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



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Name of journal: World Journal of Transplantation

ESPS Manuscript NO: 18452

Manuscript Type: Original Article
Retrospective Study

Effectiveness of repeated transplantations of hematopoietic stem cells in spinal cord injury
Bryukhovetskiy AS et al. Long-term effects of cell therapy of SCI
Andrey S Bryukhovetskiy, Igor S Bryukhovetskiy
Andrey S Bryukhovetskiy, Federal Research Center for Specialized Types of Medical Assistance and Medical Technologies of FMBA of Russia, 115682 Moscow, Russia
Andrey S Bryukhovetskiy, NeuroVita Clinic of Restorative and Interventional Neurology and Therapy, 115478 Moscow, Russia
Igor S Bryukhovetskiy, School of Biomedicine, Far Eastern Federal University, 690091 Vladivostok, Russia
Author contributions: Both authors contributed equally to this work.
Institutional review board statement: The study was reviewed and approved by Ethics Committee Russian State Medical University (Moscow, Russia). Since 2005 the method was approved for clinical practice.
Informed consent statement: All study participants, or their legal guardian, provided an informed written consent prior to study involvement.
Conflict-of-interest statement: Professor Andrey S Bryukhovetskiy PhD, MD, is an employee of the Federal Research Center for Specialized Types of Medical Assistance and Medical Technologies of FMBA of Russia. Professor Andrey S Bryukhovetskiy PhD, MD, owns stocks and shares in the NeuroVita Clinic of Restorative and Interventional Neurology and Therapy. Professor Andrey S Bryukhovetskiy PhD, MD, owns patent Preparation of Autologous Hematopoietic Stem Cells, Method of Production, Cryopreservation and Application for Treatment of Traumatic Diseases of Central Nervous System, Patent of Russian Federation RU No. 2283119 C1 dated 10.09.2006; International Application No. PCT/EP 2005108721 filed on 29.03.2005 Preparation of autologous stem cells, the methods of production, cryopreservation and use for therapy of traumatic diseases of central nervous system.
Data sharing statement: Technical appendix, statistical code, and dataset available from the corresponding author at neurovitaclinic@gmail.com. Participants gave written informed consent for data sharing, and the data are anonymized.
Open-Access: This article is an open-access article which was selected by an in-house editor and fully peer-reviewed by external reviewers. It is distributed in accordance with the Creative Commons Attribution Non Commercial (CC BY-NC 4.0) license, which permits others to distribute, remix, adapt, build upon this work non-commercially, and license their derivative works on different terms, provided the original work is properly cited and the use is non-commercial. See: http://creativecommons.org/licenses/by-nc/4.0/
Correspondence to: Andrey S Bryukhovetskiy, MD, PhD, Professor, NeuroVita Clinic of Restorative and Interventional Neurology and Therapy, 23 Kashirskoye shosse, 115478 Moscow, Russia. neurovitaclinic@gmail.com

Telephone: +7-495-3249339

Fax: +7-495-9801373
Received: April 20, 2015

Peer-review started: April 21, 2015

First decision: May 13, 2015

Revised: July 16, 2015

Accepted: August 20, 2015

Article in press:

Published online:

