DIV class=3D""><A name=3DSec7></A>
<DIV class=3DHeading3>Activation of hUCB-derived MSCs with =
cytokines</DIV>
<P class=3D"">The hUCB-derived MSCs were activated in vitro with IL-2 =
(R&amp;D=20
Systems, USA), IL-15 (R&amp;D Systems), and granulocyte macrophage=20
colony-stimulating factor (GM-CSF, <SUP>LG</SUP> PhD) alone or in =
various=20
combinations. About 3&#8201;=D7&#8201;10<SUP>5</SUP> cells were =
incubated in 100-mm culture=20
dishes containing 7&nbsp;ml of DMEM with 20% FBS and various cytokines =
(IL-2=20
alone, IL-15 alone, combination of IL-2 and IL-15, GM-CSF alone, =
combination of=20
GM-CSF and IL-2) for 72&nbsp;h at 37=B0C. The concentrations of the =
activating=20
cytokines were as follows: IL-2&#8201;=3D&#8201;50&nbsp;ng/ml, =
IL-15&#8201;=3D&#8201;20&nbsp;ng/ml,=20
GM-CSF/Sargramo-stim&#8201;=3D&#8201;40&nbsp;ng/ml. The control culture =
dishes contained cells=20
in medium only, without cytokines. After this activation period, the =
cells were=20
harvested with Fisherbrand Cell-Scrapers, washed twice in DMEM, and =
counted with=20
a Careside H-2000 (Culver, CA) electronic cell counter. The cells were =
then=20
utilized for cytotoxicity assays (visual survival cell assay and =
transwell=20
inserts) and stained for immunophenotyping via flow cytometry. In =
addition, the=20
concentrations of IL-4 and interferon-&#947; (INF-&#947;) in the =
hUCB-derived MSCs and the=20
activated hUCB-derived MSCs were determined via enzyme-linked =
immunosorbent=20
assay (ELISA), as described previously [<CITE><A=20
href=3D"http://www.springerlink.com/content/l0182768h43vj125/fulltext.htm=
l#CR13">13</A></CITE>,=20
<CITE><A=20
href=3D"http://www.springerlink.com/content/l0182768h43vj125/fulltext.htm=
l#CR21">21</A></CITE>].=20
</P></DIV>
<DIV class=3D""><A name=3DSec8></A>
<DIV class=3DHeading3>Cytotoxicity assay by visual survival cell =
assay</DIV>
<P class=3D"">The same number of effector cells, which were hUCB-derived =
MSCs and=20
activated hUCB-derived MSCs with various cytokines (IL-2 alone, IL-15 =
alone,=20
combination of IL-2 and IL-15, GM-CSF alone, combinations of GM-CSF and =
IL-2),=20
were assessed with regard to their cytotoxic capability against stable =
target=20
cells (U87MG) expressing GFP. The effector cells and GFP stable target =
cells=20
were mixed to obtain the effector/target (E/T) ratios of 0:1, 1:1, 5:1, =
and 10:1=20
in 1.5&nbsp;ml of DMEM medium/well in 24-well plates. The number of =
target cells=20
of GFP-stable U87MG was 1&#8201;=D7&#8201;10<SUP>3</SUP>/ml. The plates =
were incubated for=20
5&nbsp;days at 37=B0C. The GFP density of the target cells was then =
determined via=20
confocal microscopy (Bio-RAD MRC1024) and a computerized image analysis =
system=20
(Image-Pro Plus v 5.1). All experiments were conducted in triplicate. =
</P></DIV>
<DIV class=3D""><A name=3DSec9></A>
<DIV class=3DHeading3>Cytotoxicity assay by transwell inserts</DIV>
<P class=3D"">For coculture experiments to evaluate the target cell =
suppressor=20
activity of secreted proteins from effector cells, U87MG cells=20
(2&#8201;=D7&#8201;10<SUP>4</SUP> cells) were grown in the lower well of =
6.5-mm tissue culture=20
transwell plates on porous inserts (0.4&nbsp;&#956;m pore size), after =
which the=20
hUCB-derived MSCs or hUCB-derived MSCs activated with various cytokines =
were=20
plated on the upper well of the transwell plates with increasing cell =
numbers=20
(E/T ratios of 0:1, 1:1, 5:1, and 10:1). The porous bottom of the insert =

provides independent access to both slides of a cell monolayer giving us =
a=20
versatile tool to study cell transport and other metabolic activities in =
vitro.=20
After 3&nbsp;days, the viability of the U87MG cells in the lower well =
were=20
analyzed via MTS assays (Promega). All experiments were conducted in =
triplicate.=20
</P></DIV>
<DIV class=3D""><A name=3DSec10></A>
<DIV class=3DHeading3>Immunophenotyping of hUCB-derived MSCs and =
activated=20
hUCB-derived MSCs</DIV>
<P class=3D"">The hUCB-derived MSCs (3&#8201;=D7&#8201;10<SUP>5</SUP>) =
and activated UCB-derived=20
MSCs (3&#8201;=D7&#8201;10<SUP>5</SUP>) with various cytokines (IL-2 =
