NEUROLOGICAL PROGRESS

Cell Therapy for Neonatal Hypoxia–

Ischemia and Cerebral Palsy
Laura Bennet, PhD,1 Sidhartha Tan, MD,2 Lotte Van den Heuij, MSc,1
Matthew Derrick, MBBS,2 Floris Groenendaal, MD, PhD,3 Frank van Bel, MD, PhD,3
Sandra Juul, MD, PhD,4 Stephen A. Back, MD, PhD,5 Frances Northington, MD, PhD,6
Nicola J. Robertson, PhD, FRCPCH,7 Carina Mallard, PhD,8
and Alistair Jan Gunn, MBChB, PhD1,9

Perinatal hypoxic–ischemic brain injury remains a major cause of cerebral palsy. Although therapeutic hypothermia is
now established to improve recovery from hypoxia–ischemia (HI) at term, many infants continue to survive with
disability, and hypothermia has not yet been tested in preterm infants. There is increasing evidence from in vitro and
in vivo preclinical studies that stem/progenitor cells may have multiple beneficial effects on outcome after hypoxic–
ischemic injury. Stem/progenitor cells have shown great promise in animal studies in decreasing neurological
impairment; however, the mechanisms of action of stem cells, and the optimal type, dose, and method of
administration remain surprisingly unclear, and some studies have found no benefit. Although cell-based
interventions after completion of the majority of secondary cell death appear to have potential to improve functional
outcome for neonates after HI, further rigorous testing in translational animal models is required before randomized
controlled trials should be considered.
ANN NEUROL 2012;71:589–600

Neonatal Encephalopathy at Term
N eonatal encephalopathy due to perinatal hypoxia–
ischaemia (HI) occurs in 1 to 3 per 1,000 births at
1
term and is associated with high mortality and morbid-
ity, with life-long chronic disabilities including cerebral
palsy (CP).2,3 Neuropathological injury typically involves
neuronal death in the cerebral cortex, hippocampus, and
basal ganglia–thalamus, with increasing evidence for
global white matter involvement.4
To interpret preclinical studies of neuroprotectants,
it is critical to appreciate why the latent (early recovery)
phase has been the effective therapeutic target for the
majority of potential treatments to date. Although a suf-
ficiently severe/prolonged period of HI (the primary
phase of injury) may be associated with immediate cell
death due to metabolic failure, even relatively severe
insults can be associated with transient recovery of oxida-
tive metabolism after reperfusion in a so-called latent
phase (Fig 1).5 In this latent phase, the electroencephalo-
gram and cerebral blood flow are typically suppressed,
but cerebral oxygenation is normal.6,7 After approxi-
mately 6 to 15 hours, there may be secondary deteriora-
tion, with delayed seizures, progressive mitochondrial
failure,5,8 cytotoxic edema, and secondary cell death over
many days.6 The severity of this secondary deterioration
is closely correlated with later neurodevelopmental
impairment.9 Although considerable cell death typically
occurs during the secondary phase, this is often followed
by a chronic or tertiary phase of further progressive cell
death, repair, and remodeling.10,11 It is believed that this
additional loss of neurons and glial cells is related to
chronic loss of trophic factors and synaptic input from

View this article online at wileyonlinelibrary.com. DOI: 10.1002/ana.22670

Received Jun 17, 2011, and in revised form Oct 1, 2011. Accepted for publication Nov 4, 2011.

Address correspondence to Dr Gunn, University of Auckland, Physiology and Paediatrics, Auckland, New Zealand. E-mail: aj.gunn@auckland.ac.nz

From the 1Department of Physiology, University of Auckland, Auckland, New Zealand; 2Department of Pediatrics, NorthShore University HealthSystem,
Evanston, IL; 3Department of Neonatology, University Medical Center Utrecht, Utrecht, the Netherlands; 4Department of Pediatrics, University of
Washington, Seattle, WA; 5Department of Pediatrics, Oregon Health and Science University, Portland, OR; 6Division of Neonatology and Neonatal Research
Laboratory, Johns Hopkins School of Medicine, Baltimore, MD; 7Institute for Women’s Health, University College London, London, UK; 8Institute for
Neuroscience and Physiology, Department of Physiology, Sahlgrenska Academy, University of Gothenburg, Gothenburg, Sweden; and 9Department of
Paediatrics, University of Auckland, Auckland, New Zealand.

