Safety of Autologous MSC Infusion to Treat Epilepsy
Safety of Autologous MSC Infusion to Treat Epilepsy
• The goal of this study was to evaluate the safety and efficacy of autologous MSC
 application for the therapy of drug-resistant symptomatic epilepsy. Adult (18-60 years old)
 patients (pts) of both sexes suffering from refractory epilepsy with frequent (>5 events per
 month) seizures were included in this study. The pts were randomized to the standard
 treatment with anti-epileptic drugs (control group, 30 pts) or anti-epileptic drugs plus
 autologous mesenchymal stem cells (MSCs) (study group, 30 pts). The pts in the study group
 received one intravenous injection of ex vivo expanded MSCs (40-101 x 106 cells) and one
 subsequent endolumbal injection of neuroinduced MSCs (2.7 - 8.0 x 106 cells). Both the
 unfavorable reactions to MSC infusions and the clinical effects, including complications,
 were examined. The unfavorable reactions to the MSC injections included local pain or
 hemorrhage at the site of injection and systemic reactions of the central nervous system
 (CNS; i.e., hyperthermia, fatigue, and myalgia).The possible beneficial effects of therapy in
 the two groups of pts were examined based on clinical observations and electroencephalography
 measurements (prior and 12 months after the application of the MSC-based therapy). To
 determine potential changes in disease progression, the signs of cognitive impairment,
 behavioral disorders, and particularly, changes in seizure character and frequency were
 evaluated using the National Hospital Scale of Seizure Severity. The main points of disease
 monitoring were "yes" or "no" responses (to therapy), seizure frequency (per month), and
 remission of disease. Electroencephalography (EEG) recordings were performed to evaluate
 electrical alpha, beta, theta and delta waves based on standard and additional criteria. The
 paroxismality index, the peak frequency of EEG activity, the index of slow activity, and the
 summarized points of EEG pathology signs were calculated for each patient. All assessments
 were performed for the pts in the control and study groups, and the obtained data were
 compared to identify the potential differences between the two pts groups. Therapy was
 terminated when immediate unfavorable reactions to the MSC injections were observed. The
 final observation of each patient included clinical and EEG assessments at the time point of
 12 months (or more) after the application of the MSC-based therapy.
The main goal of this phase 1 clinical trial was to evaluate the safety of an autologous
 mesenchymal stem cell (MSC) application as therapy for drug-resistant symptomatic epilepsy in
 a one-center study. It is well known that drug-resistant symptomatic epilepsy is a
 dramatically invalidating disorder for which applied stationary and outpatient therapies have
 no real clinical effect. Therefore new approaches are needed to reach remission, to stop
 disease progression, and to increase patient quality of life. For this study, adult (18-60
 years old) patients (pts) of both sexes suffering from drug-resistant symptomatic epilepsy
 with frequent (>5 events per month) seizures were included into study. Drug-resistance was
 defined by a lack of any evident clinical response (i.e., no decrease in seizure frequency or
 reduction in disease progression) of the pts with symptomatic epilepsy to carbamazepine,
 valproic acid, topiramate, lamotrigine, or phenobarbital (i.e., anti-epileptic drugs[AEDs])
 as monotherapies and in different combinations over the previous one calendar year. The
 criteria for exclusion were patient refusal to participate, unfavorable reactions to therapy,
 CNS inflammatory conditions, chronic psychoses, CNS tumors, relapses of chronic somatic or
 neurologic diseases, and blood positivity for hepatitis B or C or HIV. The plan included the
 inclusion of 30 pts in the study group and 30 pts in the control group over a period of 5
 years. The pts were randomized to standard treatment with AEDs (control group) or AEDs plus
 autologous mesenchymal stem cells (MSCs, study group). The MSCs were obtained from bone
 marrow samples of the same patient and were purified and expanded ex vivo in a specialized
 cellular biotechnology laboratory. The MSCs were characterized by immunophenotyping as
 CD90+CD105 +CD45-CD34-cells. After 3 weeks of cultivation in vitro, a portion of the MSC were
 additionally cultured for 7 days in Neurocult-XF proliferation medium to obtain neuroinduced
 MSCs. Neuroinduction was proved by the presence of the genetic markers nestin and
 neuron-specific enolase. Finally, 40-101 x 106 autologous cultured MSCs and 2.7 - 8.0 x 106
 autologous neuroinduced MSCs were harvested, resuspended in saline solution containing 5%
 autologous blood serum for injection, and, following a measurement of the viability (98%
 cells), transferred to the clinical center.

