Neural Stem Cell Transplantation in Multiple Sclerosis Patient: a Phase I Study
Phase: PHASE1
Conditions: Progressive Multiple Sclerosis
Sponsor: IRCCS San Raffaele



Clinical Trial Protocol 
STEMS 
Rev. n° 1.2 
Page 2 of 54 
Rev. date 10 March 2017 
 
 
Other collaborators involved: 
Co-Investigator 
Massimo Filippi, MD & Mara Rocca, MD 
Division of Neuroscience- San Raffaele Hospital - Via Olgettina 60 - 20132 Milan 
Filippi.massimo@hsr.it  -  rocca.mara@hsr.it 
Co-Investigator 
Fabio Ciceri, MD & Greco Raffaella, MD 
Hematology and BMT Unit - San Raffaele Hospital - Via Olgettina 60 - 20132 Milan 
ciceri.fabio@hsr.it  -  greco.raffaella@hsr.it 
Co-Investigator 
Andrea Falini, MD 
Division of Neuroscience - San Raffaele, Via Olgettina 60, 20132 Milan, Italy 
falini.andrea@hsr.it 
Co-Investigator 
Leocani Letizia, MD 
Department of Neurology - San Raffaele Hospital - Via Olgettina 60 - 20132 Milan 
leocani.letizia@hsr.it 
Co-Investigator 
 
Vittorio Martinelli, MD 
Department of Neurology - San Raffaele Hospital - Via Olgettina 60 - 20132 Milan 
martinelli.vittorio@hsr.it 
Co-Investigator 
 
Lucia Moiola, MD, PhD 
Department of Neurology - San Raffaele Hospital - Via Olgettina 60 - 20132 Milan 
miola.lucia@hsr.it 
Co-Investigator 
 
Marta Radaelli, MD, PhD 
Department of Neurology - San Raffaele Hospital - Via Olgettina 60 - 20132 Milan 
radaelli.marta@hsr.it 
Co-Investigator 
 
Francesca Sangalli, MD,  
Department of Neurology - San Raffaele Hospital - Via Olgettina 60 - 20132 Milan 
sangalli.francesca@hsr.it 
Statistician 
Maria Pia Sormani, PhD 
Professor of Biostatistics - University of Genua - Via Balbi 5 - 16126 Genua 
mariapia.sormani@unige.it 
Pharmacovigilance 
Camilla Ferri, pharmacist 
San Raffaele Hospital - Via Olgettina 60 - 20132 Milan 
ferri.camilla@hsr.it 
 
Gianvito Martino, MD 
Division of  Neuroscience - San Raffaele Hospital - Via Olgettina 60 - 20132 Milan 
martino.gianvito@hsr.it 
Clinical Research Associate 
Bombelli Giovanna - Bonopane Marco 
San Raffaele Hospital - Via Olgettina 60 - 20132 Milan 
bombelli.giovanna@hsr.it - bonopane.marco@hsr.it 
Biologist 
Elena Brambilla, BS 
Division of Neuroscience - San Raffaele Hospital - Via Olgettina 60 - 20132 Milan 
brambilla.elena@hsr.it 
Data Manager 
Cinzia Pozzoni – Simona Albanesi 
Department of Neurology - San Raffaele Hospital - Via Olgettina 60 - 20132 Milan 
pozzoni.cinzia@hsr.it 
Clinical Research Nurse 
Elena Cavone 
Department of Neurology - San Raffaele Hospital - Via Olgettina 60 - 20132 Milan 
cavone.elena@hsr.it 
Regulatory Affairs 
Bonopane Marco 
San Raffaele Hospital - Via Olgettina 60 - 20132 Milan 
bonopane.marco@hsr.it 
 
Data Safety & Monitoring 
Committee composition 
Paolo Muraro, MD 
Department of Neurodegeneration - Imperial College London – South Kensington – SW7 2AZ 
p.muraro@imperial.ac.uk 
 
Xavier Montalban, MD 
Department of Neuroimmunology - Vall d’Hebron University Hospital - Passeig de la Vall d'Heb-
ron 119-129, 08035 Barcelona, Spain 
cem-cat@cem-cat.org 
 
Antonio Uccelli, MD 
Department of Neurology - University of Genua - Via De Toni 5 - 16132 Genua 
auccelli@neurologia.unige.it 

Clinical Trial Protocol 
STEMS 
Rev. n° 1.2 
Page 3 of 54 
Rev. date 10 March 2017 
 
 
Table of Content 
1 
Synopsis ............................................................................................................................................. 5 
1.1 
General Information ........................................................................................................................... 5 
1.1.1 
FLOW-CHART STUDIO CLINICO ........................................................................................................ 9 
1.2 
Treatment and Assessment Schedule ............................................................................................ 10 
2 
Lists ................................................................................................................................................. 12 
2.1 
Abbreviations and definitions ......................................................................................................... 12 
2.2 
Tables .................................................................................................................................................... 14 
2.3 
Figures/Flow-charts ........................................................................................................................... 14 
3 
Introduction: background information and scientific rationale ....................................... 15 
3.1 
Background information .................................................................................................................. 15 
3.2 
The treatment of Progressive Multiple Sclerosis ........................................................................ 15 
3.3 
Somatic Neural stem precursor cells ............................................................................................. 16 
3.4 
Rationale of NPCs for the treatment of MS ................................................................................ 17 
3.5 
Rationale of first dose of NPCs for the treatment of MS .......................................................... 18 
3.6 
Risk benefit assessment .................................................................................................................... 18 
3.6.1 
Risk benefit definitions ............................................................................................................................... 18 
3.6.2 
Risk benefit ratio ........................................................................................................................................... 19 
4 
Objectives ....................................................................................................................................... 21 
4.1 
Primary Objective.............................................................................................................................. 21 
5 
Study design and Endpoints ...................................................................................................... 22 
5.1 
Description of Study Design ............................................................................................................ 22 
5.2 
Duration of study ............................................................................................................................... 22 
5.3 
Primary End-point ............................................................................................................................. 22 
5.4 
Research endpoints ............................................................................................................................ 22 
5.5 
Safety criteria ...................................................................................................................................... 22 
5.5.1 
Safety profile (hNPC cells) ....................................................................................................................... 22 
5.5.2 
Adverse Event (AE) ..................................................................................................................................... 25 
5.5.3 
Adverse Drug Reaction (ADR) ................................................................................................................ 27 
5.5.4 
Serious adverse event (SAE) or Serious Adverse Reaction (Serious ADR) .............................. 28 
5.5.5 
Unexpected adverse reaction (UAR) ...................................................................................................... 28 
5.5.6 
Suspected unexpected serious adverse reaction (SUSAR) .............................................................. 28 
5.5.7 
Pharmacovigilance Procedure .................................................................................................................. 28 
5.5.8 
Follow-up of AE / SAE / ADR ................................................................................................................ 29 
5.5.9 
Pregnancy ........................................................................................................................................................ 29 
5.6 
Research criteria ................................................................................................................................ 29 
6 
Study Enrolment and Withdrawal .......................................................................................... 31 
6.1 
Inclusion criteria ................................................................................................................................ 31 
6.2 
Exclusion criteria ............................................................................................................................... 31 
6.3 
End of Study Phase ............................................................................................................................ 31 
6.3.1 
Follow-up procedure for withdrawn patients ....................................................................................... 32 
7 
Treatment Plan ............................................................................................................................. 33 
7.1 
Experimental Treatment/Drug ....................................................................................................... 33 
7.2 
Immunosuppression: ......................................................................................................................... 33 
7.3 
Advanced Therapy Investigastional Medicinal Product definition ....................................... 34 
7.4 
Preparation of the drug product .................................................................................................... 35 
7.5 
ATIMP packaging and labelling .................................................................................................... 35 

Clinical Trial Protocol 
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Rev. n° 1.2 
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Rev. date 10 March 2017 
 
7.6 
ATIMP supply, storage and accountability ................................................................................. 35 
7.7 
NIMPs packaging and labelling...................................................................................................... 35 
7.8 
NIMPs supply, storage and accountability .................................................................................. 35 
7.9 
Further information, emergency procedures .............................................................................. 35 
7.10 
Methods of assigning patients to treatment groups ................................................................... 35 
7.11 
Concomitant medication accepted ................................................................................................. 36 
7.12 
Prohibited Concomitant Therapy .................................................................................................. 36 
7.13 
Other medical procedures ................................................................................................................ 37 
8 
Overall design ............................................................................................................................... 38 
8.1 
Sample size ........................................................................................................................................... 38 
8.2 
Protocol schedule and timelines...................................................................................................... 38 
8.3 
Study Procedure and Study Flow-chart ....................................................................................... 38 
8.3.1 
Enrolment phase............................................................................................................................................ 38 
8.3.2 
Screening Phase ............................................................................................................................................ 39 
8.3.3 
Pre-treatment phase ...................................................................................................................................... 40 
8.3.4 
Treatment Phase ............................................................................................................................................ 40 
8.3.5 
Follow-up Phase............................................................................................................................................ 41 
9 
Ethical and Regulatory Considerations .................................................................................. 44 
9.1 
Regulatory and ethical compliance ................................................................................................ 44 
9.2 
Responsibilities of IRB/IEC/REB .................................................................................................. 44 
9.3 
Responsibilities of Competent Authority(ies) ............................................................................. 44 
9.4 
Responsibilities of the Investigator ................................................................................................ 44 
9.5 
Patient information and informed consent .................................................................................. 45 
9.6 
Minimizing distress ............................................................................................................................ 45 
9.7 
Minimizing Risk ................................................................................................................................. 46 
10 Data management ........................................................................................................................ 47 
10.1 
Definition of source data and source documents ........................................................................ 47 
10.2 
Documentation of data in the case report form (CRF) ............................................................. 47 
10.3 
Data management .............................................................................................................................. 47 
10.4 
Record keeping ................................................................................................................................... 48 
10.5 
Independent Data Safety and Monitoring Committee .............................................................. 48 
11 Statistical considerations ............................................................................................................ 49 
12 Quality assurance and quality control .................................................................................... 49 
12.1 
Definitions ............................................................................................................................................ 49 
12.2 
Monitoring ........................................................................................................................................... 49 
12.3 
Deviation from study protocol. ....................................................................................................... 49 
12.4 
Audits and inspections ...................................................................................................................... 49 
12.5 
Reference institutes/extramural review board (ERB) ............................................................... 49 
13 Formalities/Regulatory aspects................................................................................................. 50 
13.1 
Legal regulations and guidelines .................................................................................................... 50 
13.2 
Patient insurance ................................................................................................................................ 50 
13.3 
Financial aspects ................................................................................................................................ 50 
13.4 
Final study report .............................................................................................................................. 50 
13.5 
Declaration on the property of the data ....................................................................................... 50 
13.6 
Publication policy ............................................................................................................................... 50 
14 Feasibility declaration ................................................................................................................. 50 
15 Bibliography .................................................................................................................................. 51 

Clinical Trial Protocol 
STEMS 
Rev. n° 1.2 
Page 5 of 54 
Rev. date 10 March 2017 
 
 
1 
Synopsis 
1.1 
General Information 
Title (Acronym) 
STEMS 
Sponsor 
Ospedale San Raffaele 
Principal Investigator 
Gianvito Martino 
Sponsor 
Representa-
tive 
Giancarlo Comi 
 
Objectives 
Primary Objective  
 
 
 
 
The primary objective of the study is to evaluate the feasibility, safety 
and tolerability of intrathecally administered hNPCs, at an escalating 
dose ranging from 0.7x106±10% cells to 5.7x106±10% cells/kg of 
body weight, in patients affected by progressive MS. 
 
 
Endpoints 
Primary Endpoints  
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
Research Endpoints 
 
 
 
 
The following safety endpoints will be used to address the primary 
objectives: 
 
a) SHORT TERM (0-24 hours) 
- 
overall survival 
- 
overall safety and tolerability measured by AE recording 
- 
changes in neurological conditions not related to disease 
- 
proportion of successful intrathecal administration procedure 
(feasibility) 
 
Day 0 = hNPCs administration 
 
b) MID TERM (day 1-day 14) 
- 
overall survival 
- 
overall safety and tolerability measured by AE recording 
- 
changes in neurological conditions not related to disease 
 
c) LONG TERM (day 15 - week 96) 
- 
overall survival 
- 
overall safety and tolerability measured by AE recording 
- 
long term incidence of malignancies 
- 
percentage of subjects with treatment–related mortality de-
fined as death due to procedure and not to the course of dis-
ease 
- 
evaluation of changes in quality of life measures 
- 
changes in neurological conditions 
 
The research endpoints are evaluated to obtain preliminary data on 
the effect of hNPCs in patients affected by progressive MS. 
 

Clinical Trial Protocol 
STEMS 
Rev. n° 1.2 
Page 6 of 54 
Rev. date 10 March 2017 
 
 
Criteria of assessment 
for 
safe-
ty/efficacy/effectiveness 
 
The safety profile will be monitored from the baseline visit to the last 
follow-up visit. 
 
The SHORT TERM safety profile will be evaluated trough: 
 
- Vital signs: During the injection of hNPCs vital signs including ox-
ygen saturation will be monitored before and for 24 hours after 
hNPCs injection. 
- Physical and Neurological examination will be performed before 
the hNPCs injection and before the discharge of the patient. 
- Cerebrospinal fluid examination (CSF): a CSF examination will be 
performed in concomitance with hNPCs administration. 
- Routine Clinical Laboratory tests will be performed before the 
hNPCs injection and before the discharge of the patient. 
 
MID TERM (DAY 1 – DAY 14) safety profile will be evaluated 
trough: 
- Vital signs including oxygen saturation will be monitored during 
visit at week 1 and week 2. 
- Physical and Neurological examination will be performed during 
visit at week 1 and week 2. 
- examination: a neurological examination will be performed during 
visit at week 1 and week 2. 
- Haematological evaluation: will be performed during visit at week 
1 and week 2. 
- Routine Clinical Laboratory tests will be performed during visit at 
week 1 and week 2 (see appendix for detailed tests). 
 
The LONG TERM safety profile will be evaluated trough: 
At every scheduled visit the following procedures will be performed: 
- Vital signs  
- Physical examination 
- Neurological examination 
- Clinical Laboratory tests  
- Haematological evaluations 
- Instrumental follow-up: 
 Week 48 and week 96 electrocardiogram, chest Xray; 
 Week 96 abdominal ultrasound. 
- Administration of MsQuol54 will be performed at 24, 48 and 96 
weeks. 
- Brain and spinal cord MRI with gadolinium: will be performed at 
12, 24, 48 and 96 weeks. 
- Cerebrospinal fluid examination (CSF): a CSF examination will be 
performed 12 weeks ± 2 weeks after hNPCs administration. 
 
 

Clinical Trial Protocol 
STEMS 
Rev. n° 1.2 
Page 7 of 54 
Rev. date 10 March 2017 
 
 
Protocol design 
 
This is a prospective, monocentric, national, therapeutic exploratory, 
phase I, not randomized, open label, not controlled, single dose esca-
lation clinical trial 
  
 
Experimental drug de-
scription 
(dose/regimen/etc) 
 
The Drug Product (DP) can be classified as an Advanced Therapy 
Investigastional Medicinal Product (ATIMP) belonging to the class 
of Cell Therapy Medicinal Products. The ATIMP consists of human 
Neural Precursor Cells (hNPCs) re-suspended in their final formula-
tion medium as defined in IMPD. 
The product will be administered intrathecally through lumbar punc-
ture. 
 
Dosage indications 
The protocol will consist of a total of four TC (from A to D), each of 
which receiving a single escalating dose of hNPCs injected intrathe-
cally as it follows: 
 
- TC-A: 0.7x106 ± 10% cells/kg of body weight; 
- TC-B: 1.4x106 ± 10% cells/kg of body weight; 
- TC-C: 2.8x106 ± 10% cells/kg of body weight; 
- TC-D: 5.7x106 ± 10% cells/kg of body weight. 
 
