Mesenchymal Stem Cells for Bronchopulmonary Dysplasia: Phase 1Dose-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)-derivedmesenchymal 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, intratrachealtransplantation 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 highdose (2 � 107 cells/kg). We compared their adverse outcomes, including BPD severity, with those of historicalcase-matched comparison group.Results Intratracheal MSC transplantation was performed in 9 preterm infants, with a mean gestational age of25.3 � 0.9 weeks and a mean birth weight of 793 � 127 g, at a mean of 10.4 � 2.6 days after birth. The treatmentswere 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 growthfactor b1 in tracheal aspirates at day 7 were significantly reduced compared with those at baseline or at day3 posttransplantation. BPD severity was lower in the transplant recipients, and rates of other adverse outcomesdid not differ between the comparison group and transplant recipients.Conclusion Intratracheal transplantation of allogeneic hUCB-derived MSCs in preterm infants is safe andfeasible, and warrants a larger and controlled phase II study. (J Pediatr 2014;164:966-72).

See editorial, p 954 andrelated article, p 973

he number of very preterm infants at high risk for developing bronchopulmonary dysplasia (BPD) is increasing, because

Tadvances in neonatal intensive care have increased these infants’ chance of survival.1 Given the lack of effective measuresto 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 immunocompetentanimals attenuates hyperoxia-induced lung injury, such as impaired alveolarization, inflammatory response, increasedapoptosis, and fibrosis.7-12 Human umbilical cord blood (hUCB) is considered a better source of MSCs than other potential

From the 1Department of Pediatrics, Samsung MedicalCenter, Sungkyunkwan University School of Medicine;and 2Biomedical Research Institute, MEDIPOST Co, Ltd,Seoul, Korea

*Contributed equally.

Funded by the Korean Health and Medical TechnologyR&D Program, Ministry for Health, Welfare and FamilyAffairs, Republic of Korea (A102136). Human umbilicalcord blood–derived mesenchymal stem cells were sup-plied by MEDIPOST Co, Ltd; the sponsor had noinvolvement in study design, the collection, analysis, orinterpretation of data; writing of the report; or the deci-sion to submit the manuscript for publication. W.O. is aboard member and stockholder of MEDIPOST Co, Ltd.Samsung Medical Center and MEDIPOST Co, Ltd have

sources, such as bone marrow or adipose tissue because of their ready availabilityand greater proliferative capacity and less antigenicity than other cell types.13 Inprevious translational studies to determine the optimal route,7 dose,8 andtiming9 of transplantation of hUCB-derived MSCs in a neonatal hyperoxiclung injury model in rat pups, we found that the protection of MSCs againstneonatal hyperoxic lung injury was persistent, and that no long-term toxicities,adverse effects, or tumorigenicity were present at 70 days posttransplantation.14

Collectively, these findings offer hope that transplantation of hUCB-derivedMSCs will be effective in treating BPD. The safety and efficacy of MSC transplan-tation for prevention of BPD has not been tested previously, however. We reporta phase I dose-escalating clinical study on the safety and feasibility of transplan-tation of hUCB-derived MSCs in preterm infants with BPD.

issued or filed patents for “Method of treating lung dis-eases using cells separated or proliferated from umbilicalcord blood” under Y.C., W.P., and Yoon Sun Yang (notaffiliated with this article) (application PCT/KR2007/000535). S.Ahn, H.Y., and S.Sung declare no conflicts ofinterest.

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/).

BPD Bronchopulmonary dysplasia

HGF Hepatic growth factor

hUCB Human umbilical cord blood

IL Interleukin

IVH Intraventricular hemorrhage

KFDA Korean Food and Drug

Administration

MMP Matrix metalloproteinase

MSC Mesenchymal stem cell

PDA Patent ductus arteriosus

SAE Serious adverse event

TGF Transforming growth factor

TNF Tumor necrosis factor

VEGF Vascular endothelial growth factor

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Vol. 164, No. 5 � May 2014

Methods

This study was a phase I, open-label, single-arm, single-center trial to evaluate the safety and feasibility of intratra-cheal allograft transplantation of hUCB-derived MSCs inpreterm infants. The protocol was approved by the KoreanFood and Drug Administration (KFDA; MP-CR-006) andby the Institutional Review Board of the Samsung MedicalCenter in Seoul, Korea (2010-09-092). The primary goalwas to demonstrate the safety of intratracheal allografttransplantation of hUCB-derived MSCs in preterm infantsat high risk of developing BPD. The secondary goal wasto evaluate the feasibility and potential efficacy of MSCtransplantation for BPD in comparison with historicalcase-matched comparison group.

