Mesenchymal Stem Cells for Bronchopulmonary Dysplasia: Phase 1
Dose-Escalation Clinical Trial*
Yun Sil Chang, MD, PhD1,*, So Yoon Ahn, MD1,*, Hye Soo Yoo, MD1, Se In Sung, MD1, Soo Jin Choi, MD, PhD2,
Won Il Oh, MD, PhD2, and Won Soon Park, MD, PhD1
Objective To assess the safety and feasibility of allogeneic human umbilical cord blood (hUCB)-derived
mesenchymal stem cell (MSC) transplantation in preterm infants.
Study design In a phase I dose-escalation trial, we assessed the safety and feasibility of a single, intratracheal
transplantation of hUCB-derived MSCs in preterm infants at high risk for bronchopulmonary dysplasia (BPD).
The first 3 patients were given a low dose (1 107 cells/kg) of cells, and the next 6 patients were given a high
dose (2 107 cells/kg). We compared their adverse outcomes, including BPD severity, with those of historical
case-matched comparison group.
Results Intratracheal MSC transplantation was performed in 9 preterm infants, with a mean gestational age of
25.3 0.9 weeks and a mean birth weight of 793 127 g, at a mean of 10.4 2.6 days after birth. The treatments
were well tolerated, without serious adverse effects or dose-limiting toxicity attributable to the transplantation.
Levels of interleukin-6, interleukin-8, matrix metalloproteinase-9, tumor necrosis factor a, and transforming growth
factor b1 in tracheal aspirates at day 7 were significantly reduced compared with those at baseline or at day
3 posttransplantation. BPD severity was lower in the transplant recipients, and rates of other adverse outcomes
did not differ between the comparison group and transplant recipients.
Conclusion Intratracheal transplantation of allogeneic hUCB-derived MSCs in preterm infants is safe and
feasible, and warrants a larger and controlled phase II study. (J Pediatr 2014;164:966-72).
See editorial, p 954 and
related article, p 973
T
he number of very preterm infants at high risk for developing bronchopulmonary dysplasia (BPD) is increasing, because
advances in neonatal intensive care have increased these infants’ chance of survival.1 Given the lack of effective measures
to prevent or ameliorate this common and serious disorder,2,3 BPD remains a major cause of mortality and lifelong
morbidity in preterm infants.4-6
Several recent studies have shown that xenotransplantation of mesenchymal stem cells (MSCs) in immunocompetent
animals attenuates hyperoxia-induced lung injury, such as impaired alveolarization, inflammatory response, increased
apoptosis, and fibrosis.7-12 Human umbilical cord blood (hUCB) is considered a better source of MSCs than other potential
sources, such as bone marrow or adipose tissue because of their ready availability
and greater proliferative capacity and less antigenicity than other cell types.13 In
previous translational studies to determine the optimal route,7 dose,8 and
From the Department of Pediatrics, Samsung Medical
Center, Sungkyunkwan University School of Medicine;
timing9 of transplantation of hUCB-derived MSCs in a neonatal hyperoxic
and Biomedical Research Institute, MEDIPOST Co, Ltd,
lung injury model in rat pups, we found that the protection of MSCs against
Seoul, Korea
*Contributed equally.
neonatal hyperoxic lung injury was persistent, and that no long-term toxicities,
Funded by the Korean Health and Medical Technology
adverse effects, or tumorigenicity were present at 70 days posttransplantation.14
R&D Program, Ministry for Health, Welfare and Family
Affairs, Republic of Korea (A102136). Human umbilical
Collectively, these findings offer hope that transplantation of hUCB-derived
cord blood–derived mesenchymal stem cells were supMSCs will be effective in treating BPD. The safety and efficacy of MSC transplanplied by MEDIPOST Co, Ltd; the sponsor had no
involvement in study design, the collection, analysis, or
tation for prevention of BPD has not been tested previously, however. We report
interpretation of data; writing of the report; or the decision to submit the manuscript for publication. W.O. is a
a phase I dose-escalating clinical study on the safety and feasibility of transplanboard member and stockholder of MEDIPOST Co, Ltd.
tation of hUCB-derived MSCs in preterm infants with BPD.
Samsung Medical Center and MEDIPOST Co, Ltd have
1
2
BPD
HGF
hUCB
IL
IVH
KFDA
966
Bronchopulmonary dysplasia
Hepatic growth factor
Human umbilical cord blood
Interleukin
Intraventricular hemorrhage
Korean Food and Drug
Administration
MMP
MSC
PDA
SAE
TGF
TNF
VEGF
Matrix metalloproteinase
Mesenchymal stem cell
Patent ductus arteriosus
Serious adverse event
Transforming growth factor
Tumor necrosis factor
Vascular endothelial growth factor
issued or filed patents for “Method of treating lung diseases using cells separated or proliferated from umbilical
cord blood” under Y.C., W.P., and Yoon Sun Yang (not
affiliated with this article) (application PCT/KR2007/
000535). S.Ahn, H.Y., and S.Sung declare no conflicts of
interest.
Registered with ClinicalTrials.gov: NCT01297205.
0022-3476/$ - see front matter. Copyright ª 2014 The Authors.
Published by Elsevier Inc.
http://dx.doi.org/10.1016/j.jpeds.2013.12.011
*
This is an open access article under the CC BY-NC-ND license
(http://creativecommons.org/licenses/by-nc-nd/3.0/).
