Cell Transplantation, Vol. 18, pp. 985–998, 2009 0963-6897/09 $90.00 + .00Printed in the USA. All rights reserved. DOI: 10.3727/096368909X471279Copyright 2009 Cognizant Comm. Corp. E-ISSN 1555-3892

www.cognizantcommunication.com

Human Umbilical Cord Blood Cell Grafts for Brain Ischemia

Dong-Hyuk Park,*† Cesar V. Borlongan,* Alison E. Willing,* David J. Eve,*L. Eduardo Cruz,‡ Cyndy D. Sanberg,§ Yong-Gu Chung,† and Paul R. Sanberg*¶

*Center of Excellence for Aging & Brain Repair, Department of Neurosurgery & Brain Repair,University of South Florida College of Medicine, Tampa, FL, USA

†Department of Neurosurgery, Korea University Medical Center, Korea University College of Medicine, Seoul, Korea‡Cryopraxis and Silvestre Laboratory, Cryopraxis, BioRio, Polo de Biotechnologia do Rio de Janeiro, Rio di Janiero, Brazil

§Saneron CCEL Therapeutics, Inc., Tampa, FL, USA¶Office of Research and Innovation, University of South Florida, Tampa, FL, USA

Irreversible and permanent damage develop immediately adjacent to the region of reduced cerebral bloodperfusion in stroke patients. Currently, the proven thrombolytic treatment for stroke, tissue plasminogenactivator, is only effective when administered within 3 h after stroke. These disease characteristics shouldbe taken under consideration in developing any therapeutic intervention designed to widen the narrow thera-peutic range, especially cell-based therapy. Over the past several years, our group and others have character-ized the therapeutic potential of human umbilical cord blood cells for stroke and other neurological disordersusing in vitro and vivo models focusing on the cells’ ability to differentiate into nonhematopoietic cellsincluding neural lineage, as well as their ability to produce several neurotrophic factors and modulate im-mune and inflammatory reaction. Rather than the conventional cell replacement mechanism, we advancealternative pathways of graft-mediated brain repair involving neurotrophic effects resulting from release ofvarious growth factors that afford cell survival, angiogenesis, and anti-inflammation. Eventually, these multi-ple protective and restorative effects from umbilical cord blood cell grafts may be interdependent and act inharmony in promoting therapeutic benefits for stroke.

Key words: Angiogenesis; Human umbilical cord blood cells; Inflammation; Middle cerebral artery occlusion;Neurogenesis; Stroke; Transplantation

INTRODUCTION time compounded by considerable risks such as intra-cranial hemorrhage and recurrent stroke (84). Eventu-ally, only 3–5% of stroke patients reach the hospitalA report from the American Heart Association Statis-

tics Committee and Stroke Statistics Subcommittee within this therapeutic range and of those only 29% areeligible for r-tPA (80), indicating that only a few strokeranks stroke third among all other causes of death be-

hind heart diseases and cancer in the US (96). Each year patients have an opportunity at full restoration withrapid thrombolytic management. This unfortunate clini-about 600,000 new patients experience first stroke at-

tacks, and 180,000 are recurrent attacks (96). Among cal status clearly solicits new treatment modality with awider therapeutic spectrum for stroke management.ischemic stroke survivors who were at least 65 years

of age, 50% suffered from hemiparesis and 26% were The ischemic stroke cell death cascades involve twomajor pathophysiological events. Glutamate excitotoxi-dependent on others to assist in their daily activities

(96). Currently, the only Food and Drug Administration- city develops in the ischemic tissue due to the suddendrop in oxygen and nutrients that begins immediatelyapproved thrombolytic drug for acute ischemic stroke is

recombinant-tissue plasminogen activator (r-tPA), which upon loss of blood flow (106). Subsequently, indirectinflammatory responses, such as generation of free oxy-acts by dissolving the blood clot in the vessel and restor-

ing proper blood flow. However, there is only a 30–35% gen radicals and nitric oxide (24,71), activation of mi-croglia within tissue hypoxia and necrosis (68,107), andsuccess rate for recovery, even though r-tPA is adminis-

tered within 3 h after the initial onset of an embolic invasion of other inflammatory cells by way of chemo-taxis and leakage across the compromised blood–brainstroke (75,84), and the drug effectiveness decreases with

Online prepub date: April 29, 2009.Address correspondence to Paul R. Sanberg, Center of Excellence for Aging & Brain Repair, Department of Neurosurgery & Brain Repair,University of South Florida College of Medicine, MDC 78, 12901 Bruce B. Downs Boulevard, Tampa, FL 33612, USA. Tel: 813-974-3154; Fax:813-974-3078; E-mail: psanberg@health.usf.edu