 
Main selection criteria: 
 
 
Inclusion criteria 
 
1. Signature of the Informed Consent Form (ICF) by the patient or 
patients’ legal tutors 
2. Age 18 to 55 years 
3. Diagnosis of 
a. Progressive MS as per the revised MC Donald 2010 criteria with 
a progressive course according to 2013 Lublin phenotypes clas-
sification (PMS) with failure or intolerance to all approved ther-
apies according to the disease course or without any alternative 
approved therapy 
4. Evidence of progression of the disease defined by an increase of ≥ 
0.5 EDSS points in the last 12 months 
5. Disease duration 2 to 20 years (included) 
6. EDSS ≥ 6.5 
7. Presence of oligoclonal band in the cerebrospinal fluid (CSF) is re-
quired for Primary Progressive MS 
 
 
 
 
 
 

Clinical Trial Protocol 
STEMS 
Rev. n° 1.2 
Page 8 of 54 
Rev. date 10 March 2017 
 
 
Exclusion criteria 
 
They will be excluded from the study patients: 
1. with any active or chronic infection or diseases other than MS, in-
cluding but not limited to infection with HIV1-2, Hepatitis B or 
Hepatitis C and tuberculosis or immune deficiency syndromes; 
2. treated with any immunosuppressive therapy, including but not lim-
ited to natalizumab and fingolimod, within the 3 months prior to 
screening; 
3. treated with interferon-beta or glatiramer acetate within the 30 days 
prior to screening; 
4. treated with corticosteroids within the 30 days prior to screening; 
5. if relapse occurred during the 30 days prior to screening; 
6. with contraindications for or intolerance to any medication, treat-
ments and procedures that will be used in the study; 
7. pregnant or in lactation or of childbearing age who are not willing 
to use a contraceptive method effective* for the entire duration of 
the study; 
8. who, in the opinion of the investigator, showing any condition that 
would preclude study participation. 
*  refer to guideline 
http://www.hma.eu/fileadmin/dateien/Human_Medicines/01 
About_HMA/Working_Groups/CTFG/2014_09_HMA_CTFG_Contr
aception.pdf 
 
 
Sample size 
 
Max 24 patients (max 6 patients for each cohort) 
 
Accrual/duration time-
line 
 
Start: 2017 
 
Recruitment period: 
 
For each subject the study phase will be completed after 96 weeks 
follow up after ATIMP administration. 
 
 
Statistical 
considera-
tions 
 
Descriptive statistics (including means, standard deviations, and me-
dians for continuous variables and proportions for discrete variables) 
will be used to summarize data as appropriate. A statistical analysis 
plan (SAP) with detailed statistical methods and techniques used for 
data analysis will be prepared before conducting the analysis. 
 
 

Clinical Trial Protocol 
STEMS 
Rev. n° 1.2 
Page 9 of 54 
Rev. date 10 March 2017 
 
 
 
1.1.1 FLOW-CHART STUDIO CLINICO 
 
 
 
 
 
 
Figure 1 
 
 
 
Follow up 
Treatment 
Pre-
treatment 
Screening / 
Baseline 
Enrolment 
 
 
 •Immuno-
suppression 
•Clinical, 
laboratory and  
instrumental 
evaluation for  
inclusion/ 
exclusion 
criteria  
assessment 
•Informed 
consent  
signature 
 
 
 
•ATIMP  
injection 
•Evaluation of 
safety from 
day 0 to 2 
weeks 96 

Clinical Trial Protocol 
STEMS 
Rev. n° 1.2 
Page 10 of 54 
Rev. date 10 March 2017 
 
1.2 
Treatment and Assessment Schedule 
 
Tests and assess-
ment 
Screening/ 
Baseline 
(from -28th 
to -7th day) 
Pretreatment 
(from end of screening 
phase to day  -1) 
Day 0 
hNPCs 
Injec-
tion 
V1 
Week 
1 
V2 
Week 
2 
V3 
Week 
3 
V4 
Week 
4 
V5 
Week 
8 
V6 
Week 
12 
V7
W 
16 
V8 
W 
20 
V9 
Week 
24 
V10
W 
30 
V11 
Week 
36 
V12
W 
42 
V13 
Week 
48 
V14
W 
54 
V15 
Week 
60 
V16 
W 
66 
V17 
Week 
72 
V18 
W 
78 
V19  
Week 
84 
V20 
W 
90 
V21 
Week 
96 
V22  
Week 
104 
FU 
 
 
Day 1 
(Friday) 
Day-1 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
Informed Consent 
X 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
Inclusion / 
exclusion criteria 
X 
X 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
Medical History 
X 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
General Physical 
examination with 
Height and Weight 
X 
X 
 
X1 
X 
X 
X 
X 
X 
X 
 
 
X 
 
X 
 
X 
 
X 
 
X 
 
X 
 
X 
X 
Vital Signs2 
X 
X 
X 
X3 
X 
X 
X 
X 
X 
X 
 
 
X 
 
X 
 
X 
 
X 
 
X 
 
X 
 
X 
X 
Routine Clinical 
Laboratory tests 
X4 
X5 
X6 
 
 
X5 
 
X4 
X5 
X4 
Emo 
1 
Emo 
1 
X4 
Emo 
1 
X5 
Emo 
1 
X4 
Emo 
1 
X5 
Emo 
1 
X5 
Emo 
1 
X5 
Emo 
1 
X4 
X5 
VitB12/folates/TSH 
X 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
X 
 
 
 
 
 
 
 
X 
X 
Infectivological 
screening 
X7 
 
 
 
 
 
 
X8 
 
X8 
 
 
X8 
 
 
 
X8 
 
 
 
 
 
 
 
X8 
X8 
Neurological exam-
ination with EDSS 
X 
 
X 
 
 
 
 
X 
 
X 
 
 
X 
 
X 
 
X 
 
X 
 
X 
 
X 
 
X 
 
Haematological 
evaluation 
X 
X 
 
 
 
 
 
X 
 
X 
 
 
X 
 
X 
 
X 
 
X 
 
X 
 
X 
 
X 
X 
Research Samples/ 
Immunological 
analysis9 
X 
 
 
X 
 
 
 
X 
 
X 
 
 
 
 
X 
 
 
 
 
 
 
 
 
 
X 
 
Pregnancy Test 
X15 
 
X 
 
 
 
 
X 
 
X 
 
 
X 
 
X 
 
X 
 
X 
 
X 
 
X 
 
X 
 
ECG 
X 
 
 
 
 
 
 
 
 
 
 
 
X 
 
 
 
X 
 
 
 
 
 
 
 
X 
 
Chest X ray 
X 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
X 
 
 
 
 
 
 
 
X 
 
Abdominal 
Ultrasound 
X 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
X 
 
Treatment –
Tacrolimus and 
blood levels moni-
toring10 
 
 
 
 
 
 
 
 
 
X---------------------------------------------patient continues treatment----------------------------------------------------------------------X 
 
Prophilactic 
Treatment11 
 
 
 
 
 
 
 
 
 
X---------------------------------------------patient continues treatment----------------------------------------------------------------------X 
 
Treatment – 
Prednisone 
 
 
X-----------------decalage12------------------X 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
CSF examination 
 
 
 
X13 
 
 
 
 
 
X 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
Treatment – hNPCs 
 
 
 
X 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
Brain MRI 
X 
 
 
 
 
 
 
 
 
X14 
 
 
X 
 
 
 
X 
 
 
 
 
 
 
 
X 
 
Spinal Cord MRI 
X 
 
 
 
 
 
 
 
 
X14 
 
 
X 
 
 
 
X 
 
 
 
 
 
 
 
X 
 

Clinical Trial Protocol 
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Rev. n° 1.2 
Page 11 of 54 
Rev. date 10 March 2017 
 
 
 
 
Table 1 
1: General Physical examination on Day 0 will be performed before hNPCs Injection and the day after before discharge. 2: Pulse rate, systolic and diastolic blood pressure, respiration rate and axillary temperature. 3: Vital signs (pulse 
rate, systolic and diastolic blood pressure, respiration rate and axillary temperature) will be obtained before hNPCs injection and then after 15’, 60’ and 3 hours after the end of injection. 4: Complete blood cell count; Extensive clinical 
laboratory evaluations: PCR, LDH, Total Protein, albumin, IgG, IgA, IgM, AST, ALT, GGT, ALP, Na+, K+, Ca++, urea, uric acid, alkaline phosphatase, Total Bilirubin, Direct Bilirubin, Glucose, creatinine, creatinine clearance, ferritin, 
Serum Iron, transferrin, transferring saturation, PT, PTT, urin analyisis. 5: complete blood cell count, blood chemistry and urin analysis: PCR, AST, ALT, GGT, Na+, K+, Ca++, Total Bilirubin, Direct Bilirubin, Glucose, creatinine, 
creatinine clearance, LDH, urin analyisis; 6: Blood chemistry (note 5) will be performed at day -1 only if not performed within seven days. 7: infectivological panel at screening: HBs-Ag, HBs-Ab, HBe-Ag, HBc-IgG, HBc-IgM, HBV-
DNA, HCV-Ab, HCV-RNA, HIV-Ab, VZV-IgG, VZV-IgM, HSV1-IgG, HSV1-IgM, EBV-IgG, EBV-IgM, EBV-DNA, CMV-IgG, CMV-IgM, CMV-DNA, Toxo-IgG, Toxo-IgM, HSV2-IgG, HSV2-IgM, Galactomannan detection, 
TPHA, Quantiferon. 8: infectivological panel during follow-up: CMVDNA, EBV-DNA Galactomannan detection. 9: explained in detail in the main test, overall 25 mLs heparinized blood+ 5 mLS serum. 10: tacrolimus: will be 
administered as explained in the text and monitored as explained in the specified chapter. 11: prophilactic treatment are explained in the specified chapter. 12: Oral prednisone (deltacortene) will be administered only from day -1 till week 
4 as explained in the text. 13: CSF examination will be performed at the same time of hNPCs administration. 14: At week 12 we will perform a brain and total spinal cord MRI with gadolinium for safety reason heparinized blood+ 5 mLS 
serum. 15: The pregnancy test will be performed always by urine, except the control before inclusion that will be done by serum. Emo1 = Complete blood cell count 
 
 
T25FW, SDMT, 
9-HPT 
X 
 
 
 
 
 
 
 
 
 
 
 
X 
 
 
 
X 
 
 
 
X 
 
 
 
X 
 
OCT, VEP, SEP, 
MEP 
X 
 
 
 
 
 
 
 
 
 
 
 
X 
 
 
 
X 
 
 
 
 
 
 
 
X 
 
MSQol 
 
X 
 
 
 
 
 
 
 
 
 
 
X 
 
 
 
X 
 
 
 
X 
 
 
 
X 
 
Concomitant Medi-
cation revision 
X 
X 
X 
X 
X 
X 
X 
X 
X 
X 
 
 
X 
 
X 
 
X 
 
X 
 
X 
 
X 
 
X 
X 
AEs, SAEs record 
 
X 
X 
X 
X 
X 
X 
X 
X 
X 
 
 
X 
 
X 
 
X 
 
X 
 
X 
 
X 
 
X 
X 
Relapse Assesment 
 
X 
X 
X 
X 
X 
X 
X 
X 
X 
 
 
X 
 
X 
 
X 
 
X 
 
X 
 
X 
 
X 
X 

Clinical Trial Protocol 
STEMS 
Rev. n° 1.2 
Page 12 of 54 
Rev. date 10 March 2017 
 
 
2 
Lists 
2.1 
Abbreviations and definitions 
A 
ADR         Adverse Drug Reaction 
AE          Adverse Event 
Anti-CD 20   Rituximab 
ATIMP       Advanced Therapy Investigational Medicinal Product 
B 
BMSCs       Bone Marrow Stem Cells 
C 
CBMP       Cell-Based Medicinal Product 
CMV        Cytomegalovirus 
CNS         Central Nervous System 
CRF         Case Report Form 
CRP         C-reactive protein 
CSF         Cerebrospinal fluid 
CTCAE      Common Terminology Criteria for Adverse Events 
D 
ddPCR       droplet digital Polymerase Chain Reaction 
DMDs       Disease-Modifying Drugs 
DMSO       Dimethyl Sulfoxide 
E 
EAE         Experimental Autoimmune Encephalomyelitis 
EBV         Epstein Barr Virus 
EDSS        Expanded Disability Status Scale 
rh-EGF       recombinant human Epidermal Growth Factor 
EKG         Electrocardiography 
ESV         Erythrocyte Sedimentation Rate/Velocity 
F 
rh-bFGF      recombinant human basic Fibroblast Growth Factor 
FK506       Tacrolimus 
FSS          Functional System Scores 
G 
GA          Glatiramer Acetate 
Gd           Gadolinium 
GMP         Good Manufacturing Practice 
H 
hNPCs       human Neural stem/Precursor Cells 
HSA         Human Serum Albumin 
HSCT        Human Stem Cell Transplantation 
I 
IMP         Investigational Medicinal Product 
InSPE        Institute of Experimental Neurology 
i.t.           intrathecally 
i.v.           intravenously 

Clinical Trial Protocol 
STEMS 
Rev. n° 1.2 
Page 13 of 54 
Rev. date 10 March 2017 
 
J 
JCV         John Cunningham Virus 
K 
L 
M 
MCB         Master Cell Bank 
MEP         Motor Evoked Potentials 
MHC-I       Major Histocompatibility Complex-I 
MHC-II      Major Histocompatibility Complex-II 
MRI         Magnetic Resonance Imaging 
MS          Multiple sclerosis 
MSFC        Multiple Sclerosis Functional Composite 
MTR         Magnetization Transfer Ratio 
N 
sNPCs        somatic Neural stem/Precursor cells 
O 
P 
PB           Peripheral Blood 
PCR         Polymerase Chain Reaction 
PCS         Primary Cell Stock 
PML         Progressive Multifocal Leukoencephalopathy 
PP-MS       Primary Progressive Multiple Sclerosis 
PTLD        Post Transplant Lymphoproliferative Disease 
Q 
R 
RRMS       Relapsing-Remitting Multiple Sclerosis 
S 
S1P          Sphingosine-1-phosphate 
SAE         Serious Adverse Event 
SAR         Serious Adverse Reaction 
SCI          Spinal Cord Injury 
SEP          Sensory Evoked Potentials 
SP-MS       Secondary Progressive Multiple Sclerosis 
SUSAR      Suspected Unexpected Serious Adverse Reaction 
SVZ         SubVentricular Zone 
T 
TC           Treatment Cohorts 
U 
UAR         Unexpected Adverse Reaction 
V 
VEP          Visual Evoked Potentials 
W 
wpc          weeks post-conception 
Y 
Z 

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2.2 
Tables 
Table 1 ........................................................................................................................................... 10 
Table 2 – SAE/ADR timeline ........................................................................................................ 27 
Table 3 – List of parameters and its source ................................................................................... 47 
 
 
 
 
 
2.3 
Figures/Flow-charts 
Figure 1 – Trial Flow-chart ........................................................................................................... 9 
Figure 2 – Benefit assessment…………………………………………………………………..19 
 