Vol. 164, No. 5 May 2014
Methods
This study was a phase I, open-label, single-arm, singlecenter trial to evaluate the safety and feasibility of intratracheal allograft transplantation of hUCB-derived MSCs in
preterm infants. The protocol was approved by the Korean
Food and Drug Administration (KFDA; MP-CR-006) and
by the Institutional Review Board of the Samsung Medical
Center in Seoul, Korea (2010-09-092). The primary goal
was to demonstrate the safety of intratracheal allograft
transplantation of hUCB-derived MSCs in preterm infants
at high risk of developing BPD. The secondary goal was
to evaluate the feasibility and potential efficacy of MSC
transplantation for BPD in comparison with historical
case-matched comparison group.
Patients were enrolled at Samsung Medical Center between
February 10, 2011, and September 14, 2011. Because this was
a first-in-human trial for intratracheal allograft transplantation of hUCB-derived MSCs in preterm infants, intensive
and cautious external monitoring was maintained, and the
KFDA and Acrovan Co, Ltd (Anyang, Korea) served as
external monitors of the study.
The informed consent document was reviewed with both
the parents and principal investigator or study staff at least
twice. Full understanding was confirmed, and written
informed consent was obtained from both parents, with
particular attention given to the understanding that testing
was for safety, with neither an expectation nor a promise of
therapeutic benefit. In accordance with the original study
scheme (Figure 1), the target sample size was a minimum
of 9 patients. The first 3 patients were assigned to receive
low-dose MSCs (1 107 cells/kg), and the next 6 were
assigned to receive high-dose MSCs (2 107 cells/kg).
Inclusion criteria included preterm infants at high-risk for
developing BPD15 with a gestational age of 23-29 weeks and
birth weight of 500-1250 g, and patients (at postnatal day
5-14) needing continuous ventilator support that could not
be decreased owing to significant respiratory distress within
24 hours before enrollment. Patients were excluded for severe
congenital anomalies, lung hypoplasia, severe septic shock, or
severe (grade $3) intraventricular hemorrhage (IVH)16
(Appendix; available at www.jpeds.com).
Transplantation of hUCB-Derived MSCs
Pneumostem, passage 6 hUCB-derived MSCs (Medipost,
Seoul, Korea) were prepared in compliance with good
manufacturing practices, at concentration of 5 106 cells/
mL in normal saline. A dose of 1 107 cells (2 mL)/kg or
2 107 cells (4 mL)/kg were administered intratracheally
Figure 1. Study design. If there was no occurrence of dose-limiting toxicity, then the target minimum sample size was 9 patients.
The first 3 patients were assigned to receive low-dose MSCs (1 107 cells/kg; dose A), and the next 6 were assigned to receive
high-dose MSCs (2 107 cells/kg; dose B). If there were any dose-limiting toxicity in the dose A group (low-dose group), then an
extra enrollment of 3 cases in the dose A group would be needed. If dose-limiting toxicity occurred more than twice in the dose
B group, then the maximum tolerated dose would have been determined to be dose A without additional evaluation of dose B.
DLT, dose-limiting toxicity; MTD, maximum tolerated dose.
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via a gavage tube in 2 fractions into the left and right lungs
(Appendix).
Assessment of Safety
Safety was defined primarily as the absence of treatmentrelated serious adverse events (SAEs) according to the
Consolidated Standards of Reporting Trials,17 and secondarily as the absence of dose-limiting toxicity, defined as
death within 6 hours after MSC transplantation or
anaphylactic shock related to the MSC injection. After a
single intratracheal MSC transplantation, all patients were
regularly and intensively assessed until 84 days post-MSC
transplantation according to schedule (Appendix). For
comparison of adverse outcomes for further safety
evaluation, a historical nested case-control group was
established (Appendix). The clinical data for the
comparison group were for the same postnatal day to the
index day as that for the MSC recipients. BPD was defined
according to the National Institutes of Health workshop
severity-based diagnostic criteria.18
Temporal Profile of Tracheal Aspirate Cytokines
and Growth Factors
Tracheal aspirate fluid was collected before and after MSC
transplantation for assessment of changes in cytokines and
growth factors known to be associated with the development or prevention of BPD. Samples were collected only
when suctioning was clinically required during routine
care. The following cytokines and growth factors were
measured: interleukin (IL)-1, IL-6, IL-8, IL-10, matrix
metalloproteinase (MMP)-9, transforming growth factor
(TGF)-b, tumor necrosis factor (TNF)-a, vascular endothelial growth factor (VEGF), and hepatic growth factor
(HGF).
Statistical Analyses
Data are expressed as mean SD. To compare continuous
variables and BPD severity between study patients and the
matched comparison group, statistical comparisons
between groups were performed using 2-way ANOVA
and generalized estimating equations. Stratified logistic
regression analysis was used to compare other nominal
variables. The temporal profile of growth factors and
cytokines in the tracheal aspirate fluid was assessed using
the paired t test. A P value <.05 was considered statistically
significant. SPSS version 17 (SPSS Inc, Chicago, Illinois)
was used for all statistical analyses.
Results
Three infants received low-dose (1 107 cells/kg) MSCs, and
6 infants received high-dose (2 107 cells/kg) MSCs. Gestational age, birth weight, and postnatal age of MSC transplant
recipients were 25.3 0.9 weeks (range, 24.0-26.6 weeks),
793 127 g (range, 630-1030 g), and 10.4 2.6 days (range,
7-14 days), respectively (Table I). The estimated risk of death
or moderate/severe BPD15 at enrollment ranged from 54.1%
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Vol. 164, No. 5
to 91.4%, with an average of 74.4% 10.0% (Table II;
available at www.jpeds.com). Clinical variables, including
gestational age, birth weight, Apgar scores, and respiratory
severity scores, were not significantly different between the
MSC-treated group and the matched comparison group, or
between the low-dose and high-dose MSC subgroups
(Table III; available at www.jpeds.com).