V
C 2012 American Neurological Association 589
ANNALS of Neurology
FIGURE 1: Schematic representation of the evolution of
neural injury after exposure to hypoxia–ischemia.
neighboring cells,12 and loss of or failure of recruitment
of neural stem cells (NSCs) and glial progenitor cells
within the brain.13
In term infants with moderate to severe encephalop-
athy, it is now well established that mild to moderate hypo-
thermia induced in the first 6 hours after birth, and contin-
ued for at least 72 hours, can significantly improve survival
without disability.3,14 However, many infants still survive
with disability with current treatment protocols.3 Promising
adjunctive treatments including erythropoietin and xenon
are now undergoing phase 2 clinical trial.15,16 In preterm
infants, the precise timing of neural injury is often unclear,
and there are unresolved safety concerns around cooling.17
Although there is increasing clinical evidence that maternal
treatment with magnesium sulfate before early preterm
birth is associated with a small reduction in risk of CP in
surviving infants,18 there is still considerable uncertainty.19
Thus, additional, late interventions in the tertiary phase in
both term and preterm infants might have potential to
decrease chronic cell loss and maldevelopment due to lack
of survival signals, and help repair.
The complex etiology of HI calls for a therapy that
intervenes on multiple levels, including promoting cell
survival, regeneration, and functional reorganization.
Over the past decade, the possibility of utilizing progeni-
tor or stem cells as a therapy for HI has gained increas-
ing support. There are many different types of stem cells,
derived from embryonic, fetal, placental, and adult tis-
sues. Their promise derives from the potential to replace
lost neurons, protect host cells, and promote their growth
and differentiation, as well as modulate the host immune
response, all of which might decrease disability after HI.
Parents of children with CP have been a driving force for
the use of stem cells to treat HI brain injury. A handful
of sensational cases of apparent recovery have been
reported in the lay press, leading to inflated expectations.
Clinicaltrials.gov now lists 7 clinical trials for neonatal
590
HI or CP, 6 of them active, mostly using human umbili-
cal cord blood cells (hUCBCs). More than 200 patients
have been infused with hUCBCs for neonatal brain
injury and childhood motor deficits including CP, almost
entirely in uncontrolled studies (Fig 2).20 These data also
highlight the remarkable range of volumes given to
patients, consistent with the lack of knowledge of the
optimal dose of hUCBCs.
The safety of autologous cord blood cells appears
to be relatively good. A minor issue for late treatment is
that preserved cells are stored with dimethyl sulfoxide
(DMSO), and despite washing, thawed cells typically
include some residual DMSO, which can be associated
with acute adverse reactions.21 For example, in a pilot
study in children with acquired brain disorders aged 6
days to 9.5 years, 76% of them with CP,22 3 of 184
patients developed anaphylactic reactions despite prophy-
lactic antihistamines, bronchodilators, and steroids; all
adverse symptoms resolved rapidly after stopping the cell
infusions and appropriate medical treatment.
In contrast, although a detailed discussion is outside
the scope of this paper, the potential for nonautologous
embryonic or pluripotent stem cells to differentiate into
all cell types is associated with potential risk of terato-
mas.23,24 Embryonic stem cells have been reported to
form teratomas in mice of the same genetic back-
ground,24 or in immunodeficient animals.23 In contrast,
immunocompetent mice (or xenogenic rats) efficiently
immunorejected the cells through a natural killer cell
mechanism.25 These balanced risks of tumors and rapid
rejection need further detailed evaluation.
Analysis Strategy
For the present review, an international group of
researchers in perinatal neuroprotection have examined
FIGURE 2: (A) Number of patients with newborn brain
injury and children with motor deficits who received human
umbilical cord blood cells (hUCBCs). The drop in 2010 coin-
cided with the beginning of a randomized clinical trial at
Duke that was responsible for the majority of hUCBC
administration. (B) The volume of hUCBCs given extends
over a wide range. The number of cells is not known. Data
are derived from the Cord Blood Registry.20
Volume 71, No. 5