alone, IL-15 alone,=20
combination of IL-2 and IL-15, GM-CSF alone, combination of GM-CSF and =
IL-2)=20
were incubated for 72&nbsp;h. After 72&nbsp;h, the cells were harvested =
and=20
washed in ice-cold phosphate-buffered saline. To characterize the =
cell-surface=20
expression of the typical marker proteins, these cells were labeled with =
the=20
following antihuman antibodies: CD3-FITC, CD4-FITC (BD Bioscience, USA), =
CD8-PE,=20
and CD161-PE (BD Bioscience). About 10,000 cells were measured using a =
FACScan=20
flow cytometer (Becton Dickinson), and the results were analyzed with =
CellQuest=20
software (Becton Dickinson). The same results were obtained in three =
independent=20
experiments. </P></DIV>
<DIV class=3D""><A name=3DSec11></A>
<DIV class=3DHeading3>Culture supernatant analysis via ELISA</DIV>
<P class=3D"">The hUCB-derived MSCs (3&#8201;=D7&#8201;10<SUP>5</SUP>) =
with or without various=20
cytokines (IL-2 alone, IL-15 alone, combination of IL-2 and IL-15, =
GM-CSF alone,=20
combination of GM-CSF and IL-2) were incubated for 72&nbsp;h. The =
concentration=20
of IL-4 and INF-&#947; protein secreted form the cells was then =
quantified using=20
ELISA assay kits (R&amp;D Systems). The 96-well plates were coated with=20
antihuman IL-4 and INF-&#947; monoclonal antibodies. The =
manufacturer=92s protocols=20
were followed in all cases, and the absorbance of each well was =
determined at a=20
wavelength of 450&nbsp;nm using a microplate reader (MR5000; Dynatech=20
Laboratories, Chantilly, VA). Chemokine protein concentrations were =
calculated=20
on the basis of the standard curves. All experiments were conducted in=20
triplicate. </P></DIV>
<DIV class=3D""><A name=3DSec12></A>
<DIV class=3DHeading3>Statistical analysis</DIV>
<P class=3D"">For the visual survival cell assay and transwell inserts, =
as well as=20
the ELISA assay, differences between groups were determined via the =
Mann=96Whitney=20
test. <I>p</I> values less than 0.05 were considered to be significant.=20
</P></DIV></DIV>
<DIV class=3D""><A name=3DSec13></A>
<HR>

<DIV class=3Dheading2>Results</DIV>
<DIV class=3D""><A name=3DSec14></A>
<DIV class=3DHeading3>Cytotoxicity of hUCB-derived MSCs alone against =
malignant=20
glioma cells</DIV>
<DIV class=3DPara>
<DIV class=3D"">The in vitro cytotoxic properties of effector cells =
(hUCB-derived=20
MSCs alone) against target cells (GFP stable U87MG cell line) were =
assessed via=20
visual survival cell assays. In this study, we were able to estimate the =
GFP=20
density of the surviving target cells because the surviving target cells =

expressed green fluorescence (Fig.&nbsp;<A=20
href=3D"http://www.springerlink.com/content/l0182768h43vj125/fulltext.htm=
l#Fig1">1</A>a).=20
After 5&nbsp;days, the GFP density of the target cells mixed with no =
effector=20
cells were measured at 27.1&#8201;=B1&#8201;4.7 (SD) and mixed with =
effector cells=20
(hUCB-derived MSCs) at 25.6&#8201;=B1&#8201;2.1, =
19.5&#8201;=B1&#8201;1.0, and 15.6&#8201;=B1&#8201;5.6 at E/T ratios of=20
1:1, 5:1, and 10:1. This indicates that unactivated hUCB-derived MSCs =
evidence=20
cytotoxicity against malignant glioma cells at E/T ratios of 1:1, 5:1, =
and 10:1=20
(<I>p</I>&#8201;&lt;&#8201;0.05; Fig.&nbsp;<A=20
href=3D"http://www.springerlink.com/content/l0182768h43vj125/fulltext.htm=
l#Fig1">1</A>b).=20

<DIV class=3DFigure><A name=3DFig1></A><IMG=20
alt=3DMediaObjects/381_2007_515_Fig1_HTML.gif=20
src=3D"http://www.springerlink.com/content/l0182768h43vj125/MediaObjects/=
381_2007_515_Fig1_HTML.gif"></DIV>
<DIV class=3DCapt><SPAN class=3DCaptNr>Fig.&nbsp;1&nbsp;</SPAN>After =
5&nbsp;days,=20
the GFP density of the survival target cells (U87MG) cultured with =
effector=20
cells (hUCB-derived MSCs) at E/T ratios of 0:1, 1:1, 5:1 and 10:1 were =
analyzed.=20
<B>a</B> Surviving target cells, which express GFP, detected by =
fluorescence=20
microscope after coculture with effector cells at ratios of 0:1, 1:1, =
5:1, and=20
10:1. When the ratio of effector cells was increased, the number of =
surviving=20
GFP stable target cells decreased. Original magnifications, =D7200. =
