Adult Mesenchymal Stromal Cells to Regenerate the Neonatal Brain: the PASSIoN Trial (Perinatal Arterial Stroke Treated With Stromal Cells IntraNasally)
Phase: PHASE1
Conditions: Perinatal Arterial Ischemic Stroke, Neonatal Stroke
Sponsor: UMC Utrecht

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RESEARCH PROTOCOL 
Version 5.1 , April 2020 
 
 
 
Adult mesenchymal stromal cells to  
regenerate the neonatal brain: the PASSIoN trial  
(Perinatal Arterial Stroke treated with Stromal cells 
IntraNasally) 

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PROTOCOL TITLE ‘Adult mesenchymal stromal cells to regenerate the neonatal brain: the 
PASSIoN trial (Perinatal Arterial Stroke treated with Stromal cells IntraNasally)’ 
 
Protocol ID 
NL59265.000.16  (previous NL48431.000.14) 
Short title 
PASSIoN 
EudraCT number  
2014-001912-20  
Version 
5.1 
Date 
16-04-2020 
Coordinating investigator/ 
project leader 
Manon Benders, MD PhD  
Department of Neonatology, UMCU 
Lundlaan 6 
3584 EA Utrecht  
Internal mail no KE 04.123.1  
P.O. Box 85090 
3508 AB  Utrecht 
Telephone: +31 (0)88 7554546 
Principal investigators   
 
Manon Benders, MD PhD  
Department of Neonatology, UMCU 
Lundlaan 6 
3584 EA Utrecht  
Internal mail no KE 04.123.1  
P.O. Box 85090 
3508 AB  Utrecht 
Telephone: +31 (0)88 7554546 
 
Niek van der Aa, MD, PhD 
Department of Neonatology, UMCU 
Lundlaan 6 
3584 EA Utrecht  
Internal mail no KE 04.123.1  
P.O. Box 85090 
3508 AB  Utrecht 
Telephone: +31 (0)88 7554546 
 

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Research Group 
Dept. of Neonatology, UMCU: 
Frank van Bel, MD PhD 
Linda de Vries, MD PhD 
Floris Groenendaal, MD PhD 
Nienke Wagenaar, MD 
Lisanne Baak, MD 
 
NIDOD Laboratory: 
Cora Nijboer, PhD 
 
MD Anderson Cancer Center, Houston: 
Cobi Heijnen, PhD  
Annemiek Kavelaars, PhD 

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Sponsor  
University Medical Center Utrecht 
Division Woman and Baby 
Lundlaan 6 
3584 EA Utrecht 
Subsidising party 
Zon Mw 
Laan van Nieuw Oost-Indië 334 
2593 CE Den Haag 
Independent physician 
Laurens Jaap Kappelle, MD, PhD 
Dept. of Neurology, UMCU 
Heidelberglaan 100 
3584 CX Utrecht 
Internal mail no. G.03.232  
P.O. Box 85500 
3508 GA Utrecht 
Telephone: 088-7556865  
Email:  l.kappelle@umcutrecht.nl 
Laboratory sites  
NIDOD laboratory 
Division Woman and baby UMCU 
Internal mail no. KC 03.068.0 
Lundlaan 6 
3584 EA Utrecht 
Pharmacy  
Cell Therapy Facility, UMCU 
Heidelberglaan 100 
3584 CX Utrecht 
Contact: Prof. dr. A.C.G. Egberts 
A.C.G.Egberts@umcutrecht.nl  
GCP STATEMENT 
 
This trial will be conducted in compliance with  this protocol, Good Clinical Practices and applicable regulatory requirements. 
 
CONFIDENTIALITY STATEMENT 
 
The information in this document contains trade secrets and commercial information that are privileged or confidential and may 
not be disclosed unless such disclosure is required by law. In any event, persons to whom the information is disclosed must be 
informed that the information is privileged or confidential and may not be further disclosed by them. These restrictions on 
disclosure will apply to all future information supplied to you which is indicated as privileged or confidential. 

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PROTOCOL SIGNATURE SHEET 
 
Name 
Signature 
Date 
Sponsor or legal representative: 
UMC Utrecht 
 
For non-commercial research, 
Head of Department: 
M.J.N.L Benders, MD PhD 
Head of Department Neonatology, UMCU 
 
16-04-2020 
Coordinating Investigator/Project 
leader/Principal Investigator: 
M.J.N.L Benders, MD PhD 
Head of Department Neonatology, UMCU 
 
16-04-2020 
 
 

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TABLE OF CONTENTS 
 
INTRODUCTION AND RATIONALE ..................................................................................... 12 
1. 
OBJECTIVES ................................................................................................................. 18 
2. 
STUDY DESIGN ............................................................................................................. 19 
2.1 
Description ............................................................................................................... 19 
2.2 
Duration ................................................................................................................... 19 
2.3 
Setting ..................................................................................................................... 19 
2.4 
Study Procedures .................................................................................................... 19 
2.5 
Schematic diagram of study design ......................................................................... 22 
3. 
STUDY POPULATION ................................................................................................... 23 
3.1 
Study Population (base) .......................................................................................... 23 
3.2 
Inclusion criteria ....................................................................................................... 23 
3.3 
Exclusion criteria ..................................................................................................... 23 
3.4 
Sample size calculation ........................................................................................... 23 
4. 
TREATMENT OF SUBJECTS ........................................................................................ 25 
4.1 
Investigational product/treatment ............................................................................ 25 
4.2 
Use of co-intervention (if applicable) ....................................................................... 25 
4.3 
Escape medication (if applicable) ............................................................................ 25 
5. 
INVESTIGATIONAL PRODUCT ..................................................................................... 26 
5.1 
Name and description of investigational product(s) ................................................. 26 
5.2 
Summary of findings from non-clinical studies ........................................................ 26 
5.3 
Summary of findings from clinical studies ............................................................... 26 
5.4 
Summary of known and potential risks and benefits ............................................... 26 
5.5 
Description and justification of route of administration and dosage ........................ 29 
5.5.1 
Description and justification of source and nature of MSCs ............................. 31 
5.6 
Dosages, dosage modifications and method of administration ............................... 32 
5.7 
Preparation and labelling of Investigational Medicinal Product ............................... 32 
5.8 
Drug accountability .................................................................................................. 32 
6. 
METHODS ...................................................................................................................... 33 
6.1 
Study parameters/endpoints .................................................................................... 33 
6.1.1 
Main study parameter/endpoint ........................................................................ 33 
6.1.2 
Secondary study parameters/endpoints ........................................................... 33 
6.1.3 
Other study parameters .................................................................................... 34 
6.2 
Randomisation, blinding and treatment allocation ................................................... 34 
6.3 
Study procedures .................................................................................................... 34 
6.4 
Withdrawal of individual subjects ............................................................................. 35 
6.4.1 
Specific criteria for withdrawal .......................................................................... 35 
6.5 
Replacement of individual subjects after withdrawal ............................................... 35 
6.6 
Follow-up of subjects withdrawn from treatment ..................................................... 35 
6.7 
Premature termination of the study ......................................................................... 35 
The study may be terminated prematurely for safety or futility reasons if there are AEs or 
SAEs as defined by chapter 7. ........................................................................................... 35 

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We will use the following stopping rules: ............................................................................ 35 
7. 
SAFETY REPORTING ................................................................................................... 37 
7.1 
Section 10 WMO event ............................................................................................ 37 
7.2 
AEs, SAEs and SUSARs ......................................................................................... 37 
7.2.1 
Adverse events (AEs) ....................................................................................... 37 
7.2.2 
Serious adverse events (SAEs) ....................................................................... 37 
7.2.3 
Suspected unexpected serious adverse reactions (SUSARs) ......................... 38 
7.3 
Annual safety report ................................................................................................ 39 
7.4 
Follow-up of adverse events .................................................................................... 39 
7.5 
Data Safety Monitoring Board (DSMB) .................................................................... 39 
8. 
STATISTICAL ANALYSIS .............................................................................................. 41 
8.1 
Study parameter(s) .................................................................................................. 41 
8.2 
Other study parameters ........................................................................................... 41 
8.3 
Interim analysis (if applicable) ................................................................................. 41 
9. 
ETHICAL CONSIDERATIONS ....................................................................................... 42 
9.1 
Regulation statement ............................................................................................... 42 
9.2 
Recruitment and consent ......................................................................................... 42 
9.3 
Objection by minors or incapacitated subjects (if applicable) .................................. 42 
9.4 
Benefits and risks assessment, group relatedness ................................................. 42 
9.5 
Compensation for injury ........................................................................................... 43 
9.6 
Incentives (if applicable) .......................................................................................... 43 
10. 
ADMINISTRATIVE ASPECTS, MONITORING AND PUBLICATION ......................... 44 
10.1 
Handling and storage of data and documents ......................................................... 44 
10.2 
Monitoring and Quality Assurance ........................................................................... 44 
10.3 
Amendments ........................................................................................................... 44 
10.4 
Annual progress report ............................................................................................ 44 
10.5 
End of study report .................................................................................................. 45 
10.6 
Public disclosure and publication policy .................................................................. 45 
There are no specific agreements concerning public disclosure or publication policy. ...... 45 
11. 
STRUCTURED RISK ANALYSIS ............................................................................... 46 
11.1 
Potential issues of concern ...................................................................................... 46 
11.2 
Synthesis ................................................................................................................. 53 
12. 
REFERENCES ............................................................................................................ 56 

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LIST OF ABBREVIATIONS AND RELEVANT DEFINITIONS  
 
ABR 
ABR form, General Assessment and Registration form, is the application 
form that is required for submission to the accredited Ethics Committee (In 
Dutch, ABR = Algemene Beoordeling en Registratie) 
ADC 
AE 
Apparent diffusion coefficient 
Adverse Event 
AR 
BSITD-III 
BPD 
Adverse Reaction 
Bayley Scales of Infant and Toddler development version 3   
Bronchopulmonary dysplasia 
CA 
Competent Authority 
CCMO 
 
CTF 
Central Committee on Research Involving Human Subjects; in Dutch: 
Centrale Commissie Mensgebonden Onderzoek 
Cell Therapy Facility 
CV 
Curriculum Vitae 
DSMB 
DTI 
DWI 
Data Safety Monitoring Board 
Diffusion Tensor Imaging 
Diffusion Weighted Imaging 
EU 
European Union 
EudraCT 
FA 
European drug regulatory affairs Clinical Trials  
Fractional Anisotrophy 
GCP 
GMFCS 
GVHD 
HAI 
HI 
Good Clinical Practice 
Gross Motor Function Classification System  
Graft-versus-host disease  
Hand Assessment in Infancy 
Hypoxic-ischemia 
HINE 
IB 
Hammersmith Infant Neurological Examination 
Investigator’s Brochure 
IC 
Informed Consent 
IMP 
Investigational Medicinal Product  
IMPD 
MCA 
Investigational Medicinal Product Dossier  
Middle cerebral artery 
METC 
 
MSC  
NNRN 
Medical research ethics committee (MREC); in Dutch: medisch ethische 
toetsing commissie (METC) 
Mesenchymal stromal cell 
Dutch Neonatal Research Network; in Dutch: Nederlands Neonataal 

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Research Netwerk 
(S)AE 
(Serious) Adverse Event  
SPC 
Summary of Product Characteristics (in Dutch: officiële productinfomatie 
IB1-tekst) 
Sponsor 
The sponsor is the party that commissions the organisation or performance 
of the research, for example a pharmaceutical 
company, academic hospital, scientific organisation or investigator. A party 
that provides funding for a study but does not commission it is not 
regarded as the sponsor, but referred to as a subsidising party. 
SUSAR 
PAIS 
UMCU 
Suspected Unexpected Serious Adverse Reaction 
Perinatal arterial ischemic stroke 
University Medical Centre Utrecht 
Wbp 
WKZ 
Personal Data Protection Act (in Dutch: Wet Bescherming Persoonsgevens) 
Wilhelmina Children’s Hospital 
WMO 
Medical Research Involving Human Subjects Act (in Dutch: Wet Medisch-
wetenschappelijk Onderzoek met Mensen 
 
 