985

986 PARK ET AL.

barrier, contribute to disease progression (34,41,137). granulocyte colony-stimulating factor stimulation, pa-tients with malignancies are often unable to receive au-Thus, a more successful and effective treatment over

current thrombolytic therapy must have a multifaceted tografts due to the risk of reinfection with tumorgeniccells (81). The “timing” of intervention is critical foranti-inflammatory and neuroprotective ability to respond

to all of these insults (80) as well as a longer range of successful stroke outcome, and therefore it may not befeasible to recover, manipulate, and process quality con-opportunity that increases the survival rate and improves

functional recovery. This demand leads to exploration trolled autologous BM cells in a good manufacturingpractice (cGMP) environment in time.of new therapeutic strategies targeting the restorative

stage over the acute narrow therapeutic window, such as An efficacious substitute to BM cells is human um-bilical cord blood (hUCB), which has already been usedcell-based therapeutics designed to rescue against cell

loss and simultaneously provide anti-inflammatory and/ clinically for several years to reconstitute BM and bloodcell lineages in children with various hematological ma-or neurotrophic factors to combat the secondary neuro-

degeneration inherent in stroke. lignant and nonmalignant diseases (105). Since the firsthUCB cell transplantation to treat a 5-year-old childThe advent of stem cells catapulted the field of cell

therapy into the forefront of possible treatments for neu- who contracted Fanconi anemia in 1988 (44), a numberof hUCB transplants have been performed worldwide,rological disorders, including stroke. Of particular inter-

est is the availability of adult stem cells as donor cells many of them with unrelated donors (69,118,124). LikeBM, hUCB use has thus far been limited to hematopoi-for transplantation. Tissue-specific adult autologous stem

cells such as neural stem cells (NSCs), bone marrow etic malignancies, marrow failure, and immunodeficiencydisorders, but numerous reports reveal the many advan-(BM), or peripheral blood stem cells stand as attractive

alternative sources for cell-based therapies for stroke tages of hUCB cells for cellular therapies compared toBM (81). This includes the fact that hUCB cells, even(15) because they have the capacity for self-renewal and

can generate cells other than themselves, resembling the from allogeneic origin, are immature, and produce alower incidence of graft rejection, GvHD, and posttrans-multipotent features of primitive stem cells (i.e., embry-

onic or fetal origin). For example, adult NSCs can dif- plant infections (64,118). These characteristics have beenattributed to higher numbers of immature progenitors inferentiate into non-central nervous system derivatives,

such as blood (9) or skeletal muscle cells (38) and vice hUCB compared to those of adult marrow stem cells, onthe basis of telomere length (120). Moreover, the imma-versa. This plasticity of adult stem cells opens new ap-

proaches for their application in the treatment of various turity of cord blood stem cells and their immune naıvetehave been implicated in the optimal effects of these cellsneurodegenerative disorders. Nonetheless, clinical appli-

cation of adult NSCs, like embryonic or fetal stem cells, for hematopoietic and somatic organ therapy.The following sections present recent findings on therequires a careful preclinical evaluation of their safety

and efficacy, as well as bioethical issues (27,50). Addi- specific “stemness” properties and therapeutic potentialof hUCB as a unique, safe, and efficacious donor celltional technical difficulties exist, such as ensuring purity

of neural cultures and the harvesting of needed popula- for transplantation in stroke.tions for transplantation (91,99). Accumulating evidencehas revealed the therapeutic potential of hematopoietic UNIQUE FEATURES OF hUCBstem cells in the treatment of not only blood-related dis-

Cell Compositioneases but also various unrelated disorders, even neurode-generative diseases. Of these, BM is the current gold As a source of pluripotent stem cells, hUCB has been

reported to be a source of hematopoietic stem cells, en-standard source of hematopoietic progenitor cells usedto reconstitute blood lineages after myeloablative ther- dothelial cell precursors, mesenchymal progenitors, and

multipotent/pluripotent lineage stem cells (7,35,63). He-apy in a number of malignant and nonmalignant blooddiseases (90). Several reports have demonstrated that matopoietic stem cells from hUCB are rich in the most

primitive cells and are capable of long-term reconstitu-BM stem cells can give rise to non-blood lineage tissuesincluding neural cells (36,86,97). Some disadvantages of tion of blood lineages (11,79,115). While the number of

myeloid progenitors in hUCB is similar to the numberBM as donor cells, however, include the relative pro-longed timing between procurement of and treatment in BM (11), hUCB cells have a greater colony-forming

ability (78) and can be expanded in long-term cultureswith BM cells (61), the considerable cost of the harvest-ing procedure (61), and the low availability of human in vitro using different growth factors and have longer

telomeres than adult cells (120). Moreover, it has beenleukocyte antigen (HLA)-matched donors with BM,which is crucial to avoid graft-versus-host disease (GvHD) revealed that hUCB transplants are better at restoring

the host hematopoietic progenitor cell reservoir than(3). Although these limitations can be avoided by trans-plantation of autologous BM or peripheral blood with adult BM stem cells (37). Even a single hUCB sample

HUMAN UMBILICAL CORD BLOOD CELL GRAFTS FOR BRAIN ISCHEMIA 987

can supply enough hematopoietic stem cells to provide of GvHD and viral transmission. Additionally, such cel-lular components could also allow for relatively flexibleboth short- and long-term engraftment (105).