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3 
Introduction: background information and scientific rationale 
3.1 
Background information 
Multiple sclerosis (MS) is an acquired inflammatory and neurodegenerative immuno-mediated 
disorder of the central nervous system (CNS), characterized by inflammation, demyelination and 
primary or secondary axonal degeneration (Confavreux & Vukusic, 2006). It is a complex multi-
factorial disorder, in which environmental factors are hypothesized to interact with genetically 
susceptible individuals. Genetic susceptibility plays a role in disease initiation and currently uni-
dentified environmental factors are also involved. 
The prevalence rate of MS varies between races and geographical latitude, ranging from more 
than 100 cases out of 100,000 in Northern and Central Europe to 50 cases out of 100,000 in 
Southern Europe. 
Three major clinical forms of MS are recognized: primary progressive multiple sclerosis (PP-
MS), secondary progressive multiple sclerosis (SP-MS), and relapsing-remitting multiple sclero-
sis (RRMS) (Confavreux, Vukusic et al., 2000). RR-MS and SP-MS are probably different stag-
es of the same disease while PP-MS may imply different processes (Lublin, Reingold et al., 
2014). RR-MS represents the more frequent form of the disease (80-85%). It is characterized by 
the occurrence of acute episodes of neurological dysfunction named relapses, followed by varia-
ble recovery and periods of clinical stability. The majority of patients who start with RR-MS 
eventually develop SP-MS, 50% at 10 years and 90% at 25 years (Kragt, van der Linden et al., 
2006). During the progressive phase subjects develop sustained and progressive clinical deterio-
ration with or without superimposed relapses(Lassmann, 2007a). PP-MS is mainly characterized 
by a progressive accumulation of disability since the onset of the disease (Bramow, Frischer et 
al., 2010, Lassmann, 2007b). 
The disease shows heterogeneity with respect to its pathogenesis, clinical manifestations, prog-
nosis and, most interestingly, with respect to its pathology. The essential structural features of 
MS are focal demyelination associated with inflammation and axonal damage (Frischer, Bramow 
et al., 2009). Acute inflammation, present in the early phases of the disease, produces demye-
lination and axonal loss. 
The etiology of MS is unknown, although an autoimmune process has been implicated. It is hy-
pothesized that CNS auto-reactive T cells are stimulated in the peripheral circulation to become 
active and proliferate. The expression of adhesion molecules on the surface of T cells allows ad-
hesion to activated endothelial cells with subsequent migration of T cells into the CNS compart-
ment. These cells upon interacting with CNS myelin antigens again proliferate, and initiate a 
pro-inflammatory cascade within the brain that results in either target-directed immune damage 
or bystander damage. Important cellular and humoral elements are T-lymphocytes, macrophages, 
microglial cells, metalloproteinases and cytokines, including interferon-gamma, and tumor ne-
crosis factor (TNF)-alpha. 
3.2 
The treatment of Progressive Multiple Sclerosis 
There are currently no approved therapies to slow down or prevent the accumulation of disability 
that occurs independently of relapses (Kurtzke, 1983). The current standard of care for patients 
with SP-MS is to use immunomodulatory drugs to reduce the frequency and severity of relapses, 
to provide mental health care for treatment of depression and anxiety, physical therapy to help 
preserve ambulatory function, and occupational therapy to help adapt to the loss of upper ex-
tremity and other functions. Short courses of corticosteroids are occasionally given but their effi-
cacy is not proven, while the side effects associated with their long-term use are well established. 
Chemotherapeutic agents, such as mitoxantrone, are occasionally used in cases of rapidly pro-

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gressing SP-MS (Martinelli, Cocco et al., 2011) but without conclusive evidence that they slow 
down the progression of disability independently of their effects on relapses (Fox, 2006). 
In marked contrast to relapsing forms of MS, the majority of therapeutic clinical studies for pro-
gressive MS have failed to show a benefit compared to placebo. Only the European study of in-
terferon beta-1b for treatment of relapsing SP-MS (Kappos, 1998, Kappos, Polman et al., 2001) 
and the MIMS study of mitoxantrone for treatment of relapsing or rapidly worsening MS (in-
cluding SP-MS) (Hartung, Gonsette et al., 2002) have demonstrated positive results, but they en-
rolled subjects that looked a lot more like RRMS than SP-MS. 
Recently, Ocrelizumab has been demonstrated to induce beneficial effects on PP-MS patients, 
but without conclusive evidence that they slow down the progression of disability independently 
of its effects on the inflammatory processes related to the disease. The poor results of available 
treatment obtained in SP and PP-MS are related to the fact that they are effective in the early in-
flammatory phase of MS, whereas they have a very poor impact on disease course during the 
progressive phase when inflammation fades and neurodegeneration leads to disability. A possi-
ble explanation is that other mechanisms, in part independent from early inflammation mainly 
mediated by cells of the acquired immunity (Magliozzi, Howell et al., 2007, Serafini, Rosicarelli 
et al., 2004), may lead to neurodegeneration (Lassmann, 2007a); this could arise from an exhaus-
tion of the properties of self-repair by local precursors cells harmed by chronic inflammation and 
consequent degeneration of axons and neural cells. 
The lack of efficacious treatments for these patients is indicative of the high unmet need for ef-
fective alternative therapeutically strategies with different mechanisms of action to manage the 
irreversible progression of disability. 
3.3 
Somatic Neural stem precursor cells 
Somatic neural stem/precursor cells (NPCs) represent a heterogeneous population of mitotically-
active, self-renewing and multipotent cells of both the developing and the adult CNS showing 
complex patterns of gene expression that may vary both in space and time (Temple, 2001). Neu-
ral stem/precursor cells can be isolated and established as growth factor-dependent ‘virtually 
perpetual’ cell lines from both the developing and adult human CNS, the subventricular zone 
(SVZ) and hippocampal regions of the adult mammalian brain being found to harbor undifferen-
tiated neural cells which proliferate in vitro in response to rh-bFGF and rh-EGF and in many 
ways appear similar to their embryonic counterparts. 
In defined-medium growth condition, these cells can be grown in vitro for long periods of time, 
while retaining the potential to differentiate into post-mitotic daughter neural cells. There is still 
much debate over how to classify these germinal cells – whether they are stem cells, progenitors 
or precursors – particularly when they are removed from the developing or adult CNS and grown 
in vitro. A number of papers confirm that cells derived from the embryonic (fetal) human CNS 
can be isolated and cloned in culture and grown for extended periods of time in vitro. These cells 
display extensive self-renewal capacity and functional stability, ability to maintain a steady 
growth profile over time, multipotency, and a constant potential for neuronal and glial differenti-
ation over years of continuous in vitro culturing. Human NPCs are also cryopreservable and in-
corporate into the developing and adult rodent CNS. Tumor formation is not observed, even 
when human NPCs are transplanted into immuno-deficient hosts (Garitaonandia, Gonzalez et al., 
2016, Mazzini, Gelati et al., 2015, Pluchino, Gritti et al., 2009). 
As a consequence of their inherent biology, human NPCs from fetal human CNS can establish 
stable, transplantable non-immortalized cell lines in vitro by epigenetic stimulation (Vescovi, 
Parati et al., 1999). In particular, due to their capability to generate in vitro large numbers of cells 
in standardized and quality-controlled preparations, these lines enable the development of clini-
cal strategy of neural transplantation (Martino, Franklin et al., 2010). 

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3.4 
Rationale of NPCs for the treatment of MS 
Inflammation and degeneration are the prototypical pathological processes promoting irreversi-
ble tissue damage and clinical disability in CNS disorders. To prevent irreversible tissue damage, 
the CNS has evolutionarily developed a series of molecular and cellular mechanisms of repair 
that are activated in response to chronic inflammation and neurodegeneration. Endogenous so-
matic neural stem/precursor cells (NPCs) – the self-renewing and multipotent cells of the CNS 
capable of driving neurogenesis and gliogenesis in adult life – migrate into damaged areas to 
promote functional and structural tissue repair. 
However, reactive innate ‘protective’ CNS mechanisms are not robust enough to promote the 
full recovery of the cytoarchitecture of the damaged CNS. In order to foster and reset such 
mechanisms, NPC transplantation strategies have been developed and may represent one of the 
possible and most promising approaches to generate new therapeutic options for still incurable 
chronic neurological disorders. 
Several preclinical and clinical studies using cells obtained either from embryonic, fetal or adult 
tissues are already available (Chen, Sakowski et al., 2016, Chu, Kim et al., 2003, Feldman, Bou-
lis et al., 2014, Jeong, Chu et al., 2003, Park, Teng et al., 2002). Altogether, these studies indi-
cate that transplantation of NPCs is nowadays promising based on the ability of transplanted 
NPCs to adapt their behavior and fate to the CNS microenvironment and to promote neuropro-
tection via different and articulated strategies (Pluchino, Zanotti et al., 2005a). Despite the initial 
perception that transplantation of NPCs could only serve to replace the lost cells, recent experi-
mental studies have shown that transplanted NPCs could also exert additional neuroprotective, as 
well as immunomodulatory, “bystander” effects capable of promoting tissue repair by preventing 
tissue damage, interfering with the pathogenic process, or rescuing endogenous neural cells 
(Muller, Snyder et al., 2006). Thus, NPCs transplantation might represent an ideal therapeutic 
strategy also in neurological disorders characterized by neurodegenerative processes secondary 
to inflammation as MS. The “bystander” effect is a multistep process depending on the timing of 
cell injection and the route of cell transplantation. Once injected intraparenchymally or systemi-
cally [intravenously (i.v.) or intrathecally (i.t.)], NPCs show a certain degree of pathotropism (i.e. 
migration and homing to pathological sites) (Aboody, Brown et al., 2000). Upon migration into 
inflamed CNS areas, transplanted NPCs survive in close proximity to blood vessels where they 
interact with CNS-infiltrating blood borne inflammatory cells, endothelial cells and CNS-
resident astrocytes and microglia. Within perivascular areas, these latter cell populations secrete 
a series of molecules that are capable, on one hand, to recapitulate the microenvironment of the 
prototypical SVZ germinal niche, and, on the other hand, to inhibit the NPC cell cycle by upreg-
ulating the expression of cell cycle-dependent kinase inhibitors. As a consequence of these cellu-
lar interactions, transplanted NPCs retain an undifferentiated phenotype forming an atypical ec-
topic perivascular niche. There, undifferentiated NPCs continue to express and produce a wide 
array of constitutive transmembranal and secreted molecules that, in turn, promote tissue repair 
through immunomodulation and stimulation of endogenous reparative mechanisms (e.g. trophic 
support, cellular plasticity) (Lu, Jones et al., 2003). 
In experimental autoimmune encephalomyelitis (EAE), the animal model of MS, intrathecal (i.t.) 
or intravenous (i.v.) transplantation of NPCs inhibits T-cell proliferation and promotes the apop-
tosis of encephalitogenic CNS-infiltrating T cells (Einstein, Karussis et al., 2003, Pluchino, 
Quattrini et al., 2003, Pluchino, Zanotti et al., 2005b). Similarly to other stem cell types (i.e. 
mesenchymal stem cells), NPCs can exert a bystander immunomodulatory effects also outside 
the CNS upon systemic transplantation. Indeed, in EAE mice, i.v. injected NPCs may also inhibit 
the initiation and maintenance of the inflammatory events occurring within the secondary lym-
phoid organs. The immunomodulatory properties of NPCs seem to be a constitutive and evolu-

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tionarily conserved signature. Indeed, fetal human NPCs constitutively express a considerable 
amount of immune related genes (around 18% of the total number of expressed genes) and inhib-
it T-lymphocyte proliferation as well as dendritic cell maturation in vitro. Indeed, in nonhuman 
primates with EAE, systemically transplanted fetal human NPCs ameliorate disease severity, 
while persisting long-term within the host CNS and in the draining lymph nodes. NPCs also have 
the ability to stimulate endogenous reparative mechanisms. This effect is mainly attributed to the 
capacity of undifferentiated NPCs to secrete a plethora of trophic factors able to protect and pre-
vent neural cell damage and to re-establish the functional interactions between neural and glial 
cells. In animal models of both MS and spinal cord injury (SCI) (Fujiwara, Tanaka et al., 2004), 
enhanced endogenous remyelination following NPC transplantation has been consistently report-
ed (Pluchino, Furlan et al., 2004). 
In diseases in which the stem cell-mediated bystander effect is desirable – for example, diseases 
characterized by primary inflammation (MS, SCI and ischemic stroke) – we still need to under-
stand how to optimize the treatment in order to avoid unwanted side-effects. This is particularly 
true in diseases such as MS, in which repeated inflammatory insults determine the coexistence of 
variable proportions of both neurodegenerative and regenerative processes. As recently suggest-
ed, treatment optimization might be achieved by tightly controlling the timing and route of cell 
administration. An early time-window for NPC transplantation seems to be the most appropriate 
approach, as the expression of genes coding for molecules supporting tissue growth predomi-
nates over genes coding for molecules exerting an antiplasticity effect occurring soon after CNS 
damage. 
Concerning the administration route, orthotopic transplantation should be preferred as recent da-
ta indicate that NPCs – but also other sources of somatic stem cells (i.e. BMSCs) – might form 
tumors, in response to microenvironment mediated signals, when implanted in heterotopic tis-
sues (Melzi, Antonioli et al., 2010). 
 
3.5 
Rationale of first dose of NPCs for the treatment of MS 
The number of cells to be injected into patients via lumbar puncture has been chosen according 
to the data obtained in the preclinical trial we conducted in non human primates (Pluchino et al., 
2009). The maximal dose of cells intracisternally injected in non human primates was 5x106/kg 
cells per animal. This is the very same maximal dose we will reach in humans, as we will start 
our dose escalating protocol from 0.7x106/kg cells per patient and, if no side effects will be rec-
orded, we will proceed up to 5.7x106/kg cells per patients. 
 
3.6 
Risk benefit assessment 
3.6.1 Risk benefit definitions 
Risk is defined as the probability of physical, psychological, social, or economic harm occurring 
as a result of participation in a research study. Both the probability and magnitude of possible 
harm in human research may vary from minimal to considerable. 
Minimal risk exists where the probability and magnitude of harm or discomfort anticipated in the 
proposed research are not greater, in and of themselves, than those ordinarily encountered in dai-
ly life or during the performance of routine physical or psychological examinations or tests. 
Risk above this standard is more than minimal (moderate, maximal) and that imposes limitations 
on the conduct of the research and increases the requirements for monitoring. It also requires 
more stringent approval processes when studying children or otherwise vulnerable populations. 
Increased risk should be accompanied by the probability of appropriately increased benefits. 

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Benefit applies to the potential of the research treatment to ameliorate a condition or treat a dis-
ease. This can apply to an individual participant or to a population. In research as in clinical 
medicine, results cannot be guaranteed but, as a consequence of prior work, a benefit may appear 
to be a reasonable expectation. Since this is research, an advantage for the treatment groups can-
not be presupposed. Since the risks have not been fully evaluated, a statement of individual bene-
fit should be made most cautiously if at all. The investigator should always distinguish between 
research and treatment and never lure the patient into participating in hopes of remission or cure. 
 
3.6.2 Risk benefit ratio 
The final goal of this study and future studies is to develop a curative treatment which can be 
offered to any patient affected by Progressive MS. The proposed clinical trial is designed to ap-
propriately assess the safety of the ATIMP and applied procedures. The potential risks associated 
to the use of a novel technology and a reduced toxicity of immunosuppresion regimen are well 
balanced by the potential benefit of a potentially curative therapy for patients to whom cannot be 
offered therapeutic options. 
3.6.2.1 Benefit 
This approach might represent one of the first cell-based therapy promoting neuroprotection in 
diseases in which neurodegeneration is the final outcome of the tissue pathology. This is because, 
despite the initial perception that transplantation of NPCs could only serve to replace the lost 
cells, recent experimental studies from our own and other groups have shown that transplanted 
NPCs could also exert additional neuroprotective, as well as immunomodulatory, bystander ef-
fects capable of promoting tissue repair by preventing tissue damage, interfering with the patho-
genic process, or rescuing endogenous neural cells (Kokaia et al., 2012). As a matter of fact, 
such results do support the concept that somatic stem cells might promote CNS repair via several 
and articulated strategies in inflammatory and degenerative disorders such as MS (see Figure 2). 
 