<B>b</B>=20
Calculated surviving target cell density decreased significantly in =
relation to=20
the E/T ratio, indicating that the hUCB-derived MSCs alone might =
evidence=20
cytotoxic capacity against target cells (U87MG). <I>Bars</I> indicate =
SE.=20
<I>Asterisk</I>, <I>p</I>&#8201;&lt;&#8201;0.05 </DIV>
<HR>
</DIV></DIV></DIV>
<DIV class=3D""><A name=3DSec15></A>
<DIV class=3DHeading3>Comparison of the cytotoxicity of activated =
hUCB-derived=20
MSCs to hUCB-derived MSCs alone</DIV>
<DIV class=3DPara>
<DIV class=3D"">After 5&nbsp;days, the GFP density of the surviving =
target cells=20
(U87MG) cultured with effector cells (activated or unactivated =
hUCB-derived=20
MSCs) was compared. The density of the surviving target cells =
(Fig.&nbsp;<A=20
href=3D"http://www.springerlink.com/content/l0182768h43vj125/fulltext.htm=
l#Fig2">2</A>a),=20
after being cultured with the activated hUCB-derived MSCs by various =
cytokines=20
(IL-2 alone, IL-15 alone, combination of IL-2 and IL-15, GM-CSF alone,=20
combination of GM-CSF and IL-2), was significantly lower =
(<I>p</I>&#8201;&lt;&#8201;0.05)=20
than that observed after culturing with the hUCB-derived MSCs alone at =
E/T=20
ratios of 1:1, 5:1, and 10:1 (Fig.&nbsp;<A=20
href=3D"http://www.springerlink.com/content/l0182768h43vj125/fulltext.htm=
l#Fig2">2</A>b).=20
This indicated that activated hUCB-derived MSCs might prove more =
cytotoxic=20
against target cells than the unactivated hUCB-derived MSCs.=20
<DIV class=3DFigure><A name=3DFig2></A><IMG=20
alt=3DMediaObjects/381_2007_515_Fig2_HTML.gif=20
src=3D"http://www.springerlink.com/content/l0182768h43vj125/MediaObjects/=
381_2007_515_Fig2_HTML.gif"></DIV>
<DIV class=3DCapt><SPAN class=3DCaptNr>Fig.&nbsp;2&nbsp;</SPAN>After =
5&nbsp;days,=20
the GFP density of the survival target cells (U87MG) cultured with =
effector=20
cells was compared. <B>a</B> The surviving GFP-expressing cells could be =

determined via fluorescence microscopy after coculturing the =
hUCB-derived MSCs=20
with or without cytokine activation. When the ratio of effector cells =
was=20
increased, the number of surviving cells decreased in all groups. =
Compared with=20
the unactivated hUCB-derived MSCs, the activated hUCB-derived MSCs with =
cytokine=20
activation killed more target cells. Original magnifications, =D7200. =
<B>b</B>=20
These graphs show the comparative cytotoxicity between the hUCB-derived =
MSCs=20
without activation and activated hUCB-derived MSCs. The density of =
surviving=20
target cells after culturing with activated hUCB-derived MSCs at all E/T =
ratios=20
was significantly lower (<I>p</I>&#8201;&lt;&#8201;0.05) than after =
culturing with=20
hUCB-derived MSCs alone after 5&nbsp;days. These results demonstrate the =
greater=20
cytotoxicity of activated hUCB-derived MSCs with various cytokines =
against=20
target cells after 5&nbsp;days. <I>Bars</I> indicate SE. =
<I>Asterisk</I>,=20
<I>p</I>&#8201;&lt;&#8201;0.05 </DIV>
<HR>
</DIV></DIV></DIV>
<DIV class=3D""><A name=3DSec16></A>
<DIV class=3DHeading3>Cytotoxicity of hUCB-derived MSCs with or without =
cytokine=20
activations by transwell inserts</DIV>
<DIV class=3DPara>
<DIV class=3D"">To confirm that the observed cytotoxicity of =
hUCB-derived MSCs was=20
attributable to the release of soluble proteins, unactivated =
hUCB-derived MSCs=20
or hUCB-derived MSCs activated with various cytokines were grown in the =
upper=20
well of transwell plates in increasing numbers (E/T ratios of 1:1, 5:1, =
and=20
10:1), and target (U87MG) cells were grown in the lower well. A =
semiporous=20
membrane (0.4-&#956;m pore) separated the cells. The hUCB-derived MSCs =
with or=20
without activation against target cells (<I>p</I>&#8201;&lt;&#8201;0.05) =
evidenced=20
significant cytotoxicity in an E/T ratio-dependent pattern (Fig.&nbsp;<A =

href=3D"http://www.springerlink.com/content/l0182768h43vj125/fulltext.htm=
l#Fig3">3</A>).=20
This is attributable to the fact that the U87MG cells died as the =
results of=20
soluble proteins secreted from the unactivated and activated =
hUCB-derived MSCs.=20