Within the mononuclear fraction of hUCB, the com- donor–recipient matching requirements, hence leadingto a shorter preparatory period for treatment (81). Rochaponent is primarily composed of lymphocytes and mono-

cytes (87). In an electron microscopic comparison of et al. found that GvHD incidence was significantlylower in children receiving hUCB transplants comparedhUCB, peripheral blood, and BM cells, hUCB cells had

the more immature morphology of myelomonocytic cells to BM recipients, when both sources were from anHLA-identical sibling (95). Furthermore, even unrelatedwith small numbers of mature neutrophils (77). Com-

pared with adult peripheral blood, hUCB resembles the HLA-mismatched UCB recipients have a lower GvHDincidence than HLA-identical BM recipients (94).B-cell population but with a lower absolute number of

T cells (CD3+) and a higher CD4+/CD8+ ratio (49,87). Recently, we characterized in vitro two different sub-populations of mononuclear hUCB cells: adherent andMoreover, when comparing the characteristics of B-cell

differentiation in vitro from hematopoietic progenitors floating (19). A significant number of progenitor/stemand neural cell antigens were found to be expressed on(CD34+) between cord blood and peripheral blood, B-

cell precursors differentiated from cord blood are more cells in the floating population, whereas the adherentcell population mainly contained lymphocytes (�53%)immature (51). The relative immunological immaturity

of hUCB reveals a higher proportion of immature T cells expressing hematopoietic antigens. These results suggestthat a nonhematopoietic subpopulation of cells exists(CB45RA+), but decreased numbers of mature memory

(CD45RO+) (29,49) and CD56+ cytotoxic (29) T cells within mononuclear hUCB cells and seems to have thepotential to become neural cells.compared with other adult sources. Moreover, cord blood

lymphocytes express cytokines and their receptors such The mesenchymal stem cell (MSC) has also beenfound as a small fraction of UCB cells (30,46,133). Itas interleukin (IL)-2, IL-6, IL-7, tumor necrosis factor-

α (TNF-α), and interferon-γ (IFN-γ) at lower levels than was recently confirmed that MSCs and MSC-like pro-genitors can be isolated from amniotic fluid, placenta,in adult blood cells (45,139) and produce greater amounts

of the anti-inflammatory cytokine IL-10 (45,89), which and Wharton’s jelly (30). MSCs derived from hUCBshow impressive plasticity and differentiate into cells ofdownregulates expression of CD86 on dendritic cells

(DCs), and may prevent initiation of the T-cell-mediated all germ-line derivatives (57,70,133). Under appropriateconditions for culturing and differentiation, hUCBimmune reaction (12). These increased levels of IL-10

may stimulate regulatory T cells, which in turn inhibit MSCs could differentiate into osteogenic cells and chon-drogenic cells (66). After incubation in neural differenti-antigen-specific immune responses (5). Monocytes in

hUCB are also immature because hepatocyte growth ation medium, hUCB MSCs expressed the neural cellsurface antigen A2B5, the neurofilament polypeptide NFfactor (HGF) activates adult monocytes, but cord blood

monocytes have no response to HGF, indicating they 200, the oligodendrocyte precursor marker 04, interme-diate filament proteins characteristic of neural differenti-cannot induce integrin-α5 subunit or intercellular adhe-

sion molecule-1, both of which are necessary for normal ation (nestin and vimentin), as well as the astrocytemarker glial fibrillary acidic protein (GFAP) and thefunction (59). Moreover, hUCB monocytes have a re-

duced expression of HLA-DR compared to adult cells neural progenitor marker TuJ-1 (32). Additionally, su-pernatants from cultured hUCB MSCs promoted sur-(113). In addition, cell subpopulations differ between

adult blood and cord blood; for example, cord blood has vival of NT2N neural cells and peripheral blood mono-nuclear cells cultured under conditions designed to inducehigher numbers of natural killer (NK) cells (29), which

are able to inhibit T-cell proliferation and reduce TNF- cell stress and limit protein synthesis (32). We alsoshowed immunomodulatory effects of the hUCB MSCsα production (33). On the contrary, cytotoxicity from

NK cells is lower in hUCB compared to adult blood after coculture with murine splenocytes (32). Taken to-gether, these results indicate that hUCB MSCs also pos-(28). DCs are the sentinel cells of the body and initiate

immune responses within the lymph nodes. The DCs in sess multiple utilities that may contribute to their thera-peutic potency in the treatment of neurological diseases.cord blood reflect lymphoid DCs that are most likely to

be responsible for colonizing neonatal tissue, while thereEx Vivo Expansion and Surface Antigensare more myeloid DCs in adults (128). The lymphoid

DCs induce anti-inflammatory T-helper 2 cell responses, There is only a single chance to collect hUCB cells,and the number of mononuclear cells extracted from thatwhich, along with the naive T cells, may promote down-

regulation of immune responses (4,128). These imma- isolated donation is finite (81). Therefore, many research-ers are currently interested in the ex vivo expansion ofture immunological properties in hUCB cells appear to

result in a prolonged immunodeficient state after hUCB cord blood progenitor cells to provide a sufficient amountof stem cells for adult transplantation (72). Expansion istransplantation (42,114). This leads to a low incidence

