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HUCB-Derived MSCs in Canine Cerebral Ischemia 3555

Cell-based therapies have emerged as an alternative collected with the mothers’ consent into a blood collection
for treating neurodegenerative disorders. Several studies bag containing citrate phosphate dextrose as anticoagulant and
suggest that stem cells can differentiate into neuronal or processed within 24 hr. A fraction of mononuclear cells
glial cells in vivo and in vitro. Stem cells harvested from (MNC) was separated by centrifugation in a Ficoll-Paque Plus
bone marrow and cord blood can exhibit neuronal or gradient (Amersham Biosciences, Uppsala, Sweden), washed
glial cell properties under defined culture conditions with Hank’s balanced salt solution (HBSS; Jeil Biotechservices,
(Sanchez-Ramos et al., 2000; Bicknese et al., 2002; Daegu, Korea), and resuspended in low-glucose DMEM
Vendrame et al., 2004). Transplanted stem cells reach is- (Invitrogen, Grand Island, NY), 20% fetal bovine serum (FBS;
chemic lesions in the brain and differentiate into neuro- JRH Biosciences, Lenexa, KS), 2 mM L-glutamine, 1 mM
nal and glial cells, secrete cytokines and neurotrophic sodium pyruvate, and 1% antibiotics/antimycotics (Life Tech-
factors, and reveal their therapeutic efficacy by promot- nologies, Gaithersburg, MD) comprising 100 U/ml penicillin,
ing functional recovery and reducing infarction volumes 100 lg/ml streptomycin, and 25 lg/ml amphotericin B. After
(Li et al., 2000; Chen et al., 2001a,b; Iihoshi et al., 7 days, nonadherent cells were discarded, and adherent cells
2004; Nomura et al., 2005; Honma et al., 2006; Horita were continued in culture with two medium changes per
et al., 2006). Use of human umbilical cord blood week. Cells were maintained at 378C in a humidified atmos-
(HUCB)-derived mesenchymal stem cells (MSCs) pro- phere containing 5% carbone dioxide, with a change of cul-
vides another approach for treating cerebral ischemic ture medium every 3 days. Approximately 60% of confluent
injury. HUCB is abundant in adult stem and progenitor cells were detached with 0.1% trypsin-EDTA and replated at
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3
cells (both hematopoietic and nonhematopoietic) and is a density of 2 3 10 cells/cm in culture flasks. Charateriza-
a desirable potential source for transplantation (Newman tion and differentiation of isolated human UC-derived MSCs
et al., 2003). Several studies have suggested the efficacy were performed in another study using the same cell source
of HUCB-derived MSCs in cerebral ischemic injury in and isolation technique (Kang et al., 2004; Hong et al., 2005;
rodent models (Willing et al., 2003; Borlongan et al., Kang et al., 2005). It was also demonstrated that these human
2004; Vendrame et al., 2004). UC-derived MSCs are capable of differentiating into neural
Many of these studies were performed in rodents cells in vitro (Jeoung et al., 2004). The HUCB-derived MSCs
with intravenous or intrastriatal injection of stem cells. were provided for pure research purposes by The Seoul Cord
However, local intracerebral injection induces local brain Bank (Histostem Co., Ltd., Seoul, Korea).
damage, and, in particular, multiple injections may not
be clinically acceptable (Gage et al., 1995; Chen et al.,
2001a). Intravenous injection is a simple approach for Experimental Animals
cell therapy and can potentially target pathological sites Ten adult beagles weighing 9.2–16.0 kg were included
in a number of brain disorders (Chen et al., 2001a). in this study. Cerebral brain ischemia was induced in all 10
However, intravenous injection requires a high dose of dogs, divided into two groups: 1) The HUCBC group
stem cells to have a therapeutic effect in a rat model (HUCB-derived MSCs transplanted 1 day after cerebral ische-
(Vendrame et al., 2004). Other experimental models, mic induction) and 2) the control group (phosphate-buffered
such as canine, rabbit, and pig models, would require an saline injection 1 day after cerebral ischemic induction). All
even greater number of cells to examine the effects of animals were intensively cared for, and experimental proce-
stem cells in vivo. In addition, a higher dose will be dures were conducted in accordance with the guidelines
necessary for intravenous injection in a clinical setting. approved by the Institutional Animal Care and Use Commit-
Therefore, intraarterial injection of stem cells to a tar- tee (IACUC) of Konkuk University, Seoul, Korea.
geted organ could be a more effective approach for stem
cell therapy.
Few studies have attempted the intraarterial deliv- Cerebral Ischemia by Middle Cerebral Artery Occlusion
ery of stem cells to the brain. Furthermore, the basilar Through Injection of Thrombic Emboli
artery route has not been previously studied as an injec- Food was withheld for 8 hr before the procedure to
tion route. Endovascular intervention techniques are minimize complications of anesthesia. Anesthesia was induced
essential for intraarterial delivery of stem cells; therefore, with propofol (Anefol; Hana Pharm, Seoul, Korea), injected
animal models larger than rodents are indispensable for intravenously at a dose of 6 mg/kg. The dogs were intubated,
this approach. The purpose of this study was to examine and anesthesia was maintained by mechanical ventilation with
the effects of HUCB-derived MSCs delivered through 1.5% isoflurane (Forane; Choongwae, Seoul, Korea) in 100%
the basilar artery in a canine thromboembolic brain oxygen. The dogs were pretreated with antibiotics (cefradine,
ischemic model. Safdin; Daehan Newpharm, Seoul, Korea) and 500 U heparin
(Choongwae) intravenously prior to the procedure. The dogs
were placed in dorsal recumbency on the operating table, and
MATERIALS AND METHODS
the surgical area was prepared aseptically. The right femoral
Human UCB Harvest and Preparation of MSCs artery was surgically exposed, and a 6F sheath (RCF 6.0-35-J
HUCB-derived MSCs were prepared according to Introducer Set; Cook Medical, Bloomington, IN) was inserted
methods previously described (Kang et al., 2005), with some into the femoral artery. Heparinized normal saline (1 U/ml)
modification. In brief, human full-term UCB samples were was infused continuously throughout the procedure.

Journal of Neuroscience Research