Name of journal: World Journal of Transplantation esps manuscript no: 18452 Manuscript Type: Original Article Retrospective Study

MATERIALS AND METHODS

The 12 year trial was performed under the branch program of the Russian Academy of Medical Sciences New Cell Techniques to Medicine, with the approval and under the supervision of the Scientific Board and Ethics Committee of the Russian State Medical University (Moscow, Russia). The trial was launched 2002 and was not registered in the international database for their absence. It is an open parallel controlled trial (phase I/II) that followed IMITE protocol (Switzerland). The trial included 202 SCI patients (1008 case histories) that made trial group 1, see Table 1. According to the protocol, we evaluated the control group that included 20 SCI patients matched by age, sex and level of injury, see Table 2. The enrolled patients signed the Informed Consent. Trial participants met the following eligibility criteria: SCI occurred at least 12 mo prior to the inclusion into the trial; age between 15 and 60; adequate end organ function; adequate bone marrow function, negative pregnancy test; written, voluntary, informed consent. Exclusion criteria were acute infections, severe hematologic disorders; contraindications for MRI, pregnancy or breast feeding, grade III/IV cardiac problems as defined by the New York Heart Association Criteria; severe and/or uncontrolled medical diseases; known diagnosis of human immunodeficiency virus (HIV) infection; previous radiotherapy to  25 % of the bone marrow; major surgery within 6 weeks prior to study entry; known malignant tumours. All patients received conventional pharmaceutical treatment and intensive rehabilitation: exercise therapy, physiotherapy and massage. The suspension of HSCs and hematopoietic precursors (HPs) was intrathecally administered to the patients of the main arm every three months for 3-5 years. To produce HSCs and HPs suspension the stem cells (SC) and progenitor cells (PC) are mobilized to peripheral blood by 8 subcutaneous injections of granulocyte-colony stimulating factor (G-CSF) every 10-12 h for 4 d. First three days the G-CSF dose is 2.5 μg per kg of body weight, the last day the dose is doubled. The stem cells and precursors are harvested at day 5 in blood cell separator (COBE-spectra, Gambro BCT, United States), using a disposable system for separation and standard solutions. The separation lasts 3-4 h, depending on the speed of the procedure, weight of the patient and blood test results. The red blood cells are removed from the obtained material in a conventional way, and the received leukoconcentrate is examined. On average, the volume of the material varies from 300 to 400 mL. The material is evaluated according to total number of nuclear cells (NCs) in the sediment and according to CD34⁺ cells per a kilogram of the patient’s weight. The NCs in the sediment are determined by counting in Gorjaev’s chamber. The percentage of CD34⁺ is determined by flow cytometry method by FACScan (Becton Dickinson, United States). Previously we have provided a detailed analysis of the preparation^[10]. The standardized and certified HSCs and HPs were uniformly dispensed in 20 tubes and cryopreserved by adding DMSO in 5% final concentration, frozen down at a rate of 1 °C/min up to a temperature point of –80 ºC or –120 ºC in a programmed freezer and further stored in liquid nitrogen or liquid nitrogen vapor. The cell material is characterized in Figure 1 and Table 3. Before administration the cells are thawed in +37 ⁰C water bath and washed by double centrifugation with 0.9% NaCl. According to CD34+ count, an average dose of the cells is 5.8 × 10⁶ in a tube. The main trial group received intrathecal administrations (no less than 20) of the HSCs and HPs suspension. The autologous HSCs and HPs were harvested once in 101 patients (50%), twice in 68 patients (33.7%), and three times in 33 patients (16.3%). Totally, during the whole period of observation, the patients received 1790 intrathecal transplantations of autologous HSCs and HPs. The control group patients received analogous treatment, excluding intrathecal administration of HSCs and HPs.

The patients were clinically and paraclinically evaluated according to the protocol. Evaluation of neurologic condition included tests for locomotion and sensation, bladder and bowel functions, level of injury and its completeness/incompleteness. Safety evaluation was based on the frequency of adverse events, particularly adverse events leading to discontinuation of treatment and on the number of abnormal laboratory values.

Neurological response was assessed every 3 mo, by an examination performed by a neurologist and recorded according to ASIA scale and FIM scale. Changes from baseline in neurological status grades and body weight were summarised at defined intervals and produced in the tables of summary statistics.

MRI scan of the CNS and urodynamic tests were performed every 6 mo. Motor evoked potentials (MEP), Somatosensory evoked potentials (SEP) examinations were performed every 3 mo. Urodynamic tests were performed every 6 mo. To evaluate motor activity we used specifically developed scale of clinical restoration of motor function^[9,10] that estimated muscle force in the extremities, range of active movement and movement pace, to calculate the total score of motor activity. Additionally, motor restoration was evaluated with the Medical Research Council Scale that estimates (from 0 to 5 points, depending on the degree of manifestation) the range of active and passive movements, as well as the strength of a body and extremities. Sensitive disorders were evaluated with specifically developed scale of sensation restoration^[10] that included 2-point testing of pain, temperature and deep sensation on dermatome on each side, and evaluation of the feeling of “heaviness” in resting muscles and after training in the lower and upper extremities, abdomen and back. Completeness/incompleteness of SCI was assessed according to neurologic symptoms: lower paraplegia, conduction anesthesia and urine retention. Minimal movements or hypoesthesia below the level of injury were evaluated as an incomplete injury (no injury equals 0, an incomplete functional injury of spinal cord equals 1, a complete functional injury of spinal cord is 2).

The function of bladder was evaluated by specifically developed clinical scale to estimate the restoration of bladder function that included 3-point assessment of urination feeling and 5-point assessment of urine retention^[10]. The total score, denoting absence of neurologic bladder disorders, equals 8 points. All patients passed complex urodynamic tests. Besides, the effectiveness of the intrathecal transplantation of HSCs and HPs in chronic SCI was evaluated with ASIA index, FIM index and the International Standards for Neurological Classification of Spinal Cord Injury (ISCSCI-92).

The main criteria of effectiveness were improvement of neurologic symptoms (motor, sensitive and bladder and bowel function). The expectation period for the improvement to manifest was individual in every case, depending on the scope of injury, years post injury and functional impairment. The results of the therapy manifested from 1-3 d to 24-36 mo post transplantation and were evaluated by the clinical indexes of ASIA and FIM. Patients were considered in response if at least one of the following criteria were met: (1) An unequivocal improvement of SSEP, MEP; (2)

An unequivocal sign of tissue regeneration at MRI; (3) An unequivocal improvement of UT; and (4) Changes from baseline in neurological status grades (ASIA, FIM).

The statistical review of the study was performed by the biomedical statistician of the School of Biomedicine, Far Eastern Federal University. The material was statistically processed with SPSS 13 software. Statistical significance of the data was evaluated with Student’s coefficient, and ANOVA analysis of variance and ² method. The data were considered statistically significant at P < 0.05.