988 PARK ET AL.

essential in providing adequate dosage for both children CD34 for the selection and expansion of hematopoieticcells for transplantation (65,135). It has been shown thatand adults, reducing the time to engraftment, thereby

increasing success rate, as well as for recurrent therapy about 80% of CD34+ cells express CD133 and more than97% of CD133+ cells are CD133+/CD34+ in fresh cordin the event of initial graft failure (81). Unfortunately,

current methods of expanding hUCB cells do not pre- blood (48). Although CD133+ cells comprised 0.7% ofthe total mononuclear hUCB cells (74), expansion ofserve the quality of the hematopoietic progenitor cells

through to the end product (56,93), but also cannot make CD133+ and CD133+/CD34+ cells was significantlyhigher than that from the CD34+ cells (48). These find-up for the cells lost in the storage process, let alone aug-

ment them to a suitable dosing regimen (52,73,117,134). ings suggest that CD133+ cells may be a more primitivehematopoietic progenitor/stem cell than CD34+ cellsMoreover, simply globally multiplying the cells has

been proven to not reduce the time to engraftment (56). (39). Furthermore, CD133+ cells have been identified infetal brain and are considered to be NSCs (110,119),Instead, relative proportions of cell subpopulations that

home to marrow, form colonies, and repopulate blood even though it is not yet known whether the CD133+

cells found in hUCB are phenotypically and functionallylineages are required (81). Thus, recently, several stud-ies about ex vivo expansion of different cell subpopula- identical to the NSCs found in fetal brain (81). In the

meantime, determining a definitive phenotype for hUCBtions of hUCB have been reported. Huang et al. (54)found that ex vivo expansion of hematopoietic stem MSC has proven to be controversial, unlike that of BM.

Robinson et al. (93) defined the MSC as positive forcells (CD34+) of hUCB with hUCB MSCs was moreeffective than without MSCs. Wei et al. (127) reported CD16, CD73, CD90, and CD105 and negative for CD31,

CD34, CD45, CD80, and HLA-DR, whereas Yang et al.that T cells and NK cells as well as CD34+ cells couldbe expanded from hUCB in the same medium with the (133) characterized the MSC as having positive markers

for CD13, CD29, CD44, and CD90 and negative mark-combination of cytokines. The expanded mononuclearcells could reconstitute hematopoiesis and eliminate ers for CD14, CD31, CD34, CD45, CD51/61, CD64,

CD106, and HLA-DR. On the other hand, we showedminimal residue leukemia disease in transplanted mice.Shirvaikar et al. (103) showed that the combination of that hUCB MSCs were negative for CD3, CD11b,

CD19, CD34, and CD45 in culture (32).thrombopoietin with IL-3 used for expansion of mega-karyocytic progenitors from cord blood not only resulted

Proliferation and Differentiation Into Neuralin optimal proliferation but also protected these cellsand Other Lineagesfrom apoptosis. Thus, these cells could be employed to

supplement cord blood grafts and speed up platelet re- While embryonic primitive cells have highly prolifer-ative potential and resistance to rejection and apoptosis,covery in transplant recipients (103).

Meanwhile, there are major problems arising from which therefore may contribute to deleterious fibrosisand malignancies, cord blood stem cells are relativelythe choice of an optimal stem cell population for ex vivo

expansion and definition of the desired characteristics of quiescent under normal circumstances (116). Thus, theintrinsic proliferative and differentiatable potential ofthe expanded stem cells to be used for transplantation

(102). Expanded cells need to maintain telomerase length these quiescent stem cells needs to be stimulated by fac-tors within the environment (23), without which the cellscontinuously in order to preserve their pluripotent capac-

ity (134). The telomerase activity of hematopoietic pro- do not proliferate unrestrained. Several in vitro and invivo reports have been published showing the multipo-genitor cells is high and tends to be most active during

proliferation (20). Determining which cells should be tent nature of hUCB cells, including neural cells, thushelping to advance the potential usefulness of them inexpanded is also tricky (81). The level of maturity of a

cell is identified by the presence of, or a lack of, a com- treating neurodegenerative diseases. Bicknese et al. (8)and Buzanska et al. (13) demonstrated that hUCB cellsbination of cell surface antigens. These are potential

markers for the selection of stem cells important for could differentiate to express βIII-tubulin, GFAP, andgalactosylceramide (GalC). Of particular interest, Bu-expansion and implantation. For example, the mononu-

clear fraction of hUCB contains roughly 1% CD34+ cells zanska et al. (13) used the CD34−/CD45− nonhematopoi-etic mononuclear cell fraction of hUCB to obtain neural(81). This CD34+ population (CD34 being a marker des-

ignating the cell as having a role in early hematopoiesis) stem cell-like cells. From these, a clonogenic line ofhUCB NSCs, which expressed nestin and GFAP, was de-in hUCB can be defined as more primitive than those

found in BM because a higher proportion of them are rived. In the presence of neuromorphogen/retinoic acid,40% of the hUCB NSCs expressed βIII-tubulin and mi-negative for CD38, a marker for prelymphoid cells

(14,25). Another human antigen, CD133, has also been crotubule-associated protein (MAP)-2 (neuronal marker),30% expressed GFAP and S100β (astrocytic marker),identified as a marker for primitive hematopoietic pro-

genitor/stem cells that may prove to be a substitute to and 11% expressed GalC (oligodendrocytic marker). Re-