However, no significant differences were observed between the =
unactivated=20
hUCB-derived MSCs and the activated hUCB-derived MSCs groups.=20
<DIV class=3DFigure><A name=3DFig3></A><IMG=20
alt=3DMediaObjects/381_2007_515_Fig3_HTML.gif=20
src=3D"http://www.springerlink.com/content/l0182768h43vj125/MediaObjects/=
381_2007_515_Fig3_HTML.gif"></DIV>
<DIV class=3DCapt><SPAN class=3DCaptNr>Fig.&nbsp;3&nbsp;</SPAN>Effect of =

hUCB-derived MSCs with or without activation on the survival of target =
(U87MG)=20
cells using Transwell containing a semiporous membrane. U87MG cells=20
(2&#8201;=D7&#8201;10<SUP>4</SUP>) were seeded on the lower well, and =
after 48&nbsp;h,=20
hUCB-derived MSCs or activated hUCB-derived MSCs were plated in the =
upper well=20
at the indicated cell numbers. The viability of target cells in the =
lower well=20
was assayed via MTS assays 5&nbsp;days after the effector cells =
(unactivated=20
hUCB-derived MSCs or activated hUCB-derived MSCs) were plated. The =
effector cell=20
treatment resulted in a significant reduction in target cell (U87MG) =
survival in=20
an E/T ratio-dependent pattern, but no significant differences were =
observed=20
between unactivated hUCB-derived MSCs and activated hUCB-derived MSCs =
groups.=20
<I>Bars</I> indicate SE. <I>Asterisk</I>, <I>p</I>&#8201;&lt;&#8201;0.05 =
</DIV>
<HR>
</DIV></DIV></DIV>
<DIV class=3D""><A name=3DSec17></A>
<DIV class=3DHeading3>Phenotypic changes of hUCB-derived MSCs before and =
after=20
cytokine activation</DIV>
<DIV class=3DPara>
<DIV class=3D"">To determine the immunophenotypic changes in =
hUCB-derived MSCs=20
before and after cytokine activation (IL-2 alone, IL-15 alone, =
combination of=20
IL-2 and IL-15, GM-CSF alone, combination of GM-CSF and IL-2), flow =
cytometry=20
was conducted. The results of these analyses are provided in =
Fig.&nbsp;<A=20
href=3D"http://www.springerlink.com/content/l0182768h43vj125/fulltext.htm=
l#Fig4">4</A>a=20
(CD8-cytotoxic T cell marker, CD4-helper T cell marker) and b =
(CD161-natural=20
killer cell marker, CD3-common T cell marker). We noted no significant =
increases=20
in the populations of CD8 cells, CD4 cells, CD161 cells, and CD3 cells =
after=20
activation with various cytokines (Fig.&nbsp;<A=20
href=3D"http://www.springerlink.com/content/l0182768h43vj125/fulltext.htm=
l#Fig4">4</A>a,b).=20

<DIV class=3DFigure><A name=3DFig4></A><IMG=20
alt=3DMediaObjects/381_2007_515_Fig4_HTML.gif=20
src=3D"http://www.springerlink.com/content/l0182768h43vj125/MediaObjects/=
381_2007_515_Fig4_HTML.gif"></DIV>
<DIV class=3DCapt><SPAN class=3DCaptNr>Fig.&nbsp;4&nbsp;</SPAN>The =
frequencies of=20
various immune effector cells (CD8 cells, CD4 cells, CD161 cells, and =
CD3 cells)=20
in hUCB-derived MSCs before and after activation with various cytokines =
for=20
72&nbsp;h, as determined by flow cytometry. <B>a</B> Flow cytometric =
analyses of=20
cells showed that there was no increase of CD4 or CD8 cells, regardless =
of=20
stimulation with various cytokines. <B>b</B> Similar results were =
observed in a=20
cell population of CD3 or CD161 cells between the unactivated =
hUCB-derived MSCs=20
or activated hUCB-MSC cells. <I>Bars</I> indicate SE. <I>Asterisk</I>,=20
<I>p</I>&#8201;&lt;&#8201;0.05 </DIV>
<HR>
</DIV></DIV></DIV>
<DIV class=3D""><A name=3DSec18></A>
<DIV class=3DHeading3>Concentrations of proteins secreted from =
hUCB-derived MSCs=20
before and after cytokine activations</DIV>
<DIV class=3DPara>
<DIV class=3D"">We compared the amounts of secreted IL-4 and INF-&#947; =
protein=20
concentrations from the culture supernatants of hUCB-derived MSCs with =
or=20
without activation. Seventy-two hours after activation with various =
cytokines=20
(IL-2 alone, IL-15 alone, combination of IL-2 and IL-15, GM-CSF alone,=20
combination of GM-CSF and IL-2), IL-4 (Fig.&nbsp;<A=20
href=3D"http://www.springerlink.com/content/l0182768h43vj125/fulltext.htm=
l#Fig5">5</A>a)=20
and INF-&#947; (Fig.&nbsp;<A=20
href=3D"http://www.springerlink.com/content/l0182768h43vj125/fulltext.htm=
l#Fig5">5</A>b)=20
protein levels in the supernatants were determined via ELISA. =
Significantly=20