 The pts in the study group received one slowly delivered (over 5-10 minutes) intravenous
 injection of ex vivo expanded autologous MSCs in a volume of 20 ml, and 5-7 days later, each
 patient in the study group received an additional slowly delivered endolumbal injection of
 the neuroinduced MSCs in a volume of 5 ml. Both unfavorable reactions to the MSC injections
 (over one day following the performance of the procedures) and the early (up to one month)
 and late (up to 6 months) clinical effects, including complications, were evaluated.
 Unfavorable reactions to the MSC injections included local pain or hemorrhage at the site of
 injection and systemic reactions of the central nervous system (CNS, i.e., hyperthermia,
 fatigue, and myalgia). Later potential unfavorable systemic reactions of the CNS and vascular
 system, including infectious and noninfectious complications of the progression of the
 disease to be controlled, were examined. All of the events were documented in medical cards.
 In cases in which these events exhibited dangerous characteristics, they were declared to
 members of the monitoring board (the center's ethical committee) for the evaluation of the
 exclusion of the patients from the study or the termination of the clinical study.

 Also evaluated were the possible beneficial effects of MSC-based therapy in the pts of the
 study group. These effects were detected via clinical observations at selected therapy time
 points (i.e., 3 and 12 months after the application of the MSC-based therapy) and
 electroencephalography measurements prior to and one year after the therapy. To determine the
 possible changes in disease progression, signs of cognitive impairment, behavioral disorders,
 and changes in seizure characteristics and frequency were evaluated. For the evaluation of
 cognitive impairment, we used the Mini-Mental State Examination. The handicapping effect of
 disease on daily life was scoring using the Subjective Handicap of Epilepsy Scale. State
 anxiety and depression were evaluated using the Hospital anxiety and depression scale .
 Changes in seizure characteristics and frequency were evaluated using the National Hospital
 Scale of Seizure Severity. The processes of voluntary attention and performance were studied
 with Schulte tables according to the methods of Kraepelin. Schulte tables were used to study
 sensorimotor reaction times and the distribution and stability of attention. The "Account of
 Kraepelin" method was used to study health, fatigue and the stability of attention. The
 assessment of short-term memory was performed via the method of memorizing 10 words; this
 method aims to determine the volume and speed of oral-aural memory.

 Main clinical characteristics that are used for disease monitoring were "yes" or "no"
 responses (regarding therapy), seizure frequency (per month), and remission of disease at the
 early (3 months) and late (12 month and more) time points after therapy. To evaluate partial
 responses to therapy, the numbers of pts who exhibited 50% reductions in seizure frequency
 were assessed. Seizure type (i.e., generalized tonic-clonic, partial complex, simple partial,
 and multiple types of seizures) was also evaluated along with changes in seizure type during
 the treatment course.

 Electroencephalography recordings were performed at admission and across the monitoring
 period using a Mizar EEG 201 encephalography system with biopotential registration from 16
 body points according to the "10-20" scheme. The observed electrical alpha, beta, theta and
 delta waves were analyzed in 3-min segments (with further recalculation for each 1-minute
 segment) prior to (at admission) and 1 year after MSC application. The peak frequency of the
 alpha waves was also calculated. Both the spontaneous state and the state after the loading
 probe (hyperventilation and photostimulation) were evaluated for each patient. The
 characteristics of the electroencephalographies (EEG) were attributed to local and diffuse
 cortical alterations. We evaluated the paroxysmality index, quantities of local and
 generalized spikes of epileptiform waves per minute, peak frequency of EEG activity, index of
 slow activity, and summarized points of EEG pathology signs. EEGs with epileptiform activity
 included spikes, spike-slow waves, and high-amplitude spikes. The EEG recordings were
 performed based on standard and additionally proposed criteria . All of the assessments were
 performed for the pts in the control and study groups, and the obtained data were compared to
 determine the potential differences due to the additionally performed MSC-based therapy. A
 patient's therapy was terminated at any time point if immediate unfavorable reactions to the
 MSC injections were observed. The final observations of the patients included clinical and
 EEG assessments at 12 months (or more) after the application of the MSC-based therapy. The
 summarized data for the pts in the control and study groups were collected in an electronic
 database for further analysis and interpretations.
Inclusion Criteria:

 -  Clinical diagnosis of symptomatic epilepsy,

 -  Disease progression for the last 1-3 years,

 -  Resistance of epilepsy to therapy with carbamazepine, valproic acid, topiramate,
 lamotrigine, and phenobarbital (anti-epileptic drugs/AEDs) as monotherapies or
 combination therapies;

 -  Signed informed consent

 Exclusion Criteria:

 -  Central nervous system inflammatory disorders (meningoencephalitis of viral or
 parasite origin),

 -  Chronic decompensated psychoses ,dementia, social disadaptation,

 -  Central nervous system tumours.

 -  Blood positivity for hepatitis B or C or HIV infection;

 -  According to the judgment of the researchers, subjects who were unable to complete the
 study or may not have been able to comply with the requirements of this study (due to
 administrative or other reasons).