Figure 2 – Benefit assessment

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Transplanted NPCs adapt their fate and function(s) to specific environmental needs occurring as 
a result of different pathological conditions and therefore are capable of supporting neuroprotec-
tion not only by promoting cell replacement but also via the so-called bystander effect that is 
mainly aimed at rescuing endogenous neural cells. The bystander effect is made possible by the 
fact that NPCs sense the inflammatory environment (pathotropism) and, within such an envi-
ronment, promote tissue homeostasis and repair by releasing, at the site of tissue damage (ectop-
ic atypical perivascular niche), a milieu of constitutively expressed molecules capable of im-
munomodulation and trophic support. The net and final effect is neuroprotection because trans-
planted NPCs not only rescue damaged cells but also promote cellular and molecular plasticity. 
Thus, NPC transplantation might represent an ideal therapeutic strategy also in neurological dis-
orders characterized by multifocal and diffuse inflammatory and degenerative processes caused 
by the maladaptive interactions among different cell types 
3.6.2.2 Risk 
The risks the patients enrolled in the trial can be subjected to might be due to: 
(1) the procedure of cell injection per se (discussed in details other sections of this project); 
(2) allergic reactions to the medicinal product (discussed in details other sections of this project); 
(3) infections due the non sterility of the cell product: this risk has been very much minimized by 
the extensive in vitro examinations of the medicinal product we performed (see IMPD); 
(4) tumor formation: this risk has been minimized by the in vivo extensive assessment we have 
performed using supra maximal doses of cells infused in immunosuppressed mice (see preclini-
cal in vivo toxicy data). 
3.6.2.3 Ratio 
In conclusions, while the risk patients enrolled in the trial can be subjected to the risks that we 
usually take when we explore a new potential therapy, the benefits might overtly overcome the 
risks since the therapeutic approach we are proposing might represent one of the very first – if 
not the first - neuroprotetective therapies for conditions in which neurodegeneration is due to 
neuroinflammation, i.e. progressive MS patients. 
Moreover, if efficacious such therapy might also represent an approach to treat several other neu-
rodegenerative/neuroinflammatory CNS disorders such as stroke and spinal cord injury. 

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4 
Objectives 
4.1 
Primary Objective 
The primary objective of the study is to evaluate the feasibility, safety and tolerability of in-
trathecally administered hNPCs, at an escalating dose ranging from 0.7x106±10% cells to 
5.7x106±10% cells/kg of body weight, in patients affected by progressive MS. 

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5 
 Study design and Endpoints 
5.1 
Description of Study Design 
This is a prospective, monocentric, national, therapeutic exploratory, phase I, not randomized, 
open label, not controlled, single dose escalation clinical trial. 
5.2 
Duration of study 
Each subject will participate in the study for approximately 96 weeks. Participation will include 
a screening evaluation between -28 and -7 days before the ATIMP administration. A follow-up 
visits will be performed from 1 to 96 weeks. 
5.3 
Primary End-point 
The following safety endpoints will be used to address the primary objectives: 
 
a) SHORT TERM (0-24 hours) 
- 
overall survival 
- 
overall safety and tolerability measured by AE recording 
- 
changes in neurological conditions not related to disease 
- 
proportion of successful intrathecal administration procedure (feasibility) 
 
Day 0 = hNPCs administration 
 
b) MID TERM (day 1-day 14) 
- 
overall survival 
- 
overall safety and tolerability measured by AE recording 
- 
changes in neurological conditions not related to disease 
 
c) LONG TERM (day 15 - week 96) 
- 
overall survival 
- 
overall safety and tolerability measured by AE recording 
- 
long term incidence of malignancies 
- 
percentage of subjects with treatment–related mortality defined as death due to procedure 
and not to the course of disease 
- 
evaluation of changes in quality of life measures 
- 
changes in neurological conditions 
 
5.4 
Research endpoints 
The research endpoints are evaluated to obtain preliminary data on the effect of hNPCs in pa-
tients affected by progressive MS. 
 
5.5 
Safety criteria 
5.5.1 Safety profile (hNPC cells) 
The safety profile of hNPCs cells are unknown. Other heterogenic clinical studies on Parkinson 
disease, ataxia-teleangectasia, Huntington disease, spinal cord injury and inherited cerebellar 
atrophy were conducted and two cases of late onset graft-derived tumours were observed. To 

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date hNPCs cells have never been used in Multiple Sclerosis. However, two phase I trials and 
one phase II trial have been performed in adult patients affected by Amyotrophic Lateral Sclero-
sis and other two phase I trials have been performed in children affected by neuronal ceroid 
lipofuscinosis and and Pelizaeus-Merzbacher disease. The total number of patients exposed to 
hNPCs are 42. In all these studies a surgical injection of stem cell directly in the brain or the 
spinal cord was performed. Overall no side effects or adverse events related to hNPCs were re-
ported. Also anatomo-pathological studies conducted in subjects who died during the follow-up 
of the previous studies for the underlying disease confirmed the feasibility and the therapeutic 
potential of this treatment. 
Preclinical studies in non-human primates with hNPCs did not shown any safety issue. 
 
The safety profile will be monitored from the baseline visit to the last follow-up visit. 
 
The SHORT TERM safety profile will be evaluated trough: 
 
- Vital signs: During the injection of hNPCs vital signs including oxygen saturation will be mon-
itored before and then after 15 minutes, 60’ and 3 hours after the end of injection. Vital signs 
will be then monitored before discharge. 
Particularly, during the treatment visit acute injection toxicity and allergic reactions will be 
monitored, and it will be defined as any of the following events within three hours post injec-
tion: 
- Systolic blood pressure (mmHg) <90 or >180, or >33% decrease or increase from baseline 
- Diastolic blood pressure (mmHg) <50 or >100, or >33% decrease or increase from baseline 
- Heart rate: <60 or >33% decrease; >110 or >33% increase 
- Oxygen saturation: hypoxia (grade ≥2 according to CTCAE) 
- Body temperature (°C) >37.5°C or >1°C increase 
- Any other toxic side effect (according to CTCAE) 
 
- Physical examination will be performed before the hNPCs injection and before the discharge 
of the patient. 
- Neurological examination will be performed before the hNPCs injection and before the dis-
charge of the patient. 
All new or worsening neurological signs and symptoms will be monitored, evaluated and rec-
orded. In the presence of newly identified or worsening of neurological sign or symptom the in-
vestigator should assess whether the patient is experience a relapse of MS or another neurologi-
cal (non-MS related) disorder. 
 
- Cerebrospinal fluid examination (CSF): a CSF examination will be performed in concomitance 
with hNPCs administration. 
- Routine Clinical Laboratory tests will be performed before the hNPCs injection and before the 
discharge of the patient (see appendix for detailed tests). 
 
The following procedures might be performed, at investigator discretion, in the presence of a 
new or worsening neurological or systemic condition: 
- Haematology and biochemistry examination complete of Erythrocyte Sedimentation 
Rate/Velocity (ESV) and CRP 
- Chest X ray 
- Brain and spinal cord Magnetic Resonance Imaging (MRI) with gadolinium 
- Lumbar puncture 

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- Electrocardiography (EKG) 
 
MID TERM (DAY 1 – DAY 14) safety profile will be evaluated trough: 
 
- Vital signs including oxygen saturation will be monitored during visit at week 1 and week 2. 
- Physical examination will be performed during visit at week 1 and week 2. 
- Neurological examination: a neurological examination will be performed during visit at week 1 
and week 2. 
All new or worsening neurological signs and symptoms will be monitored, evaluated and rec-
orded. In the presence of newly identified or worsening of neurological sign or symptom the in-
vestigator should assess whether the patient is experience a relapse of MS or another neurologi-
cal (non-MS related) disorder. 
- Haematological evaluation: will be performed during visit at week 1 and week 2. 
- Routine Clinical Laboratory tests will be performed during visit at week 1 and week 2 (see ap-
pendix for detailed tests). 
 
The following procedure might be performed, at investigator discretion, in the presence of a new 
or worsening neurological or systemic condition: 
- Haematology and biochemistry examination complete of ESV e CRP 
- Chest X ray 
- Brain and spinal cord MRI with gadolinium 
- Lumbar puncture 
- EKG 
 
The LONG TERM safety profile will be evaluated trough: 
At every scheduled visit the following procedures will be performed: 
- Vital signs  
- Physical examination 
- Neurological examination 
- Clinical Laboratory tests 
- Haematological evaluations 
- Instrumental follow-up: 
 Week 48 and week 96 electrocardiogram, chest Xray; 
 Week 96 abdominal ultrasound. 
- Administration of MsQuol54 will be performed at 24, 48 and 96 weeks. 
- Brain and spinal cord MRI with gadolinium: will be performed at 12, 24, 48 and 96 weeks. 
- Cerebrospinal fluid examination (CSF): a CSF examination will be performed 12 weeks ± 2 
weeks after hNPCs administration. 
 
All new or worsening neurological signs and symptoms will be monitored, evaluated and rec-
orded. 
In the presence of newly identified or worsening of neurological sign or symptom the investiga-
tor should assess whether the patient is experience a relapse of MS, any neurological disorder 
related to the recent injection procedure or any other neurological (non-MS related) disorder. 
 

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The following procedure might be performed, at investigator discretion, in the presence of a new 
or worsening neurological or systemic condition: 
- Haematology and biochemistry examination complete of ESV e CRP 
- Chest X ray 
- Brain and spinal cord MRI with gadolinium 
- Lumbar puncture 
- EKG 
 
5.5.2 Adverse Event (AE) 
An adverse event (AE): any untoward medical occurrence in a patient or clinical trial subject 
administered a medicinal product and which does not necessarily have a causal relationship with 
this treatment. An adverse event (AE) can therefore be any unfavourable and unintended sign 
(including an abnormal laboratory finding), symptom, or disease temporally associated with the 
use of a medicinal (investigational) product, whether or not related to the medicinal (investiga-
tional) product. 
In this protocol the follow AEs (not related to the IMP) are expected: 
 
AEs related to the use of tacrolimus: 
The most common adverse events reported with use of tacrolimus are listed below. 
Nervous System: very common (frequency >10): headache and tremors. Common (> 1:100 < 
1/10): paresthesias or dysesthesias mental status changes, seizures, encephalopathy, dizziness, 
impaired writing, and peripheral neuropathy. Late neurotoxicity was highly associated with se-
vere infections and multi-organ failure. 
Respiratory System: common: pleural effusion, dyspnea, lung disorders, cough, pharyngitis rhi-
nitis and sinusitis. 
Renal System: very common: renal function impairment; common: renal insufficiency acute re-
nal failure oliguria, nephrotoxicity, kidney tubular necrosis, urinary disturbances. 
Psychiatric Symptoms: very common: insomnia; common: nightmares, depression, irritability, 
agitation, anxiety, hallucinations, abnormal dreams. 
Immunologic System: Immunologic side effects have included infectious complications as a re-
sult of immunosuppression. Cytomegalovirus is the most frequently encountered viral pathogen 
in patients treated with tacrolimus. Bacterial infections, primarily gram-positive and gram-
negative aerobes, and fungal infections are also encountered. Progressive multifocal leu-
koencephalopathy (PML) related to a JC virus infections have been reported. 
Hematologic System: common: anemia, leukopenia, leukocytosis, thrombocytopenia hematocrit 
alterations. 
Cardiovascular system: very common: hypertension; common: tachycardia hemorrhage, myo-
cardial infarction, thrombosis, hypotension, phlebitis. 
Gastrointestinal system: very common: diarrhea, nausea; common: elevations in liver function 
tests cholangitis cholestatic jaundice hepatitis gastrointestinal hemorrhage stomatitis, gastroin-
testinal perforation. 
Metabolic adverse events: very common: hyperkalemia, hyperglycemia, diabetes mellitus; 
common: hypomagnesemia, hypokalemia, hyperlipidemia, acidosis, hypocalcemia, hypophos-
phatemia, hyponatremia, anorexia. 
Oncologic adverse events: in one study of 936 transplant patients receiving tacrolimus as prima-
ry immunosuppressive therapy, post transplant lymphoproliferative disease (PTLD) occurred in 
15 (1.6%) patients. Serologic evidence of Epstein-Barr virus infections was found in all 15 pa-
tients. Oncologic side effects, or the development of new malignancies, are of particular concern 

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in post transplant patients. Lymphoproliferative disorders are most commonly encountered. 
Lymphadenopathy and monoclonal and polyclonal B cell hyperplasia as well as malignant, often 
fatal, B cell lymphomas have been reported. 
Hypersensitivity: Hypersensitivity side effects including allergic reaction have been reported 
infrequently. 
 
AEs related to chronic steroids treatment methylprednisolone and prednisone: 
 
Expected AEs include: systemic hypertension; glaucoma; fluid retention; hyperglycemia and di-
abetes; mood and emotional disorders, central obesity; osteoporosis; cataracts; increased risk of 
infections; suppressed adrenal gland hormone production; thin skin, easy bruising and slower 
wound healing. 
 
AEs related to lumbar puncture and injection procedure: 
Lumbar puncture: is a minimally invasive, diagnostic or therapeutic procedure that involves the 
removal of a small amount of cerebrospinal fluid (CSF) - the fluid that surrounds the brain and 
spinal cord - followed by the injection of neural cells into the lumbar region of the spinal col-
umn. In this study, the quantity of collected CSF will be replaced by the same amount of cell cul-
ture medium. 
The most frequent adverse events related to this procedure is the orthostatic headache and/or 
back pain. The frequency of these events is about 15-30%. Normally they resolve in few days 
and they can be treated with bed rest, anti-inflammatory drugs and hydration. The injection of 
any substance in the spinal canal could induce an inflammatory/irritative reaction with highly 
variable severity and duration of the AE. This kind of reaction is unpredictable. 
Overall, rare adverse events (<1%) are related to the simple Lumbar Puncture Procedure and 
consist in: risk of infection; risk of bleeding (epidural or subdural hematoma); risk of cranial 
nerve palsy and cauda equina syndrome; hypotension, bradycardia with or without syncope. 
 
Infective Complication due to Immunosuppressive treatments 
To address the risk of opportunistic infections and organ toxicity due to combined steroid and 
tacrolimus treatment, periodic clinical, biochemical (full blood count, renal and liver function) 
drug level (FK506), immunological and virological monitoring (PCR for cytomegalovirus-
CMV and Epstein virus-EBV) will be instituted at fixed intervals (see details in the specific 
chapter) and depending on patient’s clinical and biochemical condition and previous findings, 
for the entire duration of the immunosuppressive treatment. 
Pre-emptive CMV reactivation will be treated by standard treatment for immunosuppressed 
patients (i.e. ganciclovir or foscarnet). Immunosuppression reduction or discontinuation can be 
considered depending on severity. 
EBV reactivation will be treated with immunosuppressive drug reduction or discontinuation 
and/or anti-CD20 (Rituximab) depending on the copy level and patient’s clinical condition. 
Recurrent episodes of CMV reactivation will be addressed with reduction of the immunosup-
pressive drug tacrolimus. Immunosuppression discontinuation will be considered depending 
on severity. 
Bacterial infection will be treated according to standards. Repeated infections will be ad-
dressed by reduction or discontinuation of immunosuppression depending on site of infection, 
severity and patient’s condition. 
 

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The safety profile related to concomitant medication will be assessed trough general physical 
examination, neurological visit and trough blood examination specifically hematology and 
chemistry parameters. At every visit signs and symptoms of possible infective events will be 
recorded. 
 
For all the concomitant medication and procedure used in the protocol only AEs exceeding the 
grade 3 of severity according of the international definition of AEs will be reported (see also 
CTCAE). 
 
5.5.2.1 Documentation of (serious) AE 
Between screening and last follow–up visit the patient will be asked and examined by the inves-
tigator for the occurrence of AEs. All AEs will be documented using CTCAE terms (see Appen-
dix), including 
- 
Severity (listed under) 
- 
Onset date 
- 
Stop date 
- 
Causality assessment with study drug 
- 
Action taken 
- 
Outcome 
The severity of AEs is defined as (see also CTCAE): 
- 
Grade 1 mild 
- 
Grade 2 moderate 
- 
Grade 3 severe 
- 
Grade 4 life-threatening 
- 
Grade 5 death 
All clearly related signs, symptoms and abnormal diagnostic procedures should be grouped to-
gether and recorded as a single term in the CRF. 
5.5.2.2 Forwarding 
All the Serious AE (SAE) and the ADR will be notified, as specified in Table 2. 
 
 
Documentation 
Report 
Time 
Serious  
(AE, ADR) 
Patient’s chart 
CRF  
SAE-Form 
Investigator forwards SAE-
form 
to 
Local 
E.C. 
 
