Safety and efficacy outcomes after intranasal administration of neural stem cells in cerebral palsy : a randomized phase 1/2 controlled trial

This is a self-archived version of an original article. This version may differ from the original in pagination and typographic details. Author(s): Title: Year: Version: Copyright: Rights: Rights url: Please cite the original version: CC BY 4.0 https://creativecommons.org/licenses/by/4.0/ Safety and efficacy outcomes after intranasal administration of neural stem cells in cerebral palsy : a randomized phase 1/2 controlled trial © The Author(s) 2023. Published version Lv, Zhongyue; Li, Ying; Wang, Yachen; Cong, Fengyu; Li, Xiaoyan; Cui, Wanming; Han, Chao; Wei, Yushan; Hong, Xiaojun; Liu, Yong; Ma, Luyi; Jiao, Yang; Zhang, Chi; Li, Huanjie; Jin, Mingyan; Wang, Liang; Ni, Shiwei; Liu, Jing Lv, Z., Li, Y., Wang, Y., Cong, F., Li, X., Cui, W., Han, C., Wei, Y., Hong, X., Liu, Y., Ma, L., Jiao, Y., Zhang, C., Li, H., Jin, M., Wang, L., Ni, S., & Liu, J. (2023). Safety and efficacy outcomes after intranasal administration of neural stem cells in cerebral palsy : a randomized phase 1/2 controlled trial. Stem Cell Research & Therapy, 14, Article 23. https://doi.org/10.1186/s13287022-03234-y 2023 Lvetal. Stem Cell Research & Therapy (2023) 14:23 https://doi.org/10.1186/s13287-022-03234-y RESEARCH Safety andefficacy outcomes afterintranasal administration ofneural stem cells incerebral palsy: arandomized phase 1/2 controlled trial Zhongyue Lv1,2†, Ying Li1,2†, Yachen Wang1,2, Fengyu Cong3,4, Xiaoyan Li1,2, Wanming Cui5, Chao Han1,2, Yushan Wei6, Xiaojun Hong7, Yong Liu8, Luyi Ma9, Yang Jiao1,2,10, Chi Zhang3, Huanjie Li11, Mingyan Jin3, Liang Wang1,2, Shiwei Ni1,2 and Jing Liu1,2* © The Author(s) 2023. 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The Creative Commons Public Domain Dedication waiver (http:// creat iveco mmons. org/ publi cdoma in/ zero/1. 0/) applies to the data made available in this article, unless otherwise stated in a credit line to the data. Open Access Stem Cell Research & Therapy Abstract Background Neural stem cells (NSCs) are believed to have the most therapeutic potential for neurological disorders because they can differentiate into various neurons and glial cells. This research evaluated the safety and efficacy of intranasal administration of NSCs in children with cerebral palsy (CP). The functional brain network (FBN) analysis based on electroencephalogram (EEG) and voxel-based morphometry (VBM) analysis based on T1-weighted images were performed to evaluate functional and structural changes in the brain. Methods A total of 25 CP patients aged 3–12 years were randomly assigned to the treatment group (n = 15), which received an intranasal infusion of NSCs loaded with nasal patches and rehabilitation therapy, or the control group (n = 10) received rehabilitation therapy only. The primary endpoints were the safety (assessed by the incidence of adverse events (AEs), laboratory and imaging examinations) and the changes in the Gross Motor Function Measure-88 (GMFM-88), the Activities of Daily Living (ADL) scale, the Sleep Disturbance Scale for Children (SDSC), and some adapted scales. The secondary endpoints were the FBN and VBM analysis. Results There were only four AEs happened during the 24-month follow-up period. There was no significant difference in the laboratory examinations before and after treatment, and the magnetic resonance imaging showed no abnormal nasal and intracranial masses. Compared to the control group, patients in the treatment group showed apparent improvements in GMFM-88 and ADL 24 months after treatment. Compared with the baseline, the scale scores of the Fine Motor Function, Sociability, Life Adaptability, Expressive Ability, GMFM-88, and ADL increased significantly in the treatment group 24 months after treatment, while the SDSC score decreased considerably. Compared with baseline, the FBN analysis showed a substantial decrease in brain network energy, and the VBM analysis showed a significant increase in gray matter volume in the treatment group after NSCs treatment. Conclusions Our results showed that intranasal administration of NSCs was well-tolerated and potentially beneficial in children with CP. †Zhongyue Lv and Ying Li are contributed equally to this work as a Co-first author *Correspondence: Jing Liu [email protected] Full list of author information is available at the end of the article Page 2 of 14 Lvetal. Stem Cell Research & Therapy (2023) 14:23 Trial registration: The study was registered in ClinicalTrials.gov (NCT03005249, registered 29 December 2016, https:// www. clini caltr ials. gov/ ct2/ show/ NCT03 005249) and the Medical Research Registration Information System (CMR-20161129-1003). Keywords Neural stem cells, Cerebral palsy, Intranasal administration, Electroencephalogram, Clinical trials, Functional brain network Introduction Cerebral palsy (CP) is the leading cause of physical disability in children [1, 2], which is a group of nonprogressive permanent central nervous system (CNS) diseases that affect the motor, postural, and coordination functions [3]. The global prevalence of CP was around 2.0–3.5 per 1000 live births [4, 5], and among them, 80% of cases are classified as spastic [6]. The electroencephalography (EEG) and neuroimaging abnormalities have been observed in most CP patients [7]. Recently, stem cell therapy has shown favorable function in repairing nerve damage in CP [8, 9]. Some stem cell therapies have been trialed in patients with CP [10– 12] and have improved functional recovery. However, many aspects of stem cell therapy remain unknown, such as the best route of cells administration, the most appropriate type of stem cells, the accurate assessments of clinical efficacy, and the neuropathological mechanism of stem cell therapy in humans [13]. Several stem cell transplantation schemes have been applied in CP patients, like intrathecal administration (brain stereotactic surgery, lumbar puncture) and intravenous/intraarterial injection. Still, they reflect some drawbacks, such as procedure-related adverse events (AEs) [13]. A new and better therapeutic strategy with well-tolerated, high efficacy, and few AEs will maximize the therapeutic effect of stem cells for children with CP. Preclinical studies show that intranasal administration of stem cells for neurological disorders is a promising cellular delivery method and can reduce the incidence of AEs in patients [14]. Neural stem cells (NSCs) are believed to have the most therapeutic potential for neurological disorders because they can differentiate into various neurons and glial cells. In hypoxia–ischemia neonatal models, intranasal delivery of NSCs improved brain injury and neurological outcomes [15]. A clinical observation reported that the brain injury area of preterm infants with intracranial hemorrhage reduced significantly after receiving a nasal drip of breast milk (including nutritional factors and breast milk stem cells), which further supported the feasibility of intranasal delivery of stem cells [16]. However, the clinical trial of intranasal administration of stem cells in treating neurological disease has not been reported. As we all know, motor function deficits occur as a result of brain function and structure injury. Although clinical scale evaluation could intuitively reflect the improvements of some symptoms, it is difficult to provide direct evidence for the alterations in brain function and structure. EEG is a commonly used method to evaluate brain function in the clinic. The sleep EEG was a reliable index of the brain structural and functional changes that could reflect the brain cortical reorganization and synapses pruning of neural activity in children and adolescents [17, 18]. However, there is no way for clinicians to assess brain function by quantitatively analyzing EEG data. Neurons in the brain form a complex network. Graph theorybased functional brain network (FBN) provides a new perspective for EEG quantitative analysis [19]. The FBN has important topological properties, such as degree, global efficiency, clustering coefficient, characteristic path length, and brain network energy, which provides a new method to quantify brain function [20]. And this helps to explain the topological principle of functional reconstruction of the nervous system and brain [21, 22] and may be a new promising method for analyzing brain function changes in CP patients after stem cell therapy. Magnetic resonance imaging (MRI) is the most commonly used technology to reveal structural brain changes in the clinic. We all know that ischemia and hypoxia are the major