Patients were enrolled at SamsungMedical Center betweenFebruary 10, 2011, and September 14, 2011. Because this wasa first-in-human trial for intratracheal allograft transplanta-tion of hUCB-derived MSCs in preterm infants, intensiveand cautious external monitoring was maintained, and theKFDA and Acrovan Co, Ltd (Anyang, Korea) served asexternal monitors of the study.

The informed consent document was reviewed with boththe parents and principal investigator or study staff at least

Figure 1. Study design. If there was no occurrence of dose-limitinThe first 3 patients were assigned to receive low-dose MSCs (1 �high-dose MSCs (2� 107 cells/kg; dose B). If there were any doseextra enrollment of 3 cases in the dose A group would be neededB group, then the maximum tolerated dose would have been deteDLT, dose-limiting toxicity; MTD, maximum tolerated dose.

twice. Full understanding was confirmed, and writteninformed consent was obtained from both parents, withparticular attention given to the understanding that testingwas for safety, with neither an expectation nor a promise oftherapeutic benefit. In accordance with the original studyscheme (Figure 1), the target sample size was a minimumof 9 patients. The first 3 patients were assigned to receivelow-dose MSCs (1 � 107 cells/kg), and the next 6 wereassigned 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 andbirth weight of 500-1250 g, and patients (at postnatal day5-14) needing continuous ventilator support that could notbe decreased owing to significant respiratory distress within24 hours before enrollment. Patients were excluded for severecongenital anomalies, lung hypoplasia, severe septic shock, orsevere (grade $3) intraventricular hemorrhage (IVH)16

(Appendix; available at www.jpeds.com).

Transplantation of hUCB-Derived MSCsPneumostem, passage 6 hUCB-derived MSCs (Medipost,Seoul, Korea) were prepared in compliance with goodmanufacturing practices, at concentration of 5 � 106 cells/mL in normal saline. A dose of 1 � 107 cells (2 mL)/kg or2 � 107 cells (4 mL)/kg were administered intratracheally

g toxicity, then the target minimum sample size was 9 patients.107 cells/kg; dose A), and the next 6 were assigned to receive-limiting toxicity in the dose A group (low-dose group), then an. If dose-limiting toxicity occurred more than twice in the dosermined to be dose A without additional evaluation of dose B.

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via a gavage tube in 2 fractions into the left and right lungs(Appendix).

Assessment of SafetySafety was defined primarily as the absence of treatment-related serious adverse events (SAEs) according to theConsolidated Standards of Reporting Trials,17 and second-arily as the absence of dose-limiting toxicity, defined asdeath within 6 hours after MSC transplantation oranaphylactic shock related to the MSC injection. After asingle intratracheal MSC transplantation, all patients wereregularly and intensively assessed until 84 days post-MSCtransplantation according to schedule (Appendix). Forcomparison of adverse outcomes for further safetyevaluation, a historical nested case-control group wasestablished (Appendix). The clinical data for thecomparison group were for the same postnatal day to theindex day as that for the MSC recipients. BPD was definedaccording to the National Institutes of Health workshopseverity-based diagnostic criteria.18

Temporal Profile of Tracheal Aspirate Cytokinesand Growth FactorsTracheal aspirate fluid was collected before and after MSCtransplantation for assessment of changes in cytokines andgrowth factors known to be associated with the develop-ment or prevention of BPD. Samples were collected onlywhen suctioning was clinically required during routinecare. The following cytokines and growth factors weremeasured: interleukin (IL)-1, IL-6, IL-8, IL-10, matrixmetalloproteinase (MMP)-9, transforming growth factor(TGF)-b, tumor necrosis factor (TNF)-a, vascular endo-thelial growth factor (VEGF), and hepatic growth factor(HGF).

Statistical AnalysesData are expressed as mean � SD. To compare continuousvariables and BPD severity between study patients and thematched comparison group, statistical comparisonsbetween groups were performed using 2-way ANOVAand generalized estimating equations. Stratified logisticregression analysis was used to compare other nominalvariables. The temporal profile of growth factors andcytokines in the tracheal aspirate fluid was assessed usingthe paired t test. A P value <.05 was considered statisticallysignificant. SPSS version 17 (SPSS Inc, Chicago, Illinois)was used for all statistical analyses.