Abstract

AIM: To evaluate the short and long-term effects of the complex cell therapy of 202 cases of spinal cord injury (SCI).
METHODS: The main arm included 202 cases of SCI and the control arm included 20 SCI cases. For the therapy the hematopoietic stem cells (HSCs) and progenitor cells (PCs) were mobilized to peripheral blood by 8 subcutaneous injections of granulocyte colony-stimulating factor (G-CSF) for 4 d and are harvested at day 5. The cells were administered to the main arm intrathecally every 3 mo for a long term (3-5 years) according to the internal research protocol IMITE. MRI of the site of injury and urodynamic tests were performed every 6 mo. Motor evoked potentials (MEP), somatosensory evoked potentials (SSEP) were evaluated every 3 mo. The patients were evaluated with ASIA index, FIM index, the Medical Research Council Scale, the International Standards for Neurological Classification of Spinal Cord Injury (ISCSCI-92) and specifically developed scales. The function of bladder was evaluated by a specifically developed clinical scale. The long-term clinical outcomes were assessed for the SCI patients who received no less than 20 intrathecal transplantations of HSCs and hematopoietic precursors (HPs).
RESULTS: The restoration of neurologic deficit after HSCs and HPs transplantations was proved stable and evident in 57.4% of the cases. In 42.6% cases no neurologic improvement has been observed. In 50% of the cases the motor restoration began after the first transplantation, which is confirmed in average by 9.9 points improvement in neurologic impairment as compared to the baseline (P < 0.05). Repair of the urinary system was observed in 47.7% of the cases. The sensitivity improved from baseline 124.3 points to 138.4 after the first and to 153.5 points after the second transplantations of HSCs and HPs (P < 0.05, between the stages of research). The evaluation with ASIA index demonstrated regress of neurologic symptoms in 23 cases. Motor progress was also assessed with the ISCISCI-92 motor and sensory scores, and the data coincided with those received with the specifically developed scale. The number of the patients with the signs of locomotive repair was 56.9%. No life threatening complications or adverse effects have been observed.
CONCLUSION: The method is safe, effective and considerably improves the life quality of SCI patients. The therapy is approved for clinical use as the treatment of choice.
Key words: Spinal cord injury; Paraplegia; Tetraplegia; Hematopoietic stem cells; Stem cells; Cell therapy
© The Author(s) 2015. Published by Baishideng Publishing Group Inc. All rights reserved.
Core tip: The work summarizes the 12 year experience of stem cell therapy for chronic spinal cord injury. The unique preparation of autologous hematopoietic stem cells and hematopoietic precursors was multiply administered to 202 patients. The article analyzes short and long-term benefits, short and long-term complications and the instruments that were used for their evaluation.
Bryukhovetskiy AS, Bryukhovetskiy IS. Effectiveness of repeated transplantations of hematopoietic stem cells in spinal cord injury. World J Transplant 2015; In press

INTRODUCTION

A global incidence rate of traumatic spinal cord injury (SCI) is estimated as 23 cases per million[1]. Regional incidence rates vary from 15 (Australia) to 40 (United States) cases per 1 million of population[1]. The average age at injury increased from 28.7 years in the 1970s to 42.6 years since 2010[2], still, the incidence of traumatic SCI peaks in young people[1,3].

Although spinal fractures constitute only 0.44% of all injury types, the percentage of spinal traumas has dramatically increased (over 200-fold) for the past 7 decades. The analysis predicted 800 of new spinal cord injuries (SCI) per 10 million of population.

For the past two decades the therapeutic advances hold a lot of promise for the patients with SCI, but none of the available therapies led to restoration of the morphological structure of spinal cord and its functions. Various therapeutic programs improve outcomes and life quality of the injured only in a few cases, but still they remain unable to repair severe neurologic deficit and restore lost functions. Surgical approaches to repair SCI are aimed at orthopedic restoration of vertebral canal anatomy, and their results remain controversial. To date, an SCI is a final verdict that entails impossibility to return to the previous way of life, to restore previous working capacity and reproductive functions, resulting in tremendous social and economic losses. The total direct costs of SCI in the United States alone are estimated at about 7.7 billion USD[4].

Inefficiency of the available SCI therapies was used to be explained by the absence of regeneration potential of adult neurons, and the opportunity to restore damaged neural cells has only recently been proved[5]. By now, the first steps to develop new neurorestorational therapy of SCI have been made[6,7], although no universally acknowledged methods to restore the spinal cord after the injury are observed. Novel cell techniques and tissue engineering methods can provide the solution; so, according to the Stem Cell Summit (2009) data, 34 million of patients received transplantations of stem cells of various origin, and 1 million of them were SCI patients[8]. However, outcomes and long-term consequences of such transplantations remain as yet unknown.