HUMAN UMBILICAL CORD BLOOD CELL GRAFTS FOR BRAIN ISCHEMIA 989

tinoic acid plus brain-derived neurotrophic factor has press neural antigens after in vitro exposure to definedmedia supplemented with a cocktail of growth and neu-also been shown to induce neural differentiation in hUCB

NSC culture. During 7 days of coculture with neuromor- rotrophic factors (20). When floating mononuclear hUCBcells were exposed to a neurogenic environment throughphogens, rat astrocytes, or hippocampal slices, 80% of

cells expressed βIII-tubulin and 64% coexpressed MAP- treatment with growth factors and neurotrophins, theyhad increased proliferation, expression of neural anti-2 (60). Goodwin et al. (46) demonstrated a subset of

mononuclear cells from hUCB, which had been main- gens, and also survived longer in vivo (20).Once the cell type of interest has been determinedtained in continuous culture for more than 6 months

without antigen expression for hematopoietic differenti- and an adequate population of these cells has been ob-tained, the next step would be to transplant the cells.ation. When these cells were exposed to osteogenic, adi-

pogenic agents, or epidermal and basic fibroblast growth This raises the question of what happens to the cellsfollowing transplantation and this is discussed in thefactors (EGF and bFGFs), they expressed bone, fat, and

neural markers, respectively. These data suggest that next section.hUCB contains a cell population that is capable of ex-

Fate of Grafted Cells: What and Why?pressing antigens of multiple lineages, demonstratingthat the plasticity of these cells makes them very impor- The cells used for transplantation into humans are

preferred to be of human origin. Although xenograftingtant for cellular therapeutics with the goal of system re-pair (39). Confirming the presence of a cell population of human cells into the rodent is a widely accepted re-

search paradigm that often yields promising results (1,in hUCB with multipotent ability, McGuckin et al. (76)developed a negative immunomagnetic selection method 125), this presents a dilemma in in vivo research with

human-originated stem cells. While hUCB cells showthat depletes hematopoietic lineage marker-expressingcells from hUCB, thereby isolating a discrete lineage- the inability to generate cytotoxic T cells that respond to

allogenic antigens, and to produce the proinflammatorynegative stem cell population (0.1% of mononuclearhUCB). These selected lineage-negative hUCB cells ex- cytokines such as IFN-γ and TNF-α, transplantation of

hUCB cells into adult or aged rat brains was associatedpanded into primitive nonadherent hematopoietic pro-genitors and simultaneously produced adherent cells with with vigorous rejection and strong immunosuppression

was required to protect the graft (126). The limited sur-neuroglial progenitor cell morphology over 8 weeks.Gene expression analysis showed upregulation of primi- vival of the grafts in in vivo models possibly results

from immune rejection. Therefore, recently we per-tive neuroglial progenitor cell markers for GFAP, nestin,musashi-1, and necdin. formed studies using hUCB cells to determine their abil-

ity to survive in vivo and the effect of the immune re-Our group also revealed that the mononuclear hUCBcells under specific conditioned media including retinoic sponse on the survival by transplantation into the normal

striatum of immunodeficient NOD SCID mice (125).acid and nerve growth factor (NGF) expressed variousneural markers for early neural precursors (musashi-1, First, in vitro, long-term culture (60–211 days) of hUCB

cells resulted in several different populations of cells in-nestin, TuJ1), mature neurons (NeuN, MAP-2), andastrocytes (GFAP) (98). Moreover, cells exposed to reti- cluding neuron-like cells that were positive for TuJ1 and

nestin. Five days after transplantation of cells of thisnoic acid and NGF treatment increased TuJ1 and GFAPexpression by approximately twofold (98). Furthermore, neuronal phenotype, many differentiated hUCB cells ex-

pressing characteristic neuronal proteins were detected.Zigova et al. (138) showed that TuJ1 and GFAP immu-nopositive cells from hUCB mononuclear cells after However, at 1 month postgrafting, hUCB cells were no

longer detected without evidence of T-cell-mediated re-treatment with retinoic acid and NGF survived in thesubventricular zone of the rat neonatal forebrain. Addi- jection, such as CD4 and CD8 lymphocytes and activa-

tion of microglia and astrocytes (125). These findingstionally, we have demonstrated that in standard growthmedium such as Dulbecco’s modified Eagle medium suggest that the disappearance of implanted cells was

not due to a T-cell-mediated immune response. The(DMEM), mononuclear hUCB cells also express neuralmarkers, such as nestin, TuJ1, MAP-2, and GFAP (40). hUCB cells can survive for long periods of time in vitro

and they can also exhibit neural characteristics depen-Interestingly, at 2 weeks after plating, colocalization ofnestin, MAP-2, and various cytoplasmic expressions of dent on their environmental conditions, but the same

does not appear to be true in vivo even though the causeTuJ1 by the cells was observed. Moreover, the increasednumber of cells expressing CD133 antigen after 7 days of this discrepancy is unclear (125). A number of fac-

tors, such as the donor cell’s condition and the recipi-in culture probably gives rise to cells that show imma-ture and mature neural characteristics at the same time ent’s environment, might simultaneously affect survival

of the transplanted cells.(39). Recently, similar to the above studies, we haveexamined the ability of mononuclear hUCB cells to ex- In summary, on the basis of several previous studies