the evidence for therapeutic potential for neonatal HI in
term infants with different stem/progenitor cell prepara-
tions, including NSCs, NSCs derived from fetal tissues,
mesenchymal stem cells (MSCs), multipotent astrocytic
stem cells (MASCs), and multipotent adult-derived pro-
genitor cells (MAPCs), as well as hUCBCs. MASCs are
a special type of astrocyte derived from the subependy-
mal zones of postnatal day 1 (P1) to P6 rats, which
retain NSC-like properties, including the ability to form
neurons as well as glia.26 MAPCs are pluripotent progen-
itor cells, typically isolated from the bone marrow of
adult rodent donors,27 that retain the ability to differen-
tiate into neuronal lineages. Studies were searched for on
PubMed using the strategy of (perinatal brain injury OR
(hypoxia–ischemia AND neonatal brain injury)) AND
(cell therapy OR progenitor cells OR cord blood cells).
For each relevant paper identified in this way, we then
performed a related search. The results are summarized
in the Table.
Experimental HI
All of the studies that we identified used some modifi-
cation of the Vannucci model of unilateral carotid ar-
tery ligation followed by a period of moderate hypoxia
in neonatal rats or mice.28 This functional paradigm of
perinatal injury is associated with infarction in the mid-
dle artery territory, typically with no overt cell death in
the hemisphere contralateral to ligation. Most rodents
are highly altricial. Although P7 in the rat or mouse
was originally chosen because this is the age of peak
brain growth, which occurs at term in humans,29 at P7
rodents are just starting cortical myelination, compara-
ble with 34 weeks gestation in humans. On balance,
cortical maturity of the rat has been estimated to be
most comparable to the term human at P12 to P13.30
Thus, the studies analyzed here represent acute, well-
defined perinatal HI that is most characteristic of brain
injury at term, but at a mildly preterm stage of brain
maturation.
NSCs
NSCs have been the cell type most commonly studied in
preclinical models of neonatal HI, because of their
potential to differentiate into glial and neuronal lineages,
and even endothelial cells. In early experiments, cortical
grafts from fetal rats ((embryonic day) E13) were trans-
planted 7 days after HI at P7, and the animals were sac-
rificed 2 to 6 weeks after transplantation.31 There was no
effect on the extent of brain damage, although >80% of
grafts survived. In contrast, when a cell suspension from
the cortical sensorimotor region of fetal rats (E16) was
May 2012
Bennet et al: Stem Cells and HI
transplanted 3 days after HI at P7, motor deficits were
reduced from 3 to 9 weeks of age.32 At 10 to 12 weeks,
grafts could be identified in approximately 2=3 of animals,
and showed a neurochemical profile broadly consistent
with normal neocortex, although typical neocortical
layers did not develop even for grafts integrated into the
sensorimotor cortex, and the morphology of some phe-
notypes was different from controls.
Park et al showed that NSCs transplanted 7 days
before HI integrated and became quiescent, and did not
differentiate until they were subjected to HI.33 After HI,
NSCs migrated to the pemumbra of the infarct, where
the cells differentiated into neurons and oligodendro-
cytes, suggesting that additional environmental cues are
needed for these cells to migrate and differentiate.
Unfortunately, the authors did not assess whether there
was any functional benefit. Imitola et al demonstrated
injury-mediated homing along a stromal cell-derived fac-
tor 1a (SDF-1a) gradient toward the site of injury.34
Supporting a critical role for this factor, NSCs expressed
the SDF receptor, CXC chemokine receptor 4, and in
vitro cells increased migration and proliferation when
SDF-1a was added to culture.34
Similarly, intracerebral injection of MASCs har-
vested from mice (P1–P6) in P7 rats was associated with
diffuse migration and an astrocytic morphology in non-
hypoxic controls, whereas after HI cells migrated toward
the area of injury, and some MASCs in the injured area
developed neuronal or astrocytic phenotypes.35 In con-
trast, Sato et al found that the area of cortical infarction
was reduced when NSCs cultured from E14 fetal rats
were transplanted together with chondroitinase ABC 24
hours after HI in P7 rats, but not when NSCs were
administered alone.36 The authors postulated that coinfu-
sion facilitated NSC migration and adherence, allowing
the transplanted NSCs to more efficiently support host
cells with neurotrophins, or may have facilitated access of
neurotrophins to host neurons.
NSCs derived from human embryonic stem cells
were also able to disperse, integrate, and differentiate
well when grafted into the forebrain of P7 rats 24 hours
after HI, and showed significant sprouting.37 There was
upregulation of genes involved in neurogenesis, gliogene-
sis, and neurotrophic support, with increased microglia
in the transplanted hemispheres. Four weeks after treat-
ment, there were small improvements in contralateral
limb use and rotarod performance.37 Similarly, intraven-
tricular injection of mouse ESCs 2 to 3 days after HI in
P7 mice was associated with survival of transplanted cells
expressing neural markers after 8 months of recovery,
and improved escape latency on the Morris water maze
test at 2 and 8 months after HI.38
591
592
TABLE : Outcome of Cell-Based Interventions in Neonatal HI
ANNALS