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SUMMARY 
 
Rationale: Perinatal arterial ischemic stroke (PAIS) is an important perinatal cause of long-
lasting neurodevelopmental problems. Recent studies report an incidence of PAIS of 1 per 
2300 full-term infants born alive. Adverse consequences of PAIS include hemiplegia, 
cognitive dysfunction, epilepsy and speech problems. In 50-75% of infants, neonatal stroke 
leads to abnormal neuromotor and -developmental outcome or epilepsy. The estimated 
annual mortality rate of neonatal stroke is 3.49/100,000 annually. Current treatment options 
for PAIS mainly focus on controlling convulsions and associated infections. There is no 
treatment available that leads to reduction of neonatal brain damage in this severely affected 
group of infants. This leads to life-long consequences of PAIS and forms a large burden for 
patients and society. The overall aim of this project is to meet this need by developing a cell 
based treatment strategy.  
Animal models of neonatal brain injury provide evidence for the feasibility and efficacy of 
intranasal mesenchymal stromal cell (MSC) application in the treatment of PAIS. Additionally, 
results from human trials with MSCs in the treatment of adult stroke or other pathologic 
conditions provide evidence that MSC treatment is safe. This project aims at making the first 
step towards clinical application of MSCs to treat PAIS. Successful completion of this project 
will provide the first evidence of the safety and feasibility of MSCs to treat brain damage in 
newborn infants. 
Objective: This study will assess safety and feasibility of bone marrow-derived allogeneic 
MSCs, administered by the nasal route, in neonates who suffered from PAIS.  
Study design: A phase I/II, open-label, single-arm, single-center intervention study in the 
NICU at the Wilhelmina children’s Hospital / University Medical Centre in Utrecht of (near-
)term newborns ≥36 weeks of gestation within the first week of onset of presenting clinical 
symptoms.  
Study population: All (near-)term newborns ≥36 weeks of gestation with or without clinical 
symptoms of PAIS but with a magnetic resonance imaging (MRI) confirmed PAIS (in the 
MCA region) will be eligible for this study. Following written parental consent, 10 patients will 
be included in our study. 
Intervention: One dose of 50x10^6 MSCs via the nasal route as soon as possible after 
confirmation of the MCA-stroke, but within the first week of onset of presenting clinical 
symptoms. Within 30 minutes after cleaning the nose with saline, using standard procedures 
operative at the NICU, the MSC will be delivered.  
Main study parameters/endpoints: Our primary objective is to determine if MSC treatment 
in neonates with PAIS is safe in the acute setting, including vital signs at time of treatment, 
blood sampling before and after MSC treatment and occurrence of any adverse events. 

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Secondly we will investigate subacute and long-term safety of MSC treatment at 3 months, in 
terms of SAEs (such as infections) and cerebral tumorigenicity on MRI. Follow-up 
assessment at 3 months is part of regular care for neonates with PAIS.  
Nature and extent of the burden and risks associated with participation, benefit and 
group relatedness: The extra burden of the present study for the included infants is 
considered to be very limited to non-existent given the fact that besides the nasal treatment 
to apply the MSCs, treatment is not different compared to the standard acting treatment 
protocol for newborns with PAIS. Nasal application of MSCs has been considered as non-
invasive. With respect to possible risks of the present MSC treatment, the most important 
potential risk factors such as inflammatory actions of MSC therapy with allogeneic human 
MSCs and an increased risk for malignancies, have been intensively investigated in our own 
preclinical studies and appear to be non-present at long-term follow up. Additionally, human 
trials on MSC treatment for adult stroke or other pathologic conditions do not provide 
evidence for these or other serious adverse events or risks. No single indication has been 
found in experimental research in the developing animal models that above mentioned 
complications occur in a higher incidence as compared to non-MSC treated animals, 
whereas possibility for a substantial better short- and long-term outcome seems very realistic 
on the basis of previous experimental research data. This study will assess safety bone 
marrow-derived allogeneic MSCs, administered by the nasal route, in human neonates with 
PAIS. 

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INTRODUCTION AND RATIONALE 
 
Introduction 
Perinatal arterial ischemic stroke (PAIS) or cerebral infarction is an important perinatal cause 
of long-lasting neurodevelopmental problems. Recent studies report an incidence of PAIS of 
1 per 2300 full-term live births.(Kirton & DeVeber 2009) PAIS has been diagnosed mostly in 
the first week of life.  
 
PAIS occurs most often in de middle cerebral artery (MCA) with a predilection for the left 
MCA.(Fernández-López et al. 2014; Benders et al. 2014; Kirton et al. 2011; van der Aa et al. 
2014) The most important risk factors are associated with pathological changes that manifest 
during pregnancy and delivery. Important maternal risk factors include gestational diabetes, 
pregnancy-induced hypertension and pre-eclampsia, while perinatal factors associated with 
neonatal stroke are ventouse delivery, emergency Caesarean section, perinatal infections 
and hypoglycaemia.(Fernández-López et al. 2014; Kirton et al. 2011; Harteman et al. 2012; 
Martinez-Biarge et al. 2016) Placental pathology has also been linked to PAIS.(Elbers et al. 
2011) Coagulation disorders are associated with neonatal stroke, such as genetic 
polymorphisms of prothrombotic factors (e.g. Factor V Leiden, prothrombin, lipoprotein (a), 
methylenetetra-hydrofolate reductase or acquired prothrombotic states (e.g. activated protein 
C). These factors are increasing the risk for PAIS when combined with other risk factors such 
as perinatal asphyxia, congenital heart diseases or neonatal infections.(Fernández-López et 
al. 2014)   
 
The disease manifests itself most often with one-sided convulsions in the first week after 
birth, often accompanied with (asymmetric) hypotonia, lethargy and apnea.(Kirton et al. 
2011; van der Aa et al. 2014) When stroke is suspected clinically and on cranial ultrasound, 
this is usually confirmed by magnetic resonance imaging (MRI). Neuromotor outcome 
becomes more evident over time and depends largely on the involved cerebral structures. 
Especially involvement of the internal capsule and the basal ganglia is associated with 
impaired motor outcome.(figure 1) 
 
Figure 1: Representative MRI of a full-term infant 
with a main branch MCA infarct on the left. The 
first scan was performed during the first week. 
The diffusion weighted images (left) shows 
restricted diffusion of the cortex, subcortical white 
matter and basal ganglia. A second MRI at 3 
months of age (right) shows a large cavity with 
lack of myelination of the posterior limb of the 
internal capsule. 

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Adverse consequences of PAIS include unilateral spastic cerebral palsy (USCP) cognitive 
dysfunction, epilepsy and speech problems. In 50-75% of infants, neonatal stroke leads to 
abnormal neuromotor and -developmental outcome or epilepsy.(Fernández-López et al. 
2014) Recurrent seizures after PAIS are also often reported, with larger areas of stroke being 
more likely to be associated with postneonatal epilepsy and lower cognitive outcome. 
(Wusthoff et al. 2011; van Buuren et al. 2013) The estimated annual mortality rate of 
neonatal stroke is 3.49/100,000.(Lynch & Nelson 2001) 
 
In adult stroke, treatment focuses on thrombolytic therapy and is, after exclusion of 
hemorrhagic stroke, standard part of care given within a timeframe of 6-8 hours after the 
event. Newborns usually present with less specific symptoms, which results in a later 
diagnosis of PAIS compared to adult stroke. Since neonatal stroke is usually diagnosed later, 
early treatment options, such as hypothermia used in perinatal asphyxia, with a therapeutic 
window of six hours after birth, cannot be used in neonatal stroke. Current treatment options 
for PAIS mainly focus on supportive care, such as controlling hypoglycaemia, treatment of 
convulsions and associated infections. There is no specific treatment available that leads to 
reduction of neonatal brain damage in this severely affected group of infants. This leads to 
life-long consequences of PAIS and forms a large burden for patients and society. The 
overall aim of this project is to meet this need by assessing the safety and feasibility of  a cell 
based treatment strategy for neonates with PAIS. 
 
Rationale 
 
Mesenchymal stromal cells (MSCs) can be isolated from embryonic and fetal tissues, such 
as placental tissue, fetal blood, umbilical cord blood and stroma (Wharton’s Jelly). 
Furthermore, they can also be isolated from adult tissues like bone marrow. MSCs can 
differentiate into mesodermal tissue cells (e.g. bone, cartilage, fat), but in vitro experiments 
have demonstrated that MSCs are, during and after hypoxia-ischemia, also capable to 
stimulate the formation of neurons, astrocytes, oligodendrocytes and microglia.(Kohyama et 
al. 2001; Dezawa et al. 2004)  
 
From neonatal rat models of brain injury, we have learned that hypoxic- ischemic (HI) brain 
injury induces changes in neurovascular environment that promote neurogenesis.(Kadam et 
al. 2008; Ong et al. 2005; Yang & Levison 2007; Donega, van Velthoven, Nijboer, Kavelaars, 
et al. 2013) However, after HI brain injury, there is no induction and maintenance of long-
term neurogenesis. Therefore it is crucial to assist the brain in regeneration after injury and 

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also to reinforce development of the maturating brain. In animal HI-model studies, it has 
been demonstrated that transplantation of murine adult bone marrow-derived MSCs 
markedly improves neuroregeneration. Treatment with MSCs stimulated formation of new 
neurons and oligodendrocytes and reduced infarct size by >45%. Although intracranial MSC 
treatment stimulated formation of new brain cells and differentiation of these cells into new 
neurons and oligodendrocytes, these newly formed cells were not of transplant origin. These 
findings indicate that the neuroregenerative effect of MSCs after ischemic brain damage 
mainly relies on stimulation of endogenous repair mechanisms.(van Velthoven et al. 2010c) 
This is confirmed by data from our group that show that MSC treatment has profound effects 
on the expression of growth and differentiation factors in the brain. For example, MSC 
treatment at 10 days after HI induces upregulation of 35 genes encoding factors that are 
mainly involved in cell growth and proliferation. MSC treatment at 3 and 10 days after 
induction of HI brain damage induces expression of 28 additional factors and these are 
predominantly involved in cell differentiation and nervous system development.(van 
Velthoven et al. 2010c; van Velthoven et al. 2011) These results  show that the first dose of 
MSC stimulates cerebral cell proliferation, whereas the second injection enhances 
differentiation and maturation of newly formed cells and integration in a functional 
network.(van Velthoven et al. 2010c) 
 
Based on these findings, we hypothesized that MSC of donor origin will only transiently 
survive in the brain and that the neuroregenerative effects of MSC are mediated 
predominantly by inducing changes in the growth factor milieu of the brain. 
The corticospinal tract is the primary transmission route for voluntary movement of the 
forepaws. We showed that MSC treatment could also partially restore the damaged 
corticospinal motor tract which led to improved sensorimotor outcome.(van Velthoven et al. 
2010c; van Velthoven et al. 2010a)  
 
MSCs have many advantages over other types of stem cells or progenitors. First of all, they 
can easily be obtained from infants and adults.(Le Blanc & Mougiakakos 2012) Additionally, 
as described above, MSC treatment have a long therapeutic window, as they can be 
administered at least until 10 days after brain injury to improve behavioral outcome and 
lesion volume in neonatal HI mice. This is of great value, since the only available therapies 
for HI injury have to be given within a few hours. Besides, MSCs are hardly detectable in the 
brain after 3 days (Donega, van Velthoven, Nijboer, van Bel, et al. 2013), so the risk of Host-
versus-Graft is very low. It is also very unlikely that MSCs will cause other adverse effects if 
they are no longer present in the brain after a short period of time. Our most recent study 
showed no malignancies in the brains or in other organs of mice treated with MSCs when 

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assessed at 14 months after start of MSC treatment at 10 days after neonatal HI.(Donega et 
al. 2015)  Furthermore, MSCs have low immunogenicity because they do not express major 
histocompatibility complex (MHC) class-II antigens.(Rocha et al. 2000; Uccelli et al. 2008; 
Fleiss et al. 2014) This makes them excellent therapeutic candidates for allogeneic 
treatment.  
 