The available experience is minimally documented and rather obscure, due to insufficient theoretical and experimental evidence of cell technologies, as well as underdeveloped methods of their application, when the fate of transplanted cells, their further differentiation and transformation are unclear. The crucial question of cancer development, triggered by the transplantation of stem cells, also remains unanswered. The myths and fears of possible negative consequences of stem cell therapy significantly interfere with the research and progress in the area.

We have transplanted cells for SCI for 25 years both in research and in clinical practice and have accumulated substantial experience of victories and defeats administrating allogeneic and xenogeneic fetal neural and mesenchymal cells, isolated from animal and human embryos of 10-24 gestation weeks, as well as embryonic stem neural cells, obtained from human blastocyst. This experience is summed up in our book[9], and to date, we have refused from the clinical application of allogeneic and xenogeneic cell material for SCI. We believe the future of the SCI therapy to belong to the suspensions, prepared from autologous stem and progenitor cells, as under the SCI condition the organism specifies and individually tailors the cells for the treatment of their own SCI, along with the advantage of null immunologic and transplantation side effects and absence of undesirable paramedical ethic, legal and religious aspects[10]. The only option to use the allogeneic stem cells for SCI is haploidentical stem cells or those of close relatives, and only after the HLA typing.

In the present article we would like to determine the basic parameters for the beginning of the cell therapy for SCI and the criteria to terminate it in clinical practice.



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.


RESULTS

General efficacy of the intrathecal transplantation of HSCs and HPs

Clinical efficacy was evaluated after three years of therapy by standard neurologic examination and registration of the results in specifically developed forms. The analysis of the registered data demonstrated efficacy of the intrathecal transplantation of HSCs and HPs in 57.4% of the patients, concerning motor and sensitive restoration, as well as repair of bowel and bladder functions (Figure 2). As it can be seen from Figure 2, we observed no neurologic improvement in 42.6% cases, which can be explained by underdeveloped inclusion/exclusion criteria. To date, it is clear that the method demands rigorous screening of the patients for this therapy that will further entail the development of clearer indications and contraindications for the intrathecal transplantation of HSCs and HPs. The size of lesion, its location, type and anatomic continuity of bone structures were of prior importance in this therapy. The analysis of ineffective cases of HSCs and HPs transplantation showed that in major part of the cases (25.2%) the size of spinal cord (SC) lesion exceeded 50% of the spinal cord cross-wise and one segment long-wise, according to MRI. Other reason for the inefficacy of the intrathecal transplantation of HSCs and HPs seems to be the unnoticed moderate or slight disorder of CSF circulation, associated with CSF hypertension, instability of the spinal segment in the injury site and/or scars and cicatrices of the spinal cord that hinder the circulation of CSF. Refusal of the patients from rehabilitative therapy (40.6% of cases) has also significantly contributed to the inefficacy of the therapy. The patients considered administered transplantations sufficient for the recovery and neglected the rehabilitation. In 10.6% cases, the patients negated positive results of the therapy, although the medical exercise instructors and attending doctors observed neurologic progress. Only video records that were taken in the beginning of the treatment and in the course of it, served a decisive argument to confirm functional repair. The therapy that took from 5 to 8 years showed that these patients demonstrated good clinical results of SC functions’ repair. However, this trial included only the patients who received no less than 20 transplantations of HSCs and HPs. In other cases (8.2%) the reason of inefficacy remained unclear, prompting necessity of further research. Moreover, we did not find correlation between the number of transplanted HSCs and HPs and transplantation efficacy [P = 0.1 (P > 0.1)], which was also confirmed by the absence of difference between the number of the transplanted cells to the patients with no effect and those with positive effect, resulting from HSCs and HPs transplantation (5.3 ± 0.9 × 106, as compared to 106.4 ± 0.9 × 106, P > 0.1, respectively). The hypothesis that the process of repair after intrathecal administration of HSCs and PCS depends on the amount of the cells (5.3  0.9  106 as compared to 106.4  0.9  106) was not confirmed at a 90% significance level.
Evaluation of motor function repair: The efficacy of the intrathecal transplantation of HSCs and HPs was evaluated with the help of the assessment of neurologic condition that included 5-point test of muscle strength, active movements and pace of movements of the extremities on both sides. Total score for no neurologic disorder is 300 points. As seen from Figure 3A, 56.9% of the cases demonstrated improvement of neurologic symptoms, accompanied by muscle strength and muscle tone build-up, visual contractions of some groups of muscles, frequently unilateral, and further development of movements in lightweight positions. Largely, the active movements appeared 12-18 mo later during exercises on press machines. Accordingly, in 50% of the patients the motor restoration began after the first HSCs and HPs transplantation, which is confirmed in average by 9.9 points improvement in neurologic impairment as compared to the baseline (P < 0.05) (Figure 3B). Repeated HSCs and HPs transplantations further enhanced neurologic improvement, that made 142.5 ± 9.7 points (P < 0.05, as compared to baseline and first HSCs and HPs transplantation results). Usually, intensive exercise led to strengthening of extremities’ muscles, increase of range and pace of the movements, stabilization of the knee joints, ability to stand independently in the knee supporting position and development of the elements of walking with assisting devices (walkers). It should be noted that 91.2% reported no restoration of motor functions for several years, and development of the first controllable movements was extremely important for the patients and served an incentive for further training. However, the improvement of the muscle strength was often admitted by the patient no earlier than in 6-12 mo and became objective reality by the end of the second or even third year. By the sixth year, the patients are deeply convinced in the effectiveness and practicability of the therapy.

