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SUPPLEMENTARY MATERIAL Intrathecal administration of autologous mesenchymal stromal cells for spinal cord injury: safety and efficacy of the 100/3 guideline

JESÚS VAQUERO1, MERCEDES ZURITA1, MIGUEL A. RICO1, CONCEPCION AGUAYO1, CELIA BONILLA1, ESPERANZA MARIN2, NOEMI TAPIADOR3, MARTA SEVILLA3, DAVID VAZQUEZ4, JOAQUIN CARBALLIDO4, CECILIA FERNANDEZ1, GREGORIO RODRIGUEZ-BOTO1, MERCEDES OVEJERO5 & THE NEUROLOGICAL CELL THERAPY GROUP FROM PUERTA DE HIERROMAJADAHONDA HOSPITAL*

1Neurosurgery Service, University Hospital Puerta de Hierro-Majadahonda, Autonomous University, Madrid, Spain; 2Clinical Neurophysiology Service, University Hospital Puerta de Hierro-Majadahonda, Autonomous University, Madrid, Spain; 3Rehabilitation Service, University Hospital Puerta de Hierro-Majadahonda, Autonomous University, Madrid, Spain; 4Urology Service, University Hospital Puerta de Hierro-Majadahonda, Autonomous University, Madrid, Spain; and 5Statistic Unit, Sermes CRO, Madrid, Spain

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Trial registration ClinicalTrials.gov NCT02570932, EudraCT: 2014-005613-24

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INDEX

____________________________________________Page_______ 1,- List of investigators and collaborators 3

The Neurological Cell Therapy Group 2.- Supplementary information about the clinical trial 4

Selection and exclusion criteria Clinical explorations Patients’ flow chart

3.- Graphical Abstract of Efficacy 7 4.- Study populations 8 5.- Patients’ Results 8 6.- Individual improvements 9 7.- Neurophysiological findings 13 8.- Improvement in Patient 04 14 9.- Efficacy of cell therapy on motor power in patients with SCI at cervical level 17 ______________________________________________________

3 1.- List of investigators and collaborators The Neurological Cell Therapy Group (NCTG) This study is a result of the utilization of human and material resources of Hospital Puerta de Hierro-Majadahonda. This hospital belongs to the Spanish Public Health System and is characterized by high technology and advances in the field of organ transplantation research. The following members from the Puerta de Hierro belong currently to the NCTG. Service of Neurosurgery and Neurological Cell Therapy Unit: Jesús Vaquero, Mercedes Zurita, Celia Bonilla, Miguel A. Rico, Concepción Aguayo, Alicia Rodríguez, Paula Martínez, Silvia de la Calle, Marta V Fernández, Cecilia Fernández, Gregorio Rodríguez-Boto, Laura de Reina, Anwar Saab, Carlos Cotua, Xavier A Santander, Raquel Gutiérrez, Cristóbal Saldaña, Rasha Hassan, Celia Ortega, Alicia Madrid and Montaña Mariscal. Service of Clinical Neurophysiology : Esperanza Marín, Luis F. López, Alberto Pérez, Edwin E, Ebrat, Marta Vaquero, Mónica Martín, Isabel Mayoral and Dolores Canales. Service of Urology: Joaquín Carballido, David Vazquez and Rosario Serrano. Service of Radiology: Gertrudis Saucedo. Service of Rehabilitation: Noemi Tapiador and Marta Sevilla. Service of Hematology: Rafael Cabrera, Maria E Martínez Begoña Pérez de Camino, Ana Alarcón, Daniel Naya, Rosalía Alonso, Jose R Alamo and Irene Romera. Service of Anesthesiology: Inmaculada Mourelle, Carmen Sánchez, Rocío Segovia, Alberto Gutiérrez, Verónica Guillo, Sara del Valle and Paula Rey. Service of Nuclear Medicine: Jorge Mucientes and Begoña Rodríguez.

4 2.- Supplementary information about the Clinical Trial

This was a prospective phase 2 clinical trial. There were no controls because the patients

had a chronic spinal cord injury (SCI) without expectation of change for this disease. The

main objective was to evaluate the potential efficacy of the treatment by means of clinical

scales and urodynamic, neurophysiological and neuroimaging studies. The secondary

objective was to assess safety.

Selection and exclusion criteria Selection criteria:

Patients of both sexes, aged between 18 and 70 years, with a chronic and established SCI (ASIA A, B, C or D). For the purpose of this study, an established SCI has been considered when there is no functional recovery after a minimum period of 6 months prior to cell therapy.

Possibility to study the characteristics of SCI by Magnetic Resonance Image (MRI), urodynamic and neurophysiological techniques.