higher (<I>p</I>&#8201;&lt;&#8201;0.05) IL-4 concentrations were =
detected in the culture=20
supernatants from all activated hUCB-derived MSCs. The INF-&#947; levels =
in=20
unactivated hUCB-derived MSCs were also high. However, the levels of =
INF-&#947; from=20
the activated hUCB-derived MSCs were significantly higher than were seen =
in the=20
unactivated hUCB-derived MSCs (<I>p</I>&#8201;&lt;&#8201;0.05).=20
<DIV class=3DFigure><A name=3DFig5></A><IMG=20
alt=3DMediaObjects/381_2007_515_Fig5_HTML.gif=20
src=3D"http://www.springerlink.com/content/l0182768h43vj125/MediaObjects/=
381_2007_515_Fig5_HTML.gif"></DIV>
<DIV class=3DCapt><SPAN class=3DCaptNr>Fig.&nbsp;5&nbsp;</SPAN>These =
graphs show the=20
ELISA analysis of culture supernatants from hUCB-derived MSCs with or =
without=20
cytokine activation. <B>a</B> A significantly higher concentration of =
IL-4 was=20
detected in the culture supernatants from all activated hUCB-derived =
MSCs.=20
<B>b</B> INF-&#947; evidenced similar changes in concentration, and the =
highest were=20
observed in the culture supernatants from activated hUCB-derived MSCs =
with a=20
combination of IL-2 and IL-15. <I>Bars</I> indicate SE. <I>Asterisk</I>, =

<I>p</I>&#8201;&lt;&#8201;0.05 </DIV>
<HR>
</DIV></DIV></DIV></DIV>
<DIV class=3D""><A name=3DSec19></A>
<HR>

<DIV class=3Dheading2>Discussions</DIV>
<P class=3D"">The principal objective of our study was to explore the =
effective in=20
vitro cytotoxicity of hUCB-derived MSCs against malignant glioma cells, =
and=20
activated hUCB-derived MSCs were shown to have more cytotoxic effects =
than the=20
unactivated hUCB-derived MSCs. In addition, the activated hUCB-derived =
MSCs=20
secrete more immune response-related proteins (IL-4 and INF-&#947;) than =
the=20
unactivated hUCB-derived MSCs. </P>
<P class=3D"">We were also able to determine the cytotoxic ability of =
the=20
unactivated hUCB-derived MSCs against malignant glioma cells =
(Fig.&nbsp;<A=20
href=3D"http://www.springerlink.com/content/l0182768h43vj125/fulltext.htm=
l#Fig1">1</A>b).=20
In contrast to our results, other authors have asserted that MSCs =
suppressed=20
immune responses [<CITE><A=20
href=3D"http://www.springerlink.com/content/l0182768h43vj125/fulltext.htm=
l#CR8">8</A></CITE>,=20
<CITE><A=20
href=3D"http://www.springerlink.com/content/l0182768h43vj125/fulltext.htm=
l#CR16">16</A></CITE>].=20
However, some previous reports, including ours, have reported that =
coculture=20
with MSCs inhibits the growth of rat glioma cells [<CITE><A=20
href=3D"http://www.springerlink.com/content/l0182768h43vj125/fulltext.htm=
l#CR13">13</A></CITE>,=20
<CITE><A=20
href=3D"http://www.springerlink.com/content/l0182768h43vj125/fulltext.htm=
l#CR25">25</A></CITE>].=20
We attempted to activate hUCB-derived MSCs using various cytokines. To =
achieve=20
this, the authors activated hUCB-derived MSCs with IL-2 alone, IL-15 =
alone, a=20
combination of IL-2 and IL-15, GM-CSF alone, and a combination of GM-CSF =
and=20
IL-2. Activated hUCB-derived MSCs evidenced more effective cytotoxicity =
against=20
human malignant glioma cells than did the unactivated hUCB-derived MSCs=20
(Fig.&nbsp;<A=20
href=3D"http://www.springerlink.com/content/l0182768h43vj125/fulltext.htm=
l#Fig2">2</A>b).=20
These results are consistent with the results of earlier studies, in =
which rat=20
MSCs with or without activation were assessed with regard to their =
cytotoxicity=20
against rat malignant glioma cells [<CITE><A=20
href=3D"http://www.springerlink.com/content/l0182768h43vj125/fulltext.htm=
l#CR13">13</A></CITE>].=20
</P>
<P class=3D"">In an attempt to determine whether the soluble proteins =
secreted=20
from hUCB-derived MSCs play roles in this cytotoxicity against malignant =
glioma=20
cells, we conducted another assay using transwell inserts. We were able =
to=20
verify that the observed cytotoxicity of hUCB-derived MSCs was =
attributable to=20
an increased release of soluble proteins (E/T ratios of 1:1, 5:1, and =
10:1;=20
Fig.&nbsp;<A=20
href=3D"http://www.springerlink.com/content/l0182768h43vj125/fulltext.htm=
l#Fig3">3</A>).=20