Within 24 h 
Non-serious 
(AE, ADR) 
Patient’s chart 
- 
- 
 
Table 2 – SAE/ADR timeline 
5.5.3 Adverse Drug Reaction (ADR) 
An Adverse reaction (ADR): all untoward and unintended responses to an investigational medic-
inal product related to any dose administered. The phrase responses to an investigational medic-
inal product means that a causal relationship between the investigational medicinal product and 
an adverse event is at least a reasonable possibility, i.e. the relationship cannot be ruled out. 

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5.5.3.1 Attribution Definitions 
Not related: An adverse event that is not related to the use of the investigational product. 
Doubtful: An AE for which an alternative explanation is more likely, e.g., concomitant drug(s), 
concomitant disease(s), or the relationship in time suggests that a causal relationship is unlikely. 
Possible: An AE that might be due to the use of the investigational product. An alternative ex-
planation, e.g., concomitant drug(s), concomitant disease(s), is inconclusive. The relationship in 
time is reasonable; therefore, the causal relationship cannot be excluded. 
Probable: An AE that might be due to the use of the investigational product. The relationship in 
time is suggestive (e.g., confirmed by de-challenge). An alternative explanation is less likely, 
e.g., concomitant drug(s), concomitant disease(s). 
Very likely: An AE that is listed as a possible adverse event reaction, and cannot be reasonably 
explained by an alternative explanation, e.g., concomitant investigational drug(s), concomitant 
disease(s). The relationship in time is very suggestive (e.g., it is confirmed by de-challenge and 
re-challenge). 
5.5.4 Serious adverse event (SAE) or Serious Adverse Reaction (Serious ADR) 
SAE or SAR: any untoward expected medical occurrence or effect that at any dose results in 
death, is life-threatening, requires hospitalisation or prolongation of existing hospitalisation, re-
sults in persistent or significant disability or incapacity, or is a congenital anomaly or birth de-
fect. 
5.5.5 Unexpected adverse reaction (UAR) 
UAR: an adverse reaction, the nature or severity of which is not consistent with the applicable 
product information (e.g. investigator’s brochure for an unauthorised investigational product or 
summary of product characteristics for an authorised product). 
5.5.6 Suspected unexpected serious adverse reaction (SUSAR) 
SUSAR: an adverse event assessed as serious and unexpected and for which there is a reasona-
ble suspected causal relationship with an investigational medical product. 
5.5.7 Pharmacovigilance Procedure 
Serious Adverse Event: The Investigator shall report all SAE immediately to the Sponsor and 
Ethics Committee. The Investigator must fill the SAE report no later than 15 days. For reported 
deaths of a subject, the Investigator shall supply the Sponsor and Ethics Committee with any ad-
ditional information requested. 
Suspected Unexpected Serious Adverse Reaction (SUSAR): In case of a Serious ADR that is 
unexpected, the Investigator/Sponsor must report the reaction as soon as possible (and in any 
case no later than 7 days for reactions that are fatal or life-threatening, and no later than 15 days 
for all other SUSARs) to the competent Regulatory Authorities and to the competent Ethics 
Committee (and that relevant follow-up information is subsequently communicated within an 
additional eight days). 
Periodic reporting: Once a year throughout the trial, the Investigator should provide to Compe-
tent Regulatory Authorities and the Ethics Committee an Annual Safety Report, that is, a listing 
of all suspected Serious ADRs, which have occurred over this period and a report of the sub-
jects’ safety. 
Adverse event recording: All AEs/ADRs regardless of seriousness or relationship to Medicinal 
Product or expectedness are to be recorded on the corresponding page(s) included in the Case 
Report Form. Whenever possible, symptoms should be group as a single syndrome or diagnosis. 
The Investigator should specify the date of onset, maximal intensity, action taken with respect to 

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Medicinal Product, corrective therapy given, outcome and his/her opinion as to whether there is 
a reasonable possibility that the event was caused by the study Medicinal Product. 
The investigator shall keep detailed records of all adverse events, which are reported. These re-
ports must be provided to the competent Authorities if they so request. 
5.5.8 Follow-up of AE / SAE / ADR 
The Investigator should take all appropriate measures to ensure the safety of the patients, nota-
bly he/she should follow up the outcome of any event (clinical signs, laboratory values or other, 
etc.) until the return to normal or until consolidation of patient conditions. 
In the case of any Serious Adverse Event or Serious Adverse Drug Reaction, the patient must 
be followed up until clinical recovery is complete and laboratory results have returned to normal, 
or until progression has been stabilized. This may imply that follow-up will continue after the 
patient left the study and if the Ethical Committee requests additional investigations. 
5.5.9 Pregnancy 
Pregnant and lactating women will not be included in the study. Females must have a negative 
pregnancy test prior to be included in the study and will be required to use a reliable and effec-
tive form of birth control during the study. Male participants will also be required to prevent 
pregnancy in their partners. The birth control methods which may considered effective are: ab-
stinence, oral contraceptives either combined hormonal or progestogen alone, injectable proges-
togen, subcutaneous contraceptive implants of etonogestrel or levonorgestrel, estrogenic vaginal 
ring, percutaneous contraceptive patches, intrauterine device or system (IUD) that has a certified 
failure rate less than 1% per year, male partner sterilization only with the declaration that he is 
the sole partner, male condoms combined with a female diaphragm either with or without a vag-
inal spermicide. 
The birth control methods which are considered unacceptable in clinical trials are: periodic ab-
stinence (calendar, symptothermal, post-ovulation methods), withdrawal (coitus interruptus), 
spermicides only, and lactational amenorrhoea method (LAM).For information refer to guide-
lines 
http://www.hma.eu/fileadmin/dateien/Human_Medicines/01 
About_HMA/Working_Groups/CTFG/2014_09_HMA_CTFG_Contraception.pdf 
 
At every study visit the sexual activity of participants of reproductive age will be re-assessed. If 
a subject who was previously sexually inactive becomes sexually active, s/he will be counseled 
about the need to use a reliable and effective form of birth control. Female subjects of childbear-
ing potential will also be required to undergo pregnancy tests at regular intervals, including and 
especially prior to hNPCs administration. Women who had a positive pregnancy test will not 
treated with hNPCs. 
 
All pregnancies that are identified during the study must be followed to conclusion and the out-
come of each must be reported. The investigator should be informed immediately of any preg-
nancy whether occurring in a female participant or the female partner of a male participant. The 
investigator should report all pregnancies to Sponsor within the same timeframe (24 hours) as 
SAEs, using the SAE report form. Monitoring of the participant should continue until the con-
clusion of the pregnancy, and a follow-up SAE report form detailing the outcome of the preg-
nancy should be submitted to the Sponsor. 
5.6 
Research criteria 
In order to gather preliminary information on the efficacy of the experimental treatment clinical 
and MRI parameters will be recorded throughout the study. 

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We will evaluate: 
- the proportion of disability progression-free patients at week 96; 
- the longitudinal changes from baseline to visit week 96 in T25FW, 9-HPT, SDMT 
Disability in MS is commonly measured by the Expanded Disability Status Scale (EDSS). The 
EDSS is based on a standard neurological examination, incorporating seven functional systems 
(visual, pyramidal, cerebellar, brainstem, sensory, bowel and bladder and cerebral) rated and 
stored as functional system scores (FSS). Each FSS is and ordinal clinical rating scale ranging 
from 0 to 5 or 6. These ratings are then used in combination with ambulation capacity to deter-
mine the EDSS score that ranges from 0 (normal) to 10 (death due to MS). 
Sustained disability progression is defined as an increase of > 0.5 EDSS points in the last 12 
months. This change has not to be attributable to another etiology (i.e. fever, concurrent illness, 
MS relapse or concomitant medication) and has to be confirmed at a regularly scheduled visit 
after at least 12 weeks after the initial disease progression. 
 
Brain MRI parameters: MRI is a useful tool for monitoring MS lesions. Different MRI derived 
parameters are used to assess paraclinical evidence of drug efficacy. 
The following MRI parameters will be evaluated to address the research objective: 
- brain and spinal cord atrophy as defined by percentage in brain and spinal cord volume change 
from baseline to week 96; 
- number of new T2 brain and number of spinal cord lesions and changes in T2 lesion volume at 
week 96; 
- number of new T1 hypointense lesions and changes in T1 hypointense lesion volume between 
baseline and week 96; 
- total Gd-enhancing lesions in the brain and spinal cord MRI between baseline and week 96; 
- other MR parameters including thalamic, cortical, white matter and normal appearing brain tis-
sue average magnetization transfer ratio (MTR) – ancillary sub-study-. 
 
- Multimodal Evoked Potentials are useful for the evaluation of sensory, motor and visual 
pathways; Motor Evoked Potentials (MEP), Visual Evoked Potentials (VEP) and Sensory 
Evoked Potentials (SEP) will be performed at screening, at six and 12 months follow-up and 
then at two years follow-up (week 24, 48 and 96). We will evaluate changes in amplitude and 
velocity conduction of MEP, SEP and VEP between the baseline and week 96. 
 
- Optical coherence tomography (OCT) is a useful tool, especially in the field of research, to 
measure retinal nerve fiber layer (RNFL) thickness as a marker of axonal loss and to detect 
neurodegeneration in vivo and in a non-invasive way. OCT will be performed at screening, at 
six and 12 months follow-up and then at two years follow-up (week 24, 48 and 96). We will 
evaluate RNFL thickness changes between the baseline and week 96. 
 
- to determine immune response in patients treated during immunosuppressive therapy with 
FK506, cellular and humoral immunological assays will be performed. Immune reconstitution 
by PB immunophenotyping (lymphocyte subpopulations) will be obtained on blood at screening, 
at weeks 4th, 12th, 24th, 48th and 96th. 
 
- We will collect data on donor-recipient chimerism on blood samples and CSF (approximately 
at screening, at weeks 4th and 12th; further evaluations only if clinically useful) with ddPCR as-
says. It will be performed using the Bio-Rad QX100 system. 
- A dosage of tacrolimus on CSF will be performed at week 12th to collect data of CNS drug de-
livery. 

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6 
Study Enrolment and Withdrawal 
6.1 
Inclusion criteria 
1. Signature of the informed consent by the patient or patients’ legal tutors 
2. Age 18 to 55 years 
3. Diagnosis of 
a. Progressive MS as per the revised MC Donald 2010 criteria with a progressive course ac-
cording to 2013 Lublin phenotypes classification (PMS) with failure or intolerance to all ap-
proved therapies according to the disease course or without any alternative approved therapy 
4. Evidence of progression of disease defined by an increase of ≥ 0.5 EDSS points in the last 12 
months 
5. Disease duration 2 to 20 years (included) 
6. EDSS ≥ 6.5 
7. Presence of oligoclonal band in the CSF is required for Primary Progressive MS 
6.2 
Exclusion criteria 
They will be excluded from the study patients:  
1. with any active or chronic infection or diseases other than MS including but not limited to in-
fection with HIV1-2, Hepatitis B or Hepatitis C and tuberculosis or immune deficiency syn-
dromes; 
2. treated with any immunosuppressive therapy, including but not limited to natalizumab and fin-
golimod, within the 3 months prior to screening; 
3. treated with interferon-beta or glatiramer acetate within the 30 days prior to screening; 
4. treated with corticosteroids within the 30 days prior to screening; 
5. if relapse occurred during the 30 days prior to screening; 
6. with contraindications for or intolerance to any medication, treatments and procedures that will 
be used in the study; 
7. pregnant or in lactation or of childbearing age who are not willing to use a contraceptive meth-
od effective* for the entire duration of the study; 
8. who, in the opinion of the investigator, showing any condition that would preclude study partic-
ipation. 
* refer to guideline http://www.hma.eu/fileadmin/dateien/Human_Medicines/01 
About_HMA/Working_Groups/CTFG/2014_09_HMA_CTFG_Contraception.pdf 
 
6.3 
End of Study Phase 
For single subject: regular termination. 
The participation of the patient in the study is regularly terminated: 
 ﻅEnd of the follow-up. 
 ﻅIn case of death 
For single subject: premature termination. 
The patient may drop out of the clinical study at any time without stating reasons. This may not 
have any negative consequences for the patient’s further treatment. 
Possible reasons for withdrawal of the patient are: 
 ﻅWithdrawal of the consent. 
 ﻅAny clinical condition that in the Investigator’s opinion could become dangerous for 
the patient and prevent the good conduction of the clinical trial. 
 ﻅProtocol violation that could compromise the quality of study data. 

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 ﻅLack of co-operation/compliance of the patient. 
 ﻅOccurrence of new diseases that could influence the treatment efficacy, for which the 
study medication is contraindicated or that are treated with a medication that is not 
permitted as a concomitant medication. 
 ﻅLack of experimental treatment administration due to failure to release the Medicinal 
Product lot. 
 ﻅLost to the follow-up. 
The patients may withdraw from the study, if they decide to do so, at any time and for any rea-
son. Patients who have been withdrawn from the study cannot be re-included in the trial. 
6.3.1 Follow-up procedure for withdrawn patients 
All withdrawn patients should be recorded by the Investigator in the appropriate pages of the 
CRF when considered as confirmed. If possible, the patients should be assessed using the proce-
dure planned for the end of study visit. 
If an event causing dropout corresponds to the definition of a Serious AE/ADR, the investigator 
carries out the pharmacovigilance procedure described. 
For patients considered lost to follow-up, the CRF must be filled in up to the last visit performed. 
The Investigator should make every effort to re-contact and to identify the reason why the pa-
tient failed to attend the visit and to determine his/her health status. 
In case of death autopsy will be strongly encouraged for evaluation of pathology and presence of 
transplanted cells.  
6.3.1.1 Natural study termination 
At the end of total follow-up. 
6.3.1.2 Premature Termination or Suspension of study 
This study may be temporarily suspended or prematurely terminated if there is sufficient reason-
able cause or if any of the stopping criteria described in this protocol occurs. Circumstances that 
may warrant termination or suspension include, but are not limited to: 
 determination of unexpected, significant, or unacceptable risk to participants; 
 demonstration of efficacy that would warrant stopping; 
 insufficient compliance to protocol requirements; 
 data that are not sufficiently complete and/or evaluable; 
 determination of futility; 
 reconsideration of the risk/benefit ratio. 
 
Written notification, documenting the reason for study suspension or termination, will be pro-
vided by the suspending or terminating party to <investigator, funding agency, the sponsor and 
regulatory authorities>. 
If the study is prematurely terminated or suspended, the PI will promptly inform the Institutional 
Review Boards (IRBs) and/or Independent Ethics Committees (IECs), will provide the reason(s) 
for the termination or suspension and will instruct additional procedures to be followed in order 
to ensure that adequate consideration is given to the protection of the patient’s interests. 
For patients who have received the ATIMP, a long term safety follow-up it is required by law. 
Study may resume once concerns about safety, protocol compliance, data quality are addressed 
and satisfy the sponsor, IRB and/or CA. 

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7 
Treatment Plan 
7.1 
Experimental Treatment/Drug 
The protocol will consist of a total of four treatment cohorts (TC), labeled from A to D, each of 
which receiving a single escalating dose of allogenic hNPCs injected intrathecally, as it follows: 
- TC-A: 0.7x106 ± 10% cells/kg of body weight; 
- TC-B: 1.4x106 ± 10% cells/kg of body weight; 
- TC-C: 2.8x106 ± 10% cells/kg of body weight; 
- TC-D: 5.7x106 ± 10% cells/kg of body weight. 
 