Results

Three infants received low-dose (1� 107 cells/kg) MSCs, and6 infants received high-dose (2 � 107 cells/kg) MSCs. Gesta-tional age, birth weight, and postnatal age of MSC transplantrecipients 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 deathor moderate/severe BPD15 at enrollment ranged from 54.1%

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to 91.4%, with an average of 74.4% � 10.0% (Table II;available at www.jpeds.com). Clinical variables, includinggestational age, birth weight, Apgar scores, and respiratoryseverity scores, were not significantly different between theMSC-treated group and the matched comparison group, orbetween the low-dose and high-dose MSC subgroups(Table III; available at www.jpeds.com).

SAEsDetails of SAEs, recorded for up to 84 days after MSCtransplantation, are presented in Table I. The 9 infants whoreceived MSC therapy were discharged alive. Intratrachealtransplantation of hUCB-derived MSCs took <5 minutes. Allpatients tolerated the procedure well without immediatecomplications within 6 hours after transplantation orimmediate respiratory and cardiovascular compromise(Figure 2; available at www.jpeds.com); however, 6 patientssubsequently developed SAEs. The most common event waspatent ductus arteriosus (PDA) ligation, occurring in 4 of the9 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-doseMSC transplantation group (Table I) had congenital systemiccandidiasis, followed by necrotizing enterocolitis requiringsurgery and, eventually, periventricular leukomalacia.

Adverse OutcomesThere were no significant differences in SAEs between thelow-dose and high-dose MSC transplantation groups orbetween the MSC-treated group and matched-comparisongroup (Table IV) except in BPD severity,18 which wassignificantly lower in the MSC transplant group (regressioncoefficient, 1.7; 95% CI, 0.11-3.29; P = .036). The durationof intubation after MSC transplantation ranged from 3 to45 days, with the longest duration in the patient withcongenital systemic candidiasis. There were no significantdose-dependent or timing-dependent differences in theduration of intubation between the MSC transplantationand comparison groups. Postnatal dexamethasone use waslower in the MSC transplantation group compared with thecomparison group (67% vs 100%). The mean start day andcumulative dose of dexamethasone did not differ betweenthe 2 groups (11.0 � 2.8 days after MSC transplantationand 2.7 � 2.5 mg/kg in the MSC transplantation group vs11.0 � 2.7 days after the index day and 3.7 � 2.3 mg/kg inthe comparison group).Serial echocardiography performed by a pediatric

cardiologist before and after MSC transplantationrevealed no significant changes in cardiac function ordevelopment of pulmonary hypertension. Analysis of serialchest radiographs, including those taken at posttransplanta-tion day 84, showed no visible mass-like lesions in either lungfield (Figure 3; available at www.jpeds.com).

Daily Changes in Respiratory Severity ScoreFigure 4 shows temporal profiles of respiratory severityscores of individual patients in the MSC transplantation

Chang et al

Table I. Clinical data of enrolled patients

Variables

Patient

A1 A2 A3 B1 B2 B3 B4 B5 B6

Gestational age, wk 25+6 25+3 24+3 24+0 24+4 25+4 25+0 26+4 26+1

Birth weight, g 770 870 720 630 740 850 650 1030 880Apgar score at 1 min 6 5 4 5 3 5 4 8 5Apgar score at 5 min 9 7 7 8 5 8 6 9 8Sex F M M F M F F M MDelivery C C C C NV NV NV C CPathologically confirmed chorioamnionitis Y Y - Y Y - - Y -Antenatal steroid use Y Y Y Y - Y Y Y YRespiratory distress syndrome Y Y Y Y Y Y Y Y YEarly-onset sepsis - - - - Y - - - -PDA Y Y Y Y Y Y Y Y YTherapies administered between birth and MSC injectionNumber of surfactant doses 1 1 1 1 2 2 1 2 1Use of indomethacin or ibuprofen Y Y - Y - - Y - YSurgical ligation of PDA - Y Y - Y Y - Y -

Age at MSC injection (postnatal day) 7 13 12 10 10 9 8 14 14SAEs with major morbiditiesDeath within 6 hours after MSC transplantation - - - - - - - - -Anaphylaxis after MSC transplantation - - - - - - - - -BPD severity Mild Mild Mild Mild Moderate Mild Moderate Moderate MildIntubation duration after MSC transplantation 12 5 7 11 45 3 16 11 23Duration of nasal continuous positive airway pressure 32 29 49 48 28 25 50 40 18Pneumothorax Y - - - - - - - -PDA ligation Y - - Y - - Y - Y

Late-onset sepsis - - - - - - Y - -Necrotizing enterocolitis (stage $2) - - - - Y - - - -Periventricular leukomalacia - - - - Y - - - -IVH ($ grade 3) - - - - - - - - -Retinopathy of prematurity (stage $3) - - - - - Y - - -

F, female; M, male; C, cesarean delivery; NV, spontaneous vaginal delivery; Y, yes.