As our research demonstrated, the intrathecal transplantations of HSCs and HPs led to gradual recovery of the lost movements in chronic SCI patients, only being accompanied by specific rehabilitation. Still, rehabilitation without HSCs and HPs transplantation before enrollment into the program produced only limited effect.


Post HSCs and HPs transplantation changes of motor activity depending on the level of injury

The motor improvement was mostly observed at Th3-Th8 level of injury, specifically in 81.3% of the cases (Figure 3C). Meanwhile, cervical and lumbar SCI cases showed lesser benefit from the therapy, and functional restoration was less illustrative (Figure 4). However, the level Th3-Th8 cases demonstrated considerable repair.

Due to baseline diversity, the comparison of the clinical data between the levels of injury was done in per cent and showed maximal improvement of Th3-Th8 SCI cases after the second and consequent HSCs and HPs transplantations. After the first HSCs and HPs transplantation neurologic improvement was observed only in the cases of cervical injury, which can be explained by the fact that the first feeling of the slight changes in motor functionality (mostly of upper limbs) was much brighter in this category of the patients. By 5-8-th transplantations the quadriplegics were able to turn in their beds independently, the strength in upper extremities and back increased, and they did not require fixation to a wheelchair with the belts or any other devices. However, three years after the first transplantation, the most positive results were observed in lumbosacral cases and, strangely enough, in cervical SCI. At least, the improvement of life quality was more obvious in quadriplegics, both for the patient and for their relatives.

Accordingly, these data report more vigorous repair of motor functions at Th3-Th8 level of SCI after HSCs and HPs transplantations. Although, the represented data show limited opportunity for the restoration at the level of cervical and lumbar enlargement, we observed the benefits of cell transplantations at these levels. Follow-up of the SCI patients after the HSCs and HPs transplantations demonstrated neurologic progress in 61.1%, and it was associated with strengthening of the muscles, development and/or increase of motor activity, regress of sensitive disorders, and improvement of bowel and bladder functions. The most notable clinical effect was achieved in locomotion. In most cases, the changes in motor functions were minimal after the first HSCs and HPs transplantation and manifested in lightweight positions. Further intensive rehabilitation led to strengthening of extremities muscles, increase of pace and range of movements during exercise tests. After the second HSCs and HPs transplantation 33 patients were able to stabilize knee joints, to stand in knee supporting position independently and developed some elements of walking with assisting devices (walkers). It should be noted that 96% of the patients demonstrated no signs of neurologic restoration for several years before HSCs and HPs therapy. One of the patients from the United States restored independent automatic walk in a month of the therapy that included 4 administrations of the HSCs and HPs, and left the hospital on their own feet, although their previous treatment in the US lasted 5 years. The similar recovery was observed in the patient from Bosnia and Herzegovina, when two administrations were enough to restore the walking function after 6 years of ineffective therapies in various clinics of the world.