SAEs
Details of SAEs, recorded for up to 84 days after MSC
transplantation, are presented in Table I. The 9 infants who
received MSC therapy were discharged alive. Intratracheal
transplantation of hUCB-derived MSCs took <5 minutes. All
patients tolerated the procedure well without immediate
complications within 6 hours after transplantation or
immediate respiratory and cardiovascular compromise
(Figure 2; available at www.jpeds.com); however, 6 patients
subsequently developed SAEs. The most common event was
patent ductus arteriosus (PDA) ligation, occurring in 4 of the
9 patients (44%). One case of pneumothorax (11.1%)
developed directly related to PDA ligation. One patient
(gestational age 24.6 weeks, weight 740 g) in the high-dose
MSC transplantation group (Table I) had congenital systemic
candidiasis, followed by necrotizing enterocolitis requiring
surgery and, eventually, periventricular leukomalacia.
Adverse Outcomes
There were no significant differences in SAEs between the
low-dose and high-dose MSC transplantation groups or
between the MSC-treated group and matched-comparison
group (Table IV) except in BPD severity,18 which was
significantly lower in the MSC transplant group (regression
coefficient, 1.7; 95% CI, 0.11-3.29; P = .036). The duration
of intubation after MSC transplantation ranged from 3 to
45 days, with the longest duration in the patient with
congenital systemic candidiasis. There were no significant
dose-dependent or timing-dependent differences in the
duration of intubation between the MSC transplantation
and comparison groups. Postnatal dexamethasone use was
lower in the MSC transplantation group compared with the
comparison group (67% vs 100%). The mean start day and
cumulative dose of dexamethasone did not differ between
the 2 groups (11.0 2.8 days after MSC transplantation
and 2.7 2.5 mg/kg in the MSC transplantation group vs
11.0 2.7 days after the index day and 3.7 2.3 mg/kg in
the comparison group).
Serial echocardiography performed by a pediatric
cardiologist before and after MSC transplantation
revealed no significant changes in cardiac function or
development of pulmonary hypertension. Analysis of serial
chest radiographs, including those taken at posttransplantation day 84, showed no visible mass-like lesions in either lung
field (Figure 3; available at www.jpeds.com).
Daily Changes in Respiratory Severity Score
Figure 4 shows temporal profiles of respiratory severity
scores of individual patients in the MSC transplantation
Chang et al
ORIGINAL ARTICLES
May 2014
Table I. Clinical data of enrolled patients
Patient
Variables
A1
A2
A3
B1
B2
B3
B4
B5
B6
Gestational age, wk
Birth weight, g
Apgar score at 1 min
Apgar score at 5 min
Sex
Delivery
Pathologically confirmed chorioamnionitis
Antenatal steroid use
Respiratory distress syndrome
Early-onset sepsis
PDA
Therapies administered between birth and MSC injection
Number of surfactant doses
Use of indomethacin or ibuprofen
Surgical ligation of PDA
Age at MSC injection (postnatal day)
SAEs with major morbidities
Death within 6 hours after MSC transplantation
Anaphylaxis after MSC transplantation
BPD severity
Intubation duration after MSC transplantation
Duration of nasal continuous positive airway pressure
Pneumothorax
PDA ligation
Late-onset sepsis
Necrotizing enterocolitis (stage $2)
Periventricular leukomalacia
IVH ($ grade 3)
Retinopathy of prematurity (stage $3)
25+6
770
6
9
F
C
Y
Y
Y
Y
25+3
870
5
7
M
C
Y
Y
Y
Y
24+3
720
4
7
M
C
Y
Y
Y
24+0
630
5
8
F
C
Y
Y
Y
Y
24+4
740
3
5
M
NV
Y
Y
Y
Y
25+4
850
5
8
F
NV
Y
Y
Y
25+0
650
4
6
F
NV
Y
Y
Y
26+4
1030
8
9
M
C
Y
Y
Y
Y
26+1
880
5
8
M
C
Y
Y
Y
1
1
Y
12
1
Y
10
2
Y
10
1
Y
7
1
Y
Y
13
2
Y
14
1
Y
14
Mild
12
32
Y
Y
-
Mild
5
29
-
Mild
7
49
-
Mild
11
48
Y
-
Moderate
45
28
Y
Y
-
Mild
3
25
Y
Y
-
2
Y
9
8
Moderate
16
50
Y
Y
-
Moderate
11
40
-
Mild
23
18
Y
-
F, female; M, male; C, cesarean delivery; NV, spontaneous vaginal delivery; Y, yes.
and comparison groups. After MSC transplantation, no
patient demonstrated an obvious exacerbation of
ventilator dependency as a result of the transplantation
procedure. The MSC transplantation group had generally
lower values after MSC transplantation compared with
the comparison group, especially on day 3
posttransplantation, but the difference did not reach
statistical significance (1.4 1.4 vs 3.3 2.0 in
comparison group; mean difference,
1.05; 95% CI,
2.27 to 0.15; P = .05) (Figure 4, C).