May 2014 ORIGINAL ARTICLES

and comparison groups. After MSC transplantation, nopatient demonstrated an obvious exacerbation ofventilator dependency as a result of the transplantationprocedure. The MSC transplantation group had generallylower values after MSC transplantation compared with

Table IV. Comparison of outcomes in the MSC transplantat

MSC

Low-dose(1 � 107 cells/kg) (n =

Death at discharge, n (%) 0/3BPD, n (%) 3/3BPD severity, n (%)Mild 3/3Moderate 0/3Severe 0/3

Duration of intubation, d, mean � SDTotal duration 19.7 � 1.2Duration after MSC transplantation or index date 8. 3 � 3.5

Duration of nasal continuous positive airway pressure,d, mean � SD

36.7 � 10.8

Pneumothorax, n (%) 1/3PDA ligation after MSC transplantation or index day, n (%) 1/3Retinopathy of prematurity (grade $3), n (%) 0/3Late-onset sepsis, n (%) 0/3IVH (grade $3), n (%) 0/3Periventricular leukomalacia, n (%) 0/3Necrotizing enterocolitis (stage $2b), n (%) 0/3Postnatal steroid use for BPD, n (%) 2/3

*Statistical comparison performed with the generalized estimating equations test.

Mesenchymal Stem Cells for Bronchopulmonary Dysplasia: Phas

the comparison group, especially on day 3posttransplantation, but the difference did not reachstatistical significance (1.4 � 1.4 vs 3.3 � 2.0 incomparison group; mean difference, �1.05; 95% CI,�2.27 to 0.15; P = .05) (Figure 4, C).

ion group and matched comparison group

transplantation group

Matched-comparisongroup (n = 18) P value3)

High-dose(2 � 107 cells/kg) (n = 6)

Total(n = 9)

0/6 0/9 (0.0) 0/18 (0.0)6/6 9/9 (100) 18/18 (100)

.037*3/6 6 (67) 5 (28)3/6 3 (33) 5 (28)0/6 0 (0) 8 (44)

29.0 � 15.1 25.9 � 12.8 33.6 � 12.9 .1918.2 � 14.7 14.8 � 12.8 22.6 � 13.5 .1934.8 � 13.1 35.4 � 11.7 43.2 � 18.7 .29

0/6 1 (11) 0 (0) .673/6 4 (44) 6 (33) .861/6 1 (11) 9 (50) .261/6 1 (11) 3 (17) .890/6 0 (0) 0 (0)1/6 1 (11) 1 (6) 1.001/6 1 (11) 2 (6) 1.004/6 6 (67) 18 (100) .07

e 1 Dose-Escalation Clinical Trial 969

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 the2 groups. Day 0 refers to the day of MSC transplantation, or the index day in the matched comparison group matched to theday of MSC transplantation. MSC, MSC transplantation group; Comparison, matched comparison group. Data are presentedas mean � SEM.

THE JOURNAL OF PEDIATRICS � www.jpeds.com Vol. 164, No. 5

Temporal Profiles of Cytokines and Growth Factorsfrom Tracheal Aspirate FluidTemporal profiles of cytokines and growth factors fromtracheal aspirate fluid are shown in Figure 5 (availableat www.jpeds.com). Levels of MMP-9 from the trachealaspirate at day 7 posttransplantation were significantlyreduced compared with baseline (P = .02). IL-6, IL-8,TNF-a, and TGF-b levels were significantly lower atday 7 posttransplantation than at day 3 post-transplantation.

970

Discussion

Intratracheal transplantation of low-dose (1 � 107 cells/kg) orhigh-dose (2 � 107 cells/kg) allogeneic hUCB-derived MSCswas not associated with immediate SAEs or dose-limitingtoxicity in our cohort of extremely preterm infants at highrisk for developing BPD. The incidence of SAEs within 84days posttransplantation did not differ between theMSC trans-plant recipients andhistorical case-matched comparisongroup.