Another advantage of MSCs, is that they have proven to be effective in brain injury when 
administered intranasally. This is beneficial since it has been reported that systemically 
administered human bone marrow-derived MSCs migrate into peripheral organs but were not 
able to induce neuroprotective effects in the brain after cerebral ischemia.(Steiner et al. 
2012) After systemic administration MSCs will also migrate to peripheral organs, leading to 
loss of MSCs in these organs (trapping).(Li et al. 2015; Liu 2011; Jiang et al. 2011) This may 
result in reduced migration of MSCs into the brain and will consequently diminish their 
neuroregenerative effects on the brain.(Li et al. 2015; Liu 2011; Jiang et al. 2011; Steiner et 
al. 2012) In newborns, trapping of MSCs in other organs will be more important than in 
adults. MSCs are attracted by areas of injury in the organ systems,(Wang et al. 2014) PAIS 
is related to fetal distress and subsequent hypoxia-ischemia.(Harteman et al. 2012; Martinez-
Biarge et al. 2016) Other risk factors for PAIS are maternal fever, hypoglycemia and early-
onset sepsis/meningitis. (Harteman et al. 2012) These risk factors, as well as hypoxia-
ischemia, may lead to diffuse damage to other organ systems. When MSCs are administered 
systemically, they may be attracted by systemic damage, leading to even more trapping of 
the MSCs in other injured organs. This makes it difficult to predict if and how many MSCs will 
migrate to the brain when given systemically. Studies from our group and others have shown 
that MSCs when given intranasally migrate within two hours after administration to the 
ischemic brain region.(Donega, Nijboer, van Tilborg, et al. 2014) Intranasal administration of 
MSCs reduces lesion volume loss and improves sensorimotor outcome.(van Velthoven et al. 
2010b; Wei et al. 2015). Efficacy of intranasal versus intracranial MSC treatment on lesion 
size and motor behavior was similar in the neonatal HI mouse model.(van Velthoven et al. 
2012)  Studies of our group in the newborn baboon have shown that intranasally 
administered MSCs have the potential to migrate to the injured brain region of primates 
(unpublished observations), in accordance with earlier shown studies in mice and rats. 
Overall, the intranasal administration route provides an effective alternative allowing MSC 
treatment for brain injury without invasive methods and minimal burden for the patient.   
 
Even though all evidence on efficacy and feasibility of MSCs in the treatment of neonatal 
brain injury is based on animal models, there is evidence that human MSCs have the same 
potential. Human MSCs were found to improve sensorimotor function and decrease gray and 

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white matter volume loss in our mouse model of HI brain injury.(Donega, Nijboer, Braccioli, et 
al. 2014) These findings are comparable to those of murine MSCs and strongly support the 
therapeutic potential of hMSCs for neonatal brain injury. Additionally, this study confirms the 
efficacy of (human) MSCs when administered intranasally.  
 
There are no clinical trials published on MSC treatment for neonatal stroke. However, there 
are some studies available describing the treatment of adult stroke with MSCs. These 
studies may support the evidence for the use of MSCs in neonatal stroke, but it is important 
to realize that results from adult trials can not be directly translated to neonatal care for 
several reasons. First of all, the newborn brain has much better plasticity capacity than the 
brain of older children and adults. This causes the newborn to recover more easily after 
(severe) brain injury than an adult.(Bower 1990; Schneider 1970) Secondly, effectiveness of 
MSC treatment reduces with increasing age, because endogenous neurogenesis capacity 
also reduces with age. This causes neonates to have more potential to regenerate their 
damaged neurons and/or networks compared to adults.(Titomanlio et al. 2011; Bondolfi et al. 
2004; Kuhn et al. 1996; Leuner et al. 2007) Lastly, neonatal stroke (PAIS) is a different 
disease, with different pathology and symptomatology, than adult stroke.(Comi & Johnston 
2009) 
 
However, the results from adult trials can be used to support safety and feasibility of MSC 
treatment in neonatal stroke. Clinical trials with MSC treatment for adult patients with 
ischemic stroke have shown some evidence for the efficacy of MSCs in the treatment of 
ischemic stroke. These studies described patients who were treated with MSC at 7-133 days 
post-stroke.(Bang et al. 2005; Honmou et al. 2011) Although the evidence is sparse, there 
are some trials, mainly uncontrolled open-label studies, that have demonstrated clinical 
neurological improvement in patients with acute and chronic stroke after treatment with 
MSCs.(Bang et al. 2005; Lee et al. 2010) Most importantly, these trials have not 
demonstrated any serious adverse events, such as neurological deterioration, systemic 
infections nor tumor development, related to MSC transplantation. This is also concluded 
from a meta-analysis on over 1200 patients treated with MSCs for various pathological 
indications. Only transient fever within the first days seemed to be related to MSC 
transfusion.(Lalu et al. 2012)  
There is only one clinical trial describing the use of MSCs in treatment of neonates.(Chang et 
al. 2014) Chang et al. assessed in a phase I dose-escalation trial the safety and feasibility of 
intratracheal administration of allogeneic umbilical cord-derived MSCs in preterm infants with 
high risk for bronchopulmonary dysplasia (BPD). Importantly, there were no serious adverse 
events or toxicity related to a higher dose of 2 x 10^7 cells/kg.(Chang et al. 2014) These 

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findings were more recently confirmed by another group treating 3 premature infants with 
approximately 2 x 10^6/kg bone marrow-derived MSCs intravenously.(Henckel et al. 2015) 
The authors concluded that intravenous MSC treatment was feasible, well tolerated and no 
effects on pulmonary and cerebral blood flow could be detected.(Henckel et al. 2015) An 
ongoing study in Korea is treating preterm infants with intraventricular hemorrhage with 
allogeneic umbilical cord MSCs, and preliminairy results are promising and show no adverse 
effects.(Presented at Pediatric Academic Societies Meeting, Baltimore, Monday May 2, 2016 
- 8:00 am, E-PAS2016:3090) 
Overall, MSC treatment under several pathologic conditions, including stroke, in adults and 
children appeared to be safe.  
 
In summary, based on the available evidence there seems to be a solid basis for clinical 
translation. Animal models of neonatal brain injury provide evidence for the feasibility, 
efficacy and safety of intranasal MSC application in the treatment of PAIS. Additionally, 
results from human trials with MSCs in the treatment of adult stroke or other pathologic 
conditions, including BPD in neonates, provide evidence that MSC treatment is safe. This 
project aims at making the first step towards clinical application of MSCs to treat PAIS. 
Successful completion of this project will provide the first evidence of safety of MSCs to treat 
brain damage in newborn infants. 

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1. OBJECTIVES 
 
Our primary objective is to determine if MSC treatment, in neonates with PAIS, is safe in the 
acute setting, including vital signs at time of treatment, blood sampling before and after MSC 
treatment and occurrence of any adverse events or toxicity. Secondly we will investigate 
safety of MSC treatment at 3 months, in terms of SAEs and tumorigenicity on MRI.   
 
Primary Objective 
• To determine if MSC treatment in (near-)term infants with PAIS is safe in the acute 
setting. 
Safety is defined primarily as the absence of treatment-related serious adverse events 
(SAEs) according to the Consolidated Standards of Reporting Trials (chapter 7.2), 
secondly as the absence of dose-limiting toxicity, defined as death within 24 hours after 
MSC transplantation or anaphylactic shock related to the MSC administration. At least, all 
patients will be regularly and intensively assessed, including vital signs, before and 24 
hours after treatment, until discharge from our hospital. Also, blood sampling will be 
performed before and 24 hours after treatment with MSCs to determine infection and 
inflammation parameters. Acute safety will be determined until discharge. 
Secondary Objectives  
• To determine if MSC treatment in (near-)term infants with PAIS is safe in the subacute 
setting. 
o 
Subacute safety is defined primarily as the absence of treatment-related 
serious adverse events (SAEs) according to the Consolidated Standards of 
Reporting Trials (chapter 7.2) until the age of 3 months. Patients will then be 
asked to report on other SAEs, including infections.  
• To determine if MSC treatment in (near-)term infants with PAIS is safe at 3 months in 
terms of cerebral tumorigenicity.  
o 
To assess safety of MSC treatment on the brain, infants will be scanned 
using MRI prior to MSC treatment and at 3 months of age. Long-term adverse 
effects on the brain in terms of tumorigenicity will be determined using this MRI, 
which is part of regular stroke follow-up program.   
 

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2. STUDY DESIGN 
 
2.1 Description 
We will perform a single-center, open-label intervention study in the NICU of the Wilhelmina 
Children’s Hospital (WKZ) / University Medical Center in Utrecht (UMCU), the Netherlands to 
investigate the feasibility and safety of neonatal treatment with allogeneic MSCs in (near-) 
term neonates with PAIS. Since this is a phase 1/2 safety/feasibility pilot clinical trial, we will 
assess occurrence of adverse events. 
 
2.2 Duration 
The duration of the study for each subject from inclusion until study end will be a maximum of 
3 months, comprising a 1 week intervention period and follow-up until 3 months. Subject 
recruitment will be (approximately) 12-24 months, at the longest, depending on when the 
intended number of subjects is included. This study is expected to last for approximately 2.5 
years, but will only end after all 10 subjects have been included, treated and have been seen 
for follow-up (MRI and neurodevelopmental testing) at 3 months of age. 
2.3 Setting 
This study will be executed in the NICU of the WKZ/UMCU, the Netherlands, where (near-) 
term infants suspected of PAIS following parental consent for the study  are admitted. Infants 
of the other participating hospitals suspected of having PAIS will be transferred to the UMCU 
for further diagnostic procedures (MRI) and subsequent treatment.  
 
2.4 Study Procedures 
Recruitment and inclusion 
The NICU of the WKZ is known to have most expertise in neonatal stroke. Therefore, infants 
suspected of having PAIS will be transferred from hospitals in our region of care and also 
from other hospitals outside our region, to the UMCU and admitted to our NICU, allowing 
infants to be part of this study. Patients are eligible for our study if they meet our in- and 
exclusion criteria, PAIS may have been suspected with cranial ultrasound and will be 
confirmed within 7 days after onset of symptoms by MRI (including 3D-T1, T2 weighted, 
diffusion weighted imaging [DWI] and DTI sequences) and this PAIS is characterized by a 
predominantly unilateral ischemic lesion within the territory of the MCA, with involvement of 
the corticospinal tracts, cortex, white matter and basal ganglia. Following written parental 
consent, patients will then be included in our study. Parental consent will be obtained as 
soon as possible after admission at our NICU. Patients with PAIS that are diagnosed more 
than one week after birth are not eligible for enrolment.  
 

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Intervention period 
 After inclusion in our study, patients will receive MSCs as soon as possible after 
confirmation of the MCA-stroke, but within the first week of onset of presenting clinical 
symptoms. Steps to be undertaken before MSC administration include:  
- 
A bacterial culture taken from both nostrils before administration of MSCs.  
- 
Blood samples will be collected before administration of MSCs. Blood 
samples are taken routinely in the hospital for clinical purposes, but some extra 
material (0.5-1 mL) will be collected to determine infection markers CRP, 
procalcitonin and complete blood count. These samples will be combined with routine 
blood sampling and taken from routinely placed (arterial) catheters. 
- 
30 minutes prior to delivery of the MSCs, the nose will be cleaned with 
saline using standard procedures operative at the NICU.  
After these procedures have been performed, infants will receive one dose of 50 x 10^6 
MSCs intranasally. We will use a sterile plastic (e.g. 1,0 mL) syringe containing the MSCs to 
slowly drip the MSCs alternatively in both nostrils.  
 
24 hours after MSC administration, blood samples will be collected again. Blood samples are 
taken routinely in the hospital for clinical purposes, but some extra material (0.5-1 mL) will be 
collected to determine infection markers CRP, procalcitonin and complete blood count. 
These samples will be combined with routine blood sampling and taken from routinely placed 
(arterial) catheters. 
 
During their stay on the NICU, vital functions of the infant will be monitored, as part of 
standard clinical care. Cranial ultrasound and neurologic examination will be performed as 
part of standard clinical care for neonates with PAIS. These procedures will at least all be 
performed before and 24 hours after MSC treatment, until discharge from the NICU. 
 
All subjects will remain on the NICU of the study center during treatment and for at least 4 
days after intervention to monitor acute safety. However, if a subject is transferred to a level 
II or level I neonatal unit at another hospital during the intervention period, the protocol will be 
continued under supervision of the principal investigator (PI) and delegated staff. 
 
Safety monitoring  
All potential risks are described in chapter 11.1e. These can be summarized in five 
categories: administration reactions; bacteraemia; systemic infections; post-administration 
viral infections and ectopic tissue formation. For all infants, these risks will be closely 
monitored and planned actions are described in chapter 11.2. Additionally, all infants will be 
monitored more closely by (extra) blood sampling, meaning blood sampling before and 24 

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hours after MSC administration. Also, we will only continue this study after the first three 
infants had their second MRI (at three months of age) to assess the size of the infarction and 
other brain abnormalities on MRI (at three months of age), see paragraph 6.7. 
 