Possibility of follow-up after treatment and to fulfill a protocol of physical therapy during

the follow-up period. Hematologic and laboratory parameters within the normal range according to laboratory

standards, accepting, however, slight deviations that are considered not significant in the context of the treatment to be performed.

Written informed consent, in accordance with good clinical practice guidelines and local

legislation, obtained before any study procedure. Absence in the genetic study of abnormalities that might pose a potential risk in the

process of cell expansion.

Exclusion criteria:

Pregnancy and/or lactation. Systemic disease representing an additional risk to treatment. Patients with questions about his possible cooperation in rehabilitation treatments, or

negative report in psychological evaluation. Current neoplastic disease or in the previous 5 years (diagnosed or treated).

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Patients treated with hematopoietic growth factors or requiring maintained

anticoagulation. Neurodegenerative disease. History of substance abuse, psychiatric illness or allergy to protein products used in the

process of cell expansion. Positive serology to human immunodeficiency virus (HIV) or syphilis. Active hepatitis B or hepatitis C.

If they exist, in the opinion of the investigator, medical, social or psychosocial factors

that may negatively influence the study.

Clinical explorations

Clinical assessment in all patients before starting cell therapy, with study of ASIA scale,

in different subsections: ASIA grade, motor score (MS) and sensitivity scores Pin Prick Score (PPS), PPS, and Light Touch Score, (LTS). The sum of scores in MS, PPS and LTS and sensitivity is the total ASIA score.

IANR-SCIFRS scale, from the "International Association of Neurorestoratology" that values the specific functional deficit of SCI.

Visual Analog Scale (VAS) for assessment of neuropathic pain.

Penn Scale and the modified Ashworth Scale, to assess spasms and spasticity, respectively.

Scale of Geffner, to study bladder function.

NBD scale, to study neurogenic bowel dysfunction.

Study of somatosensory-evoked potentials (SSEPs) and motor-evoked potentials (MEPs) elicited by magnetic stimulation over the scalp, and electromyographic (EMG) recording of potentials in infralesional muscles. SSEPs were studied with a Medelec Synergy 10-channel equipment (v.14.0, Viasys Healthcare). For the study of MEPs induced by transcranial magnetic stimulation, we used Rapid2 Magstim equipment. EMG recordings were made with MedelecSYNERGY-10 equipment.

Urodynamic studies, using Solar Luna equipment (Medical Measurement Systems Inc.). Possibility of voluntary micturition, first sensation at filling, maximum bladder capacity and detrusor pressure at filling, together with bladder compliance and postmictional residue were mainly assessed.

Neuroimage studies (MRI) were performed in order to evaluate possible morphological changes of spinal cord after treatment. We used a 3T imager (XR InteraAchieva 3T,

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Philips Healthcare). Lesions were studied on T1-and T2-weighted images and on MR-myelography images, using turbo-spin-echo sequences.

Patients’ Flow Chart

Flow chart.

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3. Graphical Abstract of Efficacy

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4. Study populations

Study sets.

Set Total Screening failure

Total 11 0

Intention to treat 11 0

Per protocol 9 0

Safety 11 0

5. Patients’ Results

Patients’ results

Patient ASIAIANR-SCIFRS

PENN ASHWORTH EVA NBD Geffner AE

01 2

02 0

03 1

04 005 5

06 0

07 0

08 0

09 0

10 111 0

% improvement 77.78 88.89 33.33 22.22 77.78 77.78 55.56Note. Green: Patients who improve. Yellow: Patients who neither improve nor worsen. Red: Patients who worsen. Grey: Patients not included in the Per Protocol set. AE: Adverse Events.

6. Individual improvements

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Individual improvements.ASIA total score

Individual improvements.

Individual improvements: IANR-SCIFRS scale

Individual improvements: PENN scale

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Individual improvements.Individual improvements: ASHWORTH scale

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Individual improvements: VAS scale

Individual improvements.

Individual Improvements: Geffner Scale

Individual improvements: NBD Scale

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13 7. Neurophysiological findings

Neurophysiological findings.

Visit 10 % Patients who improve

SSEP 55.56%

MEP 55.56%

Sensitivity conduction 33.33%

Motor conduction 22.22%

Voluntary muscle contraction 44.44%

Infralesional active muscle reinnervation 44.44%

B

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8. Improvement in Patient 04 In this patient, the second dose was administered with a delay of 4 months because the cells did not meet the minimum established. The results of this patient on the efficacy of the treatment are represented below graphically.

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9. Efficacy of cell therapy on motor power in patients with SCI at cervical level

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