contributor to CP [23] and could lead to neuronal and glial injury. Previously, the research group used the diffusion tensor imaging (DTI) sequence of MRI to evaluate the effect of stem cell therapy on the integrity of white matter (the primary concentration area of glial cells). However, it is unknown whether stem cell therapy affects the changes of neurons in the human body. The survival and functional recovery of neurons are crucial for functional neurological repair. The soma of neurons is located in the gray matter. The morphometric changes of the brain are conventionally tested using voxel-based morphometry (VBM). T1-weighted image analysis of MRI may provide reliable evidence for the recovery of neurons and provide a reference for studying the neuropathological mechanism of stem cell therapy in the human body. Herein, we conducted an open-label randomized controlled clinical trial to evaluate the safety and efficacy of intranasal administration of NSCs for CP for the first Page 3 of 14 Lvetal. Stem Cell Research & Therapy (2023) 14:23 time. And we performed FBN analysis based on sleep EEG and VBM analysis based on T1-weighted images before and after treatment to evaluate the effects of NSCs on brain function and gray matter structure. Materials andmethods Study design This phase 1/2, randomized, controlled clinical trial was designed to evaluate the safety and efficacy of intranasal delivery of NSCs loaded by degradable patches. According to the principle of continuous enrollment, we randomly assigned all 25 patients who met the enrollment conditions from January 2018 to March 2019, including 15 patients in the treatment group (receiving NSCs transplantation three times and rehabilitation therapy; Patients 1–15) and 10 patients in the control group (receiving rehabilitation therapy only; Patients 16–25). This study was registered with ClinicalTrials. gov (NCT03005249) and the Medical Research Registration Information System (CMR-20161129–1003). And it was approved by the Ethics Committee of the First Affiliated Hospital of Dalian Medical University. The reference number of the ethics approval was LCKY2016-60. Eligibility andscreening Eligible participants, aged between 3 and 12years, were clinically presenting with moderate to severe paralysis characterized by spastic CP induced by ischemic hypoxia. The Gross Motor Function Classification System (GMFCS) levels were II-V (Patient recruitment conditions are shown in Additional file1). All the subjects had participated in regular rehabilitation training for no less than one year but had no apparent improvements in clinical symptoms 3–6months before treatment. All the participants’ guardians signed informed consent before participating in the study. The NSCs nasal patch complex preparation The allogeneic NSCs were isolated from aborted human fetal forebrain tissue. These tissues were collected from healthy pregnant women who had requested an artificial abortion. All the donors voluntarily signed informed consent forms, and the tests for human immunodeficiency virus, syphilis, hepatitis B, and other pathogenic microorganisms were negative. All donor tissue was stored in sealed containers of sterile 0.9% normal saline (NS) and transported to the stem cell preparation center registered with the China Food and Drug Administration (CFDA) via a cold chain within 6h of abortion. The whole procedure was in accordance with the current international Good Manufacturing Practices process for separation, extraction, amplification, passage, and detection. The NSCs were passaged every 7–10 days and carried out using passages 4–8 for our clinical research. Before clinical application, cytopathic tests were performed for endotoxins, mycoplasma, chlamydia, and viruses. And the proportions of positive antigens expression were calculated by Flow Cytometry assay of NSCs biomarkers (Vimentin, Nestin, Notch-1, SOX2, SSEA1and Musashi-1). Immunofluorescence staining was used to detect the differentiation ability of NSCs into neurons, astrocytes, and oligodendrocytes. Observation of interaction between patches and NSCs was performed by overlaying images of cells and materials and then analyzed by confocal photographing. 