AEs and SAEs occurring during the study will be recorded in the manner described under 
Section 7.2. All SAEs will be followed up until resolution or until the event is considered 
stable. SAEs resulting in a life threatening condition or death will be reported to the 
regulatory authorities and the EC in line with the local regulatory requirements. In addition 
the investigator will report any SAEs resulting in death and/or a life-threatening situation and 
any unexpected related SAEs to the regulatory authorities and the Ethics Committee (EC) in 
line with the local regulatory requirements. An independent data safety and monitoring board 
(DSMB) has, in addition to an internal safety review committee, been established to carry out 
further safety surveillance (see Paragraphs 7.2.3 and 7.4). 
 
MRI measurements  
For secondary parameters, subjects will be scanned on a 3 Tesla MR scanner as soon as 
possible after clinical suspicion of PAIS and again 3 months after the MSC treatment. Further 
details on MRI measurements can be found in chapter 6. All infants will return to the WKZ, 
UMCU for a MRI scan at 3 months of age.  
 
Follow-up  
After the intervention period, subjects will be followed until 3 months corrected age. At 3 
months neurological assessment will be performed as part of the regular stroke follow-up 
program. This will also include filming spontaneous and endogenous movements (General 
Movements). Additionally, fidgety movements will be assessed. Neuromotor development will 
also be assessed using the HINE. After parental consent, results from these examinations 
will be used for this study. Subjects in this study will also be followed for at least 2 years of 
age at regular time points. This follow-up is part of regular care for neonates with PAIS, but 
these results are not part of this study protocol and are beyond the scope of this study.  
 

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2.5 Schematic diagram of study design  
 
 

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3. STUDY POPULATION 
3.1 Study Population (base)  
Newborn infants born at (near-)term, meaning ≥36+0 weeks of gestation, will be included 
in the study. Subjects will be recruited from all Dutch NICUs collaborating in the Dutch 
Neonatal Research Network (NNRN). 
 
3.2 Inclusion criteria 
In order to be eligible to participate in this study, a subject must meet all of the following 
criteria: 
• 
(Near-)Term infants, ≥36+0 weeks of gestation, admitted to one of the 
Dutch NICUs, diagnosed with PAIS, confirmed by MRI within 7 days after 
presentation with clinical symptoms.  
• 
PAIS as characterized by a predominantly unilateral ischemic lesion within 
the territory of the MCA, with involvement of the corticospinal tracts, cortex, white 
matter and basal ganglia. 
• 
Written informed consent from custodial parent(s).  
 
3.3 Exclusion criteria 
A potential subject who meets any of the following criteria will be excluded from 
participation in this study: 
• 
Any proven or suspected congenital anomaly, chromosomal disorder,  
           metabolic disorder. 
• 
Presence of an infection of the central nervous system. 
• 
No realistic prospect of survival, (e.g. severe brain injury), at the discretion 
          of the attending physician.  
 
3.4 Sample size calculation 
 
We aim to include 10 infants diagnosed with PAIS on MRI, who will be treated with MSCs 
via the nasal route.  
 
Since this is a proof-of-principle study, the sample size is not only calculated based on 
expected power, but on the feasibility to be successful in recruitment of individuals needed 
for this study in order to complete the protocol in 2 years. It is expected that about 180,000 
infants will be born alive per year.(StatLine 2015) Since the incidence of PAIS is 1/2300 there 
will be about 80 PAIS patients in the Netherlands yearly. A study from our group showed that 
about 26% of all PAIS patients involved the main MCA branch.(Harteman et al. 2012) Based 

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on this expectation, we think it will be feasible to recruit 10 PAIS patients in 12-24 months. 
We furthermore expect only a minimal drop out of initially included patients because PAIS is 
not life-threatening and follow-up is tightly regulated in all NICUs, including the WKZ. 
 
Data analysis 
Data will be analyzed on an intention to treat basis. SAEs are planned to be compared. 
Statistical analysis is described in detail in chapter 8. 
 
 
 

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4. TREATMENT OF SUBJECTS 
4.1 Investigational product/treatment 
The treatment protocol requires that infants will receive MSCs as soon as possible after 
confirmation of the PAIS, but no later than the end of the first week of onset of presenting 
clinical symptoms.  Steps to be undertaken before MSC administration include:  
- 
A bacterial culture taken from both nostrils before administration of MSCs.  
- 
Blood samples will be collected before administration of MSCs. Blood 
samples are taken routinely in the hospital for clinical purposes, but some extra 
material (0.5-1 mL) will be collected to determine infection markers CRP, 
procalcitonin and complete blood count. These samples will be combined with routine 
blood sampling and taken from routinely placed (arterial) catheters  
- 
30 minutes prior to delivery of the cells, the nose will be cleaned with saline 
using standard procedures operative at the NICU.  
 
After these procedures have been performed, infants will receive one dose of 50 x 10^6 
MSCs intranasally. We will use a sterile plastic (e.g. 1,0 mL) syringe containing the MSCs to 
slowly drip the MSCs alternatively in both nostrils. Blood samples will be collected again at 
24 hours after administration of MSCs. 
 
The syringe with MSC will be packaged at the CT-F of the UMC Utrecht and will be delivered 
to the NICU by the clinical research nurse. 
 
4.2 Use of co-intervention (if applicable) 
Not applicable.  
 
4.3 Escape medication (if applicable) 
Not applicable.  

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5. INVESTIGATIONAL PRODUCT  
5.1 Name and description of investigational product(s) 
Allogeneic bone marrow-derived adult MSCs obtained from the Cell Therapy Facility 
(CTF) of the UMCU. The CTF will obtain MSC, marrow: a cell product containing 
mesenchymal stromal cells  derived from bone marrow. Mesenchymal stromal cells 
consist largely of mesenchymal stem cells. However, the International Society for Cellular 
Therapy (ISCT) states that ‘multipotent mesenchymal stromal cells’ (MSC) is the currently 
recommended definition for the plastic-adherent cells isolated from bone marrow and 
other tissues that have often been labelled as mesenchymal stem cells.(Dominici et al. 
2006) We therefore decided to use the same acronym (MSC) as the active substance/cell 
type necessary for this study is mesenchymal stem cells.  
 
5.2 Summary of findings from non-clinical studies 
An overview of findings from non-clinical studies on MSC treatment for PAIS is given in 
chapter 7 of the Investigator’s Brochure (IB).  
 
5.3 Summary of findings from clinical studies 
An overview of findings from clinical studies on MSC treatment for PAIS is given in 
chapter 8 of the IB.  
 
5.4 Summary of known and potential risks and benefits 
An overview of findings on potential risks and benefits of MSC treatment for PAIS is given 
in chapters 7 and 8 of the IB.  
 
In summary, we have shown in our own mouse models of neonatal ischemic infarction 
that intracerebral application of murine adult bone marrow-derived MSCs markedly 
improves outcome. Treatment with MSC reduced infarct size by >45%, stimulated 
formation of new neurons and oligodendrocytes, partially restored cortico-spinal motor 
tract activity, and improved sensorimotor outcome.(van Velthoven et al. 2010c; van 
Velthoven et al. 2010a) Additionally, our findings indicate that MSCs can be successfully 
and rapidly administered to the brain via an efficient non-invasive route, the nasal 
route.(Donega, van Velthoven, Nijboer, van Bel, et al. 2013) The MSCs subsequently 
accumulate predominantly in the infarcted area. Notably, MSCs delivered via the nasal 
route reduce infarct size and improve motor function to the same extent as MSCs 
administered intracranially.(van Velthoven et al. 2010b) 
 

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In general, risks or adverse events related to MSC treatment can be categorized in three 
groups: acute, subacute and longterm adverse events.  
 
1. Acute adverse events: Administration Reaction/Toxicity 
• 
Systolic blood pressure >33% decrease or increase from the baseline 
during or directly following the intranasal administration of the investigational product 
• 
Diastolic blood pressure >25% decrease or increase from baseline during 
or directly following the intranasal administration of the investigational product 
• 
Heart rate >33% decrease or increase from baseline during or directly 
following the infusion of the investigational product 
• 
Development of dyspnoea, vomiting, flushing, diarrhea, during or within 1 
hour from the intranasal administration of the investigational product 
• 
Fever ≥ 2 grade increase from baseline during or within 2 
hours from the intranasal administration of the investigational product 
• 
Development of exanthema, or urticaria developing during or within 2 
hours from the intranasal administration of the investigational product 
 
Allogeneic hUCB-derived MSC treatment in preterm infants with BPD appeared to be safe 
and without any acute adverse events.(Chang et al. 2014; Henckel et al. 2015) A large 
meta-analysis on clinical trials for numerous diseases did not show any evidence for acute 
severe toxicity due to MSC transplantation.(Lalu et al. 2012) This analysis used both adult 
and pediatric trials to report on a total of 1012 patients. Including eight randomized control 
trials, there was no association detected between MSC transplantation and acute 
infusional toxicity and organ system complications. There was a significant increase in 
fever with MSCs compared to the control group, but the fever was reported to be transient 
in all trials.(Lalu et al. 2012)  
 
2. Subacute adverse events:  
• 
Bacteremia due to bacterial contamination of product. Fever ≥ 2 grade 
increase from baseline during or within 2 hours from the intranasal administration of 
the investigational product AND product contamination proven by positive product 
cultures. In case of contamination patients will be treated with antibiotics in case this 
is required. As described in the IMPD, there will be extensive quality control in the 
CTF, e.g. testing for contamination of the product. This risk is therefore considered to 
be minimal. 
• 
 Systemic infection (e.g. meningitis) due to use of MSCs. We have no 
evidence from preclinical trials, nor from trials in adult stroke or neonatal BPD, that 

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MSC treatment increases the risk of infections. However, MSCs might be able to 
change inflammatory status. In order to monitor more closely, we will take extra blood 
samples from all subjects at baseline and 24 hours following MSC administration to 
determine infection markers such as CRP, procalcitonine and complete blood count.  
• 
 Post-administration virus infection. This risk is minimal and adherent to all 
blood (derived) product administration. If a patient develops a new viral infection that 
is suspected to be due to transfusion of blood products or intranasal administration of 
the investigational product, a lookback procedure should be initiated by the treating 
physician according to local regulations for all blood products received. If a MSC 
donor is proven to be the source of virus infection, this shall be considered a serious 
adverse event. All donors are being screened for infectious disease markers as 
indicated by the Wet Veiligheid en Kwaliteit Lichaamsmaterialen (WVKL), the 
transposition into Dutch Law of the EU Directive 2006/17. Please find specification of 
control of drug stubstance in chapter 2.1.S.4.1 of the IMPD. 
 
Allogeneic hUCB-derived MSC treatment in preterm infants with BPD appeared to be safe 
and without any acute or subacute adverse events.(Chang et al. 2014; Henckel et al. 2015) 
Also from literature on MSC treatment for adult stroke it was shown that MSC treatment has 
no known risks.(Bang et al. 2005; Honmou et al. 2011) A large meta-analysis on clinical trials 
for numerous diseases did not show any evidence for severe subacute adverse effects due 
to MSC transplantation.(Lalu et al. 2012) This analysis used both adult and pediatric trials to 
report on a total of 1012 patients with ischemic stroke, Crohn’s disease, cardiomyopathy, 
myocardial infarction, Graft-versus-host disease (GvHD) and healthy volunteers. Including 
eight randomized control trials, there was no association detected between MSC 
transplantation and organ system complications or infections.  
 
3. Long-term adverse events: Ectopic tissue formation and tumorigenicity 
 
Although concerns that MSCs might transform into tumorigenic cells still 
exist, there is a general agreement that bone-marrow-derived MSCs can be safely 
cultured in vitro with no risk of malignant transformation.(Bernardo et al. 2007; Uccelli 
et al. 2008) So far, there have been no reports in humans of the formation of tumors 
by in vitro-cultured cells, thus making MSCs amenable for use in transplantation.(see 
IB and (Lalu et al. 2012)) This is also shown in table 1 of the IB: none of these studies 
have reported tumorigenesis related to MSC transplantation. 
It is also advantageous that MSCs disappear within a short time frame, i.e. do not 
integrate in the brain, thereby limiting the ability to lead to tumor formation. 
 

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From animal models of neonatal stroke we can conclude that no adverse effects have 
been described for MSC treatment. Our research group has also assessed long-term 
safety of intranasal MSC treatment.(Donega et al. 2015) At 14 months following HI no 
significant lesions or neoplasia were observed in the nasal tubinates, brains or other 
peripheral organs of mice treated intranasally with MSCs. Additionally, it has been shown 
that MSCs improved sensorimotor and cognitive function for at least 9 and 14 months, 
respectively. These results provide strong evidence of long-term safety (and efficacy) of 
MSC treatment following neonatal HI in mice. (Donega et al. 2015) Also a large meta-
analysis on MSC treatment for various pathologies does not support tumorigenicity of 
MSC.(Lalu et al. 2012) Table 1 from the IB summarizes several other studies that 
performed administration of autologous and allogenic bone marrow derived MSCs in the 
treatment of several diseases. From these studies with a follow-up time of 2 months to 4 
years, it was shown that (adult) patients do not have an increased risk of long-term 
effects, including tumorigenicity, after allogeneic MSC treatment.  
 
