Mesenchymal Stem Cells for Bronchopulmonary Dysplasia: Phase 1 Dose-Escalation Clinical Trial

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

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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/).

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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% 968

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

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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.

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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

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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

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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

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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.

References 16. 1. Stoll BJ, Hansen NI, Bell EF, Shankaran S, Laptook AR, Walsh MC, et al. Neonatal outcomes of extremely preterm infants from the NICHD Neonatal Research Network. Pediatrics 2010;126:443-56. 2. Avery ME, Tooley WH, Keller JB, Hurd SS, Bryan MH, Cotton RB, et al. Is chronic lung disease in low birth weight infants preventable? A survey of eight centers. Pediatrics 1987;79:26-30. 3. Bregman J, Farrell EE. Neurodevelopmental outcome in infants with bronchopulmonary dysplasia. Clin Perinatol 1992;19:673-94. 4. Bland RD. Neonatal chronic lung disease in the post-surfactant era. Biol Neonate 2005;88:181-91. 5. Bhandari A, Panitch HB. Pulmonary outcomes in bronchopulmonary dysplasia. Semin Perinatol 2006;30:219-26. 6. Jobe AH. The new bronchopulmonary dysplasia. Curr Opin Pediatr 2011;23:167-72. 7. Chang YS, Oh W, Choi SJ, Sung DK, Kim SY, Choi EY, et al. Human umbilical cord blood–derived mesenchymal stem cells attenuate hyperoxia-induced lung injury in neonatal rats. Cell Transplant 2009; 18:869-86. 8. 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 hyperoxiainduced lung injury in neonatal rats. Cell Transplant 2011;20:1845-54. 9. Chang YS, Choi SJ, Ahn SY, Sung DK, Sung SI, Yoo HS, et al. Timing of umbilical cord blood–derived mesenchymal stem cells transplantation determines therapeutic efficacy in the neonatal hyperoxic lung injury. PLoS ONE 2013;8:e52419. 10. van Haaften T, Byrne R, Bonnet S, Rochefort GY, Akabutu J, Bouchentouf M, et al. Airway delivery of mesenchymal stem cells

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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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Vol. 164, No. 5 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.

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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.

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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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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.

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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

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