Post HSCs and HPs transplantation changes of motor activity depending on the type of injury

As expected, comparison of the results, depending on type of injury, showed better progress in the cases of incomplete SCI. Sixty percent of incomplete injury cases demonstrated improved locomotion, as compared to 46.7% of complete SCI cases (Figure 5). The patterns, identified at early period of the therapy, were fully confirmed 1-3 years post therapy beginning. They are supported by the changes of clinical condition in incomplete SCI cases, manifested in the increase of motor points from baseline 142.1 ± 5.7 to 150.5 ± 5.7 after the first transplantation, and 172.6 ± 8.1 after the second transplantation (P < 0.05) (Figure 5). In complete SCI cases neurologic improvements were minimal and made only 5 points after the first HSCs and HPs transplantation (P < 0.05). The tendency to improve to 81 ± 7.9 points was observed after the second transplantation (P < 0.1), which can be explained by the insignificant number of cases (n = 11) Due to different baseline scores of incomplete SCI and complete SCI cases, the comparison between the stages of therapy was done in percent and did not demonstrated significant difference in results after the first, or after the second, and even after the twentieth HSCs and HPs transplantations.


Post HSCS AND HPS transplantation changes of motor activity depending on years post injury

The increase of motor activity increase (Figure 6) after HSCs and HPs transplantation was observed only in the cases of 2-5 years post SCI; it was manifested in the motor activity increase from baseline 134.5 ± 7.3 points to 144.5 ± 8.6 points after the first transplantation and to 173.4 ± 10.7 after the second P < 0.05 between baseline and transplantations, respectively). Neither cases of 1-2 years post SCI, nor the cases over 5 years post injury showed statistically significant changes of clinical symptoms. These results seem to be conditioned by the inability of HSCs and HPs to realize their regeneration potential, due to residual inflammation and apoptosis in the patients with the period post SCI, varying from 1 to 2 years and due to degenerative changes in spinal cord in over 5 years old SCI cases. Still, regress of motor neurologic symptoms was observed in some of the patients with such SCI, so that in one of the cases the motor functions were considerably repaired 29 years post injury.


Testing muscle strength repair in SCI patients after HSCS AND HPS transplantation with Medical Research Council Scale

The Medical Research Council Scale was used to confirm the obtained results of motor progress after the HSCs and HPs transplantation in chronic SCI patients. The scale seems to be one of the most convenient and clear measurements of the strength of separate muscles, and originally was meant to detect locomotion deficit in the injuries of peripheral nerves. Total score for the absence of neurologic impairment makes 100 points.

As seen in Figure 7, the HSCs and HPs transplantation, accompanied by intensive rehabilitation, resulted in the increase of the muscle strength at all stages of research (P < 0.05). The second HSCs and HPs transplantation did not lead to muscle strength increase in damaged extremities. These data can be explained by insensitivity of the measurement tool to paresis improvements, the so called ceiling effect, that agrees with the data of Belova[11]. It is also confirmed by the analysis of muscle strength, the patients being distributed according to the level and type of injury (Figures 8A and B). Strengthening of the muscles was observed in the cases of more severe injuries: at the level of cervical intumescence and with complete SCI.

On the other hand, recovery of the muscle strength after HSCs and HPs transplantation repeated the pattern of the progress of motor functions, depending on the years post injury. This manifested in the slight score increase in the cases of 2-5 years post SCI after the first HSCs and HPs transplantation (from baseline 63.8 ± 4.6 points to 78 ± 7.1 points after HSCs and HPs transplantation, P < 0.05, respectively). However, after the second HSCs and HPs transplantation, muscle strength increase was registered only in the patients with 1-2 years old injury. The cases of over 5 years old SCI demonstrated no statistically valid increase of muscle strength, herewith, confirming the hypothesis of hindered motor restoration, due to degenerative changes in spinal cord in these cases. Hence, the changes in muscle strength, measured by Medical Research Council Scale, demonstrated improvement of locomotion after HSCs and HPs transplantation despite low sensitivity of the tool and consequent low increase of the score (Figure 9).