990 PARK ET AL.

in vitro and in vivo, hUCB cells, due to their unique and NeuN. On the other hand, our group compared theefficiency of intravenous versus intrastriatal transplanta-primitive nature and ability to differentiate or become

various lineages of cells including neural, may be a use- tion of mononuclear hUCB cells to evaluate which pro-duced the greatest behavioral improvements in rats withful target for cell-based therapies requiring either the re-

placement of individual cell types and/or provision of permanent MCAO (129). Spontaneous activity (MCAO-lesioned animals become significantly hyperactive) sig-missing materials in neurological disorders.nificantly decreased when cells were transplanted 24 h

PRECLINICAL STROKE RESEARCH after stroke compared with control animals. BehavioralON TRANSPLANTATION OF HUMAN improvement, as measured by spontaneous activity, was

UMBILICAL CORD BLOOD CELLS similar with both cell delivery routes. However, in thestep test at 2 months after transplantation, significant im-As mentioned, although the timing to begin treatment

for stroke is crucial (39), the therapeutic window of cur- provements were found only after intravenous deliveryof the hUCB cells. Also, in the passive avoidance test,rent stroke treatment is still restrictive. Many patients

outside of this window suffer from irreversible sequela. transplanted rats learned the task faster than nontrans-planted ones. These results suggest that intravenous injec-This current circumstance has brought serious consider-

ation to the exploration of new therapeutic interventions tion of mononuclear hUCB cells may be more effectivethan direct striatal implantation in producing long-termsuch as a cell-based therapy. Using adult BM mesenchy-

mal stem cells, functional improvement has been dem- functional benefits to the stroke animal. Next, we fo-cused on the dose effect of mononuclear hUCB cells ononstrated in a rodent stroke model (16,18,22,100). While

it has been reported that transplanted cells could suc- the behavioral recovery and infarct volume in MCAOrats (121). Twenty-four hours after MCAO, rats werecessfully migrate to areas of ischemic infarcts and differ-

entiate into neuronal and glial cell types, recently Chopp intravenously infused with 104 up to 3–5 × 107 mononu-clear hUCB cells. At 4 weeks after transplantation, thereand collegues suggested that recovery mechanisms are

likely to be due to trophic factors released by cells that was a significant recovery in behavior, when 106 ormore hUCB cells were delivered. Infarct volume mea-may promote endogenous neurogenesis, angiogenesis,

and axogenesis rather than a result of neuronal replace- surements showed an inverse relationship between celldosage and injured volume, which reached significancement (26,88,101,136). Transplanted hematopoietic stem

cells (CD34+), which were isolated from peripheral at the higher doses of mononuclear hUCB cells. Theseresults revealed an important dose relationship betweenblood, also could differentiate into neural lineage cells

and induce angiogenesis, in conjunction with improve- introduced implanted cells, behavioral improvement,and neuronal sparing, using intravenous mononuclearment of the neurological impairment in a rodent stroke

model (31,104). hUCB cell injection in the MCAO model.In addition to route and dosage, determining the ther-

The In Vivo Potential of Human Umbilical Cord apeutic window of cell transplantation for stroke is aBlood Cells major concern in the preclinical research field as well as

for clinical application. Our research group attempted tohUCB is another promising source of multipotentialstem cells that has shown affirmative effects in in vivo determine the optimal time to administer hUCB cells

after stroke. Initially, in an in vitro assay system, thestudies for the treatment of stroke. Chen et al. (17) showedthat the intravenous administration of mononuclear hUCB migration capability of mononuclear hUCB cells accord-

ing to the time after stroke was investigated (82). Thiscells at 24 h or 7 days after middle cerebral artery occlu-sion (MCAO) significantly improved neurological func- assay revealed an increased migratory activity of mono-

nuclear hUCB cells towards both the hippocampal andtion in a rat model. On histological examination, mono-nuclear hUCB cells were found mainly in the cortex and striatal extracts harvested at 24–72 h after stroke. Fur-

thermore, the extracts possessed increased levels of cyto-striatum of the injured hemisphere in the ischemic bound-ary zone, whereas few cells were observed in the contra- kine-induced neutrophil chemoattractant-1 (CINC-1) and

monocyte chemoattractant protein-1 (MCP-1) at 48 hlateral side. Some of the implanted mononuclear hUCBcells were immunoreactive for the endothelial cell marker after MCAO, suggesting participation of these sub-

stances in the cell migration. Further analysis of the is-FVIII (8%), as well as GFAP (6%), MAP-2 (3%), andNeuN (2%) by immunohistochemistry (17). Using a chemic extracts showed that growth-regulated onco-

gene/CINC-1 (the rat equivalent of human IL-8) andnonhematopoietic cell line derived from hUCB, Xiao etal. (132) also observed a decrease in infarct volume after MCP-1 were expressed in a time-dependent pattern sim-

ilar to that of the migration assays. The results from thisintravenous transplantation into rats with ischemic braininjury. Histological examination showed that some of study are promising in that the current 3-h therapeutic

window for the treatment of stroke victims, using ap-the transplanted cells were colocalized for human nuclei