Cell Type Administration Species Hypoxia Engraftment, Days Cellular/Molecular Outcome Reference

NSCs
NSCs Seeded onto a polymer P7 mice 120 min Appeared to fill area Donor cells on Some evidence Park 200239
of Neurology

scaffold, then implanted of infarction, with polymer scaffold of axonal

in cortex 7 days after HI vascularization differentiated into connection with
at 14 days. neurons and intact cortex.
oligodendrocytes. Reduced
astrogliosis and
inflammation.
NSCs transduced
3.0  105 cells in 8ll, P7 mice 120–180 Donor-derived Donor cells No functional Park 200676
with a retrovirus intracerebral, 3 days min neurons increased differentiated into outcome.
encoding NT-3 after HI from 5% to >80% cholinergic,
around the area of GABAergic, and
infarction, and 20% in glutamatergic
the infarct at 2–4 weeks. neurons.
NSCs derived 3  104 cells in 3ll, P7 mice 90 min Few cells in hippocampus Co-staining of Memory and Ma 200738
from mESC i.v., 2–3 days after HI and cerebral cortex at ESCs and NF. learning
2 and 8 months. improvement
after 2 and
8 months.
NSCs derived 3 injections of 1  106 P7 rats 90 min Engraftment at 4 weeks. Transplanted No change in Daadi 201037
from hESC cells in 2ll, cells ! NeuN, lesion size.
intrahemispheric, TuJ1, and GAD Improved use of
24 hours after HI expression, contralateral
enhanced axonal forelimb
sprouting. Increased and ameliorated
upregulation of locomotor deficits.
genes involved in
regeneration.
Rat neural 2.5  105 cells in P7 rats 120 min Some cells found N/A. Decreased lesion Sato 200836
progenitor cells 5ll, i.v., 24 hours in/around lesion at size and increased
from embryonic after HI 7 days. wet weight of brain.
forebrain
(with ChABC)

Volume 71, No. 5

 TABLE (Continued)

Cell Type Administration Species Hypoxia Engraftment, Days Cellular/Molecular Outcome Reference

May 2012
Fetal cortical Fetal neocortical P7–P8 120–150 80% graft survival N/A. No decrease Elsayed 199631
grafts transplants from rats min rate, evidence of in lesion size.
E13 rat embryos, integration at 2
7 days after HI and 6 weeks.
Fetal cortical 1  104 cells in P7 rats 150 min Grafts were N/A. Significant Jansen 199732
grafts 2ll, sensorimotor identifiable improvement
cortex, 3 days after HI and integrated in motor tests
at 21 and 29 days. from P21 to P63.
Human fetal 5  105 in 5ll, left P7 rats 90 min Human cells present The majority of No functional Qu 200577
NSCs cerebral ventricle, in SVZ, corpus transplanted cells outcome.
3 days after HI callosum, cortex, differentiated into
and hippocampus, neurons and
particularly on astrocytes.
injured side.
MSCs
Rat MSCs 4  106 in 0.5ml, P7 rats 120 min MSCs present A few MSCs No functional Guan 200446
(derived intraperitoneal, throughout expressed nestin outcome.
from bone 2 hours after HI cerebra in both protein; no
marrow) groups at 14 days. differentiation
into neurons or
astrocytes.
hMSCs derived 1  106 cells in 100ll, P7 rats 210 min Cells found equally No change in Lee 201045
from bone marrow intracardiac, distributed in both hemispheric
3 days after HI hemispheres at 6 weeks. volume, but
improved
neurologic
performance.
Murine MSCs 1  105 cells in 2ll, P9 mice 45 min <1% of BrdU-positive Reduced neuronal Improved van Velthoven
(derived from intrahemispheric, cells were donor cells. and oligodendrocyte motor skills 201042
bone marrow) 3 or 10 days after HI loss 21 days after at P19 and P30.
HI, greater BrdU
incorporation in
the ischemic
hemisphere.
Bennet et al: Stem Cells and HI