However, we were unable to observe any significant differences in =
cytotoxicity=20
between unactivated hUCB-derived MSCs and activated hUCB-derived MSCs, =
although=20
we also noted some tendencies indicative of the cytokine-dependent =
cytotoxicity=20
of hUCB-derived MSCs (Fig.&nbsp;<A=20
href=3D"http://www.springerlink.com/content/l0182768h43vj125/fulltext.htm=
l#Fig3">3</A>).=20
</P>
<P class=3D"">We were able to differentiate rat MSCs into immune =
effector cells,=20
and these activated rat MSCs exert more cytotoxic effects against rat =
glioma=20
cells than were observed in unactivated rat MSCs [<CITE><A=20
href=3D"http://www.springerlink.com/content/l0182768h43vj125/fulltext.htm=
l#CR13">13</A></CITE>].=20
We expected to see similar results using hUCB-derived MSCs; however,=20
hUCB-derived MSCs did not differentiate into immune effector cells =
(Fig.&nbsp;<A=20
href=3D"http://www.springerlink.com/content/l0182768h43vj125/fulltext.htm=
l#Fig4">4</A>a,b),=20
although activated hUCB-derived MSCs evidence more profound cytotoxic =
effects=20
against malignant glioma cells than was observed with unactivated =
hUCB-derived=20
MSCs (Fig.&nbsp;<A=20
href=3D"http://www.springerlink.com/content/l0182768h43vj125/fulltext.htm=
l#Fig2">2</A>a,b).=20
This indicated that hUCB-derived MSCs may evidence characteristics =
different=20
than those observed with rat MSCs. </P>
<P class=3D"">MSCs generate several neurotrophic factors, including the =
nerve=20
growth factor (NGF) [<CITE><A=20
href=3D"http://www.springerlink.com/content/l0182768h43vj125/fulltext.htm=
l#CR22">22</A></CITE>],=20
which can induce the differentiation and growth inhibition of rat glioma =
cells=20
in vitro [<CITE><A=20
href=3D"http://www.springerlink.com/content/l0182768h43vj125/fulltext.htm=
l#CR19">19</A></CITE>].=20
In the rat glioma model, NGF generated by MSCs could induce both =
differentiation=20
and tumor growth suppression [<CITE><A=20
href=3D"http://www.springerlink.com/content/l0182768h43vj125/fulltext.htm=
l#CR25">25</A></CITE>].=20
We have already demonstrated another cytotoxic mechanism of rat MSCs, =
which=20
involved the differentiation of rat MSCs into immune effector cells =
[<CITE><A=20
href=3D"http://www.springerlink.com/content/l0182768h43vj125/fulltext.htm=
l#CR13">13</A></CITE>];=20
however, hUCB-derived MSCs were not shown to differentiate into immune =
effector=20
cells, although these cells could be activated by a variety of =
cytokines. </P>
<P class=3D"">Higher concentrations of IL-4 and INF-&#947;, markers for =
helper T cells=20
and cytotoxic T cells, were detected from activated hUCB-derived MSCs, =
which=20
indicated that immune response-related proteins were secreted more =
prominently=20
from hUCB-derived MSCs after cytokine activation (Fig.&nbsp;<A=20
href=3D"http://www.springerlink.com/content/l0182768h43vj125/fulltext.htm=
l#Fig5">5</A>a,b).=20
The increased level of IL-4 and INF-&#947; perform a certain role in the =

cytokine-dependent pattern cytotoxicity of hUCB-derived MSCs =
(Fig.&nbsp;<A=20
href=3D"http://www.springerlink.com/content/l0182768h43vj125/fulltext.htm=
l#Fig2">2</A>b).=20
</P>
<P class=3D"">Usually, neural SCs (NSCs) are utilized as an effective =
delivery=20
vehicle for certain genes derived from a potent tropism for the =
dissemination of=20
glioma cells in vivo [<CITE><A=20
href=3D"http://www.springerlink.com/content/l0182768h43vj125/fulltext.htm=
l#CR1">1</A></CITE>,=20
<CITE><A=20
href=3D"http://www.springerlink.com/content/l0182768h43vj125/fulltext.htm=
l#CR10">10</A></CITE>,=20
<CITE><A=20
href=3D"http://www.springerlink.com/content/l0182768h43vj125/fulltext.htm=
l#CR18">18</A></CITE>].=20
NSCs can be isolated only from the fetal or adult brain or generated =
from=20
embryonic SCs. To overcome this limitation of NSCs, MSCs may be a good=20
alternative source of gene vehicles, as MSCs evidence effective tropisms =
and=20
gene delivery characteristics for the treatment of glioma [<CITE><A=20
href=3D"http://www.springerlink.com/content/l0182768h43vj125/fulltext.htm=
l#CR4">4</A></CITE>,=20
<CITE><A=20
href=3D"http://www.springerlink.com/content/l0182768h43vj125/fulltext.htm=