The intrathecal injection of hNPCs will be performed in a hospitalized setting. 
The patients will be left in Trendelenburg position for about 30 minutes and monitored for at 
least 24 hours. All patients will receive normal saline solution (500 cc). 
Body temperature, blood pressure, heart rate and oxygen saturation will be measured at baseline 
and after 15, 60, 180 minutes and then before discharge. Any toxicity will be treated at the dis-
cretion of the attending neurologist. 
All patients will receive an i.v. dose of methylprednisolone (125 mg) immediately prior to lum-
bar puncture. 
The injections of cells will be performed with lumbar puncture in lateral decubitus position re-
moving about 10 cc of CSF that will be used for chemical-physical, infectivological analysis and 
OBs/ BBB analysis. 2 CSF ml will be stored for further research analysis. CSF will be replaced 
with the 10 cc of suspension solution containing hNPCs (at 2 ml/min injection rate). The injec-
tion of stem cell will be performed in about 5 minutes. In case of traumatic lumbar puncture with 
haematic CSF, the injection of hNPCs will not be performed. A second attempt will be per-
formed after 15 ± 3 days. 
7.2 
 Immunosuppression: 
Human NPCs have been shown to be relatively low immunogenic (e.g., no expression of MHC-I 
molecules and low levels of MHC-II molecules). However, as they are human in origin their 
transplantation will be handled like other organ transplant in order to prevent the unexpected re-
jection (if any) of transplanted hNPCs and we have planned to put our patients on long term im-
mune suppression. The immunosuppression will be applied in agreement with previous studies 
with hNPCs in Amyotrophic Lateral Sclerosis and in children affected by neuronal ceroid 
lipofuscinosis and and Pelizaeus-Merzbacher disease. In all these studies the patients a long last-
ing immunosuppression was applied in order to guarantee the engraftment of stem cell. Data de-
riving by these studies confirmed that immunosuppression was well tolerated by the majority of 
patients and no opportunistic infections were reported till now. 
According to our hematologist guidelines the patients will receive and oral prednisolone as 
explained below. 
Tacrolimus (FK506) is a macrolide lactone with potent immunosuppressive activity 100 times 
that of cyclosporine by weight. The molecular mechanism of action is mediated via an inhibition 
of the phosphorylase activity of calcineurin by drug-immunophilin complex, resulting in the in-
hibition of IL-2 gene expression. 
There are many studies now showing significant efficacy of tacrolimus in GVHD prevention in 
both allogeneic HSCT. 
FK506: The initial oral dose of tacrolimus will be 0.05 mg/kg twice daily from day-7. Tacroli-
mus, if tolerated, will be given and gradually tapered till week 96. 

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A pharmacological surveillance of FK506 through drug level for consequent dose adjustment 
will be performed. 
In order to maintain a target of 5-10 ng/ml, whole blood levels of tacrolimus will be obtained: 
- two times a week until the therapeutic range was reached (first samples on day -4 and day -1); 
- weekly till week 4 after hNPCs injection; 
- at week 8, 12, 24, 36, 48 and then every 24 weeks till the end of follow-up. 
Tacrolimus will be started day -7 before hNPCs injection as immunosuppressive medication to 
prevent the rejection of the cells, and it is expected that the patients will take the drug for the end 
of the follow-up. 
Dose modifications of tacrolimus were dependent primarily on serum creatinine and the blood 
levels of tacrolimus. A dose reduction of at least 25% was mandatory for patients with a creati-
nine elevated to greater than 2.0 times baseline and at least a 50% reduction for patients with a 
creatinine greater than 3 times baseline. 
 
Oral prednisone (deltacortene) 50 mg tapering of 5 mg every five days as follows dosage: 
37,5 mg, 25mg, 12,5 mg and 5 mg. 
Any or all the immunosuppressive agents may be reduced or stop at the discretion of the investi-
gator if it is suspected or determined that the immunosuppressive agent is the cause of toxicity 
and if symptoms cannot be managed adequately with symptomatic treatment. 
Anti-infective prophylaxis will be administered for the duration of FK506/tacrolimus therapy 
with acyclovir (1 tablet-800 mg twice daily), and co-trimoxazole (1 tablet-800 mg/160 mg, 
three times/week. Folic acid (1 tablet of 5 mg) will be administered with co-trimoxazole, as 
supportive therapy to reduce co-trimoxazole myelotoxicity three times a week. 
 
7.3 
Advanced Therapy Investigastional Medicinal Product definition 
The Drug Product (DP) can be classified as an Advanced Therapy Investigastional Medicinal 
Product (ATIMP) belonging to the class of Cell Therapy Medicinal Products. 
 
The following definitions shall apply throughout this study. 
 
The Starting Materials is the Master Cell Bank (MCB) originated from cryopreserved non-
immortalized human fetal neural precursor cells (hNPCs) BI-0194-008 cell line obtained from a 
single 10-12 weeks post-conception (wpc) human fetus and manufactured as defined in the In-
vestigastional Medicinal Product Dossier (IMPD). 
 
The Drug Substance (DS) will consist of Working Cell Bank (WCB) obtained after expansion of 
MCB as defined in IMPD. 
 
The DP will be constituted by thawed WCB-hNPCs re-suspended in their final formulation me-
dium as defined in IMPD. 
 
Medicinal product manufacturing and release will be performed at “Laboratorio di Terapia Cel-
lulare e Genica Stefano Verri - ASST - Monza, Ospedale San Gerardo” (later also “Laboratorio 
Verri”), as described in the IMPD. Production will be performed according to GMP (Good Man-
ufacturing Practice) certified facility authorized by Italian Ministry of Health according to 
D.L.vo 166/2003 for the production of sterile medicinal products, the production of specific me-

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dicinal products obtained with particular technologies (i.e. cell therapy), and for primary packag-
ing operations. 
7.4 
Preparation of the drug product 
When a new patient, enrolled in the clinical protocol, is ready for treatment, cryopreserved WCB 
vials will be thawed. Freezing medium will be discarded and cells re-suspended in 10 ml of in-
jection medium and transferred to primary package(s). 
7.5 
ATIMP packaging and labelling 
The Medicinal Product will be labelled with a unique identification code of the patient and of the 
product, released by Laboratorio di Terapia Cellulare e Genica Stefano Verri, according to 
cGMP. 
7.6 
ATIMP supply, storage and accountability 
The ATIMP is produced at 37° C by “Laboratorio Verri” and it will be injected into patient's 
CSF at physiological body temperature. The DP will be shipped from “Laboratorio Verri” to 
OSR with a dedicated courier at controlled temperature (25°C ± 5°C) monitored with a data log-
ger. The ATIMP will be released to an Investigator or another member of the investigating team 
designated by Sponsor or Principal Investigator. The person receiving the DP should control the 
data logger to check the temperature and report any non-compliance. Then the ATIMP will be 
transferred in a biohazard container at room temperature to the clinical ward for injection, ac-
cording to OSR internal operating procedures. 
Any non-compliance related to the delivery and / or transport of the drug will be managed ac-
cording to OSR internal operating procedures. 
The ATIMP will kept at ambient or room-air temperatures (max 37°C) while waiting for pa-
tient’s preparation for intrathecally injection. 
The accounting of the ATIMP will be recorded in the ATIMP log in the Trial Master File (TMF).  
After injection the CRN will send to the OSR Pharmacy by fax the documentation certifying the 
receipt of the ATIMP, injection of ATIMP and waste disposal. 
The PI is responsible for the accountability and traceability of the product. 
7.7 
NIMPs packaging and labelling 
NIMPs will be provided by the local pharmacy, according to institutional guidelines. 
7.8 
NIMPs supply, storage and accountability 
NIMPs will be delivered by courier to the responsible of pharmacy. The drugs has to be stored 
following the drug-specific SPC with access only to authorised personnel. The absolute amounts 
of drug administered per day and per patient have to be documented according to institutional 
guidelines. 
The PI is responsible for supply, storage and accountability of the products. 
7.9 
Further information, emergency procedures 
No other special information is necessary. 
7.10 Methods of assigning patients to treatment groups 
The protocol will consist of a total of four TC (from A to D), each of which receiving a single 
escalating dose of hNPCs injected intrathecally as it follows: 
 
 

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- TC-A: 0.7x106 ± 10% cells/kg of body weight; 
- TC-B: 1.4x106 ± 10% cells/kg of body weight; 
- TC-C: 2.8x106 ± 10% cells/kg of body weight; 
- TC-D: 5.7x106 ± 10% cells/kg of body weight. 
 
The trial will start with TC-A and will go through the subsequent enrolment of patients to be in-
cluded in TCs from B to D. 
Each cohort will consist of three patients at minimum. However in case of safety issue the nu-
merosity of each TC will be increased to six patients, as explained below. 
We define a DLT any adverse event of grade 3 possibly related to hNPCs or to the procedure. 
However we will not consider a DLT a prolonged hospitalization due to post lumbar puncture 
headache if not associated to any meningeal sign. We will not considered DLT adverse events 
related to concomitant treatments (tacrolimus and steroids) 
After the inclusion of the first patient of the TC we will wait at least 14 days to treat the second 
patient. The same interval time will be used for all the further patients within the same TC. After 
the inclusion of all the planned patients within a TC, we will wait at least 3 months after the in-
clusion of the last patient before switching to the higher TC. 
In case of none DLT within the TC, we will wait at least 3 months after the inclusion of the last 
patient and then we will move to the higher TC. 
In case of 1 DLT within the TC, the cohort will be extended to 6 patients. If another DLT will be 
observed, the current dosage will be considered excessive ad the immediate lower dosage will be 
considered MTD. 
The safety monitoring board will review all safety date in the case of an AE ≥ 3 evaluated “pos-
sibly related to hNPC” by investigators and in any case before the shifts between TCs. 
 
This will be repeated for every TC up to the end of the study. 
 
7.11 Concomitant medication accepted 
A concomitant therapy is any drug or substance administered from the time of the subject’s writ-
ten consent until the subject’s End of Study Visit. 
Concomitant treatment with any of the following is allowed: 
- Medications necessary for the treatment of AEs 
- Any medications used to treat MS symptoms such as spasticity, bladder impairment, pain, 
or depression 
- Short courses of iv high-dose corticosteroids as a rescue medication for exacerbations of MS 
- Corticosteroids that are administered by non-systemic routes (e.g., topical, inhaled) 
Any treatment which are continued during the study and/or initiated or changed during the study 
must be recorded in source data and in the CRF. 
 
7.12  Prohibited Concomitant Therapy 
Concomitant treatment with any of the following is not allowed while receiving study treatment, 
unless as otherwise described in this protocol: 
- Any non-study drug treatments directed toward the treatment of MS such as immunomodu-
latory treatments or chronic immunosuppressant therapy except tacrolimus 
- Any investigational product, including investigational symptomatic therapies for MS and 
investigational therapies for non-MS indications 

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- Systemic steroid therapy including, but not limited to, oral corticosteroids (e.g., prednisone) 
or periodic (e.g., monthly) treatment with IV methylprednisolone, except for protocol-
defined treatment of relapses as described above and course of oral prednisolone at the 
above mentioned dosage in the first 25 days after hNPCs injection. 
Subjects who receive any of these restricted treatments may be required to permanently discon-
tinue study treatment. 
 
7.13 Other medical procedures 
All the routinely procedures used in MS patients are allowed. 
 

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8 
Overall design 
8.1 
Sample size 
Will be enrolled max 24 patients. 
 
8.2 
Protocol schedule and timelines 
Anticipated start of recruitment: April, 2017 
Anticipated stop of recruitment: March 2018 or study termination 
End of total follow-up:  
When all subjects have completed the 96 weeks follow up 
period or terminated the study 
Draft statistical report  
3-4 months after the end of total follow-up 
Integrated final report  
6 months after the end of total follow-up 
 
8.3 
Study Procedure and Study Flow-chart 
8.3.1 Enrolment phase 
Is the process of informed consent signature (described in the ethical consideration chapter). 
Starting from the signature of the informed consent by the patient or patients’ legal tutors, the 
subjects will be enrolled in the clinical trial. When the enrolment is confirmed the patient screen-
ing number will be assigned consecutively in increasing order starting with ‘001’. The list will be 
filed with the investigator’s file. After the enrolment of a patient in the study, the investigator 
sends the enrolment module to the principle investigator by fax or email to clinical research 
monitor. 
 
Those protocol phases are foreseen after signature of the informed consent: 
 
 Screening/baseline phase, during which the conditions required by the clinical protocol for 
patients’ inclusion/exclusion will be assessed (screened subject). The duration will be of 
three weeks (+/- 3 working days). At the end of the screening phase, the inclusion/exclusion 
criteria of the study will be evaluated (enclosed subject). 
 Pre-treatment phase: carried from the end of the screening phase to the day before the start of 
treatment. 
 Treatment phase: The intrathecally administration of hNPCs will be performed on day 0. Pro-
filactical treatment with Tacrolimus will be performed for 96 weeks starting from day -7 
whereas oral corticosteroids will be administered for the first 4 weeks. 
 Follow-up phase: 96 weeks after the experimental drug administration day 0.  
 
At the end specify if the end of total follow-up or single patient follow-up. 
 
At the end of the total follow-up, for all patients enrolled and treated in the study is planned a 
long term follow up, in accordance with the national applicable laws and the international guide-
lines. 
 

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8.3.2 Screening Phase 
All subjects must sign informed consent prior to conduct any study specific procedure. All the 
inclusion criteria and none of the exclusion criteria listed above respectively, must be satisfied. 
Following consent, all these evaluations will be performed within the screening phase three 
weeks (+/- 3 working days): 
- Record of general medical and MS history with concomitant medication revision 
- Inclusion Criteria and Exclusion criteria evaluation 
- Physical examination 
- Record of vital signs (blood pressure, heart rate, O2 saturation, temperature, weight, height) 
- Hematological visit 
- Haematological blood tests comprehensive of: 
  Complete blood count*, extensive clinical laboratory evaluations**, 
  *Hemoglobin, Platelets, Leukocytes, Neutrophils, Lymphocytes, Monocytes 
 **PCR, LDH, Total Protein, albumin, IgG, IgA, IgM, AST, ALT, GGT, ALP, Na+, K+, 
Ca++, BUN, uric acid, Total Bilirubin, Direct Bilirubin, Glucose, creatinine, creatinine 
clearance, ferritin, Serum Iron, transferrin, transferring saturation, INR, PTT 
- Infections screening: HBs-Ag, HBs-Ab, HBe-Ag, HBc-IgG, HBc-IgM, HBV-DNA, HCV-
Ab, HCV-RNA, HIV-Ab, VZV-IgG, VZV-IgM, HSV1-IgG, HSV1-IgM, EBV-IgG, 
EBV-IgM, EBV-DNA, CMV-IgG, CMV-IgM, CMV-DNA, Toxo-IgG, Toxo-IgM, 
HSV2-IgG, HSV2-IgM, Galactomannan detection 
- Vitamin B12 and folats evaluation 
- TSH evaluation 
- Blood group 
- Serum pregnancy test (if applicable) 
- Immunological analysis 
- Electrocardiogram 
- Chest Rx 
- Abdominal ultrasound 
- Neurological examination comprehensive of EDSS scale, T25FW, 9HPT, SDMT 
- Neurophysiological assessment (OCT, VEP, SEP, MEP) 
- Brain and Spinal cord MRI 
 
After the patient inclusion the investigator sends the enrolment module to the clinical research 
module. When the inclusion is confirmed the patient definitive number will be assigned as fol-
low: 
 
Patient number for monocentric trial (UPN) 
Screening number  
Inclusion number  
0 
0 
1 
0 
0 
1 

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8.3.3 Pre-treatment phase  
This phase will start after the ending of all the procedure of the screening/baseline phase and it 
will end the day before the treatment phase (day-1): 
 
Day 1 of pretreatment phase (Friday): 
 
- Inclusion/exclusion criteria revision 
- Record of vital signs (blood pressure, heart rate, O2 saturation, temperature, weight, height) 
- AEs, SAEs record 
- Tacrolimus administration at 0.05 mg/kg twice daily 
- Acyclovir, co-trimoxazole and pholates from the beginning of tacrolimus as prophylactic 
medication 
- Haematological evaluation 
- Physical examination 
- Pharmacological surveillance of FK506 through blood sample will be performed after three 
(Monday) and seven days (Friday). This procedure will be performed till the tacrolimus 
will reach the therapeutical range. Haematological blood test comprehensive of: com-
plete blood cell count, blood chemistry and urin analysis: PCR, AST, ALT, GGT, Na+, 
K+, Ca++, Total Bilirubin, Direct Bilirubin, Glucose, creatinine, creatinine clearance, 
LDH, PT, PTT (will be performed seven days after the beginning of tacrolimus) 
 
Day -1 
 
- Neurological examination comprehensive of EDSS scale 
- Vital signs 
- AEs, SAEs record 
- Relapse Assessment 
- Concomitant medication revision 
- Haematological blood examination: complete blood cell count, blood chemistry: will be per-
formed if not performed within seven days 
- Prednisone (Deltacortene) administration (50 mg) 
 
At the end of all these assessment, patients will be assigned to one of the four different cohort of 
treatment (A-D) (see chapter Methods of assigning patients to treatment groups). 
 