Sensation repair in SCI patients after HSCs and HPs transplantation

Sensation repair after the intrathecal transplantation of HSCs and HPs was evaluated in 71 patients by the assessment of neurologic condition that included 2-point tests of pain, temperature, deep sensation on dermatomes on both sides, as well as the assessment of the feeling of muscle “heaviness” in rest and after exercise in upper and lower extremities, abdomen and back. Total score, denoting absence of neurologic motor disorders, made 312 points.

As different from the locomotion, the repair of sensation was registered in a much fewer number of chronic SCI cases (Figure 10), the reason as yet remaining unclear. At the same time, the analysis of the obtained clinical data showed (Figure 11A) that the cell therapy led to the increase of sensitivity from baseline 124.3 points to 138.4 after the first and to 153.5 points after the second transplantations of HSCs and HPs (P < 0.05, between the stages of research).

Clinically, the repair manifested in the expansion of sensation areas, accompanied by gradual involvement of new dermatomes. Major part of the patients observed the elements of deep sensation after the first transplantation and characterized them as the “heaviness” of muscles in rest and after physical training. Further, it was noted that development of the feeling of the position of lower extremities in space preceded stabilization of knee joints and development of the first elements of walking.

Expansion of the areas of surface sensation did not depend on the level of injury, i.e. the sensation could manifest with separate dermatomes of lower and/or upper extremities, anterior chest or abdomen walls. In most of the cases the dermatomes did not restore in full, but only partially the sensation seldom restored unilaterally. Having received 5-7 HSCs and HPs transplantations, some of the patients restored sensation in all or almost all dermatomes of extremities and body. Hence, after the transplantation of HSCs and HPs, the sensation restores in chronic SCI cases, but in fewer cases than locomotion.

Case distribution, depending on the level or type of injury, demonstrated restoration of sensation in the most severe cases (complete SCI of cervical intumescence) (Figures 11B and C). These results are likely to be conditioned by low sensitivity of the measurement scale, i.e., “ceiling effect”. However, gradation of the sensation disorders was copied from widely applied measurement scales, including ISCSCI-92, and, hence, demonstrated the inefficiency of applied evaluation methods that demand upgrade.

No clinical changes were observed in the distribution of the cases, depending on the years post injury. This can be explained by lesser damage of posterolateral parts of spinal cord that agrees with the multiple data of pathomorphological tests. However, additional tests are necessary to confirm this hypothesis. Obtained clinical data of sensation repair were objectified with somatosensory evoked potentials[12].
Evaluation of bladder repair in SCI patients after HSCs and HPs transplantation

Efficacy of the rapier of bladder functions was evaluated in 72 patients with the assessment of neurologic condition that included 3-point assessment of the feeling of urination and 5-point assessment of urine retention. Total score that denotes absence of neurologic signs of urinary disorder is 8 points.

Repair of the urinary system was observed in 47.7% of the cases after the intrathecal transplantation of HSCs and HPs. Clinically, the restoration of urinary system manifested in creeping sensation in the body or unpleasant feelings in the lower abdomen that preceded involuntary urination, but complete syndrome of vegetative hyperreflexia was absent (changes in blood pressure and heart rate, arrhythmia, sweating, fever above the injury level). Many patients observed the feeling of weak “swelling” above pubic symphysis that allowed beginning of bladder training with closing urethral or cystostomic catheter. Further restoration of the capacity to retain urine for at least 1-3 min led to intermittent catheterization, or refusal from the cystostomy. In some cases, 3-5 intrathecal transplantations of HSCs and HPs resulted in full refusal from intermittent catheterizations and further complete repair of urinary function.