l#CR23">23</A></CITE>=96<CITE><A=20
href=3D"http://www.springerlink.com/content/l0182768h43vj125/fulltext.htm=
l#CR25">25</A></CITE>,=20
<CITE><A=20
href=3D"http://www.springerlink.com/content/l0182768h43vj125/fulltext.htm=
l#CR30">30</A></CITE>].=20
The authors have already addressed the potentiality of rat MSCs, which =
are=20
capable of directly killing rat malignant glioma cells [<CITE><A=20
href=3D"http://www.springerlink.com/content/l0182768h43vj125/fulltext.htm=
l#CR13">13</A></CITE>].=20
Recently, human skin-derived SCs have been shown to exert a significant =
direct=20
effect on the development of malignant glioma [<CITE><A=20
href=3D"http://www.springerlink.com/content/l0182768h43vj125/fulltext.htm=
l#CR27">27</A></CITE>].=20
Although these cells were not MSCs, these results represent an =
innovative change=20
in previous concepts (SCs as gene delivery) namely, that SCs themselves =
may be=20
therapeutically effective for the treatment of malignant glioma. The =
authors=20
attempted, for the first time, to apply hUCB-derived MSCs, not as =
vehicles for=20
gene delivery but as direct effector cells. </P></DIV>
<DIV class=3D""><A name=3DSec20></A>
<HR>

<DIV class=3Dheading2>Conclusions</DIV>
<P class=3D"">Recently, MSCs are focused to become an effective gene =
delivery=20
vehicle for malignant glioma. We have suggested conceptual changes about =
MSCs.=20
Our current study suggests a potential role of hUCB-derived MSCs with or =
without=20
cytokine activation, as a direct effector cells in malignant glioma. =
However, we=20
have to confirm the safety of these cells. Furthermore, the mechanisms =
in detail=20
underlying the cytotoxicity of hUCB-derived MSCs will require further=20
investigation. </P></DIV>
<DIV class=3DAcknowledgments><SPAN=20
class=3DAcknowledgmentsHeading>Acknowledgments&nbsp;&nbsp;</SPAN><SPAN=20
class=3D"">This work was supported by the Korea Research Foundation =
Grant funded=20
by the Korean Government (MOEHRD, Basic Research Promotion Fund,=20
RF-2006-311-E00362) and a grant (SC3112) from Stem Cell Research Center =
of the=20
21st Century Frontier Research Program funded by the Ministry of Science =
and=20
Technology, Republic of Korea.</SPAN></DIV>
<P></P>
<HR>

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db=3DPubMed&amp;dopt=3DAbstract&amp;list_uids=3D14592834"=20
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src=3D"http://www.springerlink.com/content/l0182768h43vj125/pubmed_link.g=
if"=20
      width=3D65 border=3D0></A> <A=20
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target=3D_blank><IMG=20
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href=3D"http://chemport.cas.org/cgi-bin/sdcgi?APP=3Dftslink&amp;action=3D=
reflink&amp;origin=3Dspringer&amp;version=3D1.0&amp;coi=3D1%3ACAS%3A528%3=
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  <TR>
    <TD>&nbsp;</TD></TR>
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db=3DPubMed&amp;dopt=3DAbstract&amp;list_uids=3D9417092"=20
      target=3D_blank><IMG height=3D20 alt=3DPubMed=20
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src=3D"http://www.springerlink.com/content/l0182768h43vj125/pubmed_link.g=
if"=20
      width=3D65 border=3D0></A> <A =
href=3D"http://dx.doi.org/10.1074/jbc.273.1.381"=20
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src=3D"http://www.springerlink.com/content/l0182768h43vj125/crossref_link=
.gif"=20
      width=3D65 border=3D0></A> <A=20
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href=3D"http://chemport.cas.org/cgi-bin/sdcgi?APP=3Dftslink&amp;action=3D=
reflink&amp;origin=3Dspringer&amp;version=3D1.0&amp;coi=3D1%3ACAS%3A528%3=
ADyaK1cXjvFWhug%253D%253D&amp;md5=3Dec4b4cafe8d9671038d3dc21fd0fbd53"=20
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src=3D"http://www.springerlink.com/content/l0182768h43vj125/chemport_link=
.gif"=20
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  <TR>
    <TD>&nbsp;</TD></TR>
  <TR vAlign=3Dtop>
    <TD>18.</TD>
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Wang KC,=20
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stem=20