Risk-benefit balance 
We can conclude that MSC treatment in newborns with PAIS might be beneficial and no 
subacute and long-term risks are to be expected based on available evidence. Acute 
administration reactions, such as subacute temperature increases, are potentially to be 
expected, but these are expected to be transient, self-limiting and unharmful, based on 
the available evidence. However, this is the first study in which MSCs will be administered 
intranasally in humans, so potential risks might be unknown and/or unexpected. Therefore 
we have planned to perform a pilot (phase I/II) trial to assess safety and feasibility of MSC 
treatment in newborns with PAIS. Using close monitoring and (preventative) planned 
actions, as described in chapter 11.1e and 11.2, and tightly regulated stopping rules (see 
chapter 6.7) we expect to minimize or prevent risks.  
 
5.5 Description and justification of route of administration and dosage 
 
Administration route 
MSCs will be administered intranasally to prevent loss of MSC in peripheral organs and to 
make sure MSCs will arrive rapidly and efficiently at the injured brain region. From the 
nasal mucosa, MSCs cross the cribiform plate and migrate throughout the brain using the 
rostral migratory stream.(Danielyan et al. 2009; Danielyan et al. 2011) In an animal model 
of neonatal brain injury, it was demonstrated that MSCs were detected in the olfactory 
bulb, in the subventricular zone of the lateral ventricles and more caudally in the ischemic 
damaged hippocampus within 2 hours after intranasal administration.(Donega, van 

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Velthoven, Nijboer, van Bel, et al. 2013). We also showed improved sensorimotor 
outcome and decrease of gray and white matter loss after intranasal treatment with MSCs 
compared to vehicle treatment. It was therefore concluded that the nasal route is efficient 
for MSC transplantation after neonatal brain injury.(van Velthoven et al. 2010b) This 
finding is confirmed by a study of Wei et al, who treated rats intranasally with bone 
marrow-derived MSCs. At 24 days following stroke, MSCs were found to significantly 
reduce infarct size, promote angiogenesis, neurogenesis and neurovascular repair. Rats 
treated with MSCs had improved sensorimotor and social behavioral outcome compared 
to their placebo-treated controls.(Wei et al. 2015)  
Studies of our group in the newborn baboon have shown that intranasally administered 
MSCs have the potential to migrate to the injured brain regions of primates(unpublished 
observations), in accordance with earlier shown studies in mice and rats is known that the 
olfactory anatomy of primates such as the baboon is more identical to human anatomy 
than rodent olfactory anatomy. We therefore expect the MSCs to migrate in a similar way 
in the newborn baby when administered intranasally.  
 
It was hypothesized that survival of intranasally applied MSCs is better compared to 
intracranially applied, because MSCs are allowed to adapt to the brain environment while 
they migrate to the injured area. Intracranially applied MSCs are administered directly into 
the lesion site and therefore have no possibility to slowly adapt to the toxic ischemic milieu 
in the brain.(van Velthoven et al. 2010b) We expect that the risk of anti-donor response is 
lower when the MSCs are administered intranasally compared to intravenously, because 
locally administered allogeneic MSCs appear to be non- to weakly immunogenic. 
(Danielyan et al. 2009; van Velthoven et al. 2012) . It is also hypothesized that the efficacy 
of intranasally applied MSCs is better compared to systemically applied MSCs because 
MSCs will not be attracted by other (damaged) peripheral organs, but are only allowed to 
migrate to the injured brain area. We have found chemotactic factors involved in 
regulating MSC migration to the lesion after intranasal administration, that mediate 
effective migration.(Donega, Nijboer, Braccioli, et al. 2014) 
In summary, the intranasal administration route provides an effective alternative allowing 
MSC treatment for brain injury without invasive methods and minimal burden for the 
patient.   
 
 
Administration dose 
(Near)-term infants with PAIS that are included in this study will receive,      50 x 10^6 
MSCs, intranasally. Based on mean body mass, 50 x 10^6 cells/patient is the human dose 

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equivalent to the dose that was effective in a mouse model of stroke (0.5 – 1.0 x 10^6 
cells/mouse).(van Velthoven et al. 2010b; van Velthoven et al. 2013; Donega, van 
Velthoven, Nijboer, van Bel, et al. 2013; van Velthoven et al. 2014; Donega, Nijboer, van 
Tilborg, et al. 2014) In another mouse study, intranasally applied human MSCs were most 
effective when the dosage was 2 x 10^6 MSCs per mouse.(Donega, Nijboer, Braccioli, et 
al. 2014) In human studies with adult stroke, the dose that was administered varied 
between 5 x 10^7 MSC/patient (Bhasin et al. 2011) to 12 x 10^8 MSCs/patient (Bang et al. 
2005; Honmou et al. 2011; Hess et al. 2014) However, these cells were administered 
intravenously or intra-arterially, probably leading to loss of cells in peripheral organs.  
Studies of our group in the newborn baboon (of about 1.1 kilogram) have shown that one 
intranasally administered dose of 30 x 10^6 MSCs has the potential to migrate to the 
injured brain region of primates, in accordance with earlier shown studies in mice and 
rats.(unpublished observations)  
Based on earlier evidence from our clinic, we expect to include infants with a birth weight 
ranging from 2.5 to 4.5 kilogram (Benders et al. 2014), and a dose of 50 x 10^6 seems 
appropriate for this weight.  
 
Additional products 
Not applicable 
5.5.1 
Description and justification of source and nature of MSCs 
 
Source of MSCs 
For this study, MSCs will be harvested from adult donor bone marrow. This is because MSCs 
can easily be obtained from bone marrow, expansion is possible and there is a high 
percentage (>90%) of MSC in stromal cells of the bone marrow. As described in chapter 
2.1.S.1. of the IMPD, the MSCs will be harvested from healthy volunteers. The same MSCs 
are used in another trial in the UMC Utrecht to treat GvHD (see IMPD). The CTF of the UMC 
Utrecht has gained a lot of experience with MSCs from bone marrow and results from their 
earlier studies did not show any signs of toxicity of the cells (see chapter 1 IMPD). The MSCs 
from potential donors that will be used in the trial, were also tested in animal experiments 
(see IB). Also in other trials, MSCs are widely used. There are more than 500 ongoing 
studies using MSCs to treat various diseases in adults and children (http://clinicaltrials.gov). 
 
Nature of cells 
MSCs have many advantages over other types of stem cells or progenitors. First of all, they 
can easily be obtained from infants and adults.(Le Blanc & Mougiakakos 2012) Additionally, 
as described above, MSC treatment have a long therapeutic window, as they can be 

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administered at least until 10 days after brain injury to improve functional and behavioral 
outcome and lesion volume in neonatal HI mice. This is of great value, since the only 
available therapies for HI injury have to be given within a few hours. Besides, MSCs are 
hardly detectable in the brain after 3 days (Donega, van Velthoven, Nijboer, van Bel, et al. 
2013), so the risk of Host-versus-Graft is very low. It is also very unlikely that MSCs will 
cause other adverse effects if they are no longer present in the brain after a short period of 
time. Our most recent study shows that no malignancies are found in the brains or in other 
organs of mice treated with MSCs when assessed at 14 months after start of MSC treatment 
at 10 days after neonatal HI.(Donega et al. 2015) Furthermore, MSCs have low 
immunogenicity because they do not express major histocompatibility complex (MHC) class-
II antigens.(Rocha et al. 2000; Uccelli et al. 2008) This makes them excellent therapeutic 
candidates for allogeneic treatment.  
 
5.6 Dosages, dosage modifications and method of administration 
(Near-)Term infants with PAIS that are included in this study will receive 50 x 10^6 MSCs 
in 600µL saline with 20% Human Serum Albumin (HSA) as described in section 5.5.  
Infants will be put in anti-Trendelenburg (of approximately 10°) position. Thirty minutes 
prior to delivery of the cells, the nose will be cleaned with saline using standard 
procedures operative at the NICU. After 30 minutes 0.6 mL MSCs will be slowly dripped 
into each nostril. This will be done using a sterile plastic (e.g. 1.0 mL) syringe containing 
the MSCs to slowly drip the MSCs directly in both nostrils.  
 
5.7 Preparation and labelling of Investigational Medicinal Product 
All physical, chemical and pharmaceutical properties and formulation of the MSCs are 
described in chapter 3 of the IB. Description of formulation of all reagents, solvents and 
other materials can also be found in the IB. Safety procedures during cell manufacture, 
biosafety qualification testing of cells, storage and handling are detailed in the 
Investigational Medical Product Dossier (IMPD).  
Study product will be labelled in accordance with applicable laws and regulations as 
proposed in annex 13 of the guideline Good Manufacturing Practice 
(2003/94/EG).(website http://ec.europa.eu/health/files/eudralex/vol-
4/2009_06_annex13.pdf) All labels will contain information required for regulatory as well 
as identification purposes. 
 
5.8 Drug accountability 
Not applicable. 

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6. METHODS  
6.1 Study parameters/endpoints 
6.1.1 Main study parameter/endpoint 
Safety is defined primarily as the absence of treatment-related serious adverse events 
(SAEs) according to the Consolidated Standards of Reporting Trials (chapter 7.2 and 
bron), secondly as the absence of dose-limiting toxicity, defined as death within 24 hours 
after MSC transplantation or anaphylactic shock related to the MSC administration, and 
thirdly as the absence of adverse events at 3 months of age in terms of cerebral damage 
and neurodevelopmental outcome (see secondary outcome).  
 
For acute safety, all patients will be regularly and intensively assessed, including blood 
sampling and vital signs, before and 24 hours after treatment, until discharge from our 
hospital. Also, blood sampling will be performed before and 24 hours after treatment with 
MSCs to determine inflammation parameters . Acute safety will be determined until 
discharge.  
 
6.1.2 Secondary study parameters/endpoints  
To determine if MSC treatment in (near-)term infants with PAIS is safe in the subacute 
setting, patients will be monitored until the age of 3 months. Subacute safety is defined 
primarily as the absence of treatment-related serious adverse events (SAEs) according to 
the Consolidated Standards of Reporting Trials (chapter 7.2) until the age of 3 months. 
Patients will then be asked to report on other SAEs, including infections, during the first 
three months of age. Whenever their child experiences fever, or other signs of 
infection/inflammation, there will be tested for bactermia, systemic infections and/or post-
administration virus infections.   
 
To determine if MSC treatment in (near-)term infants with PAIS is safe at 3 months in 
terms of cerebral tumorigenicity, infants will be scanned using MRI prior to MSC 
treatment and at 3 months of age. Long-term adverse effects on the brain in terms of 
tumorigenicity will be determined using this MRI, which is part of regular stroke follow-up 
program.  Scans will be assessed by a pediatric radiologist to see if there are any 
adverse events on the brain (including tumor formation).  
 
All MRI investigations will be performed on a 3.0 Tesla MR system. Infants will be sedated 
with a combination of chlorpromazine (0.5 mg/kg), pethidine (2 mg/kg), and promethazine 
(0.5 mg/kg) intramuscularly, which is part of our standard MRI procedure. During MR 

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examination the neonates will be placed in a vacuum fixation pillow (Med Vac Infant 
Immobilizer Bag, Radstadt, Austria) to prevent movement. Monitoring will be performed 
using pulse oximetry (Nonin, Minneapolis, MN) and respiration rate will be observed using 
the standard Philips equipment (Philips Medical Systems, Best, Netherlands). For hearing 
protection we will use: Minimuffs® (Natus Medical Incorporated, San Carlos, CA, USA), 
Earmuffs (EM's 4 Kids, Brisbane, Australia) and a passive acoustic noise dampener, the 
acoustic hood that properly fitted in the bore of the MRI scanner (Intera, Philips Medical, 
Best, Holland). 
 