593
594
TABLE (Continued)
ANNALS

Cell Type Administration Species Hypoxia Engraftment, Days Cellular/Molecular Outcome Reference

Murine MSCs 1  105 cells in 2ll, P9 mice 45 min Very few cells present in : host cell Reduced lesion van Velthoven
intrahemispheric, the granule cell layer of proliferation and size. Improved 201043
3 or 10 days after HI the dentate gyrus at differentiation motor skills
6 weeks. toward neurons P20-18-27.
of Neurology

and oligodendrocytes.
: immune cell
proliferation.
Murine MSCs 5  105 cells, P9 mice 45 min MSCs present in the Cocultured MSC þ ; gray and van Velthoven
intranasal, 10 days affected hemisphere brain extracts 10 white matter 201044
after HI but had not differentiated. days after HI, : loss.
CXCR4, FGF2,
SDF-1, NGF, and
NT; ; IL1b and
TGFb1 mRNA.
MASCs
mMASCs 5  103 cells in 1ll, P7 rats 120 min Cells found in lesion at Transplanted cells in No functional Zheng
somatosensory cortex, 3–21 days later. nonischemic brain outcome. 200635
24 hours and 5 days had astrocytic
after HI phenotype; a few
with neuronal
appearance in
injured cortex.
MAPCs
rMAPCs, derived 2  105 in 3ll, P7 rats 150 min Donor cells present in ; motor asymmetry Yasuhara
from bone marrow intrahippocampal, the hippocampal region. at P21 but no 200640
þ cyclosporin 7 days after HI difference in
rotarod test.
rMAPCs þ 2  105 cells in 3ll, P7 rats 150 min 0.18% of i.v., 0.51% Reduced neuronal ; motor asymmetry Yasuhara
cyclosporin i.v. (jugular) and of intracranial cells found loss in CA3 and : rotarod times 200841
intrahippocampal, in the CA3, CA2 of region of at P21 but not P14
7 days after HI hippocampus 14 hippocampus. after both i.v. and
days later. intracerebral
administration.

Volume 71, No. 5

 TABLE (Continued)

May 2012
Cell Type Administration Species Hypoxia Engraftment, Days Cellular/Molecular Outcome Reference