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db=3DPubMed&amp;dopt=3DAbstract&amp;list_uids=3D17000692"=20
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src=3D"http://www.springerlink.com/content/l0182768h43vj125/pubmed_link.g=
if"=20
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target=3D_blank><IMG=20
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href=3D"http://chemport.cas.org/cgi-bin/sdcgi?APP=3Dftslink&amp;action=3D=
reflink&amp;origin=3Dspringer&amp;version=3D1.0&amp;coi=3D1%3ACAS%3A528%3=
ADC%252BD28XpvFShtrs%253D&amp;md5=3D715cc8282070aa47eaaa7d35c6a906bb"=20
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src=3D"http://www.springerlink.com/content/l0182768h43vj125/chemport_link=
.gif"=20
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  <TR>
    <TD>&nbsp;</TD></TR>
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  <TR>
    <TD>&nbsp;</TD></TR>
  <TR vAlign=3Dtop>
    <TD>20.</TD>
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src=3D"http://www.springerlink.com/content/l0182768h43vj125/pubmed_link.g=
if"=20
      width=3D65 border=3D0></A> <A=20
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target=3D_blank><IMG=20
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href=3D"http://chemport.cas.org/cgi-bin/sdcgi?APP=3Dftslink&amp;action=3D=
reflink&amp;origin=3Dspringer&amp;version=3D1.0&amp;coi=3D1%3ACAS%3A528%3=
ADC%252BD3MXjs1aitrY%253D&amp;md5=3D887d37ea4c6b516213d3735e72394bae"=20
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src=3D"http://www.springerlink.com/content/l0182768h43vj125/chemport_link=
.gif"=20
      width=3D65 border=3D0></A> </TD></TR>
  <TR>
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  <TR vAlign=3Dtop>
    <TD>21.</TD>
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by=20
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db=3DPubMed&amp;dopt=3DAbstract&amp;list_uids=3D10798661"=20
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if"=20
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href=3D"http://chemport.cas.org/cgi-bin/sdcgi?APP=3Dftslink&amp;action=3D=
reflink&amp;origin=3Dspringer&amp;version=3D1.0&amp;coi=3D1%3ASTN%3A280%3=
ADC%252BD3c3ls1Giuw%253D%253D&amp;md5=3D21ebce0003547579e82c5b8d33871bb9"=
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.gif"=20
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  <TR>
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  <TR vAlign=3Dtop>
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href=3D"http://chemport.cas.org/cgi-bin/sdcgi?APP=3Dftslink&amp;action=3D=
reflink&amp;origin=3Dspringer&amp;version=3D1.0&amp;coi=3D1%3ASTN%3A280%3=
ADC%252BD38vls1Sgug%253D%253D&amp;md5=3D9cae5fec54b088fa30ef29fedf2269b3"=
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src=3D"http://www.springerlink.com/content/l0182768h43vj125/pubmed_link.g=
if"=20
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href=3D"http://chemport.cas.org/cgi-bin/sdcgi?APP=3Dftslink&amp;action=3D=
reflink&amp;origin=3Dspringer&amp;version=3D1.0&amp;coi=3D1%3ACAS%3A528%3=
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reflink&amp;origin=3Dspringer&amp;version=3D1.0&amp;coi=3D1%3ACAS%3A528%3=
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src=3D"http://www.springerlink.com/content/l0182768h43vj125/pubmed_link.g=
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href=3D"http://chemport.cas.org/cgi-bin/sdcgi?APP=3Dftslink&amp;action=3D=
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  <TR vAlign=3Dtop>
    <TD>27.</TD>
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href=3D"http://chemport.cas.org/cgi-bin/sdcgi?APP=3Dftslink&amp;action=3D=
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    <TD>&nbsp;</TD></TR>
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Fisher=20
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Y, Peng=20
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MD (2006)=20
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  <TR>
    <TD>&nbsp;</TD></TR></TBODY></TABLE></BODY></HTML>

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