Table IV. Comparison of outcomes in the MSC transplantation group and matched comparison group
MSC transplantation group
High-dose
Low-dose
(1 107 cells/kg) (n = 3) (2 107 cells/kg) (n = 6)
Death at discharge, n (%)
BPD, n (%)
BPD severity, n (%)
Mild
Moderate
Severe
Duration of intubation, d, mean SD
Total duration
Duration after MSC transplantation or index date
Duration of nasal continuous positive airway pressure,
d, mean SD
Pneumothorax, n (%)
PDA ligation after MSC transplantation or index day, n (%)
Retinopathy of prematurity (grade $3), n (%)
Late-onset sepsis, n (%)
IVH (grade $3), n (%)
Periventricular leukomalacia, n (%)
Necrotizing enterocolitis (stage $2b), n (%)
Postnatal steroid use for BPD, n (%)
Total
(n = 9)
Matched-comparison
group (n = 18)
P value
0/3
3/3
0/6
6/6
0/9 (0.0)
9/9 (100)
0/18 (0.0)
18/18 (100)
3/3
0/3
0/3
3/6
3/6
0/6
6 (67)
3 (33)
0 (0)
5 (28)
5 (28)
8 (44)
19.7 1.2
8. 3 3.5
36.7 10.8
29.0 15.1
18.2 14.7
34.8 13.1
25.9 12.8
14.8 12.8
35.4 11.7
33.6 12.9
22.6 13.5
43.2 18.7
.19
.19
.29
1/3
1/3
0/3
0/3
0/3
0/3
0/3
2/3
0/6
3/6
1/6
1/6
0/6
1/6
1/6
4/6
1 (11)
4 (44)
1 (11)
1 (11)
0 (0)
1 (11)
1 (11)
6 (67)
0 (0)
6 (33)
9 (50)
3 (17)
0 (0)
1 (6)
2 (6)
18 (100)
.67
.86
.26
.89
.037*
1.00
1.00
.07
*Statistical comparison performed with the generalized estimating equations test.
Mesenchymal Stem Cells for Bronchopulmonary Dysplasia: Phase 1 Dose-Escalation Clinical Trial
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Vol. 164, No. 5
Figure 4. Temporal profiles of respiratory severity scores before and after MSC transplantation in A, each enrolled patient,
B, before and after the index day in each comparison patient, and C, a comparison of mean respiratory severity scores of the
2 groups. Day 0 refers to the day of MSC transplantation, or the index day in the matched comparison group matched to the
day of MSC transplantation. MSC, MSC transplantation group; Comparison, matched comparison group. Data are presented
as mean SEM.
Temporal Profiles of Cytokines and Growth Factors
from Tracheal Aspirate Fluid
Temporal profiles of cytokines and growth factors from
tracheal aspirate fluid are shown in Figure 5 (available
at www.jpeds.com). Levels of MMP-9 from the tracheal
aspirate at day 7 posttransplantation were significantly
reduced compared with baseline (P = .02). IL-6, IL-8,
TNF-a, and TGF-b levels were significantly lower at
day 7 posttransplantation than at day 3 posttransplantation.
970
Discussion
Intratracheal transplantation of low-dose (1 107 cells/kg) or
high-dose (2 107 cells/kg) allogeneic hUCB-derived MSCs
was not associated with immediate SAEs or dose-limiting
toxicity in our cohort of extremely preterm infants at high
risk for developing BPD. The incidence of SAEs within 84
days posttransplantation did not differ between the MSC transplant recipients and historical case-matched comparison group.
Chang et al
ORIGINAL ARTICLES
May 2014
BPD severity was significantly decreased, and retinopathy of
prematurity requiring surgery was less prevalent in the MSC
transplantation group compared with the matched comparison
group. Taken together, these findings indicate that intratracheal
transplantation of up to 2 107 cells/kg of hUCB-derived
MSCs in preterm infants may be safe and feasible.
We used passage 6 hUCB-derived MSCs for human intratracheal transplantation. We previously observed karyotype
stability at up to passage 11 of hUCB-derived MSCs,7,14,19
and characteristics of low expression of major histocompatibility complex class 1 and lack of major histocompatibility
complex class 2 molecules.13,20 Furthermore, in a newborn
rat pup model of hyperoxia, no long-term adverse effects
or tumorigenicity had occurred by the tenth postnatal week
after MSC transplantation.14 Transplanted MSCs had a
low rate of engraftment in the recipient rat lung, and the
engraftment dissipated shortly after transplantation21 to
almost undetectable levels by the tenth postnatal week.14
The route of MSC transplantation is an important consideration. We previously demonstrated in newborn rats that
local intratracheal MSC transplantation is more effective
than systemic intraperitoneal administration in protecting
against hyperoxic lung injury.7 Intratracheal transplantation
has a lower burden of unexpected side effects originating
from systemic distribution of donor cells compared with
intravenous administration. Moreover, for preterm infants
receiving invasive ventilation via an endotracheal tube,
intratracheal instillation does not require an additional
procedure for local transplantation. Thus, in this study, we
administered the MSCs intratracheally in 2 fractions, using
the same method as for administration of exogenous
surfactant. No immediate clinical instability or complications were evident (Figure 2), suggesting that intratracheal
administration of MSCs is safe and feasible.
We previously showed that intratracheal delivery of MSCs
attenuated hyperoxic lung injury in newborn rats in a
dose-dependent manner, with at least 5 104 cells per rat
pup weighing approximately 10 g required to produce
anti-inflammatory, antifibrotic, and antioxidative effects.8
Although currently no guidelines are available for the extrapolation of preclinical data into clinical trials, extrapolating
from the cell dose used in the animal studies, we chose doses
of 1 107 and 2 107 cells/kg for use in this study of human
infants. In contrast to our preclinical animal study showing a
dose-dependent response,8 in this study, the high-dose MSC
group, although small, seemed to have a longer duration of
intubation and higher BPD severity scores compared
with the low-dose group, although the difference was not
statistically significant. Further studies are needed to
determine optimal MSC doses for transplantation.