8.3.4 Treatment Phase 
On day 0 patients will undergo pharmacological surveillance of FK506 through blood sample. 
IV Methylprednisolone at the dosage of 125 mg will be administer one hour prior to lumbar 
puncture. 
They will received intrathecally hNPCs through lumbar puncture in an hospitalized setting and 
the patients will be monitored for at least 24 hours. Before the injection, hNPCs will be thawed 
and DMSO will be carefully washed out; hNPCs will be than resuspended in 10 cc of saline so-
lution. 
CSF examination cohprensive of chemicophisical and infectivological will be performed. 
 

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Body temperature, blood pressure, heart rate and oxygen saturation will be measured at baseline 
and after 15, 60, 180 minutes and then before discharge. Any toxicity will be treated at the dis-
cretion of the treating neurologist. 
 
8.3.5 Follow-up Phase 
The follow-up phase will have a duration of 96 weeks. However the patients we’ll be followed 
for the further 6 years at least. 
Patients will attend control visits weekly for the first 4 weeks after hNPCs administration and 
then at 8 and 12 weeks. Starting from 12 weeks visits will be performed every 12 weeks. The 
procedures of each visit are described in details in the flow chart. 
During the follow-up phase patients will receive tacrolimus, acyclovir, co-trimoxazole and fo-
lates for the entire follow-up (till week 96) and oral prednisone for 4 weeks. 
 
The patient will undergo:  
- Neurological visit with EDSS at week 4, 12, and then every scheduled visit 
- T25FW, 9-HPT, SDMT at week 24, 48, 72, 96 
- Haematological visit at week 4, 12, and then every scheduled visit 
- General physical examination at every scheduled visit 
- Record of vital signs at every scheduled visit 
- AEs, SAEs record- Relapse Assessment at every scheduled visit 
- Concomitant medication revision at every scheduled visit 
- Haematological blood tests comprehensive of: 
   Complete blood count, extensive clinical laboratory evaluations will be performed at week 4, 
12, 24, 48 and 96 
   Complete blood tests and biochemistry, urinanalisys evaluation will be performed at week 2, 8, 
36, 60, 72 and 84 
   Complete blood count evaluation will be performed at week 16, 20, 30, 42, 54, 66, 78 and 90 
- Infectivological screening: CMV-DNA, EBV-DNA, Galactomannan detection will be per-
formed at week 4, 12, 24, 48 and 96 
- Immunological analysis: will be performed at week 4, 12, 24, 48 and 96 
- Whole blood levels of tacrolimus will be obtained as reported in the specific section, in order 
to maintain a target of 5-10 ng/ml. 
- CSF examination will be performed at week 12. During this visit the patient will be admitted to 
the hospital in the morning and will be discharged the day after (24 hours of observation after 
procedure). Complete chemico-physical evaluation, oligoclonal bands detection, CSF Cell eval-
uation, virological screening, ddPCR assays. 
- Electrocardiogram, Chest Xray will be performed at visit week 48 e 96 and abdominal ultra-
sound only at visit 96 
- Brain and spinal cord MRI at week 12, 24, 48 and 96 
- Neurophysiological assessment (OCT, VEP, SSEP, MEP) will be performed at week 24, 48 
and 96 
- Administration of MsQuol54 will be performed at 12, 24, 48, 72 and 96 weeks 
 
Additional visits will be planned if necessary. Specifically unscheduled visits will be performed 
in case of new or worsening symptoms related to the disease to exclude a relapse or in case of 
any adverse event not related to the disease. 
 
 

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END OF STUDY VISIT: 
This visit will be performed 8 week after the end of treatment visit. 
 
The patient will undergo: 
- Neurological visit with EDSS 
- Haematological visit 
- General physical examination 
- Record of vital signs 
- AEs, SAEs record- Relapse Assessment 
- Concomitant medication revision 
- Haematological blood tests comprehensive of: Complete blood tests and biochemistry, uri-
nanalisys evaluation 
- Infectivological screening: CMV-DNA, EBV-DNA, Galactomannan detection 
- Administration of MsQuol54 
 
UNSCHEDULED VISIT: 
In case of clinical, neurological deterioration or every systemic new symptom not due to MS an 
unscheduled visit will be performed. During this visit the following procedure might be per-
formed, at investigator discretion: 
- complete neurological and general examination 
- Haematology and biochemistry examination complete of ESV e CRP, CMV DNA, EBV DNA 
will be determinate every 24 weeks 
- Chest Xray 
- Brain and spinal cord MRI with gadolinium 
- Lumbar puncture 
 
MS RELAPSE VISIT  
The assessment, management and reporting of MS relapse is made by the Primary Treating Phy-
sician. 
MS relapse definition: appearance of a new neurological abnormality or worsening of previously 
stable or improving pre-existing neurological abnormality, separated by at least 30 days from 
onset of a preceding clinical demyelinating event (McDonald et al. 2001). The abnormality must 
have been present for at least 24 hours and occurred in the absence of fever (< 37.5°C) or known 
infection. MS relapse is defined as: any perceived change in symptoms or signs that persist for  
48 hours in the absence of flu or fever and that is accompanied by objective changes on EDSS 
functional subscores (FSS) that leads to either a 1.0 point change in EDSS (for baseline EDSS 3 
- 5.0) or a 0.5 change (for baseline EDSS  5.5) together with FSS changes of 2 points in any 
one FSS or 1 point in any 2 FS. Only relapses meeting the protocol definition will be considered 
as measure of efficacy. 
Diagnosing MS relapses during the study: Patients will be instructed to immediately contact the 
Primary Treating Physician if he/she develops new or re-occurring or worsening neurological 
symptoms. If a patient reports new neurological symptoms or worsening of previous symptoms, 
an unscheduled visit is to be planned as soon as possible, ideally within 7 days. During this visit, 
the treating physician will first assess whether the new/worsening neurological abnormality is 
consistent with the definition of MS relapse above. 
If so, the standard neurological examination (for the EDSS score) should be performed by the 
Independent EDSS Rater. If there is any doubt in the opinion of the treating physician, the de-
fault must always be to refer the case to the Independent EDSS Rater to perform an EDSS rating. 

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Confirmation of MS relapse: the definition of a confirmed MS relapse is one accompanied by a 
clinically relevant change in the EDSS performed by the Independent EDSS Rater, i.e. an in-
crease of at least 0.5 points on the EDSS score, or an increase of 1 point on two FSs or 2 points 
on one FS, excluding changes involving bowel/bladder or cerebral FS compared to the previous 
available rating (the last EDSS rating that did not occur during a relapse). 

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9 
Ethical and Regulatory Considerations 
9.1 
Regulatory and ethical compliance 
This clinical study was designed, shall be implemented, conducted and reported in accordance 
with: 
- the ethical principles laid down by the 18th World Medical Assembly (Helsinki, 1964) and all 
applicable amendments laid down by the World Medical Assemblies; 
- the International Conference on Harmonization (ICH) Harmonized Tripartite Guidelines for 
Good Clinical Practice (GCP); 
- all international laws and regulations; 
- all national laws and regulations of the country(ies) in which the clinical trial is performed; 
- all international or national applicable guidelines. 
 
9.2 
Responsibilities of IRB/IEC/REB 
The protocol, the patient information sheet, the data protection declaration form, the informed 
consent/assent form and all proposed clinical trial documents must be reviewed and approved by 
a properly constituted Institutional Review Board/Independent Ethics Committee/Research Eth-
ics Board (IRB/IEC/REB) before study start. 
 
It is mandatory to obtain the written and dated approval/favorable opinion, signed by the chair-
man with (IRB/IEC/REB) composition. The clinical trial (study number, clinical trial protocol 
title and version number), the documents reviewed (clinical trial protocol, Informed Consent 
Form, Investigator’s Brochure, investigator’s CV, etc.), the list of voting members and their 
qualifications and the date of the review should be clearly stated on the written ethics committee 
approval/favorable opinion. 
 
9.3 
Responsibilities of Competent Authority(ies) 
The protocol and all proposed clinical trial documents must be reviewed and approved by Com-
petent Authority(ies) of the country(ies) in which the clinical trial is performed before study start. 
It is mandatory to obtain the written and dated authorization, signed by the chairman of CA of 
the country(ies) in which the clinical trial is performed before study start. 
 
9.4 
Responsibilities of the Investigator 
Prior to study start, the investigator is required to sign a protocol signature page confirming 
his/her agreement to conduct the study in accordance with these documents and all of the in-
structions and procedures found in this protocol. The investigator undertake to perform the clini-
cal trial in accordance with this clinical trial protocol, ICH/Good Clinical Practice and the appli-
cable regulatory requirements. The investigator is required to ensure compliance with all proce-
dures required by the clinical trial protocol and with all study required procedures. The investi-
gator agrees to provide all information requested in the Case Report Form (CRF) in an accurate 
and legible manner and to give access to all relevant data and records to Sponsor monitors, audi-
tors, Clinical Quality Assurance, IRBs/IECs/REBs and regulatory authorities as required. 
 

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9.5 
Patient information and informed consent 
All patients should be informed to the fullest extent possible about the study, in language and 
terms they are able to understand. The investigator (according to applicable regulatory require-
ments), or a person designated by the investigator, and under the investigator's responsibility, 
should fully inform the patient of all pertinent aspects of the clinical trial. The patient must be 
given ample opportunity to decide whether or not to participate in this study and to ask questions 
concerning this. It must also be made clear to the patient that he/she can withdraw from the stu-
dy at any time without giving reasons and that he/she will not be in any way disadvantaged by 
this. 
 
Prior to a patient’s participation in the clinical trial, he/she MUST give consent freely and con-
sciously. 
 
Eligible patients may only be included in the study after providing written (witnessed, where 
required by law or regulation), IRB/IEC/REB-approved informed consent or, if incapable of do-
ing so, after such consent has been provided by a legally acceptable representative of the patient. 
In cases where the patient’s representative gives consent, the patient should be informed about 
the study to the extent possible given his/her understanding. If the patient is capable of doing so, 
he/she should indicate assent by personally signing and dating the written informed consent 
document or a separate assent form. 
 
Informed consent/assent must be obtained before conducting any study-specific procedures (i.e. 
all of the procedures described in the protocol). The process of obtaining informed con-
sent/assent should be documented in the patient source documents. The date when a patient’s 
informed consent/assent was actually obtained will be captured in their CRFs. Any Informed 
Consent will be part of Investigator’s file and retained with it. A copy of the signed and dated 
written Informed Consent Form must be provided to the patient. 
 
9.6 
Minimizing distress 
Repeated invasive procedures may be painful or frightening. This trial is designed for minimized 
the discomfort, the studies are designed and conducted by investigators experienced in the 
treatment of patients. 
The protocols and investigations was been designed specifically for the MS population (not 
simply re-worked from adult protocols) and approved by an IEC expert in patients. 
Practical considerations to ensure that participants’ experiences in clinical studies are positive 
and to minimize discomfort and distress include the following: 
 ﻅPersonnel knowledgeable and skilled in dealing with the MS population and its needs, 
including skill in performing MS procedures 
 ﻅA physical setting with furniture, play equipment, activities, and food appropriate for 
MS 
 ﻅThe conduct of studies in a familiar environment such as the hospital or clinic where 
participants normally receive their care 
 ﻅApproaches to minimize discomfort of procedures, such as: 
 ﻅTopical anesthesia to place IV catheters 
 ﻅIndwelling catheters rather than repeated venipunctures for blood sampling 
 ﻅCollection of some protocol-specified blood samples when routine clinical samples are 
obtained 

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9.7 
Minimizing Risk 
However important a study may be to prove or disprove the value of a treatment, participants 
may suffer injury as a result of inclusion in the study, even if the whole community benefits. 
Every effort are made to anticipate and reduce known hazards. Investigators should be fully 
aware before the start of a clinical study of all relevant preclinical and clinical toxicity of the 
medicinal product. To minimize risk in clinical studies, those conducting the study should be 
properly trained and experienced in studying the, including the evaluation and management of 
potential adverse events. 
In designing studies, every attempt are made to minimize the number of participants and of pro-
cedures, consistent with good study design. Mechanisms should be in place to ensure that a 
study can be rapidly terminated should an unexpected hazard be noted. 

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10 Data management 
10.1 Definition of source data and source documents 
Source Data: All information in original records and certified copies of original records of clini-
cal findings, observations, or other activities in a clinical trial necessary for the reconstruction 
and evaluation of the trial. Source data are contained in source documents (original records or 
certified copies). 
Source Documents: Original documents, data, and records (e.g., hospital records, clinical and 
office charts, laboratory notes, memoranda, subjects' diaries or evaluation checklists, pharmacy 
dispensing records, recorded data from automated instruments, copies or transcriptions certified 
after verification as being accurate copies, microfiches, photographic negatives, microfilm or 
magnetic media, x-rays, subject files, and records kept at the pharmacy, at the laboratories and at 
medico-technical departments involved in the clinical trial). 
All parameters asked for in the case report form (CRF) should be documented in the source doc-
uments (see the follow scheme). 
 
Parameter 
Source document 
Medical history / patient’s eligibility 
- 
patients’ charts 
Therapy 
- 
patients’ charts 
Disease status 
- 
laboratory reports  
- 
imaging reports 
Adverse events 
- 
patients’ charts 
- 
laboratory reports 
- 
doctor’s letters 
- 
nurses documentation 
Physical examination 
- 
patients’ charts 
Survival 
- 
patients’ charts, autopsy report if available 
Table 3 – List of parameters and its source 
 
For the following parameters, the CRF might be used as source document: 
Specify the parameters 
10.2 Documentation of data in the case report form (CRF) 
All relevant data collected during the study for all of the patients enrolled in the study shall be 
entered in the CRF by the responsible investigator or someone authorised by him in a timely 
manner so that they are clear and legible. The physician shall confirm the completeness, correct-
ness and plausibility of the data by his signature with the date. The entries shall be made with 
black ballpoint pen. 
The properly filled in CRF will remain in the trial centre, in case of on-line web CRF a copy of 
all pages are conserved in the trial centre. 
All the health workers, involved in the data management, have attended a specific course on use 
of CRF. 
10.3 Data management 
The data will be recorded in the CRF designed for this study. All CRF will be checked for com-
pleteness, plausibility and compliance with the ICH guidelines and the institutional SOPs. 

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10.4 Record keeping 
The investigator shall arrange for the retention of the patient identification list, the signed in-
formed consent forms and the signed data protection declaration for at least 15 years after the 
completion or discontinuation of the study. Patient files and other source data shall be kept for 
the maximum period of time permitted by the hospital. 
The sponsor will keep essential documents according to ICH-GCP, chapter 8. 
 