 
6.1.3 Other study parameters  
None.  
 
6.2 Randomisation, blinding and treatment allocation 
Not applicable.  
 
6.3 Study procedures 
A description of all procedures, techniques and tests that are used to assess the defined 
study endpoints is given in chapters 6.1.1 and 6.1.2. A schedule including the timeline of 
all assessments is given in the flowchart of chapter 2.5. In summary, all procedures that 
subjects must undergo are:  
- 
Intensive monitoring of vital signs at baseline until at least 24 hours after 
MSC treatment. 
- 
Blood samples will be collected before and 24 hours after administration of 
MSCs. Blood samples are taken routinely in the hospital for clinical purposes, but 
some extra material (0.5-1 mL) will be collected to determine infection markers CRP, 
procalcitonin and complete blood count. These samples will be combined with routine 
blood sampling and taken from routinely placed (arterial) catheters. 
- 
MRI investigation including 3D-T1, T2 weighted, DWI, DTI and MR-
spectoscropy sequences within 7 days after clinical presentation suggestive of PAIS  
- 
Repeated MRI investigation including 3D-T1, T2 weighted, DWI, DTI and 
MR-spectroscopy sequences at 3 months of age.  
 
 
These procedures are all part of clinical care for neonates with PAIS.  
 

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6.4 Withdrawal of individual subjects 
Subjects can leave the study at any time for any reason if they wish to do so without 
any consequences. The investigator or physician can decide to withdraw a subject 
from the study for urgent medical reasons. Withdrawal from this study has no 
consequences for medical treatment of the patient.  
6.4.1 Specific criteria for withdrawal  
Not applicable.  
 
6.5 Replacement of individual subjects after withdrawal 
Not applicable.  
 
6.6 Follow-up of subjects withdrawn from treatment 
Not applicable.  
 
6.7 
Premature termination of the study 
The study may be terminated prematurely for safety or futility reasons if there are AEs 
or SAEs as defined by chapter 7.  
We will use the following stopping rules: 
• 
If the region of infarction, as defined on T2 weighted MR imaging and/or 
DWI, increases by >20% after treatment with MSCs in 3 different patients.  
• 
If there appear to be any adverse effects that might be related to admission 
of MSCs in 3 different patients. These adverse effects include: 
o Systemic infections / meningitis 
o Hemorrhagic transformation of the infarction 
o Gastrointestinal hemorrhage 
o Thromboembolism 
o Formation of malignancies 
• 
Unexpected and unaccountable deterioration of clinical situation, for 
example persistent seizures, after treatment with MSCs in 3 different patients. 
Usually, stroke-related seizures will disappear within three days following their 
onset and treated with anti-epileptic drugs. 
• 
Mortality after treatment with MSCs in one patient will stop the study 
temporarily until an investigation has been performed.  
 

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Continuation of the study will only take place after the first three infants completed their 
second MRI scan at 3 months of age. This will safeguard that all stopping criteria will be 
monitored closely and the study may be terminated prematurely in time when stopping 
criteria are met. 

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7. SAFETY REPORTING 
 
7.1 Section 10 WMO event 
In accordance to section 10, subsection 4, of the WMO, the sponsor will suspend the 
study if there is sufficient ground that continuation of the study will jeopardise subject 
health or safety.  The sponsor will notify the accredited METC without undue delay of a 
temporary halt including the reason for such an action. The study will be suspended 
pending a further positive decision by the accredited METC. The investigator will take 
care that all subjects are kept informed.  
 
7.2 AEs, SAEs and SUSARs 
7.2.1 Adverse events (AEs) 
Adverse events are defined as any undesirable experience occurring to a subject 
during the study, whether or not considered related to intranasal MSC treatment. All 
adverse events reported spontaneously by the subject or observed by the investigator 
or his staff will be recorded. 
7.2.2 Serious adverse events (SAEs) 
A serious adverse event is any untoward medical occurrence or effect that at any 
dose:  
- 
results in death; 
- 
is life threatening (at the time of the event); 
- 
requires hospitalisation or prolongation of existing inpatients’ hospitalisation; 
- 
results in persistent or significant disability or incapacity; 
- 
is a congenital anomaly or birth defect; 
- 
Any other important medical event that may not result in death, be life threatening, 
or require hospitalization, may be considered a serious adverse experience when, 
based upon appropriate medical judgement, the event may jeopardize the subject 
or may require an intervention to prevent one of the outcomes listed above. 
 
The sponsor will report the SAEs through the web portal ToetsingOnline to the 
accredited METC that approved the protocol, within 15 days after the sponsor has 
first knowledge of the serious adverse events. 
 
SAEs that result in death or are life threatening should be reported expedited. The 
expedited reporting will occur not later than 7 days after the responsible investigator 

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has first knowledge of the adverse event. This is for a preliminary report with another 
8 days for completion of the report.  
7.2.3 Suspected unexpected serious adverse reactions (SUSARs) 
Adverse reactions are all untoward and unintended responses to an investigational 
product related to any dose administered. 
 
Unexpected adverse reactions are SUSARs if the following three conditions are met: 
1. the event must be serious (see chapter 7.2.2); 
2. there must be a certain degree of probability that the event is a harmful and an 
undesirable reaction to the medicinal product under investigation, regardless of 
the administered dose; 
3. the adverse reaction must be unexpected, that is to say, the nature and severity 
of the adverse reaction are not in agreement with the product information as 
recorded in: 
- 
Summary of Product Characteristics (SPC) for an authorised medicinal 
product; 
- 
IB for an unauthorised medicinal product. 
 
The sponsor will report expedited the following SUSARs through the web portal 
ToetsingOnline to the METC: 
- 
SUSARs that have arisen in the clinical trial that was assessed by the METC; 
- 
SUSARs that have arisen in other clinical trials of the same sponsor and with the 
same medicinal product, and that could have consequences for the safety of the 
subjects involved in the clinical trial that was assessed by the METC. 
The remaining SUSARs are recorded in an overview list (line-listing) that will be 
submitted once every half year to the METC. This line-listing provides an overview 
of all SUSARs from the study medicine, accompanied by a brief report highlighting 
the main points of concern.  
The expedited reporting of SUSARs through the web portal ToetsingOnline is 
sufficient as notification to the competent authority. 
 
 
The expedited reporting will occur not later than 15 days after the sponsor has first 
knowledge of the adverse reactions. For fatal or life threatening cases the term will 
be maximal 7 days for a preliminary report with another 8 days for completion of the 
report.  

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7.3 Annual safety report 
 
In addition to the expedited reporting of SUSARs, the sponsor will submit, once a year 
throughout the clinical trial, a safety report to the accredited METC and competent 
authority. 
This safety report consists of: 
- 
a list of all suspected (unexpected or expected) serious adverse reactions, along with 
an aggregated summary table of all reported serious adverse reactions, ordered by 
organ system, per study; 
- 
a report concerning the safety of the subjects, consisting of a complete safety analysis 
and an evaluation of the balance between the efficacy and the harmfulness of the 
medicine under investigation. 
 
7.4 Follow-up of adverse events 
All AEs will be followed until they have abated, or until a stable situation has been 
reached. Depending on the event, follow up may require additional tests or medical 
procedures as indicated, and/or referral to the general physician or a medical specialist. 
SAEs need to be reported until discharge after the second MRI around the age of 3 
months. As described in chapter 7 of the IB, MSCs do not survive for a very long period. 
When MSCs disappear within a short time frame and they are no longer present in the 
subject, they are not able/unlikely to lead to (serious) adverse events such as death, 
tumor formation or disturbances of the immune system. We have therefore decided that 
after a period of 3 months, AE or SAEs are very unlikely to be linked to the use of MSCs 
and conclusions regarding safety of MSCs can be drawn.    
 
7.5 Data Safety Monitoring Board (DSMB)  
A DSMB will be established to perform ongoing safety surveillance and to perform interim 
analyses on the safety data. This committee will be an independent committee. The 
DSMB is composed of 4 members from the UMCU: three are members of the internal 
DSMB of the UMCU and one member has clinical expertise as paediatric neurologist. 
Each member has no conflict of interest with the study. The task and responsibility of the 
DSMB are described in the DSMB charter. CVs of each DSMB member are added to the 
DSMB charter.  
 
The DSMB may decide to terminate the trial prematurely using the stopping criteria as 
stated in chapter 6.7 of this protocol.  
 

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The advice(s) of the DSMB will only be sent to the sponsor of the study. Should the 
sponsor decide not to fully implement the advice of the DSMB, the sponsor will send the 
advice to the reviewing METC, including a note to substantiate why (part of) the advice of 
the DSMB will not be followed. 

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8. STATISTICAL ANALYSIS 
In this study, 10 newborns will be included who will be treated with MSCs via the nasal route. 
This is a phase I/II, open-label, single-arm, single-center intervention study to assess the 
safety and feasibility of intranasal BM-MSCs in neonatal stroke patients.  
 
8.1 Study parameter(s) 
 
Safety is defined primarily as the absence of treatment-related serious adverse events 
(SAEs) according to the Consolidated Standards of Reporting Trials (chapter 7.2 and 
bron), secondly as the absence of dose-limiting toxicity, defined as death within 24 hours 
after MSC transplantation or anaphylactic shock related to the MSC administration, and 
thirdly as the absence of adverse events at 3 months of age in terms of cerebral damage 
and neurodevelopmental outcome (see secondary outcome). 
 
For acute safety, all vital signs and blood sample parameters (continuous variables) at 
baseline and at 24 hours following MSC transplantation will be compared using Wilcoxon 
signed-rank tests.  The occurrence of SAE’s, including mortality, toxicity, tumor growth, 
etc (see chapter 11.1) will be expressed in percentages and analyzed qualitatively.   
 
8.2 Other study parameters 
Not applicable. 
 
8.3 Interim analysis (if applicable) 
No interim analysis will be performed. Stopping rules are described in chapter 6.7. A 
DSMB will be established to advice on stopping, as is described in chapter 7.5 and in the 
DSMB Charter.  

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9. ETHICAL CONSIDERATIONS 
9.1 Regulation statement 
This study will be conducted according to the principles of the Declaration of Helsinki 
(version 2013: www.wma.net) and in accordance with the Medical Research Involving 
Human Subjects Act (WMO) and other guidelines, regulations and Acts.  
 
9.2 Recruitment and consent 
Parents of neonates born in a Dutch hospital and/or admitted to our NICU who are 
potentially eligible for inclusion into the study will be informed as soon as possible after 
suspicion of PAIS about the study protocol by a neonatologist involved in the study. 
These parents include those of infants with or without symptoms of PAIS (i.e.one sided 
convulsions) and a PAIS confirmed with MRI. The parents will receive both the patient 
information letter describing the research proposal as well as an informed consent form. 
As soon as possible but allowing for an appropriate period of time allowing the parents to 
read, understand and, if necessary, having received additional information if requested, 
consent for participation will be asked in case PAIS (in the MCA region) is confirmed  
within 7 days after the onset of symptoms by MRI (including 3D-T1, T2 weighted, DWI 
and DTI sequences). Following written parental consent, patients will then be included in 
our study. Patients with PAIS that are diagnosed more than one week after birth are not 
eligible for enrolment.  
 
9.3 Objection by minors or incapacitated subjects (if applicable) 
Resistance by minors will be conducted by the code of conduct accepted by the “Bestuur 
van de Nederlandse Vereniging voor Kindergeneeskunde” (NVK) at May 21 2001, and it 
will be specified in the informed consent letter. 
 
9.4 Benefits and risks assessment, group relatedness 
In this study we will include (near-)term newborns with proven PAIS. Current treatment 
options for PAIS mainly focus on controlling convulsions, hypoglycaemia and associated 
infections. There is no treatment available that leads to reduction of neonatal brain 
damage in this severely affected group of infants. This leads to life-long consequences of 
PAIS and forms a large burden for patients and society. The overall aim of this project is 
to meet this by making the first step towards clinical application of MSCs to treat PAIS. 
Successful completion of this project will provide the first evidence of the safety and 
feasibility of intranasally administered bone-marrow derived MSCs in newborn infants with 
PAIS. 

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The extra burden of the present study for the included infants is considered to be very 
limited to non-existent given the fact that besides the nasal treatment to apply the MSCs, 
treatment is not different compared to the standard acting treatment protocol for newborns 
with PAIS. Nasal application of MSCs has been considered as non-invasive. With the 
respect to possible risks of the present MSC treatment, the most important potential risk 
factors such as inflammatory actions of MSC therapy with allogeneic human MSCs and an 
increased risk for malignancies, have been intensively investigated in our own preclinical 
studies and appear to be non-present at long-term follow up. Additionally, human trials on 
MSC treatment for adult stroke or preterm infants with BPD and other pathologic 
conditions do not provide evidence for these or other serious adverse events or risks. 
However, this is the first study administering MSCs intranasally in humans, so potential 
risks might be unknown and/or unexpected. Using close monitoring and (preventative) 
planned actions, as described in chapter 11.1e and 11.2, and tightly regulated stopping 
rules (see chapter 6.7) we expect to minimize or prevent these risks.No single indication 
has been found in experimental research in the developing animal models that above 
mentioned complications occur in a higher incidence as compared to non-MSC treated 
animals, whereas possibility for a substantial better short- and long-term outcome seems 
very realistic on the basis of previous experimental research data. Therefore we have 
planned to peform a pilot (phase I/II) trial to assess safety and feasibility of MSC treatment 
in newborns with PAIS. 
 