hUCBCs
Human cord blood 1  107 cells in P7 rats 80 min Many cells in ischemic N/A. Normalized 12% Meier
mononuclear cells 500ll, intraperitoneal, hemisphere, 21 days. difference in toe 200647
24 hours after HI distances, 8%
step lengths.
Human cord blood 1  107 cells in P7 rats 120 min Few hUCB cells located No change in No effect on de Paula
mononuclear cells 100lL, i.v. (jugular), in rat brain, 21 days. volume of injured spatial memory 200951
24 hours after HI hemisphere after deficits.
21 days.
hUCBC-derived 1  105 cells in 2ll, P7 rats 150 min Differentiation into Apparent (subjective) Improved Xia 201078
mononuclear cells intracortical, astrocytes but not reduction in cortical neurological
(passage 10) þ 3 days after HI neurons, 7 days after neuronal loss at score, P24 to P38.
cyclosporine A transplant. 28 days.
Human cord blood 2  106 cells in P7 rats 90 min Few cells in ischemic Decreased cell Improved neonatal Pimentel-Coelho
mononuclear cells 200ll, intraperitoneal, cortex and striatum, death and reflexes at P11 201050
3 hours after HI 2 days. microglial but not at P14.
activation.
Human cord blood 1.5  104 cells in P7 rats 150 min Few cells in ischemic Increased growth 20–25% Yasuhara
mononuclear cells þ 200ll, i.v., hippocampus, 14 days. factors in brain, improvement 201049
mannitol 2–3 hours after HI CA1 dendrites. rotarod and
elevated body
swing tests at p14
and P21.
Human cord blood 1  107 cells in 500l, P7 rats 80 min Many cells in peri-infarct No change Improved Geissler
mononuclear cells intraperitoneal, area after 42 days. in size of sensorimotor 201148
24 hours after HI hemispheric lesion. function, cortical
maps, and receptive
fields, and reduced
hyperexcitability,
at P48.
BrdU ¼ bromodeoxyuridine; ChABC ¼ chondroitinase ABC; CXCR ¼ chemokine (C-X-C motif ) receptor; ESC ¼ embryonic stem cell; FGF ¼ fibroblast growth factor; GABA ¼
gamma-aminobutyric acid; GAD ¼ glutamate decarboxylase; h ¼ human; HI ¼ hypoxia–ischemia; i.v. ¼ intravenous; IL ¼ interleukin; MAPC ¼ multipotent adult-derived progeni-
tor cell; MASC ¼ multipotent astrocytic stem cell; MSC ¼ mesenchymal stem cell; N/A ¼ not applicable; NF ¼ neurofilament; NGF ¼ nerve growth factor; NSC ¼ neural stem
cell; NT ¼ neurotrophin; P ¼ postnatal day; SDF ¼ stromal cell-derived factor; SVZ ¼ subventricular zone; TGF ¼ transforming growth factor; Tuj1 ¼ Neuron-specific class III
beta-tubulin; UCBC ¼ umbilical cord blood cell.
Bennet et al: Stem Cells and HI

595
ANNALS of Neurology
In practical terms, these studies still represent com-
paratively early treatment. Intriguingly, there is early evi-
dence from the P7 rat that if NSCs are seeded onto a
polymer scaffold platform before transplantation, they
may be able to bridge evolving cystic lesions 7 days after
HI and establish at least some connections to host tis-
sue.39 Given the results of other studies, discussed below,
where cells were given at least as late, it is unclear
whether such a technological assist is needed.
MAPCs
MAPCs given to P7 rat pups either into the hippocam-
pus or intravenously (i.v.) 7 days after HI were associated
with a significant reduction in motor asymmetry 14 days
after injection compared to controls.40,41 In the earlier
study, intracerebral MAPC injection did not significantly
improve rotarod times (a measure of sensorimotor func-
tion),40 whereas the later study suggested a small
improvement in rotarod times 14 days (ie, P21) after
both intracerebral and i.v. administration. A potential
complicating aspect of this protocol is that all animals
were treated with daily immunosuppression throughout
the survival period. However, controls also received this
intervention. MAPCs were present in hippocampus areas
after both intracerebral and i.v. injection 14 days after
treatment (P21),40,41 with significant improvement in