Chang et al

May 2014 ORIGINAL ARTICLES

BPD severity was significantly decreased, and retinopathy ofprematurity requiring surgery was less prevalent in the MSCtransplantation group comparedwith thematched comparisongroup. Taken together, these findings indicate that intratrachealtransplantation of up to 2 � 107 cells/kg of hUCB-derivedMSCs in preterm infants may be safe and feasible.

We used passage 6 hUCB-derived MSCs for human intra-tracheal transplantation. We previously observed karyotypestability at up to passage 11 of hUCB-derived MSCs,7,14,19

and characteristics of low expression of major histocompat-ibility complex class 1 and lack of major histocompatibilitycomplex class 2 molecules.13,20 Furthermore, in a newbornrat pup model of hyperoxia, no long-term adverse effectsor tumorigenicity had occurred by the tenth postnatal weekafter MSC transplantation.14 Transplanted MSCs had alow rate of engraftment in the recipient rat lung, and theengraftment dissipated shortly after transplantation21 toalmost undetectable levels by the tenth postnatal week.14

The route of MSC transplantation is an important consid-eration. We previously demonstrated in newborn rats thatlocal intratracheal MSC transplantation is more effectivethan systemic intraperitoneal administration in protectingagainst hyperoxic lung injury.7 Intratracheal transplantationhas a lower burden of unexpected side effects originatingfrom systemic distribution of donor cells compared withintravenous administration. Moreover, for preterm infantsreceiving invasive ventilation via an endotracheal tube,intratracheal instillation does not require an additionalprocedure for local transplantation. Thus, in this study, weadministered the MSCs intratracheally in 2 fractions, usingthe same method as for administration of exogenoussurfactant. No immediate clinical instability or complica-tions were evident (Figure 2), suggesting that intratrachealadministration of MSCs is safe and feasible.

We previously showed that intratracheal delivery of MSCsattenuated hyperoxic lung injury in newborn rats in adose-dependent manner, with at least 5 � 104 cells per ratpup weighing approximately 10 g required to produceanti-inflammatory, antifibrotic, and antioxidative effects.8

Although currently no guidelines are available for the extrap-olation of preclinical data into clinical trials, extrapolatingfrom the cell dose used in the animal studies, we chose dosesof 1� 107 and 2� 107 cells/kg for use in this study of humaninfants. In contrast to our preclinical animal study showing adose-dependent response,8 in this study, the high-dose MSCgroup, although small, seemed to have a longer duration ofintubation and higher BPD severity scores comparedwith the low-dose group, although the difference was notstatistically significant. Further studies are needed todetermine optimal MSC doses for transplantation.

Optimal timing of MSC transplantation is another keyissue remaining to be clarified. In our work with newbornrats, the therapeutic efficacy of hUCB-derived MSCtransplantation was time-dependent, with greater efficacywhen given before the peak and plateau for inflammatoryresponses in hyperoxic lung injury.9 Experimental rodentdata in hyperoxic lung injury cannot be extrapolated directly

Mesenchymal Stem Cells for Bronchopulmonary Dysplasia: Phas

into human infants for BPD; however, we chose 5-14 daysafter birth with ventilator dependency as a transplantationtiming based on this animal study,9 which is a relatively earlyfor preterm infants, with sufficient stabilization after birthbut before BPD is established. In the present study, notime-dependent variation in therapeutic efficacy was evidentwhen MSCs were given between 7 and 14 days postnatally.We also found that MSC transplantation seemed to decreasethe respiratory severity score shortly after transplantation,followed by an increase by around day 7 posttransplantation;this might be considered a time when a second transplanta-tion, if necessary, might be indicated.We previously showed that inflammatory responses

mediated by proinflammatory cytokines play a pivotal rolein the development of BPD.7-9,22 Furthermore, the protectiveeffects of hUCB-derived MSC therapy against neonatalhyperoxic lung injury are mediated primarily by paracrineantiinflammatory, 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, andTGF-b1 in the tracheal aspirate fluid were significantlyreduced after MSC transplantation. However, Kotechaet al23 have shown that proinflammatory cytokines in thetracheal aspirate fluid peak at 7 days, and then subside inpreterm infants who develop chronic lung disease. Thus,without a proper matched comparison group, whether ourdata showing decreased tracheal aspirate inflammatory cyto-kines are related to immunomodulatory effects of MSCs orsimply reflect the natural course of inflammation is difficultto ascertain. In this study, VEGF and HGF tended to bereduced after MSC transplantation; these results contradictour previous results showing significant up-regulation ofhyperoxia-induced growth factors.9