9.5 Compensation for injury 
The sponsor/investigator has a liability insurance which is in accordance with article 7 of 
the WMO. 
  
The sponsor (also) has an insurance which is in accordance with the legal requirements 
in the Netherlands (Article 7 WMO and the Measure regarding Compulsory Insurance for 
Clinical Research in Humans of 24th November 2014). This insurance provides cover for 
damage to research subjects through injury or death caused by the study. 
 
The insurance applies to the damage that becomes apparent during the study or within 4 
years after the end of the study. 
 
9.6 Incentives (if applicable) 
Not applicable.  

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10. ADMINISTRATIVE ASPECTS, MONITORING AND PUBLICATION 
10.1 Handling and storage of data and documents 
Data will be handled confidentially (using coding). Where it is necessary to trace data to 
an individual subject, a subject identification-code list will be used to link the data to the 
subject. The code will not be based on the patient initials and birth date. The key to the 
code will be safeguarded by the research team (Principal Investigator), because data and 
human material will be kept for a longer period of time. The handling of personal data will 
be complied with the Dutch Personal Data Protection. 
 
10.2 Monitoring and Quality Assurance  
A detailed description of monitoring of the study is given in the Monitoringplan and the 
Monitor proposal (part K6).  
 
10.3 Amendments  
A ‘substantial amendment’ is defined as an amendment to the terms of the METC 
application, or to the protocol or any other supporting documentation, that is likely to 
affect to a significant degree: 
- the safety or physical or mental integrity of the subjects of the trial; 
- the scientific value of the trial; 
- the conduct or management of the trial; or 
- the quality or safety of any intervention used in the trial. 
 
All substantial amendments will be notified to the METC and to the competent authority. 
 
Non-substantial amendments will not be notified to the accredited METC and the 
competent authority, but will be recorded and filed by the sponsor.  
 
10.4 Annual progress report 
The sponsor/investigator will submit a summary of the progress of the trial to the 
accredited METC once a year. Information will be provided on the date of inclusion of the 
first subject, numbers of subjects included and numbers of subjects that have completed 
the trial, serious adverse events/ serious adverse reactions, other problems, and 
amendments.  
 

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10.5 End of study report 
The sponsor will notify the accredited METC and the competent authority of the end of 
the study within a period of 90 days. The end of the study is defined as the last patient’s 
last visit.  
 
In case the study is ended prematurely, the sponsor will notify the accredited METC and 
the competent authority within 15 days, including the reasons for the premature 
termination. 
 
 Within one year after the end of the study, the investigator/sponsor will submit a final 
study report with the results of the study, including any publications/abstracts of the study, 
to the accredited METC and the Competent Authority.  
 
10.6 Public disclosure and publication policy 
There are no specific agreements concerning public disclosure or publication policy. 

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11. STRUCTURED RISK ANALYSIS  
 
11.1 Potential issues of concern 
 
a. Level of knowledge about mechanism of action 
 
From neonatal rat models of brain injury, we have learned that HI brain injury induces 
changes in neurovascular environment that promote neurogenesis.(Kadam et al. 2008; 
Ong et al. 2005; Yang & Levison 2007) However, after HI brain injury, there is no 
induction and maintenance of long-term neurogenesis. Therefore, it is crucial to assist the 
brain in regeneration after injury and also to reinforce development of the maturating 
brain. In animal stroke-model studies, it has been demonstrated that transplantation of 
murine adult bone marrow-derived MSCs markedly improves neuroregeneration. 
Treatment with MSC stimulated formation of new neurons and oligodendrocytes and 
reduced infarct size by >45%. Although intracranial MSC treatment stimulated formation of 
new brain cells and differentiation of these cells into new neurons and oligodendrocytes, 
these newly formed cells were not of transplant origin. These findings indicate that the 
neuroregenerative effect of MSCs after ischemic brain damage mainly relies on 
stimulation of endogenous repair mechanisms.(van Velthoven et al. 2010c) This is 
confirmed by other data from our group that show that MSC treatment has profound 
effects on the expression of growth and differentiation factors in the brain. For example, 
MSC treatment at 10 days after HI induces upregulation of 35 genes encoding growth and 
differentiation factors that are mainly involved in cell growth and proliferation. MSC 
treatment at 3 and 10 days induces expression of 28 additional growth and differentiation 
factors and these are predominantly involved in cell differentiation and nervous system 
development.(van Velthoven et al. 2010c; van Velthoven et al. 2011) These results show 
that the first dose of MSC stimulates cerebral cell proliferation, whereas the second 
injection enhances differentiation and maturation of newly formed cells and integration in a 
functional network.(van Velthoven et al. 2010c) Importantly, we have shown recently that 
at 3 days after MSC administration, the number of MSCs in the brain sharply decreases 
indicating low survival of MSCs for longer time periods (Donega, 2014 Exp Neurol). 
 
Based on these findings, we hypothesize that MSC of donor origin will only transiently 
survive in the brain and that the neuroregenerative effects of MSC are mediated 
predominantly by orchestrating changes in the growth and differentiation factor milieu in 
the brain. The corticospinal tract is the primary transmission route for voluntary movement 
of the forepaws. We showed that MSC treatment could also partially restore the damaged 

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corticospinal motor tract which led to improved sensorimotor outcome.(van Velthoven et 
al. 2010c; van Velthoven et al. 2010a)  
 
An overview of findings from non-clinical studies on MSC treatment for neonatal stroke is 
given in chapter 7 of the IB. These studies confirm the role of MSCs in stimulating 
endogenous neuroregenerative mechanisms and thereby reducing cerebral damage, 
restoring corticospinal motor tracts and improving sensorimotor outcome.  
 
b. Previous exposure of human beings with the test product(s) and/or products with a 
similar biological mechanism 
 
At present, there are no results of clinical trials available on the treatment of neonatal 
stroke with MSCs. The only available trial on MSC administration in neonates, showed 
that allogeneic hUCB-derived MSC treatment in premature neonates with BPD was safe 
and without any adverse events.(Chang et al. 2014; Henckel et al. 2015) However, many 
studies have reported on MSC treatment for stroke in adult patients. An overview of 
findings from these trials is given in chapter 8 of the IB. In summary, these studies show 
potential of MSCs in the treatment of adult stroke as they improve functional outcome. 
Even though neonatal and adult stroke do not have a common underlying cause, they 
both result in brain damage caused by common harmful processes. These include 
mechanisms of excitotoxicity and neuroinflammation, involving microglial activation, 
proinflammatory cytokine production and toll-like receptor (TLR) activation.(Fleiss et al. 
2014) Comparable to our study, most of these trials administered MSCs days to weeks 
after the cerebral damage occurred, while brain tissue loss is usually complete after 10 
days.(van Velthoven et al. 2010a; Bonestroo et al. 2015) Therefore the main goal of all 
MSC treatment studies was not to have neuroprotective effects, but to induce endogenous 
neuroregeneration and repair. We therefore presume that treatment with MSCs in 
neonatal stroke will have the same biological mechanism of action as in adult stroke and 
will also lead to improved neuroimaging and functional outcome.  
 
c. Can the primary or secondary mechanism be induced in animals and/or in ex-vivo 
human cell material? 
 
An overview of findings from non-clinical studies on MSC treatment for neonatal stroke is 
given in chapter 7 of the IB. These studies show that MSC treatment in animal models of 
neonatal stroke reduces cerebral damage, restoring corticospinal motor tracts and 
improving sensorimotor outcome.  

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d. Selectivity of the mechanism to target tissue in animals and/or human beings 
 
Human MSCs communicate with other cells in the human body and appear to migrate to 
areas of injury in response to signals of cellular damage and apoptosis.(Kang et al. 2012; 
Vogel et al. 2010) MSCs migrate selectively into damaged brain lesions, and specific 
molecular signals, such as stromal cell- derived factor-1 (SDF-1/CXCR4), intracellular 
signaling and adhesion molecules (selectins and integrins) and proteases have been 
reported to be involved in the interaction of MSCs to reach, recognize, and function in 
cerebral ischemic tissue.(Komatsu et al. 2010; Chavakis et al. 2008; Shen et al. 2007; 
Shyu et al. 2006) It is therefore hypothesized that areas of brain injury caused by PAIS will 
attract MSCs to migrate to the ischemic brain injury site. MSCs will be administered 
intranasally to prevent loss of MSC in peripheral organs (Steiner et al. 2012) and to make 
sure MSCs will arrive rapidly and efficiently at the injured brain region.   
 
e. Analysis of potential effect 
Adverse events 
From animal models of neonatal stroke we can conclude that no adverse effects have 
been described for MSC treatment. Our research group has also assessed long-term 
safety of intranasal MSC treatment.(Donega et al. 2015) At 14 months following HI no 
significant lesions or neoplasia were observed in the nasal tubinates, brain or other 
peripheral organs of mice treated intranasally with MSCs and compared with control or 
vehicle-treated HI mice. These results provide strong evidence of the long-term safety of 
MSC treatment following neonatal HI in mice.(Donega et al. 2015)  
 
As MSC treatment has never been studied in human neonatal stroke, we can only provide 
evidence for the safety of MSC treatment in other pathological conditions. There is only 
one clinical trial describing the use of MSCs in treatment of neonates.(Chang et al. 2014) 
Chang et al assessed in a phase I dose-escalation trial the safety and feasibility of 
intratracheal administration of allogeneic umbilical cord-derived MSCs in preterm infants 
with high risk for BPD. Nine infants were included in this study: 3 patients were given 1 x 
10^7 MSCs/kg and 6 patients were given 2 x 10^7 MSCs/kg. After 7 days there was a 
decrease in inflammatory cytokines (e.g. interleukin-6 and interleukin-8) and BPD severity 
was lower in patients who received MSCs compared to their historical case-matched 
controls. Importantly, there were no serious adverse events or toxicity related to a higher 
dose.(Chang et al. 2014) More recently some preliminary findings from another trial were 
published, confirming safety and feasibility of MSC treatment in premature BPD 

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patients.(Henckel et al. 2015) Although these MSCs were administered intravenously, no 
adverse effects were found.(Henckel et al. 2015) 
 
As described in chapter 8 of the IB, there are many other clinical trials in adults in which 
MSC are used for the treatment of several diseases. Although the hypothesized mode of 
action of MSCs in different pathological conditions varies, the most important findings from 
these clinical trials refer to safety issues. In none of the trials serious adverse events 
related to MSC treatment were detected during bone marrow harvesting and 
administration. Some studies reported mild allergic reaction(Duijvestein et al. 2010), 
probably related to preservation substances in the MSC-product, or self-limiting fever 
within a few hours after MSC-transplantation.(Shi et al. 2012) In some studies patients 
had a serious adverse event, most likely not related to MSC administration.(Forbes et al. 
2014; Wang et al. 2013; Weiss et al. 2013; Haack-sørensen et al. 2014) Additionally, a 
large meta-analysis on clinical trials for numerous diseases did not show any evidence for 
severe adverse effects due to MSC transplantation.(Lalu et al. 2012) This analysis used 
both adult and pediatric trials to report on a total of 1012 patients with ischemic stroke, 
Crohn’s disease, cardiomyopathy, myocardial infarction, GvHD and healthy volunteers. 
Including eight randomized control trials, there was no association detected between MSC 
transplantation and acute infusional toxicity, organ system complications, infection, death 
or malignancy. There was a significant increase in fever with MSCs compared to the 
control group, but the fever was reported to be transient in all trials and most often low 
grade (≤38°C).(Lalu et al. 2012)  
 
No correlation between the dose of MSC and their potential efficacy or side effects 
could be observed in multiple studies (range 1-8 10^6 cells/kg body weight).(Lalu et al. 
2012) It is known that systemic complications such as fever are more common when 
MSCs were systemically (e.g. intravenously) administered. We found no evidence that 
fever occurred in our animal studies.(Donega et al. 2015) From our preclinical studies in 
mice we found that local (intracranial) administration of MSCs at 3 days after brain injury 
resulted in a decrease of pro-inflammatory cytokines IL-1b, IL-1R and IL-3.(van Velthoven 
et al. 2011)In vitro we have also found that if MSCs are cultured in a hypoxic-ischemic 
brain environment, they produce more growth factors and less pro-inflammatory cytokines 
(IL-1b, IL-6 en TGFb) than MSCs cultured in a normal brain environment.(van Velthoven 
et al. 2010b) Intranasal administration of MSCs, dampened neuro-inflammatory responses 
due to hypoxic-ischemic brain injury at 5-18 days after MSC treatment. The number of 
GFAP and Iba-1 positive cells, as markers for the neuroinflammatory response of glial 
cells, decreases after administration of MSCs, possibly as a result of anti-inflammatory 

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functions of MSCs. (Donega, Nijboer, van Tilborg, et al. 2014) In conclusion, it seems that 
local/nasal MSC treatment has rather an anti-inflammatory effect and will not induce 
inflammation. We therefore do not expect fever to complicate intranasal administration of 
MSCs, as a priori chances are low. Additionally, the danger of transient and low grade 
fever is low. 
 