Analysis of the clinical data showed that in 33.8% cases the manifestations of urinary restoration began after the first transplantation of HSCs and HPs, showing clinical improvement from baseline 0.4 ± 0.2 points to 1.2 ± 0.2 points after the first transplantation of HSCs and HPs (P < 0.05) (Figure 12). Consequent transplantations improved the urinary function further, thus increasing the score to 1.9 ± 0.4 points.

Hence, the transplantation of HSCs and HPs can lead to gradual restoration of urinary function in chronic SCI cases. Analysis of the data, depending on the level of injury (Figure 13), showed that largely, the improvement in the urinary system after HSCs and HPs transplantation was noted at Th3-Th8 level of SCI and at the level of lumbar enlargement (70%). It manifested in the increasing urinary restoration (Figure 13) from baseline 1.1 ± 0.8 points to 2.5 ± 0.8 points after the first transplantation and to 2.9 ± 0.9 points after the repeated HSCs and HPs transplantations (P < 0.05 between the therapy stages) in Th3-Th8 SCI cases. In SCI at the level of lumbar enlargement the urinary function changed from baseline 1 point to 1.9 and 2.8 after the first and the second transplantations, respectively (P < 0.05 between the therapy stages).

Despite fewer number of the SCI patients at the level of cervical intumescence, who showed the urinary system repair (36.8%), the restoration from baseline 0.1 points to 0.7 points and to 1.3 points was clinically registered after the first HSCs and HPs transplantation after the second HSCs and HPs transplantation, respectively (P < 0.05 between the therapy stages).

Thus, the urinary system after the intrathecal transplantation of the HSCs and HPs restores irrespective of the level of the spinal cord injury. However, the urinary system restores more efficiently in the cases of SCI at the level of Th3-Th8 and lumbar enlargement.

The repair of the urinary system after HSCs and HPs transplantation did not depend on the type of SCI, as shown in Figure 14A. However, in the cases of the incomplete SCI the urinary disorder at a baseline was less significant, as well as after the first transplantation. After the second transplantation, no statistically significant changes in the clinical evaluation of urinary system have been observed.

Restoration of the urinary system did not depend on period post injury, either. As seen in Figure 14B, some restoration of urinary function was observed irrespectively from years post injury. There is a clear tendency for further improvement of urinary function after 2 or 3 years of HSCs and HPs therapy, as compared to baseline.

Consequently, the intrathecal transplantation of HSCs and HPs in chronic SCI patients is an efficient method to repair urinary function. The lower levels of SCI are more prone to restore urinary function, which can be explained by closer location of urination centers in sacral spinal segments to lesion sites and, possibly, by larger concentration of HSCs and HPSs in the sites of injury. Herewith, neither the type of injury, nor years post injury, do not influence restoration of urinary function.


Evaluation HSCS and HPS transplantation efficiency in SCI patients with ASIA, FIM and ISCISCI-92

The functional repair of spinal cord was analyzed for 72 chronic SCI cases; it was measured with ASIA, ISCISCI-92 and FIM indexes at all stages of HSCs and HPSs transplantation. The evaluation with ASIA index demonstrated regress of neurologic symptoms only in 23 cases. Two patients with complete SCI (ASIA A) showed restoration of the locomotion below neurologic level of injury with muscle force of no less than 3 points (ASIA C) after HSCs and HPs transplantation, and over 3 points (1 patient, ASIA D). The ASIA B patient after HSCs and HPs transplantation showed neurologic restoration to ASIA C.

Nevertheless, the above shown analysis of clinical regress of neurologic symptoms demonstrates inefficiency of the ASIA impairment scale that was used to evaluate restoration of the spinal cord functions. On the one hand, it is associated with the specific features of restoration of spinal cord functions, and on the other, with low sensitivity of the index that gives only general estimation of regress of the neurologic damage. According to Belova[11] the ASIA index is applicable only for screening of the spinal functions during acute period of SCI. To evaluate neurologic progress in SCI, the more detailed characterization of locomotion, sensation and urination is required in every individual case.