2 × 106 cells/mL of NSCs were seeded at the materials and cultured in a 37°C incubator with 5% CO2 atmosphere for 21days to test the degradation of patch materials invitro by measuring the dry weight of cells and patch complexes. The 100%, 50%, 25%, and 0% extract of patch materials were used to culture NSCs for 14days to test the biocompatibility of NSCs and materials, and CCK-8 tests were used to detect the cytotoxicity of patch materials. The NSCs nasal patch complex transplantation Patients in the treatment group took oral Loratadine tablets (5 mg) and received intravenous Dexamethasone (4mg) 2h before transplantation. And then, they were sedated with 10% Chloral Hydrate enema (50mg/ kg). NSCs (5 × 105/kg) loaded with patches were placed on the olfactory fissure in bilateral nasal cavities by an otolaryngologist within 5min. After that, patients were required to lay supine and monitored for at least 4h. Every child received NSCs transplantation three times with an interval of 1month. Safety assessments The short-term safety profile was tested for one week after NSCs transplantation, including AEs, physical examinations, daily vital signs, dietary sleep records, physical examination records of pediatric neurologists and rehabilitation practitioners (blind method), blood biochemical examinations, ultrasonic cardiography, radiographic images of the chest and articulation/coxae, electrocardiogram, EEG, and MRI. And the long-term safety profile was monitored up to 24months after NSCs infusion. Scales analysis The clinical symptoms analysis includes the gross motor, fine motor, sleep quality, social ability, speech, life adaptability, and self-care ability. The scales used for measurements include the Gross Motor Function Measure-88 (GMFM-88), the Activities of Daily Living (ADL) scale, the Sleep Disturbance Scale for Children (SDSC), and some adapted scales derived from the Gesell Growth Page 4 of 14 Lvetal. Stem Cell Research & Therapy (2023) 14:23 Scale and the Chinese Psychological Development Scale, like the Fine Motor Function Scale (FMFS), the Sociability Scale (SS), the Life Adaptability Scale (LAS), and the Expressive Ability Scale (EAS). All scales were evaluated by neurologists and pediatricians blinded to the whole study. And the data were collected at baseline (1week before the treatment), 1month (1M), 3months (3M), 6months (6M), and 24months (24M) after NSCs treatment for all the CP patients. EEG acquisition andpreprocessing The sleep EEG data were collected by a Micromed Brain SPY Plus (Micromed, Italy) at baseline, 6M, and 24M after NSCs treatment for all the CP patients. EEG signals were recorded at a 128Hz sampling rate according to the international 10–20 placement system at the following positions: C3, C4, P3, P4, O1, O2, P7, and P8. The reference electrode was located at Cz, and the ground electrode was located at the central forehead. Electrode impedance was kept below 10 kΩ. Electrocardiogram data were recorded in a separate channel. Ten segments of EEG data in the non-rapid eye movement phase (NREM) II stage of sleep were randomly intercepted by an experienced technician, and each segment lasted 15s. EEG signal was high pass-filtered at 0.30Hz and low pass-filtered at 30Hz. Functional brain network construction FBN refers to a graph that includes the nodes and functional connections formed by the cooperation and coordination of neural activities between neurons and between parts of the nervous system. The most commonly used method to construct FBN is the Pearson correlation coefficient (PCC) (the EEG data processing flow is shown in Additional file 2). The Degree, Clustering coefficient, Characteristic path length, Global efficiency, and Brain network energy analysis were performed to compare the difference with the baseline or the control group (see Additional file3 for the analysis methods). MRI acquisition Data were acquired from each subject with a 3.0T GE Signa HDxt scanner, including T1-weighted imaging and T2-weighted imaging. Three-dimensional highresolution T1 weighted images were scanned by brain volume scanning sequence (BRAVO) with the following parameters: TR = 8.2 ms, TE = 3.2 ms, TI = 450ms, FA = 12°, FOV = 256 mm × 256 mm, acquisition matrix = 256 × 256, layer thickness = 1 mm, and the number of layers = 188. Patients with cognitive impairment were examined under sedation with 10% chloral hydrate. Voxel‑based morphological analysis VBM analysis was performed with FSL