According to multivariate analyses of 23- to 27-week-oldpreterm infants, lower gestational age and mechanicalventilation at day 7 were major predictors of BPD.15 In thepresent study, MSCs were administered to 9 extremelypreterm infants at very high risk for developing BPD(gestational age 24-26 weeks and on ventilator support, withdeteriorating respiratory condition; Table II). All 9 infantswho underwent MSC transplantation survived, and only 3of these infants developed moderate BPD. Compared withhistorical matched comparison group, MSC transplantationrecipients had significantly lower BPD severity. Meanrespiratory index at day 3 posttransplantation, duration ofintubation, duration of continuous positive airway pressure,and rates of postnatal steroid use were all lower in the MSCtransplantation group compared with the matchedcomparison group, although none of the differences wasstatistically significant. The 72% rate of moderate/severeBPD observed in the matched comparison group alsosupports a contention that the infants undergoing MSCtransplantation were at greater risk for developingmoderate/severe BPD, and that the significantly reducedBPD severity observed in that group might be attributableto the beneficial effects of MSC transplantation rather thanto patient selection bias. Overall, these findings strongly

e 1 Dose-Escalation Clinical Trial 971

THE JOURNAL OF PEDIATRICS � www.jpeds.com Vol. 164, No. 5

suggest that further phase II clinical trials of intratrachealtransplantation of hUCB-derived MSCs in preterm infantsare warranted. To assess long-term safety, a long-termfollow-up study (NCT01632475) on the MSC-treatedpreterm infants reported here is currently underway. n

We are grateful to Eun Sun Kim for her assistance with datamanagement 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

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Appendix

Trial Design

For sample size determination of this first in-human phase Iclinical trial, a “3 + 3” cohort expansion design wasconsidered at first. The KFDA recommended that thenext-higher dose escalation be expanded to 6 patients fordetermination of the maximum tolerated dose. Thus, a min-imum of 9 patients was planned to establish the safety profileof this phase I study (Figure 1). To help establish the safety ofMSC transplantation, we monitored the first transplantrecipient for adverse events for 2 weeks before enrollingother patients in each dose group. High-dose MSC therapywas not instituted until the KFDA had given approval, afterits review of the outcomes of infants who had received thelow-dose treatment. The KFDA and Acrovan Co, Ltd actedas the external monitors of this study, performing intensiveand cautious external monitoring. Acrovan Co, Ltd is aclinical research consulting company that provided clinicalmonitoring services to support this trial.

The primary outcome was the feasibility and safety ofescalating doses of hUCB-derived MSCs in preterm infants,as assessed bymonitoring for SAEs and dose-limiting toxicity.The secondary outcome was the incidence of adverse events ofhUCB-derived MSC transplantation as determined by death,BPD ($moderate), severity of BPD,1 duration of invasive ornoninvasive ventilation after injection of MSCs, duration ofhospitalization, retinopathy of prematurity (grade $3),2

periventricular leukomalacia, necrotizing enterocolitis (Bellstage $2b),3 and culture-proven sepsis. Safety, feasibility,and potential efficacy were further evaluated by comparingthe incidence of SAEs in infants undergoingMSC transplanta-tion and historical comparison infants.

Patients

Inclusion criteria included infants with a gestational age of23-29 weeks, birth weight of 500-1250 g, and postnatal age5-14 days requiring continuous ventilator support that couldnotbedecreasedowing to significant respiratory distresswithin24hours before enrollment. The criteria for aminimum level ofrequired ventilator support were defined as receipt of high-frequency ventilation or synchronized intermittentmechanicalventilation, with settings of respiratory rate >12/minute andfraction of inspired oxygen >0.25. Exclusion criteria atenrollment were substantial congenital heart disease (otherthan PDA), lung hypoplasia, severe congenital anomaly,operation within 72 hours before intended enrollment,surfactant treatment within 24 hours before intendedenrollment, shock, severe sepsis, active pulmonary hemor-rhage, severe pneumothorax, or severe IVH (grade$3).4

hUCB-Derived MSC Preparation

MSCs were produced according to proper manufacturingpractices at MEDIPOST Co, Ltd. Cell quality control and