Optimal timing of MSC transplantation is another key
issue remaining to be clarified. In our work with newborn
rats, the therapeutic efficacy of hUCB-derived MSC
transplantation was time-dependent, with greater efficacy
when given before the peak and plateau for inflammatory
responses in hyperoxic lung injury.9 Experimental rodent
data in hyperoxic lung injury cannot be extrapolated directly
into human infants for BPD; however, we chose 5-14 days
after birth with ventilator dependency as a transplantation
timing based on this animal study,9 which is a relatively early
for preterm infants, with sufficient stabilization after birth
but before BPD is established. In the present study, no
time-dependent variation in therapeutic efficacy was evident
when MSCs were given between 7 and 14 days postnatally.
We also found that MSC transplantation seemed to decrease
the respiratory severity score shortly after transplantation,
followed by an increase by around day 7 posttransplantation;
this might be considered a time when a second transplantation, if necessary, might be indicated.
We previously showed that inflammatory responses
mediated by proinflammatory cytokines play a pivotal role
in the development of BPD.7-9,22 Furthermore, the protective
effects of hUCB-derived MSC therapy against neonatal
hyperoxic lung injury are mediated primarily by paracrine
antiinflammatory, antioxidative, and antifibrotic effects,
rather than by the cells’ regenerative capacity.7-9 In this study,
the concentrations of IL-6, IL-8, MMP-9, TNF-a, and
TGF-b1 in the tracheal aspirate fluid were significantly
reduced after MSC transplantation. However, Kotecha
et al23 have shown that proinflammatory cytokines in the
tracheal aspirate fluid peak at 7 days, and then subside in
preterm infants who develop chronic lung disease. Thus,
without a proper matched comparison group, whether our
data showing decreased tracheal aspirate inflammatory cytokines are related to immunomodulatory effects of MSCs or
simply reflect the natural course of inflammation is difficult
to ascertain. In this study, VEGF and HGF tended to be
reduced after MSC transplantation; these results contradict
our previous results showing significant up-regulation of
hyperoxia-induced growth factors.9
According to multivariate analyses of 23- to 27-week-old
preterm infants, lower gestational age and mechanical
ventilation at day 7 were major predictors of BPD.15 In the
present study, MSCs were administered to 9 extremely
preterm infants at very high risk for developing BPD
(gestational age 24-26 weeks and on ventilator support, with
deteriorating respiratory condition; Table II). All 9 infants
who underwent MSC transplantation survived, and only 3
of these infants developed moderate BPD. Compared with
historical matched comparison group, MSC transplantation
recipients had significantly lower BPD severity. Mean
respiratory index at day 3 posttransplantation, duration of
intubation, duration of continuous positive airway pressure,
and rates of postnatal steroid use were all lower in the MSC
transplantation group compared with the matched
comparison group, although none of the differences was
statistically significant. The 72% rate of moderate/severe
BPD observed in the matched comparison group also
supports a contention that the infants undergoing MSC
transplantation were at greater risk for developing
moderate/severe BPD, and that the significantly reduced
BPD severity observed in that group might be attributable
to the beneficial effects of MSC transplantation rather than
to patient selection bias. Overall, these findings strongly
Mesenchymal Stem Cells for Bronchopulmonary Dysplasia: Phase 1 Dose-Escalation Clinical Trial
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suggest that further phase II clinical trials of intratracheal
transplantation of hUCB-derived MSCs in preterm infants
are warranted. To assess long-term safety, a long-term
follow-up study (NCT01632475) on the MSC-treated
preterm infants reported here is currently underway. n
Vol. 164, No. 5
11.
12.
We are grateful to Eun Sun Kim for her assistance with data
management and thank the Samsung Biomedical Research Institute,
Biostatistics Team, for their statistical support.
Submitted for publication Jul 9, 2013; last revision received Nov 13, 2013;
accepted Dec 6, 2013.
Reprint requests: Won Soon Park, MD, PhD, Department of Pediatrics,
Samsung Medical Center, Sungkyunkwan University School of Medicine, 50
Irwon-dong, Gangnam-gu, Seoul 135-710, Korea. E-mail: wonspark@skku.
edu
13.
14.
15.
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a murine model of neonatal chronic lung disease. Am J Respir Crit
Care Med 2009;180:1122-30.
Abman SH, Matthay MA. Mesenchymal stem cells for the prevention of
bronchopulmonary dysplasia: delivering the secretome. Am J Respir Crit
Care Med 2009;180:1039-41.
Le Blanc K. Immunomodulatory effects of fetal and adult mesenchymal
stem cells. Cytotherapy 2003;5:485-9.
Ahn SY, Chang YS, Kim SY, Sung DK, Kim ES, Rime SY, et al. Long-term
(postnatal day 70) outcome and safety of intratracheal transplantation
of human umbilical cord blood–derived mesenchymal stem cells in
neonatal hyperoxic lung injury. Yonsei Med J 2013;54:416-24.
Laughon MM, Langer JC, Bose CL, Smith PB, Ambalavanan N,
Kennedy KA, et al. Prediction of bronchopulmonary dysplasia by
postnatal age in extremely premature infants. Am J Respir Crit Care
Med 2011;183:1715-22.
Papile LA, Burstein J, Burstein R, Koffler H. Incidence and evolution of subependymal and intraventricular hemorrhage: a study
of infants with birth weights less than 1500 g. J Pediatr 1978;92:
529-34.
Ioannidis JP, Evans SJ, Gotzsche PC, O’Neill RT, Altman DG, Schulz K,
et al. Better reporting of harms in randomized trials: an extension of the
CONSORT statement. Ann Intern Med 2004;141:781-8.