software, which can quantitatively calculate local gray matter voxels’ size and signal intensity. Skull stripping was performed first, and then, a gray matter template was created at a resolution of 2 × 2 × 2 mm3 in standard space. And then, transform each voxel’s gray matter density values into gray matter volume values, followed by nonlinear registration. Use Gaussian smoothing kernel and image to discrete convolution operation, and perform the spatial smoothing of gray matter volume map after registration transformation (the result of 8mm smoothing kernel is selected for the following analysis in this analysis). Finally, the results of different smoothing degrees are obtained, that is, each subject’s gray matter volume map. Statistical analysis All statistical analyses were performed with the Software Package for Social Sciences for Windows v23.0 (IBM, Armonk, New York). Categorical data are presented as frequencies and percentages. Continuous data are presented as the mean ± standard deviation (SD) or median (Q1-Q3) as appropriate. To compare categorical data between groups, χ2 tests were performed. Continuous data were compared using the independent Student’s t test or Mann–Whitney U test, as appropriate. Changes of variables at 24months relative to baseline in the study and control group were compared using paired Student’s t test or Wilcoxon rank-sum test. Changes in variables at baseline, 1M, 3M, 6M, and 24M in the study group were compared using the one-way repeated-measures ANOVA or generalized linear mixed model. A two-tailed P value < 0.05 was considered statistically significant. Results Patients Figure1 summarizes the numbers of patients screened, enrolled, and excluded. Of the 300 eligible patients, 275 were excluded due to severe comorbidities or unwillingness to participate. A total of 25 patients were enrolled (further information is provided in Additional file4), of whom 15 (6.73 ± 2.12years old) were randomized to the treatment group and 10 (5.72 ± 1.97years old) to the control group. There were no significant differences between the two groups of patients in terms of baseline characteristics, including age, sex, body weight, GMFCS, and the Manual Ability Classification System (MACS) (Table1). Routine management was provided to both groups of patients after initial hospitalization and perioperatively. Six patients had mild anemia before treatment and had been given iron supplementation throughout the treatment. The last Page 5 of 14 Lvetal. Stem Cell Research & Therapy (2023) 14:23 patient completed 24months of follow-up in August 2021. Finally, 24 patients were included in the statistical analysis, and one patient was lost to follow-up. The NSCs nasal patch complex The NSCs grew spherical invitro (Fig.2a), and the flow cytometry analysis showed that NSCs biomarkers were positive (Fig.2b). When cultured invitro, NSCs can differentiate into neurons, oligodendrocytes, and astrocytes (Fig.2c). The biodegradable patch materials loaded with NSCs, which have filed a patent application (International Patent Number: PCT/CN2019/077105; Chinese patent number: PCT/CN2019/077105; 201,810,623,850.5), were made of gelatin sponge and cut into cylinders with a diameter of 8mm and a thickness of 5mm (Fig.2d). Confocal immunofluorescence microscopy showed that NSCs were uniformly distributed within the patch, as shown in Fig.2e. The test of degradability of patches before treatment showed that patches degraded utterly at 21days invitro (Fig.2f). The cytotoxic assay showed that the 100%, 50%, 25%, and 0% extract of path materials had no toxicity to NSCs growth (Fig.2g). Fig. 1 Consolidated Standards of Reporting Trials (CONSORT) flow diagram Table 1 Baseline demographics and characteristics SD: standard deviation; kg: kilogram; GMFCS: gross motor function classification system; MACS: manual ability classification system Treatment group (n, %) Control group (n, %) P value Age, years Mean ± SD 6.73 ± 2.12 5.72 ± 1.97 0.249 > 6 years 8 (57%) 5 (50%) ≤ 6 years 6 (43%) 5 (50%) Sex 1.000 Male 10 (71%) 7 (70%) Female 4 (29%) 3 (30%) Body weight (kg) 18.82 ± 4.13 22.10 ± 7.62 0.187 GMFCS 0.783 II 3(21%) 3(30%) III 4(29%) 3(30%) IV 5(36%) 4(40%) V 2(14%) 1(10%) MACS 0.162 II 5(36%) 6(60%) III 3(21%) 1(10%) IV 4(29%) 1(10%) V 2(14%) 2(20%) Page 6 of 14 Lvetal. Stem Cell Research & Therapy (2023) 14:23 The primary endpoints Adverse events All 25 patients received the intended treatment of either NSCs combined with rehabilitation or rehabilitation alone. No severe AEs related to the intranasal delivery treatments or NSCs occurred 24 months after transplantation. Most of the observed AEs were grade I and II in severity or were transient, lasting a few hours or up to 3days following transplantation (Table2). Only four AEs happened during the 24months follow-up period in the treatment group. Two patients had a low-grade fever (body temperature < 38℃) but recovered without medicine. One patient experienced nasal mucosa hemorrhage (volume < 0.1 mL) following nasal delivery and recovered following oppression hemostasis. One patient had a history of complex partial seizure (CPS) and took oral antiepileptic drugs before treatment. Three months after NSCs treatment, the patient’s CPS type and frequency Fig. 2 The NSCs Nasal Patch Complex. a: Neurospheres of human NSCs (P6) cultured in vitro under the microscope; b: The identification of human NSCs by Flow cytometry: Vimentin, Nestin, Notch-1, Sox2, SSEA1, and Musashi-1; c: Differentiation ability of NSCs into neurons (Tuj1), astrocytes (GFAP) and oligodendrocytes (Oligo1); d: Physical drawing of the nasal patch after magnification of 5 × ; e: Observation of NSCs nasal patch complexes by laser confocal microscope; f: NSCs nasal patch complex biodegradation weight-time curve in vitro: at 21d, the samples were gelatinous, all dissolved after washing to achieve complete degradation; g: The growth curve of NSCs cultured in material extracts of different concentrations (cont, 25%, 50%, 100%) was obtained from the CCK-8 test results: Cont: control Page 7 of 14 Lvetal. Stem Cell Research & Therapy (2023) 14:23 changed and were controlled by adjusting the dose of sodium valproate and increasing carbamazepine. Laboratory andimaging examinations There were no significant differences in laboratory tests between baseline and after the whole course of treatment in the treatment group (Additional file5). MRI of the nasal cavity and brain did not reveal any noticeable pathological changes in any patient, including microbleeds, mass formation associated with cell transplantation, infection, or tumor formation. Neither of the two groups showed any evidence of an increase in systemic inflammation or other immune responses. Scales analysis One patient was lost to follow-up in the treatment group because of the local COVID-19. Twenty-four patients completed the follow-up during 24months of the clinical research. We evaluated clinical improvements among the treatment and control groups using scale data at baseline and the endpoint of 24months. The scale scores in gross motor function, fine motor function, self-care ability, sociability, expressive ability, and sleep quality showed no apparent differences between the treatment and control groups at baseline (Table3). As shown in Table3, compared with the control group, patients in the treatment group had obvious improvements in the self-care ability and gross motor function evaluated by the ADL and GMFM-88 (including A, C, D, E, and TOTAL) scales at 24M after NSCs administration (P < 0.05). The score of the FMFS, SS, LAS, EAS, and GMFM-88-B showed a tendency toward an increase, and SDSC showed a trend to decrease in the treatment group, although not statistically significant. Compared with the baseline, the scale scores of the FMFS, SS, LAS, EAS, GMFM-88 (including B, C, D, E, and TOTAL), and ADL increased significantly in the treatment group at 24M, while the SDSC score decreased (P < 0.05). But in the control group, GMFM-88, ADL, and SDSC showed no statistically changes, and the FMFS and LAS decreased statistically significantly (P < 0.05). The scores of the SS and EAS showed obviously increased but were much less pronounced than the treatment group. These results suggested that intranasal administration of NSCs could improve the patient’s clinical symptoms such as movement, language, cognition, self-care ability, and even sleep quality. To explore the onset time of NSCs in improving the clinical symptoms of patients with CP and the stability of its therapeutic effect, we further analyzed the evaluation scale data of patients in the treatment group at different time points. We observed a statistically significant increase in all scales except for LAS and GMFM-88-A at different follow-up time points (Table 4 and Additional file6). Patients’ activities of self-care ability (ADL, from 34.54 ± 20.90 to 45.85 ± 24.34, P < 0.01), standing (GMFM-88-D, from 15.86 ± 10.33 to 22.85 ± 11.51, P < 0.05), and walking/running/jumping (GMFM-88-E, from 14.29 ± 13.60 to 21.77 ± 15.24, P < 0.01) began to show significant improvement as early as 1M after treatment. 