numbers of CA3 neurons at P21 with both routes of
administration.41
MSCs
Intracranial injection of mouse MSCs 3 days after HI in
P9 mice was associated with a 42% and 31% reduction
in neuronal and oligodendrocyte loss, respectively, 28
days after HI.42 There was greater proliferation of endog-
enous neural and glial cells, with reduced proliferation of
microglia. Functional and histological improvement was
seen when MSC administration was delayed until 10
days after HI.42 Further functional improvements were
associated with repeated injections of MSCs both 3 and
10 days after HI.43 Finally, this group has presented evi-
dence that in the same paradigm, MSCs given intrana-
sally 10 days after HI can improve outcome.44 Consist-
ent with the reports from other studies discussed above
that the benefit of cell treatment may not be due to new
cell formation, MSCs were present in the affected hemi-
sphere 28 days after HI, but they remained
undifferentiated.
Further supporting the potential for systemic treat-
ment to improve functional outcomes, intracardiac injec-
tion of hMSCs 3 days after HI in P7 rats was associated
with significant improvement in 1 of 2 neuromotor tests
596
14 to 40 days after treatment despite no significant
reduction in the volume of infarction.45 MSCs were
found in control brains and diffusely throughout the
lesioned and nonlesioned hemispheres; however, more
cellular differentiation was seen after HI than sham HI.
Similarly, Guan et al reported that MSCs were found
extensively throughout the brain 14 days after intraperi-
toneal infusion of MSCs 2 hours after HI in P7 rats.46
MSCs were increased in the injured hemisphere after HI
but showed little differentiation into neural cell types.
hUCBCs
Finally, there is also evidence for benefit with hUCBCs
in rodents. Meier et al reported that intraperitoneal injec-
tion of 107 hUCBCs 24 hours after HI in P7 rats was
associated with reduced spastic paresis despite no change
in the area of hemispheric infarction.47 The cells
migrated to the injured hemisphere within 3 days, but
with no evidence of differentiation 14 days after adminis-
tration. More recently, this group showed in the same
paradigm that similarly to their first report, hUCBCs
given 24 hours after HI were associated with no effect on
the size of the lesion, and yet significantly preserved
somatosensory function on the side of the lesion.48 Nota-
bly, they found reduced hyperexcitability, and restoration
of the cortical maps and receptive fields in the somato-
sensory cortex. This strongly suggests that benefit was
mediated by improved function of surviving brain tissue.
Similarly, Yasuhara et al found that i.v. administra-
tion of 1.5  104 hUCBCs, with or without cotreatment
with mannitol, 7 days after HI in P7 rats was associated
with significant improvement in motor asymmetry and
coordination 7 and 14 days later.49 In this study, only a
few cells were found in the damaged area. The authors
suggested a paracrine effect as the levels of neural growth
factor, glial cell line-derived neurotrophic factor, and
brain-derived neurotrophic factor were increased 3 days
after treatment, particularly when cells were combined
with mannitol to increase blood–brain barrier
permeability.49
Consistent with these findings, Pimentel-Coelho et
al found that intraperitoneal injection of hUCBCs in P7
rats 3 hours after HI was associated with only trivial
numbers of hUCBCs in the lesion, a small reduction in
neuronal loss in the striatum, and small improvements in
2 sensorimotor reflexes 4 days after HI, but no signifi-
cant difference from vehicle controls at 2 and 7 days.50
The improved outcomes were associated with reduced
caspase-3 cleavage around the lesion site and an overall
reduction in activated microglia and macrophages con-
sistent with antiapoptotic and anti-inflammatory actions.
Volume 71, No. 5