With respect to possible risks of the present MSC treatment, the most important potential 
risk factors such as inflammatory actions of MSC therapy with allogeneic human MSCs 
and an increased risk for malignancies, are intensively investigated in our own preclinical 
studies and were not found.(Donega et al. 2015) In addition, we found that intranasal 
administration of human MSCs enhanced secretion of anti-inflammatory 
cytokines.(Donega, Nijboer, Braccioli, et al. 2014) Additionally, human trials on MSC 
treatment for adult stroke or other pathologic conditions do not provide evidence for these 
or other serious adverse events or risks. No single indication has been found in 
experimental research in the developing animal models that above mentioned 
complications occur in a higher incidence as compared to non-MSC treated animals, 
whereas possibility for a substantial better short- and long-term outcome seems very 
realistic on the basis of previous experimental research data. However, this is the first 
study administering MSCs intranasally in humans, so potential risks might be unknown 
and/or unexpected. Therefore, we have planned to perform a pilot (phase I/II) trial to 
assess safety and feasibility of MSC treatment in newborns with PAIS. Using close 
monitoring and (preventative) planned actions, as described in chapter 11.1e and 11.2, 
and tightly regulated stopping rules (see chapter 6.7) we expect to minimize or prevent 
these risks. 
 
Summary of potential risks 
Even though there is no evidence that risks as mentioned below occur in a higher 
incidence as compared to non-MSC treated animals, these risks will be monitored as part 
of standard clinical care on the NICU. These risks are based on theoretical mechanisms 
of action, most likely to occur after systemic administration of MSCs. In general, risks or 
adverse events related to MSC treatment can be categorized in three groups: acute, 
subacute and longterm adverse effects.  
 
1. Acute adverse effects: Administration Reaction/Toxicity 
• 
Systolic blood pressure >33% decrease or increase from the baseline 
during or directly following the intranasal administration of the investigational product 

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• 
Diastolic blood pressure >25% decrease or increase from baseline during 
or directly following the intranasal administration of the investigational product 
• 
Heart rate >33% decrease or increase from baseline during or directly 
following the infusion of the investigational product 
• 
Development of dyspnoea, vomiting, flushing, diarrhea, during or within 1 
hour from the intranasal administration of the investigational product 
• 
Fever ≥ 2 grade increase from baseline during or within 2 
hours from the intranasal administration of the investigational product 
• 
Development of exanthema, or urticaria developing during or within 2 
hours from the intranasal administration of the investigational product 
 
2. Subactue adverse effects:  
• 
Bacteremia due to bacterial contamination of product. Fever ≥ 2 grade 
increase from baseline during or within 2 hours from the intranasal administration of 
the investigational product AND product contamination proven by positive product 
cultures. In case of contamination patients will be treated with antibiotics in case this 
is required. 
• 
 Systemic infection (e.g. meningitis) due to use of MSCs. We have no 
evidence from preclinical trials, nor from trials in adult stroke or neonatal BPD, that 
MSC treatment increases the risk of infections. However, MSCs might be able to 
change inflammatory status. In order to monitor more closely, we will take extra blood 
samples from all subjects at baseline and 24 hours following MSC administration to 
determine infection markers such as CRP, procalcitonine and complete blood count.  
• 
 Post-administration virus infection. This risk is minimal and adherent to all 
blood (derived) product administration. If a patient develops a new viral infection that 
is suspected to be due to transfusion of blood products or intranasal administration of 
the investigational product, a lookback procedure should be initiated by the treating 
physician according to local regulations for all blood products received. If a MSC 
donor is proven to be the source of virus infection, this shall be considered a serious 
adverse event. All donors are being screened for infectious disease markers as 
indicated by the Wet Veiligheid en Kwaliteit Lichaamsmaterialen (WVKL), the 
transposition into Dutch Law of the EU Directive 2006/17. Please find specification of 
control of drug stubstance in chapter 2.1.S.4.1 of the IMPD. 
 
3. Long-term effects: Ectopic tissue formation and tumorigenicity 
 
Although concerns that MSCs might transform into tumorigenic cells still 
exist, there is a general agreement that bone-marrow-derived MSCs can be safely 

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cultured in vitro with no risk of malignant transformation.(Bernardo et al. 2007; Uccelli 
et al. 2008) So far, there have been no reports in humans of the formation of tumors 
by in vitro-cultured cells, thus making MSCs amenable for use in transplantation.(see 
IB and (Lalu et al. 2012)) This is also shown in table 1 of the IB: none of these studies 
have reported tumorigenesis related to MSC transplantation. 
It is also advantageous that MSCs disappear within a short time frame, i.e. do not 
integrate in the brain, because when they are no longer present, they are not able to 
lead to tumor formation. 
The recent meta-analysis and our own pre-clinical study does not support 
tumorigenicity of MSC.(Lalu et al. 2012)(Donega et al. 2015) If ectopic tissue 
formation or a new malignancy is detected in a patient at any time following infusion 
of the investigational product, the coordinating investigator should be contacted. 
Information regarding the MSC donor’s HLA typing will be provided so that genetic 
testing for presence of donor-derived MSC can be performed. If MSC donor-derived 
tissue is present either as malignant tissue or in the malignancy surrounding tissue, 
this is regarded as a suspect unexpected serious adverse reaction. 
 
f. Pharmacokinetic considerations 
 
Not applicable.  
 
g. Study population 
PAIS or cerebral infarction is an important perinatal cause of long-lasting 
neurodevelopmental problems. The disease manifests itself most often with one-sided 
convulsions in the first week of life, often accompanied with (asymmetric) hypotonia, 
lethargy and apnea.(Kirton et al. 2011) When PAIS is suspected clinically this is usually 
confirmed by MRI. Neuromotor outcome becomes more evident over time and depends 
largely on the involvement of the corticospinal tracts. Adverse consequences of PAIS 
include USCP, cognitive dysfunction, epilepsy and speech problems. In 50-75% of infants, 
PAIS leads to abnormal neuromotor and -developmental outcome or epilepsy.(Fernández-
López et al. 2014) Postneonatal epilepsy after PAIS is also often reported, and is 
associated with larger PAIS.(Wusthoff et al. 2011; van Buuren et al. 2013) The estimated 
annual mortality rate of neonatal stroke is 3.49/100,000 annually.(Lynch & Nelson 2001) 
Current treatment options for PAIS mainly focus on controlling convulsions, treatment of 
hypoglycaemia and associated infections. There is no treatment available that leads to 
reduction of neonatal brain damage in this severely affected group of infants. This leads to 
life-long consequences of PAIS and forms a large burden for patients and society. The 

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overall aim of this project is to meet this need by assessing the safety and feasibility of  a 
cell based treatment strategy for neonates with PAIS. 
Patients included in this study will be given MSCs as soon as possible after confirmation 
of PAIS, but within the first week of onset of presenting symptoms. At time of 
administration of MSCs, these infants will be admitted to the NICU of our hospital where 
they can be monitored intensively.  
 
h. Interaction with other products 
The potential interactions with other medicinal products, food and other substances have 
been investigated by the CTF of the UMC Utrecht. No interacting substances were found.  
 
i. Predictability of effect 
 
In this study we will not investigate any therapeutic effects of MSCs. We will only assess 
safety in terms of (S)AEs.  
 
j. Can effects be managed? 
 
Planned pharmacovigilance actions for potential risks are described below in section 11.2 
 
11.2 Synthesis 
Adverse events, observed by the attending physician, which are expected or possibly 
related to the infusion of the product, are reported to the head of the CTF. Adverse events 
will be reported in a database, including patient characteristics and product 
characteristics. Adverse events will be evaluated with the Medical Director of the CTF in 
their weekly meetings. We will report the observed adverse events to the Dutch Health 
Inspectorate. Serious adverse events will be reported within 48 hours after the event has 
happened. 
 
Planned pharmacovigilance actions for potential risks 
 
1. Acute adverse effects: Administration Reaction, during and shortly after administration 
Planned actions: 
• 
Monitoring of heart rate and blood pressure. And development of 
dyspnoea, nausea/vomiting, flushing, diarrhea, during or within 1 hour from the 
administration of the investigational product 

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• 
Monitoring temperature during or within 2 hours from the administration of 
the investigational product. During admission, temperature will be monitored very 
strictly, because hyperthermia after hypoxia-ischemia is associated with adverse 
(neurologic) outcome.(Laptook et al. 2008; Perlman 2006) If the temperature rises 
above 37.5ºC we will use routinely clinical used precautions to prevent actual 
hyperthermia, such as lowering environmental temperature or use paracetamol. 
• 
Development of exanthema or urticaria developing during or within 2 
hours from the administration of the investigational product. 
 
 
2. Subactue adverse effects: 
• 
Bacteremia due to bacterial contamination of product. 
Planned actions: Monitoring of temperature during the following hours to days after 
intranasal administration. In case of contamination patients will be treated with antibiotics 
in case this is required. 
• 
Systemic infection due to use of MSC therapy 
Planned actions: Close monitoring of first three patients by extra blood sampling and 
determination of infection markers. In case of systemic infection patients will be treated 
with appropriate antibiotics in case this is required, according to the antibiotics protocol 
used on the NICU.  
• 
Post-administration virus infection. 
Planned actions: If a patient develops a new viral infection that is suspected to be due to 
transfusion of blood products or intranasal administration of the investigational product, a 
look-back procedure should be initiated by the treating physician according to local 
regulations for all blood products received. If an MSC donor is proven to be the source of 
virus infection, this shall be considered a serious adverse event. 
 
 
3.  Long-term adverse effects: Ectopic tissue formation and tumorigenicity 
Planned actions: If ectopic tissue formation or a new malignancy is detected in a patient at 
any time following infusion of the investigational product, information regarding the 
MSC donor’s HLA-typing will be provided so that genetic testing for presence of 
donor-derived MSC can be performed. If MSC donor-derived tissue is present either 
as malignant tissue or in the malignancy surrounding tissue, this is regarded as a 
serious adverse reaction. Treatment will be accordingly to the malignant tissue. 
 
The extra burden of the present study for the included infants is considered to be very limited 
to non-existent given the fact that besides the nasal treatment to apply the MSCs, treatment 
is not different compared to the standard acting treatment protocol for newborns with PAIS. 

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Nasal application of MSCs has been considered as non-invasive. With the respect to 
possible risks of the present MSC treatment, the most important potential risk factors such as 
inflammatory actions of MSC therapy with allogeneic human MSCs and an increased risk for 
malignancies, are intensively investigated in our own preclinical studies and appear to be 
non-present at long-term follow up. Additionally, human trials on MSC treatment for adult 
stroke or other pathologic conditions do not provide evidence for these or other serious 
adverse events or risks. However, this is the first study administering MSCs intranasally in 
humans, so potential risks might be unknown and/or unexpected. Therefore, this study will 
assess safety and feasibility of bone marrow-derived allogeneic MSCs, administered by the 
nasal route, in human neonates with PAIS. Using close monitoring and (preventative) 
planned actions, as described in chapter 11.1e and 11.2, and tightly regulated stopping rules 
(see chapter 6.7) we expect to minimize or prevent these risks.. No single indication has 
been found in experimental research in the developing animal models that above mentioned 
complications occur in a higher incidence as compared to non-MSC treated animals, 
whereas possibility for a substantial better short- and long-term outcome seems very realistic 
on the basis of previous experimental research data.  

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