3M after treatment, expressive ability (EAS, from 29.29 ± 21.41 to 36.92 ± 19.86, P < 0.05) and the overall function of gross motor (GMFM-88-TOTAL, from 52.41 ± 24.44 to 71.45 ± 22.29, P < 0.05) had been significantly improved. 6M after treatment, the patients’ social skills (SS, from 26.68 ± 18.65 to 32.82 ± 17.82, P < 0.05) improved, and other previous improvements were maintained. The improvement of the most complex hand fine motor, which requires high control of small muscles, muscle tone, and hand–eye coordination, did not begin until 24M (FMFS, from 24.00 ± 15.85 to 36.56 ± 17.86, P < 0.05). And all the significant improvements persisted until 24 M post-transplantation in the treatment group. The results suggested that NSCs intranasal Table 2 Adverse events of safety outcomes between groups Treatment group Control group Severe adverse events Seizure 1 0 Urinary tract infection 0 0 Pneumonia 0 0 Influenza 0 0 Death 0 0 Other adverse events Otitis media acute 0 0 Nasal mucosa hemorrhage 1 0 Conjunctival irritations 0 0 Constipation 0 0 Dyspepsia 0 0 Nausea, vomiting 0 0 Diarrhea 0 0 Colitis 0 0 Anorexia 0 0 Herpangina 0 0 Fever 2 0 Febrile convulsion 0 0 Irritability 0 0 Insomnia 0 0 Bronchitis 0 0 Hypoxia 0 0 Dermatitis 0 0 Urticaria 0 0 Hirsutism 0 0 Alopecia 0 0 Page 8 of 14 Lvetal. Stem Cell Research & Therapy (2023) 14:23 administration could result in long-term improvements for CP patients and the effects were more pronounced at the early stage of gross motor. The secondary endpoints Functional brain networks ofEEG To further explore the changes in brain function in CP patients subjected to NSCs transplantation, we performed a graph theory-based FBN analysis of EEG in all enrolled patients. As shown in Fig.3a, the FBN connection showed a gradual weakening trend in sleep EEG in both groups with the progress of the intervention. Then, we calculated five topological features of FBN based on graph theory, including the degree (D), clustering coefficient (C), characteristic path length (L), global efficiency (E), and brain network energy (BNE) [24, 25]. The results showed that there was a significant difference (P < 0.05) in the BNE (Fig.3b) between two groups 6M after NSCs Table 3 Clinical changes of scales between groups Data are summarized as the mean ± SD (standard deviation). GMFM: Gross Motor Function Measure; ADL: Activity of Daily Living; SDSC: Sleep Disturbance Scale for Children; FMFS: Fine Motor Function Scale; SS: Sociability Scale; LAS: Life Adaptability Scale; EAS: Expressive Ability Scale; 24M: 24months after treatment *P < 0.05 (compared with Control group); #P < 0.05 (compared with Baseline) Treatment group Control group P value FMFS Baseline 24.00 ± 15.85 26.50 ± 14.99 0.701 24 M 36.56 ± 17.86#24.56 ± 14.25#0.136 SS Baseline 25.50(8.75–45.00) 26.00(15.13–46.75) 0.546 24 M 48.50(23–53.50)#27.50(13.75–44.63)#0.238 LAS Baseline 23.50(11.13–50.63) 31.75(13.50–48.25) 0.841 24 M 54.00(22.50–56.00)#27.25(15.00–51.88)#0.238 EAS Baseline 17.50(8.00–51.75) 29.25(10.75–50.50) 0.886 24 M 54.00(19.00–56.00)#34.00(11.75–52.63)#0.395 GMFM-88-A (Lying and Rolling) Baseline 51(44.5–51) 49(38.5–49.5) 0.259 24 M 51(51–51) 48(36.5–50.5) 0.041* GMFM-88-B (Sitting) Baseline 44.5(26.25–57) 46.5(29–56.5) 0.796 24 M 59(56–60)#41.5(21.5–57.75) 0.051 GMFM-88-C (Crawling and Kneeling) Baseline 18.5(6.75–33) 7(0–35.25) 0.625 24 M 40(35–42)# 5(0–32.25) 0.016* GMFM-88-D (Standing) Baseline 17(7.5–23.5) 7.5(0–18.5) 0.172 24 M 27(25–31)#3.5(0–17) 0.002* GMFM-88-E (Walking, Running and Jumping) Baseline 11(2–25.25) 4.5(0–16.25) 0.285 24 M 31(12–47)#1.5(0–12) 0.007* GMFM-88-TOTAL Baseline 52.41 ± 24.44 47.43 ± 27.30 0.644 24 M 74.95 ± 23.84#41.94 ± 24.50 0.009* ADL Baseline 34.54 ± 20.90 40.65 ± 31.72 0.574 24 M 61.68 ± 26.16#32.81 ± 20.09 0.018* SDSC Baseline 49.43 ± 14.27 47.90 ± 19.81 0.828 24 M 39.14 ± 9.77#47.50 ± 18.54 0.164