Jobe AH, Bancalari E. Bronchopulmonary dysplasia. Am J Respir Crit
Care Med 2001;163:1723-9.
Kim ES, Chang YS, Choi SJ, Kim JK, Yoo HS, Ahn SY, et al. Intratracheal
transplantation of human umbilical cord blood–derived mesenchymal
stem cells attenuates Escherichia coli–induced acute lung injury in
mice. Respir Res 2011;12:108.
Yang SE, Ha CW, Jung M, Jin HJ, Lee M, Song H, et al. Mesenchymal
stem/progenitor cells developed in cultures from UC blood. Cytotherapy
2004;6:476-86.
Pierro M, Ionescu L, Montemurro T, Vadivel A, Weissmann G, Oudit G,
et al. Short-term, long-term, and paracrine effects of human
umbilical cord–derived stem cells in lung injury prevention and
repair in experimental bronchopulmonary dysplasia. Thorax 2012;68:
475-84.
Doyle LW, Halliday HL, Ehrenkranz RA, Davis PG, Sinclair JC. Impact
of postnatal systemic corticosteroids on mortality and cerebral palsy in
preterm infants: effect modification by risk for chronic lung disease.
Pediatrics 2005;115:655-61.
Kotecha S, Wilson L, Wangoo A, Silverman M, Shaw RJ. Increase in
interleukin (IL)-1 beta and IL-6 in bronchoalveolar lavage fluid obtained
from infants with chronic lung disease of prematurity. Pediatr Res 1996;
40:250-6.
Chang et al
ORIGINAL ARTICLES
May 2014
Appendix
Trial Design
For sample size determination of this first in-human phase I
clinical trial, a “3 + 3” cohort expansion design was
considered at first. The KFDA recommended that the
next-higher dose escalation be expanded to 6 patients for
determination of the maximum tolerated dose. Thus, a minimum of 9 patients was planned to establish the safety profile
of this phase I study (Figure 1). To help establish the safety of
MSC transplantation, we monitored the first transplant
recipient for adverse events for 2 weeks before enrolling
other patients in each dose group. High-dose MSC therapy
was not instituted until the KFDA had given approval, after
its review of the outcomes of infants who had received the
low-dose treatment. The KFDA and Acrovan Co, Ltd acted
as the external monitors of this study, performing intensive
and cautious external monitoring. Acrovan Co, Ltd is a
clinical research consulting company that provided clinical
monitoring services to support this trial.
The primary outcome was the feasibility and safety of
escalating doses of hUCB-derived MSCs in preterm infants,
as assessed by monitoring for SAEs and dose-limiting toxicity.
The secondary outcome was the incidence of adverse events of
hUCB-derived MSC transplantation as determined by death,
BPD ($moderate), severity of BPD,1 duration of invasive or
noninvasive ventilation after injection of MSCs, duration of
hospitalization, retinopathy of prematurity (grade $3),2
periventricular leukomalacia, necrotizing enterocolitis (Bell
stage $2b),3 and culture-proven sepsis. Safety, feasibility,
and potential efficacy were further evaluated by comparing
the incidence of SAEs in infants undergoing MSC transplantation and historical comparison infants.
Patients
Inclusion criteria included infants with a gestational age of
23-29 weeks, birth weight of 500-1250 g, and postnatal age
5-14 days requiring continuous ventilator support that could
not be decreased owing to significant respiratory distress within
24 hours before enrollment. The criteria for a minimum level of
required ventilator support were defined as receipt of highfrequency ventilation or synchronized intermittent mechanical
ventilation, with settings of respiratory rate >12/minute and
fraction of inspired oxygen >0.25. Exclusion criteria at
enrollment were substantial congenital heart disease (other
than PDA), lung hypoplasia, severe congenital anomaly,
operation within 72 hours before intended enrollment,
surfactant treatment within 24 hours before intended
enrollment, shock, severe sepsis, active pulmonary hemorrhage, severe pneumothorax, or severe IVH (grade $3).4
hUCB-Derived MSC Preparation
MSCs were produced according to proper manufacturing
practices at MEDIPOST Co, Ltd. Cell quality control and
quality assurance tests were conducted in accordance with
KFDA standards. hUCB was obtained from full-term
infants after informed maternal consent. hUCB was
collected in bags containing anticoagulant and processed
within 24 hours of collection. After separation over
Histopaque (density 1.077 g/cm3; Sigma-Aldrich, St Louis,
Missouri), mononucleated cells in the low-density fraction
were cultivated as reported. MSCs were characterized in
accordance with recommendations of the International
Society of Cellular Therapy. In brief, these recommendations include evidence of differentiation potential and
flow cytometry assessment confirming the expression of
CD73, CD90, and CD105 surface molecules (in >90%
of samples) and absence of CD34, CD45, and CD14
(present in <2% of samples).
The ex vivo cultured MSC manufacturing process is a
scaled adaptation of the technique described by Yang et al.5
The complete process consists of a total of 6 cell passages.
hUCB processing involves isolation steps to remove
hematopoietic elements, followed by MSC expansion from
the nucleated cells in culture medium (a-minimal essential
medium: Gibco BRL, Grand Island, New York) supplemented with 10% fetal bovine serum. The cells are cryopreserved at 150 C or colder in 10% dimethyl sulfoxide.