As shown above, the sensation recovered after the manifestations of the restoration of motor functions analysis, especially in S4-S5 segments. The sensation restored mosaically, frequently after the development of passive or active movements, and involved the segments only partially. Absence of sensation in S4-S5 segments conditioned ASIA A level of impairment, even if motor functions of certain muscles below injury level were preserved to a certain extent. In this respect, 10 patients observed restoration of muscle force in most of the muscles below the level of injury that enabled their walking with assisting devices after 4-8 transplantations, while currently, two patients are able to cover short distances independently. However, only one of these patients demonstrated restoration of sensation in S4-S5 segments.

Hence, the ASIA impairment scale is effective to assess the degree of disability, but is ineffective, when used to assess the restoration of spinal cord functions in chronic SCI after HSCs and HPs transplantation.

Motor progress was also assessed with the ISCISCI-92 motor and sensory scores, and the data coincided with those received in evaluation of motor functions by the specifically developed scale. The number of the patients with the signs of locomotive repair was 56.9%. Moreover, the motor activity rates increased from baseline 32.7 points to 37.1 after the first transplantation and to 39.9 after repeated transplantation of HSCs and HPs (P < 0.05, at each stage of transplantation) (Figure 14C).

In spite of clinical restoration of sensation in 38.6% of the patients, the ISCISCI-92 scores did not confirm these data. This is conditioned by the absence of evaluation of deep sensation in ISCISCI-92, and, as noted before, by the “ceiling effect”, when the neurologic status changes within the partial restoration of sensation.

Hence, the assessment of motor restoration with the ISCISCI-92 scores demonstrated effectiveness of the HSCs and HPs transplantation in chronic SCI patients. The ISCISCI-92 score confirms the data of our specifically developed scale to assess the clinical motor restoration of spinal cord, thus, demonstrating its applicability in practice. The advantage of our evaluation scale of clinical motor restoration over the ISCISCI-92 lies in the multi-factor analysis of the motor activity, based on standard neurologic examination. Absence of changes in sensation as measured by ISCISCI-92 scores that, however, are accompanied by the clinical signs of restoration, demands development of new tools to measure changes both in surface sensation (touch and pain) and deep sensation. Despite partial solution of this issue in the specifically developed scale of clinical motor restoration, the “ceiling effect” was not overcome in partial restoration of this function.

We would like to focus on the restoration of the functional independence after HSCs and HPs transplantation that was evaluated in 64 patients with the Functional Independence Measurement (FIM) scale. The signs of the restoration of life activity was observed in 36.2% patients and were minimal (from baseline 50.1 points to 50.5 points after the first transplantation, and to 50.7 points after repeated HSCs and HPs transplantation; P < 0.05 at all stages of therapy, respectively) (Figure 15). It is associated with moderate restoration of the spinal functions after the first HSCs and HPs transplantations that manifested mostly in locomotion. However, as shown above, further transplantations resulted in more profound clinical progress. Besides, the FIM scale, when applied to chronic SCI cases has significant disadvantage: in the cases of considerable disorders of nerve impulse conductance, the FIM displayed very low sensitivity, due to absence of detailed functional evaluation. Accordingly, the analysis of the obtained data showed very slight improvement of the FIM scores, demonstrating improvement of the functional independence conditioned by the motor function of spinal cord.
Complications of intrathecal application of HSCS AND HPS

During 12 years of follow up we observed no life threatening complications resulting from the HSCs and HPs transplantation. The complications of HSCs and HPs administration were evaluated at three stages: stage 1 after the first transplantation; stage 2 after one year of the therapy that included 5.3 ± 0.5 administrations; stage 3 two years of regular administrations (10.1 ± 1.1). The complications were summed up in Table 4. We observed one case of cancer (femoral carcinoma) of 202 followed up cases. However, according to the conclusion of the experts of the Russian Cancer Research Centre, it was not associated with HSCs and HPs transplantation. Surprisingly, in the control group of 20 cases we registered one case of spontaneous brain cancer development (pituitary adenoma) too, for which the patient was operated on.




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