10.5 Independent Data Safety and Monitoring Committee 
A DSMC is involved (specify the essential items of DSMC SOPs). 

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11 Statistical considerations 
Descriptive statistics (including means, standard deviations, and medians for continuous varia-
bles and proportions for discrete variables) will be used to summarize data as appropriate. A sta-
tistical analysis plan (SAP) with detailed statistical methods and techniques used for data analy-
sis will be prepared before conducting the analysis. 
12 Quality assurance and quality control 
12.1 Definitions 
Audit: A systematic and independent examination of trial related activities and documents to de-
termine whether the evaluated trial related activities were conducted, and the data were record-
ed, analyzed and accurately reported according to the protocol, sponsor's standard operating pro-
cedures (SOPs), Good Clinical Practice (GCP), and the applicable regulatory requirement(s). 
Monitoring: The act of overseeing the progress of a clinical trial, and of ensuring that it is con-
ducted, recorded, and reported in accordance with the protocol, Standard Operating Procedures 
(SOPs), Good Clinical Practice (GCP), and the applicable regulatory requirement(s). 
Quality Assurance (QA): All those planned and systematic actions that are established to ensure 
that the trial is performed and the data are generated, documented (recorded), and reported in 
compliance with Good Clinical Practice (GCP) and the applicable regulatory requirement(s). 
Quality Control (QC): The operational techniques and activities undertaken within the quality 
assurance system to verify that the requirements for quality of the trial-related activities have 
been fulfilled. 
12.2 Monitoring 
A comprehensive quality control can be ordered in the form of monitoring. It might include 
checking the whole course of the study, the management of documentation, the management of 
data, the management of subject enrolled the management of experimental drug and the man-
agement of biological sample. The Sponsor of the study will be in charge for monitoring. 
The investigators will assure the monitoring of the clinical study to assure conformance to pro-
tocol as well as the completeness, correctness and plausibility of the completed case report 
forms (CRF). 
12.3 Deviation from study protocol. 
Every deviation from the trial protocol must be specified and documented separately for each 
patient. The investigator must consult with the monitor and discuss the type and extent of devia-
tion as well as the possible consequences for further participation of the patient in the study. If 
the evaluability of a patient is questionable the coordinating investigator will be consulted. 
12.4 Audits and inspections 
If necessary a comprehensive quality control could be ordered in the form of an audit. It might 
include checking the whole course of the study, the documentation, statistical analysis and the 
investigators. 
The Sponsor guarantees the availability for the inspections from the regulatory agencies. 
12.5 Reference institutes/extramural review board (ERB) 
No reference institute or extramural review board is planned. 

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13 Formalities/Regulatory aspects 
13.1 Legal regulations and guidelines 
This study will be conducted in conformance with the regulations of the latest versions of the 
current applicable laws: the last version of “Declaration of Helsinki” and the principles of good 
clinical practice (ICH-GCP). 
13.2 Patient insurance 
The Sponsor certifies that it has taken out a liability insurance policy which covers the liability 
of the Investigator and his/her co-workers and which is in accordance with local laws and re-
quirements. An abstract copy of insurance certificate will be archived in Trial Master File. 
13.3 Financial aspects 
Fondazione Italiana Sclerosi Multipla (FISM), Fondazione CARIPLO and Associazione Amici 
del Centro Sclerosi Multipla Onlus (ACeSM) are financial sponsors of this clinical trial. 
Agreements (contracts) between Hospital San Raffaele (via Olgettina, 60 – Milano) and finan-
cial sponsors assigned a grant to Neuroimmunology Unit. 
This grant covers the costs of the present clinical trial. 
13.4 Final study report 
The Sponsor will be responsible for preparing a Final Clinical Study Report. 
13.5 Declaration on the property of the data 
The Sponsor of this trial declares that: 
( ﻅFirst option) All the data deriving from this protocol belong to the Sponsor. 
13.6 Publication policy 
No specific rules are determined. 
 
 
14 Feasibility declaration 
The clinical trial feasibility is evaluated based on five principal items: Potential enrolment, 
Budget, Required staff, Facility, Supplies defined as following: 
 
Potential enrolment: we estimate an enrolment of 24 patients per year, based on patient diag-
nosed or referred to our centre in the past 10 years. 
Budget: the protocol has a dedicated reasonable (grant/financing/donation), managed by hSR 
grant office. 
Required staff: our team is fully qualified, and trained, dedicated research staff is present to co-
ordinate the trial. The PI has adequate time and scheduling availability to devote to the overall 
supervision of the trial. Sub-investigators in number and qualified adequate to assist PI. Ancil-
lary or specialty staff (pharmacy, labs, diagnostics, etc) involved in the trial is adequate in num-
ber and qualification. 
Facility: adequate clinic and office space is available. The following special equipment is pre-
sent. 
Supplies: The Sponsor will supply (Case Report Forms, source documents, electronic consent 
template, packaged lab kits, pre-paid shipping, drugs, etc.). 

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15 Bibliography 
Aboody KS, Brown A, Rainov NG, Bower KA, Liu S, Yang W, Small JE, Herrlinger U, 
Ourednik V, Black PM, Breakefield XO, Snyder EY (2000) Neural stem cells display extensive 
tropism for pathology in adult brain: evidence from intracranial gliomas. Proc Natl Acad Sci U S 
A 97: 12846-51 
Bramow S, Frischer JM, Lassmann H, Koch-Henriksen N, Lucchinetti CF, Sorensen PS, Laursen 
H (2010) Demyelination versus remyelination in progressive multiple sclerosis. Brain 133: 2983-
98 
Chen KS, Sakowski SA, Feldman EL (2016) Intraspinal stem cell transplantation for 
amyotrophic lateral sclerosis. Ann Neurol 79: 342-53 
Confavreux C, Vukusic S (2006) Natural history of multiple sclerosis: a unifying concept. Brain 
129: 606-16 
Confavreux C, Vukusic S, Moreau T, Adeleine P (2000) Relapses and progression of disability 
in multiple sclerosis. N Engl J Med 343: 1430-8 
Chu K, Kim M, Jeong SW, Kim SU, Yoon BW (2003) Human neural stem cells can migrate, 
differentiate, and integrate after intravenous transplantation in adult rats with transient forebrain 
ischemia. Neurosci Lett 343: 129-33 
Einstein O, Karussis D, Grigoriadis N, Mizrachi-Kol R, Reinhartz E, Abramsky O, Ben-Hur T 
(2003) Intraventricular transplantation of neural precursor cell spheres attenuates acute 
experimental allergic encephalomyelitis. Mol Cell Neurosci 24: 1074-82 
Feldman EL, Boulis NM, Hur J, Johe K, Rutkove SB, Federici T, Polak M, Bordeau J, Sakowski 
SA, Glass JD (2014) Intraspinal neural stem cell transplantation in amyotrophic lateral sclerosis: 
phase 1 trial outcomes. Ann Neurol 75: 363-73 
Fox EJ (2006) Management of worsening multiple sclerosis with mitoxantrone: a review. Clin 
Ther 28: 461-74 
Frischer JM, Bramow S, Dal-Bianco A, Lucchinetti CF, Rauschka H, Schmidbauer M, Laursen 
H, Sorensen PS, Lassmann H (2009) The relation between inflammation and neurodegeneration 
in multiple sclerosis brains. Brain 132: 1175-89 
Fujiwara Y, Tanaka N, Ishida O, Fujimoto Y, Murakami T, Kajihara H, Yasunaga Y, Ochi M 
(2004) Intravenously injected neural progenitor cells of transgenic rats can migrate to the injured 
spinal cord and differentiate into neurons, astrocytes and oligodendrocytes. Neurosci Lett 366: 
287-91 
Garitaonandia I, Gonzalez R, Christiansen-Weber T, Abramihina T, Poustovoitov M, Noskov A, 
Sherman G, Semechkin A, Snyder E, Kern R (2016) Neural Stem Cell Tumorigenicity and 
Biodistribution Assessment for Phase I Clinical Trial in Parkinson's Disease. Sci Rep 6: 34478 

Clinical Trial Protocol 
STEMS 
Rev. n° 1.2 
Page 52 of 54 
Rev. date 10 March 2017 
 
Glass JD, Boulis NM, Johe K, et al. Lumbar intraspinal injection of neural stem cells in patients 
with amyotrophic lateral sclerosis: results of a phase I trial in 12 patients. Stem Cells 2012 
Jun;30(6):1144-51. 
Gupta N, Henry RG, Strober J, Kang S, et al. Neural Stem Cell Engraftment and Myelination in 
the Human Brain , (2012);4 Sci Transl Med 2012; 155(4):1-10. 
Hartung HP, Gonsette R, Konig N, Kwiecinski H, Guseo A, Morrissey SP, Krapf H, Zwingers T, 
Mitoxantrone in Multiple Sclerosis Study G (2002) Mitoxantrone in progressive multiple 
sclerosis: a placebo-controlled, double-blind, randomised, multicentre trial. Lancet 360: 2018-25 
Jeong SW, Chu K, Jung KH, Kim SU, Kim M, Roh JK (2003) Human neural stem cell 
transplantation promotes functional recovery in rats with experimental intracerebral hemorrhage. 
Stroke 34: 2258-63 
Kappos L (1998) Placebo-controlled multicentre randomised trial of interferon beta-1b in 
treatment of secondary progressive multiple sclerosis. European Study Group on interferon beta-
1b in secondary progressive MS. Lancet 352: 1491-7 
Kappos L, Polman C, Pozzilli C, Thompson A, Beckmann K, Dahlke F, European Study Group 
in Interferon beta-1b in Secondary-Progressive MS (2001) Final analysis of the European 
multicenter trial on IFNbeta-1b in secondary-progressive MS. Neurology 57: 1969-75 
Kokaia Z, Martino G, Schwartz M, Lindvall O (2012) Cross-talk between neural stem cells and 
immune cells: the key to better brain repair? Nat Neurosci 15: 1078-87 
Kragt JJ, van der Linden FA, Nielsen JM, Uitdehaag BM, Polman CH (2006) Clinical impact of 
20% worsening on Timed 25-foot Walk and 9-hole Peg Test in multiple sclerosis. Mult Scler 12: 
594-8 
Kurtzke JF (1983) Rating neurologic impairment in multiple sclerosis: an expanded disability 
status scale (EDSS). Neurology 33: 1444-52 
Lassmann H (2007b) New concepts on progressive multiple sclerosis. Curr Neurol Neurosci Rep 
7: 239-44 
Lassmann H (2007a) Multiple sclerosis: is there neurodegeneration independent from 
inflammation? J Neurol Sci 259: 3-6 
Li DK, Zhao G, Paty D. Randomized controlled trial of interferon-beta-1a in secondary 
progressive MS: MRI results. Neurology 2001; 56(11):1505-1513. 
Lu P, Jones LL, Snyder EY, Tuszynski MH (2003) Neural stem cells constitutively secrete 
neurotrophic factors and promote extensive host axonal growth after spinal cord injury. Exp 
Neurol 181: 115-29 
Lublin FD, Reingold SC, Cohen JA, Cutter GR, Sorensen PS, Thompson AJ, Wolinsky JS, 
Balcer LJ, Banwell B, Barkhof F, Bebo B, Jr., Calabresi PA, Clanet M, Comi G, Fox RJ, 
Freedman MS, Goodman AD, Inglese M, Kappos L, Kieseier BC et al. (2014) Defining the 
clinical course of multiple sclerosis: the 2013 revisions. Neurology 83: 278-86 

Clinical Trial Protocol 
STEMS 
Rev. n° 1.2 
Page 53 of 54 
Rev. date 10 March 2017 
 
 
Magliozzi R, Howell O, Vora A, Serafini B, Nicholas R, Puopolo M, Reynolds R, Aloisi F 
(2007) Meningeal B-cell follicles in secondary progressive multiple sclerosis associate with early 
onset of disease and severe cortical pathology. Brain 130: 1089-104 
Martinelli V, Cocco E, Capra R, Salemi G, Gallo P, Capobianco M, Pesci I, Ghezzi A, Pozzilli 
C, Lugaresi A, Bellantonio P, Amato MP, Grimaldi LM, Trojano M, Mancardi GL, Bergamaschi 
R, Gasperini C, Rodegher M, Straffi L, Ponzio M et al. (2011) Acute myeloid leukemia in Italian 
patients with multiple sclerosis treated with mitoxantrone. Neurology 77: 1887-95 
Martino G, Franklin RJ, Baron Van Evercooren A, Kerr DA, Stem Cells in Multiple Sclerosis 
Consensus G (2010) Stem cell transplantation in multiple sclerosis: current status and future 
prospects. Nat Rev Neurol 6: 247-55 
Mazzini L, Gelati M, Profico DC, Sgaravizzi G, Projetti Pensi M, Muzi G, Ricciolini C, Rota 
Nodari L, Carletti S, Giorgi C, Spera C, Domenico F, Bersano E, Petruzzelli F, Cisari C, 
Maglione A, Sarnelli MF, Stecco A, Querin G, Masiero S et al. (2015) Human neural stem cell 
transplantation in ALS: initial results from a phase I trial. J Transl Med 13: 17 
Melzi R, Antonioli B, Mercalli A, Battaglia M, Valle A, Pluchino S, Galli R, Sordi V, Bosi E, 
Martino G, Bonifacio E, Doglioni C, Piemonti L (2010) Co-graft of allogeneic immune 
regulatory neural stem cells (NPC) and pancreatic islets mediates tolerance, while inducing 
NPC-derived tumors in mice. PLoS One 5: e10357 
Muller FJ, McKercher SR, Imitola J, et al. At the interface of the immune system and the 
nervous system: how neuroinflammation modulates the fate of neural progenitors in vivo. Ernst 
Schering Res Found Workshop. 2005: 83-114. 
Muller FJ, Snyder EY, Loring JF (2006) Gene therapy: can neural stem cells deliver? Nat Rev 
Neurosci 7: 75-84 
Park KI, Teng YD, Snyder EY. The injured brain interacts reciprocally with neural stem cells 
supported by scaffolds to reconstitute lost tissue. Nat Biotechnol 2002; 20:1111-1117. 
Panitch H, Miller A, Paty D, Weinshenker B. Interferon beta-1b in secondary progressive MS: 
results from a 3-year controlled study. Neurology 2004; 63(10):1788-1795. 
Park KI, Teng YD, Snyder EY (2002) The injured brain interacts reciprocally with neural stem 
cells supported by scaffolds to reconstitute lost tissue. Nat Biotechnol 20: 1111-7 
Pluchino S, Gritti A, Blezer E, Amadio S, Brambilla E, Borsellino G, Cossetti C, Del Carro U, 
Comi G, t Hart B, Vescovi A, Martino G (2009) Human neural stem cells ameliorate 
autoimmune encephalomyelitis in non-human primates. Ann Neurol 66: 343-54 
Pluchino S, Quattrini A, Brambilla E, et al. Injection of adult neurospheres induces recovery in a 
chronic model of multiple sclerosis. Nature 2003; 422:688-694. 
Pluchino S, Zanotti L, Deleidi M, Martino G (2005a) Neural stem cells and their use as 
therapeutic tool in neurological disorders. Brain Res Brain Res Rev 48: 211-9 

Clinical Trial Protocol 
STEMS 
Rev. n° 1.2 
Page 54 of 54 
Rev. date 10 March 2017 
 
Pluchino S, Furlan R, Martino G (2004) Cell-based remyelinating therapies in multiple sclerosis: 
evidence from experimental studies. Curr Opin Neurol 17: 247-55 
Pluchino S, Zanotti L, Rossi B, et al. Neurosphere-derived multipotent precursors promote 
neuroprotection by an immunomodulatory mechanism. Nature 2005; 436:266-271. 
Riley J, Federici T, Polak M, et al. Intraspinal stem cell transplantation in amyotrophic lateral 
sclerosis: a phase I safety trial, technical note, and lumbar safety outcomes. Neurosurgery. 2012 
Aug; 71(2):405-16.  
Selden NR, Al-Uzri A, Huhn SL, et al. Central nervous system stem cell transplantation for 
children with neuronal ceroid lipofuscinosis. J Neurosurg Pediatr 2013 Jun; 11(6):643-52. 
Serafini B, Rosicarelli B, Magliozzi R, Stigliano E, Aloisi F (2004) Detection of ectopic B-cell 
follicles with germinal centers in the meninges of patients with secondary progressive multiple 
sclerosis. Brain Pathol 14: 164-74 
Temple S (2001) The development of neural stem cells. Nature 414: 112-7 
Vescovi AL, Parati EA, Gritti A, Poulin P, Ferrario M, Wanke E, Frolichsthal-Schoeller P, Cova 
L, Arcellana-Panlilio M, Colombo A, Galli R (1999) Isolation and cloning of multipotential stem 
cells from the embryonic human CNS and establishment of transplantable human neural stem 
cell lines by epigenetic stimulation. Exp Neurol 156: 71-83