In preparation for administration, the frozen MSCs were
thawed and washed with culture medium and saline. The
washing step was developed by process validation, including
no detection of residual level of bovine protein and dimethyl
sulfoxide. After the washing step, the MSCs for transplantation consisted of 1 vial containing approximately 5 million
cells suspended in 1 mL of sterile saline as an excipient,
containing no preservatives. The MSCs were further tested,
including a viability test and cell count, and then transferred
to the bedside within 4 hours. The final viability was
determined by Trypan blue testing. MSCs for transplantation
were stored at 2-8 C, with a shelf life of 24 hours from the
time of manufacture.
Transplantation of hUCB-Derived MSCs
Cell doses were determined based on preclinical efficacy
and toxicity studies showing an effective dose range of
1.0-5.0 107 cells/kg without any adverse effects6 and a
good laboratory practice study (G07228, repeat-dose toxicity
test) under the guidance of the KFDA.
The prepared hUCB-derived MSCs (1 107 cells/kg or
2 107 cells/kg), mixed with normal saline at a concentration
of 5 106 cells/mL (2 or 4 mL/kg), were drawn into a syringe
through a 22-gauge needle. The needle was removed, and a
5-French feeding tube was connected to the syringe. After
the infant was positioned on the right side with the bed flat
and with manual ventilation, one-half of the MSCs were
administered by intratracheal instillation via a gavage tube,
with the syringe tip positioned 1 cm above the end of the
endotracheal tube. This procedure was repeated with the
infant positioned on the left side.
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Assessment of Safety
Immediately after MSC transplantation, patients were monitored for SAEs and dose-limiting toxicity, respectively. SAEs
in this study are defined as any untoward medical occurrence
that results in death or is life-threatening, requires inpatient
hospitalization or prolongation of existing hospitalization, or
results in persistent or significant disability (Table I). If
applicable, SAEs were monitored, assessed, and reported on
a daily basis up to 84 days posttransplantation. As part of
routine care in the neonatal intensive care unit, vital signs
with electrocardiography were continuously monitored and
recorded in an electronic medical record system. Physical
examination and chest radiography with blood gas analysis
were performed within 12 hours before and after MSC
transplantation (and reassessed as necessary). Cranial
ultrasonography and echocardiography were performed
at baseline (screening point); at days 2, 7, and 28
posttransplantation; and at 36 weeks corrected gestational
age. Routine laboratory tests were performed as usual.
Tracheal aspirate fluid was collected at baseline and at day
3, 7, 14, and 28 posttransplantation only if the patient
remained intubated on those days.
To assess the incidence of adverse events or outcomes for
further safety and feasibility evaluation, we enrolled 2
matching neonate comparisons for every study case retrospectively after completion of the trial. For this nested
comparison group, we selected 2 matched infants for each
MSC transplantation patient according to the following
criteria: gestational age within 3 days, birth weight within
50 g, and similar ventilator modes and mean respiratory
severity scores (mean airway pressure fraction of inspired
oxygen) within 24 hours before MSC transplantation.
Because of the logistical demands of matching birth weight,
gestational age, respiratory severity scores, and ventilator
mode, the matched comparison group infants were not
consecutive cases. They were born and cared for at Samsung
Medical Center between January 2009 and November 2011.
Infants from both the MSC-treated group and the matched
comparison group were evaluated for respiratory distress
syndrome, mechanical ventilator duration (invasive/noninvasive), respiratory severity score, BPD, IVH (grade $3),4
PDA, blood culture–confirmed sepsis, retinopathy of prematurity,2 necrotizing enterocolitis (Bell stage $2b),3 and periventricular leukomalacia.
Principles of Neonatal Intensive Care Management
There were no obvious changes in the neonatal intensive care
management policies, including ventilation guidelines of
88%-95% of target saturation and 44-55 mmHg of target
PCO2, extubation criteria, PDA ligation, or postnatal steroid
use, during the period January 2009 to November 2011, when
the MSC transplantation and matched comparison group infants were born and cared for. In this study, postnatal steroid
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treatment was reserved for preterm infants who were
dependent on invasive ventilation at or after the third week
of life with deteriorating respiratory condition, who are at
the greatest risk for developing BPD and may benefit
from steroid treatment.7,8 A high-dose (0.5 mg/kg/day) or
low-dose (0.2 mg/kg/day) dexamethasone regimen was
started at the discretion of the attending neonatologist, and
then tapered within 7-10 days. An additional course of
dexamethasone was considered based on the infant’s clinical
condition.
PDA ligation was reserved for only those preterm infants
dependent on mechanical ventilation with cardiovascular
compromise, such as persistent hypotension requiring
inotropics and/or deteriorating respiratory condition,
usually after failure of medical treatment.9
Tracheal Aspirate Fluid Analysis
Tracheal aspirate fluid samples were obtained twice by suctioning the major airways after 0.5 mL of saline had been
instilled into the endotracheal tube. Samples were collected
only when suctioning was clinically required during routine
care. The supernatant was frozen at 70 C after centrifugation at 15 000 rpm for 10 minutes. Levels of HGF, TGF-b1,
and MMP-9 were calculated by enzyme immunoassay using
the Quantikine Kit (R&D Systems, Minneapolis, Minnesota).
IL-1, IL-6, IL-8, IL-10, TNF-a, and VEGF were measured
with the Milliplex MAP ELISA Kit (Millipore, Billerica, Massachusetts), according to the manufacturer’s specifications.
References
1. Jobe AH, Bancalari E. Bronchopulmonary dysplasia. Am J Respir Crit
Care Med 2001;163:1723-9.
2. International Committee for the Classification of the Late Stages of
Retinopathy of Prematurity. An international classification of retinopathy of prematurity, II: the classification of retinal detachment. Arch
Ophthalmol 1987;105:906-12.