quality assurance tests were conducted in accordance withKFDA standards. hUCB was obtained from full-terminfants after informed maternal consent. hUCB wascollected in bags containing anticoagulant and processedwithin 24 hours of collection. After separation overHistopaque (density 1.077 g/cm3; Sigma-Aldrich, St Louis,Missouri), mononucleated cells in the low-density fractionwere cultivated as reported. MSCs were characterized inaccordance with recommendations of the InternationalSociety of Cellular Therapy. In brief, these recommenda-tions include evidence of differentiation potential andflow cytometry assessment confirming the expression ofCD73, 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 removehematopoietic elements, followed by MSC expansion fromthe nucleated cells in culture medium (a-minimal essentialmedium: Gibco BRL, Grand Island, New York) supple-mented with 10% fetal bovine serum. The cells are cryo-preserved 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. Thewashing step was developed by process validation, includingno detection of residual level of bovine protein and dimethylsulfoxide. After the washing step, the MSCs for transplanta-tion consisted of 1 vial containing approximately 5 millioncells 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 transferredto the bedside within 4 hours. The final viability wasdetermined by Trypan blue testing. MSCs for transplantationwere stored at 2-8�C, with a shelf life of 24 hours from thetime of manufacture.

Transplantation of hUCB-Derived MSCs

Cell doses were determined based on preclinical efficacyand toxicity studies showing an effective dose range of1.0-5.0 � 107 cells/kg without any adverse effects6 and agood laboratory practice study (G07228, repeat-dose toxicitytest) 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 concentrationof 5� 106 cells/mL (2 or 4 mL/kg), were drawn into a syringethrough a 22-gauge needle. The needle was removed, and a5-French feeding tube was connected to the syringe. Afterthe infant was positioned on the right side with the bed flatand with manual ventilation, one-half of the MSCs wereadministered by intratracheal instillation via a gavage tube,with the syringe tip positioned 1 cm above the end of theendotracheal tube. This procedure was repeated with theinfant positioned on the left side.

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Assessment of Safety

Immediately after MSC transplantation, patients were moni-tored for SAEs and dose-limiting toxicity, respectively. SAEsin this study are defined as any untoward medical occurrencethat results in death or is life-threatening, requires inpatienthospitalization or prolongation of existing hospitalization, orresults in persistent or significant disability (Table I). Ifapplicable, SAEs were monitored, assessed, and reported ona daily basis up to 84 days posttransplantation. As part ofroutine care in the neonatal intensive care unit, vital signswith electrocardiography were continuously monitored andrecorded in an electronic medical record system. Physicalexamination and chest radiography with blood gas analysiswere performed within 12 hours before and after MSCtransplantation (and reassessed as necessary). Cranialultrasonography and echocardiography were performedat baseline (screening point); at days 2, 7, and 28posttransplantation; and at 36 weeks corrected gestationalage. Routine laboratory tests were performed as usual.Tracheal aspirate fluid was collected at baseline and at day3, 7, 14, and 28 posttransplantation only if the patientremained intubated on those days.

To assess the incidence of adverse events or outcomes forfurther safety and feasibility evaluation, we enrolled 2matching neonate comparisons for every study case retro-spectively after completion of the trial. For this nestedcomparison group, we selected 2 matched infants for eachMSC transplantation patient according to the followingcriteria: gestational age within 3 days, birth weight within50 g, and similar ventilator modes and mean respiratoryseverity scores (mean airway pressure � fraction of inspiredoxygen) within 24 hours before MSC transplantation.Because of the logistical demands of matching birth weight,gestational age, respiratory severity scores, and ventilatormode, the matched comparison group infants were notconsecutive cases. They were born and cared for at SamsungMedical Center between January 2009 and November 2011.

Infants from both theMSC-treated group and the matchedcomparison group were evaluated for respiratory distresssyndrome, mechanical ventilator duration (invasive/nonin-vasive), respiratory severity score, BPD, IVH (grade $3),4

PDA, blood culture–confirmed sepsis, retinopathy of prema-turity,2 necrotizing enterocolitis (Bell stage $2b),3 and peri-ventricular leukomalacia.