However, in contrast, de Paula et el found that intrave-
nous injection of hUCBCs 24 hours after HI in P7 rats
was associated with no significant improvement in either
volume of infarction or in spatial memory 3 weeks
later.51
Knowledge Gaps
Although the optimal dose, timing, and mechanisms of
action for stem cells in animal models are still not clear
from the studies above, they do provide reasonable evi-
dence that administration of progenitor/stem cells may
have beneficial effects on histological and behavioral out-
comes after neonatal HI. It is striking that these benefits
seem to be comparable between very distinct types of
stem cells, although some studies of similar cell types,
timing, and routes of administration found either no or
restricted/conditional benefits.31,45,51 In practical terms,
autologous hUCBCs have the major advantage of not
requiring immunosuppression,22,52 but this is balanced
by the hematogenous origin of the cells, and thus less
clear commitment to neural differentiation.
Mechanisms of Action
Early studies aimed to establish whether the cells could
engraft and replace dying cells. Although in many studies
the cells did survive and proliferate in the brain, the net
increase in cell numbers was generally negligible. Even
where differentiation was seen, it is not clear that it
resulted in fully functional and integrated neurons or
glia. It is striking that more recent studies suggest little
differentiation,53 raising the possibility of methodological
issues with cell identification in earlier studies. Much
more importantly, this suggests that the key mechanisms
are actually release of neurotrophic and immunomodula-
tory/immune suppressive factors.
Consistent with this hypothesis, recent studies have
confirmed significant functional improvements with pe-
ripheral administration after neonatal HI, despite little
evidence of functional engraftment.41,44,45,54 Evidence
from adult models of stroke and central nervous system
disease support the concept that progenitor/stem cells
can attenuate tertiary damage associated with postisch-
emic inflammation and secondary cell death due to lack
of environmental stimulation.55–61 Nevertheless, again,
not all studies support functional benefits.51,62–64
Treatment at different time points during the evolu-
tion of injury may allow a range of potentially comple-
mentary mechanisms to be targeted. If stem cells are
administered relatively soon after HI, then many cells
may be saved by attenuation of the immune response,
decreasing the release of excitotoxins, cytotoxins, and ox-
May 2012
Bennet et al: Stem Cells and HI
ygen free radicals as well as diminishing the infiltration
of leukocytes.58 For example, multipotent progenitors
derived from human amnion can reduce inflammation,
with decreased monocyte chemoattractant protein-1,
transforming growth factor (TNF)a, interleukin (IL)-1 /-
6, and profibrotic TNF-b expression and downregulation
of tissue inhibitors of matrix metalloproteinase-1 and -
2.65 These cells can attenuate white matter injury and
increase plasma concentrations of the anti-inflammatory
cytokine IL-10 in preterm fetal sheep after exposure to
lipopolysaccharide.66,67
Neurotrophic factors are secreted by progenitor cells
in vitro, including neurotrophin-3, brain-derived neuro-
trophic factor, and glial-derived neurotrophic factor,
which support and drive endogenous cell migration, pro-
liferation, and differentiation.68 In the adult rat, Borlon-
gan et al reported that systemic injection of hUCBCs
improved behavioral outcome and lesion size in associa-
tion with increased neurotrophin levels in the brain.60
Even when intravenous cell injection was combined with
a blood–brain barrier permeabilization agent, no cells
engrafted. Similarly, even intracerebral injection of MSCs
in the neonatal rat was associated with trivial survival of
injected MSCs (
1% after 18 days),54 but marked
induction of genes supporting growth and survival in the
brain.
Brain Injury Associated with Premature Birth
Premature birth is associated with a very high burden of
neurological injury, including a 15% risk of severe neuro-
logical impairment, and up to 50% risk of cognitive defi-
cits in later childhood.69 Although HI likely contributes
to at least some brain injury, chronic inflammation and
many other poorly defined factors are involved,13 the
timing remains unclear, and preterm infants most often
develop diffuse white matter injury in the first few weeks
rather than acute gray matter loss.70,71 As previously
reviewed, this is followed by long-term impairment of
the development of both gray matter and white matter
structures.13
There is increasing preclinical evidence that exoge-
nous oligodendrocyte progenitor cells and glial-restricted
precursor cells can augment recovery from adult spinal
cord injury, by augmenting the key supporting cells,
astrocytes, and oligodendrocytes.72 Given that classic
periventricular white matter injury in preterm infants is
associated with axonopathy and depletion of oligoden-
drocytes,73 these types of cells may be particularly helpful
for promoting repair of the preterm brain. However, we
were unable to identify any relevant studies of perinatal
brain injury that used these cells. Conceptually, it is im-
portant to appreciate that over the past few decades the
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prevalence of necrotic periventricular leukomalacia has
steadily decreased. Although subtle, astrogliotic lesions
remain common, and there is evidence that the myelin
deficit associated with these milder lesions may be associ-
ated with maturation arrest of oligodendrocyte precur-
sors, without axonopathy.74 The potential of these oligo-
dendrocyte progenitors to myelinate is presently unclear.
Thus, it will be critical to test whether cell-based inter-
ventions can promote normal white matter maturation
and myelination in white matter lesions.
Conclusions
All of the studies reported to date of cell administration for
neonatal HI have been performed in rodents, which are a
poor model for human brain development. Laboratory
rodents are highly altricial, and their brains are much sim-
pler (lissencephalic), with very small white matter volumes
compared with humans.75 Based on previous experience,
this study group strongly recommends that efficacy must be
demonstrated in at least 2 nonrodent species with complex,
humanlike brains before undertaking definitive, large
randomized controlled trials of cell-based interventions. In
contrast, fresh autologous volume and red blood cell-
reduced cord blood cells appear to be of sufficiently low
risk to justify early safety and feasibility studies. Although
there appears to be strong potential for cell-based interven-
tions after neonatal HI, there is still much work to be done.
Clinically, a minimally invasive strategy with cells that do
not raise ethical issues is essential. We need to determine
the most appropriate cell type, dose, mode, and timing of
administration, as well as potential long-term side effects,
to ensure safe translation into the clinical realm.
Acknowledgment
L.B. and A.J.G. were supported by the Health Research
Council of New Zealand grants 11-576 and 09-065.
Potential Conflicts of Interest
Nothing to report.
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