HUMAN UMBILICAL CORD BLOOD CELL GRAFTS FOR BRAIN ISCHEMIA 991

proved anticoagulant treatment, may be extended with potential anti-inflammatory effects of UCB therapy maybe one of the mechanisms that protect against neuronalthe use of mononuclear hUCB cell therapy to 24–72 h

poststroke event. Also, the chemokines present in the death. In continuing studies, we found that, followingMCAO, the rat’s spleen size was reduced concomitantlysupernatant could provide a sound starting point for ex-

amining the mechanisms responsible for the in vivo mi- with their CD8+ T-cell counts (123). Interestingly, MCAO-induced spleen size reduction correlated with the extentgration of mononuclear hUCB cells after stroke induc-

tion (82). More recently, we demonstrated that, in vivo, of ischemic damage; however, hUCB cell treatment res-cued the spleen weight, splenic CD8+ T-cell counts, asthe hUCB treatment window is also narrow even though

it is longer than thrombolytic agents. When we systemi- well as significantly reducing the amount of brain injury.Additionally, splenocyte proliferation assays demon-cally administered the hUCB cells at times ranging from

3 h to 30 days post-MCAO, maximal improvements strated that hUCB cell treatment opposed MCAO-asso-ciated T-cell proliferation by increasing the productionwere observed with treatment at 48 h (80). These results

might suggest that it is not critical for the cells to be of IL-10 while decreasing IFN-γ (123). Recently, we in-vestigated the expression of cytokines and chemokinespresent indefinitely after transplantation, but that they

are actually only needed within a very short time frame produced by hUCB cells under various culturing condi-tions (83). The heterogeneous cells from mononuclearto either appropriately prime the immune system or pro-

vide other secretory products to cells within the brain in fractions of hUCB consistently produced a variety ofchemokines and cytokines when grown in various cul-order to confer therapeutic effects (80).ture conditions, but in particular, IL-8, MCP-1, and IL-

Mechanisms Underlying Recovery Beyond 1α. They are produced more extensively than any otherNeural Differentiation chemokines in the human body, especially in the brain,

and have been implicated as the first line of defense inCell replacement had been believed to be the mainmechanism responsible for the functional recovery after the inflammatory reaction. These findings support the

idea that inflammatory modulation from hUCB trans-transplantation on the basis of accumulating results ofhUCB cells expressing neural phenotypes and providing plantation may be partially responsible for the functional

improvements seen in animal models of injury, includ-behavioral improvement (40,47,98,132,138), even thoughfew cord blood cells are present in the ischemic region ing stroke.

A second potential mode of action is neovasculariza-compared to the number of infused cells (21,121,122,129,130). However, there has been increasing evidence tion around ischemic tissue, promoted by hUCB. Angio-

genesis is an important healing process of wounds in-of the multifaceted therapeutic effects of hUCB such asneurotrophic, angiogenic, and anti-inflammatory actions, cluding tissue ischemia, and it can attenuate local tissue

ischemia and improve clinical outcomes (67). Further-beyond the ability of transdifferentiation into neural lin-eage, in the MCAO stroke model (Fig. 1). These effects more, angiogenesis appears to promote neurogenesis in

neurological disease (92,112). There is accumulatingmore likely come from the trophic and other factor-releas-ing capabilities of hUCB (10). Recently, it has been re- data that transplantation of hUCB cells induce neovascu-

larization, followed by functional improvement in thevealed that mononuclear hUCB-derived neuronal progeni-tor cells decreased free radicals and induced antioxidants animal ischemic disease model (53,62). VEGF was in-

creased in the experimental group (53) even though itand neurotrophic factors such as NGF, vascular endothe-lial growth factor (VEGF), and bFGF in an oxygen and was unclear whether transplanted hUCB cells directly

released it or stimulated other cells for indirect secretion.glucose deprivation insult model (2).Initially, we focused on the anti-inflammatory action UCB not only contains high numbers of CD34+ endothe-

lial progenitors cells (EPCs), which can give rise to ma-of hUCB cells. Vendrame et al. (122) demonstrated thatthere was an increase in the number of CD45+/CD11b+ ture endothelial cells and induce angiogenesis in ische-

mic tissues (85), but mononuclear cells (55) or MSCs (30)(microglia/macrophage) and CD45+/B220+ (B cell) cellsin the brain of rats with permanent MCAO, whereas mo- of UCB also can differentiate into EPCs. Taguchi et al.

(109) demonstrated that CD34+ cells derived from hUCBnonuclear hUCB cell transplantation significantly de-creased the number of these cells. The reduction in the induced angiogenesis, followed by endogenous neuro-

genesis in a mouse stroke model. Ding et al. (30) re-number of CD45+/CD11b+ cells after transplantation ofhUCB cells is particularly interesting because chronic vealed that hUCB MSCs differentiated into glial, neu-

ronal, and vascular endothelial cells as well as promotedmicrogliosis is thought to mediate neuronal damage, notonly in ischemic injury but also in other neurodegenera- the formation of new vessel angiogenesis in the rat

stroke model. Recently, we found that animals that re-tive diseases such as Parkinson’s disease (107,111). ThehUCB treatment also decreased the proinflammatory cy- ceived the monocyte/macrophage (CD14+)-depleted hUCB

preparation performed significantly to a lesser degreetokines such as TNF-α and IL-1β (122). Therefore, the