Principles of Neonatal Intensive Care ManagementThere were no obvious changes in the neonatal intensive caremanagement policies, including ventilation guidelines of88%-95% of target saturation and 44-55 mmHg of targetPCO2, extubation criteria, PDA ligation, or postnatal steroiduse, during the period January 2009 to November 2011, whenthe MSC transplantation and matched comparison group in-fants were born and cared for. In this study, postnatal steroid

treatment was reserved for preterm infants who weredependent on invasive ventilation at or after the third weekof life with deteriorating respiratory condition, who are atthe greatest risk for developing BPD and may benefitfrom steroid treatment.7,8 A high-dose (0.5 mg/kg/day) orlow-dose (0.2 mg/kg/day) dexamethasone regimen wasstarted at the discretion of the attending neonatologist, andthen tapered within 7-10 days. An additional course ofdexamethasone was considered based on the infant’s clinicalcondition.PDA ligation was reserved for only those preterm infants

dependent on mechanical ventilation with cardiovascularcompromise, such as persistent hypotension requiringinotropics and/or deteriorating respiratory condition,usually after failure of medical treatment.9

Tracheal Aspirate Fluid Analysis

Tracheal aspirate fluid samples were obtained twice by suc-tioning the major airways after 0.5 mL of saline had beeninstilled into the endotracheal tube. Samples were collectedonly when suctioning was clinically required during routinecare. The supernatant was frozen at �70�C after centrifuga-tion at 15 000 rpm for 10 minutes. Levels of HGF, TGF-b1,and MMP-9 were calculated by enzyme immunoassay usingthe Quantikine Kit (R&D Systems, Minneapolis, Minnesota).IL-1, IL-6, IL-8, IL-10, TNF-a, and VEGF were measuredwith the Milliplex MAP ELISA Kit (Millipore, Billerica, Mas-sachusetts), 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 retinop-

athy 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, OhW, 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.

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Figure 2. Temporal profiles of heart rate, oxygen saturation, mean airway pressure, and fraction of inspired oxygen before andafter hUCB-derived MSC transplantation in the 9 study patients up to 24 hours posttransplantation.

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Figure 3. Chest radiographs obtained at 84 days posttransplantation in all 9 study patients showing comparable findings inboth 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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Figure 5. Levels of cytokines and growth factors from tracheal aspirate fluid collected beforeMSC transplantation and at 3 daysand 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.

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Table II. Estimated risk of death or moderate/severe BPD in study patients at enrollment

Patient number

A1 A2 A3 B1 B2 B3 B4 B5 B6

Gestational age, wk 25 25 24 24 24 25 25 26 26Birth weight, g 770 870 720 630 740 850 650 1030 880Estimated risk of deathor moderate/severeBPD at enrollment, %*Hispanic 74.9 76 89.2 78.7 78.7 54.1 66.1 71.4 78White 65.3 66.5 91.1 82.2 82.3 60.7 71.5 76.7 82Black 62.6 63.4 91.4 82.3 82 57.6 69.6 74.8 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 groupMatched

comparison group,total (n = 18) P value

Low-dose(1 � 107 cells/kg) (n = 3)

High-dose(2 � 107 cells/kg) (n = 6) Total (n = 9)

Gestational age, wk, mean � SD 25.3 � 0.7 25.3 � 0.9 25.3 � 0.9 25.3 � 1.0 .60Birth weight, g, mean � SD 787 � 76 797 � 153 793 � 127 795 � 99 .94Apgar score, 1 min, mean � SD 5.0 � 1.0 5.3 � 2.4 5.2 � 2.0 4.5 � 1.4 .34Apgar score, 5 min, mean � SD 7.6 � 1.2 7.0 � 1.8 7.2 � 1.6 7.2 � 1.4 .93Female sex, n (%) 2/3 3/6 4 (44) 13 (72) .38Cesarean delivery, n (%) 3/3 4/6 7 (78) 14 (78) 1.00Antenatal corticosteroid use, n (%) 3/3 4/6 7 (78) 15 (83) 1.00Pathological chorioamnionitis, n (%) 2/3 2/6 2 (22) 7 (39) 1.00Respiratory distress syndrome, n (%) 3/3 6/6 9 (100) 18 (100)PDA, n (%) 3/3 6/6 9 (100) 18 (100)Medication (before MSC transplantation or index day) 2/3 3/6 5 (56) 14 (78) .43Operation (before MSC transplantation or index day) 2/3 3/6 5 (56) 9 (50) 1.00

Early-onset sepsis, n (%) 0/3 1/6 1 (11) 1 (6) .67IVH (grade $3), n (%) 0/3 0/6 0 (0) 0 (0)Age at MSC transplantation or index day for either casesor comparisons, postnatal d, mean � SD

11.3 � 3.8 10.8 � 2.5 10.4 � 2.6 11.0 � 2.8 .80

Mean respiratory severity score (1 day before MSCtransplantation or index day), mean � SD

2.1 � 0.4 3.4 � 2.8 2.9 � 0.9 2.5 � 1.1 .10

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