3. Bell MJ, Ternberg JL, Feigin RD, et al. Neonatal necrotizing enterocolitis:
therapeutic decisions based upon clinical staging. Ann Surg 1978;187:1-7.
4. Papile LA, Burstein J, Burstein R, Koffler H. Incidence and evolution of
subependymal and intraventricular hemorrhage: a study of infants with
birth weight less than 1500 g. J Pediatr 1978;92:529-34.
5. Yang SE, Ha CW, Jung M, et al. Mesenchymal stem/progenitor cells
developed in cultures from UC blood. Cytotherapy 2004;6:476-86.
6. Chang YS, Choi SJ, Sung DK, Kim SY, Oh W, Yang YS, et al. Intratracheal
transplantation of human umbilical cord blood–derived mesenchymal
stem cells dose-dependently attenuates hyperoxia-induced lung injury
in neonatal rats. Cell Transplant 2011;20:1845-54.
7. Doyle LW, Halliday HL, Ehrenkranz RA, Davis PG, Sinclair JC. Impact of
postnatal systemic corticosteroids on mortality and cerebral palsy in
preterm infants: effect modification by risk for chronic lung disease.
Pediatrics 2005;115:655-61.
8. Grier DG, Halliday HL. Management of bronchopulmonary dysplasia in
infants: guidelines for corticosteroid use. Drugs 2005;65:15-29.
9. Clyman RI, Couto J, Murphy GM. Patent ductus arteriosus: are current
neonatal treatment options better or worse than no treatment at all?
Semin Perinatol 2012;36:123-9.
Chang et al
May 2014
ORIGINAL ARTICLES
Figure 2. Temporal profiles of heart rate, oxygen saturation, mean airway pressure, and fraction of inspired oxygen before and
after hUCB-derived MSC transplantation in the 9 study patients up to 24 hours posttransplantation.
Mesenchymal Stem Cells for Bronchopulmonary Dysplasia: Phase 1 Dose-Escalation Clinical Trial
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Figure 3. Chest radiographs obtained at 84 days posttransplantation in all 9 study patients showing comparable findings in
both lung fields with mild BPD in 6 patients (A1, A2, A3, B1, B3, and B6) and with moderate BPD in 3 patients (B2, B4, and B5).
In addition, no visible mass-like lesions were observed in either lung field.
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ORIGINAL ARTICLES
Figure 5. Levels of cytokines and growth factors from tracheal aspirate fluid collected before MSC transplantation and at 3 days
and 7 days posttransplantation. Data are presented as mean SEM. *P < .05, compared with pretransplantation level. †P < .05,
compared with posttransplantation level at 3 days posttransplantation.
Mesenchymal Stem Cells for Bronchopulmonary Dysplasia: Phase 1 Dose-Escalation Clinical Trial
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Table II. Estimated risk of death or moderate/severe BPD in study patients at enrollment
Patient number
Gestational age, wk
Birth weight, g
Estimated risk of death
or moderate/severe
BPD at enrollment, %*
Hispanic
White
Black
A1
A2
A3
B1
B2
B3
B4
B5
B6
25
770
25
870
24
720
24
630
24
740
25
850
25
650
26
1030
26
880
74.9
65.3
62.6
76
66.5
63.4
89.2
91.1
91.4
78.7
82.2
82.3
78.7
82.3
82
54.1
60.7
57.6
66.1
71.5
69.6
71.4
76.7
74.8
78
82
81
*Calculated using the BPD outcome estimator from the National Institute of Child Health and Human Development (available at https://neonatal.rti.org).
Table III. Clinical characteristics of the MSC transplantation group and historical matched comparison group
MSC transplantation group
Matched
High-dose
Low-dose
comparison group,
7
(1 10 cells/kg) (n = 3) (2 10 cells/kg) (n = 6) Total (n = 9)
total (n = 18)
P value
7
Gestational age, wk, mean SD
Birth weight, g, mean SD
Apgar score, 1 min, mean SD
Apgar score, 5 min, mean SD
Female sex, n (%)
Cesarean delivery, n (%)
Antenatal corticosteroid use, n (%)
Pathological chorioamnionitis, n (%)
Respiratory distress syndrome, n (%)
PDA, n (%)
Medication (before MSC transplantation or index day)
Operation (before MSC transplantation or index day)
Early-onset sepsis, n (%)
IVH (grade $3), n (%)
Age at MSC transplantation or index day for either cases
or comparisons, postnatal d, mean SD
Mean respiratory severity score (1 day before MSC
transplantation or index day), mean SD
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25.3 0.7
787 76
5.0 1.0
7.6 1.2
2/3
3/3
3/3
2/3
3/3
3/3
2/3
2/3
0/3
0/3
11.3 3.8
25.3 0.9
797 153
5.3 2.4
7.0 1.8
3/6
4/6
4/6
2/6
6/6
6/6
3/6
3/6
1/6
0/6
10.8 2.5
25.3 0.9
793 127
5.2 2.0
7.2 1.6
4 (44)
7 (78)
7 (78)
2 (22)
9 (100)
9 (100)
5 (56)
5 (56)
1 (11)
0 (0)
10.4 2.6
25.3 1.0
795 99
4.5 1.4
7.2 1.4
13 (72)
14 (78)
15 (83)
7 (39)
18 (100)
18 (100)
14 (78)
9 (50)
1 (6)
0 (0)
11.0 2.8
2.1 0.4
3.4 2.8
2.9 0.9
2.5 1.1
.60
.94
.34
.93
.38
1.00
1.00
1.00
.43
1.00
.67
.80
.10
Chang et al