992 PARK ET AL.

Figure 1. Schematic diagram depicting how human umbilical cord blood (hUCB) grafts can beneficially influence the strokepatients. Transplanted hUCB cells may exert their beneficial effects with respect to stroke either directly on hypoxic cell death byneurogenesis (upper side) or indirectly via release of a number of factors that modulate the secondary inflammation and promotetissue repair and angiogenesis (lower side).

than those receiving the other hUCB cells (stem cell-, creased in the ischemic hemisphere of rat brain, and sig-nificantly promoted hUCB cell migration compared toT-cell-, B-cell-depleted, and whole mononuclear frac-

tion of cord blood) in a rat stroke model (131). These the contralateral side (58). Also chemokine receptorswere constitutively expressed on the surface of hUCBresults suggest that monocyte/macrophage is critical to

hUCB-induced recovery following stroke. Monocytes cells. The hUCB cell migration was neutralized withpolyclonal antibodies against MCP-1 or MIP-1α. Thesehave been known to play a key role in inducing angio-

genesis under abnormal conditions such as ischemia and results suggested that the increased host chemokines inthe ischemic area can bind surface receptors on exoge-tumor (108). Thus, these findings imply that transplanta-

tion of monocyte-depleted hUCB does not induce angio- nous hUCB, and attract systemically delivered hUCBcells into the central nervous system.genesis properly, and in turn, cannot improve neurologi-

cal dysfunction. In conclusion, stroke involves a complicated cascadeof inflammatory events that eventually lead to a signifi-A third benefit of hUCB cells is their apparent migra-

tion following implantation to ischemic regions of the cant necrotic area adjacent to the obstructed vasculature.This cascade is time dependent, and when hUCB cellsbrain. We recently showed in vivo that several chemo-

kines increase in the ischemic brain area (58). MCP- are administered intravenously or intraparenchymally 48h following stroke onset, they can prevent impending1 and macrophage inflammatory protein (MIP-1α) are

implicated in bloodstream monocyte infiltration into tis- neural cell crisis. The transplantation of hUCB cellsmost likely inhibits the apoptotic cascade, induces en-sues under pathological conditions (6,43). We found that

MCP-1 and MIP-1α expression was significantly in- dogenous and/or exogenous neurogenesis, and modu-

HUMAN UMBILICAL CORD BLOOD CELL GRAFTS FOR BRAIN ISCHEMIA 993

jected umbilical cord blood cells is not required for neu-lates the immune/inflammatory response to injury bothroprotection in stroke. Stroke 35(10):2385–2389; 2004.peripherally and locally. Furthermore, another advantage

11. Broxmeyer, H. E.; Hangoc, G.; Cooper, S.; Ribeiro,in using hUCB cells for the treatment of stroke is the R. C.; Graves, V.; Yoder, M.; Wagner, J.; Vadhan-Raj,potential restoration of blood flow to the infarcted area S.; Benninger, L.; Rubinstein, P.; et al. Growth charac-

teristics and expansion of human umbilical cord bloodbecause cord blood contains endothelial progenitor cells,and estimation of its potential for transplantation inwhich may be useful in neovascularization therapy. Es-adults. Proc. Natl. Acad. Sci. USA 89(9):4109–4113;sentially, if the present problems that hamper the appli-1992.

cation of hUCB cells to human stroke patients, such as 12. Buelens, C.; Willems, F.; Delvaux, A.; Pierard, G.;uncertain safety, imperfect expansion processes, and Delville, J. P.; Velu, T.; Goldman, M. Interleukin-10 dif-

ferentially regulates B7-1 (CD80) and B7-2 (CD86) ex-limited graft survival in the recipient, can be overcomepression on human peripheral blood dendritic cells. Eur.in the future, hUCB will be a good candidate for cell-J. Immunol. 25(9):2668–2672; 1995.based therapy, providing multiple therapeutic effects in

13. Buzanska, L.; Machaj, E. K.; Zablocka, B.; Pojda, Z.;a single transplant that no other pharmacological agent Domanska-Janik, K. Human cord blood-derived cells at-could mimic. tain neuronal and glial features in vitro. J. Cell Sci.

115(Pt. 10):2131–2138; 2002.ACKNOWLEDGMENTS: We wish to thank Nicole Kuzmin-14. Cardoso, A. A.; Li, M. L.; Batard, P.; Hatzfeld, A.;Nichols for her help. P.R.S. is a cofounder and C.V.B. and

Brown, E. L.; Levesque, J. P.; Sookdeo, H.; Panterne,A.E.W. are consultants for Saneron CCEL Therapeutics Inc.B.; Sansilvestri, P.; Clark, S. C.; et al. Release from qui-P.R.S., C.V.B., and D.J.E. were not involved in the editorialescence of CD34+ CD38− human umbilical cord bloodand peer review process for this article. This article is basedcells reveals their potentiality to engraft adults. Proc.on a presentation to the Pan Pacific Symposium of Stem CellNatl. Acad. Sci. USA 90(18):8707–8711; 1993.Research held in Taiwan, June 2008.

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