Eun Ji Lee, Min-Ju Lee, Ye Jin Ryu, Sang-Hyeon Nam, Rokhyun Kim, Sehyeon Song, Kyunghyuk Park, Young Jun Park, Jong-Il Kim, Seong-Ho Koh, Mi-Sook Chang PII: S1525-0016(24)00749-4 DOI: https://doi.org/10.1016/j.ymthe.2024.11.022 Reference: YMTHE 6643 To appear in: Molecular Therapy Received Date: 23 April 2024 Accepted Date: 15 November 2024 Please cite this article as: Lee EJ, Lee M-J, Ryu YJ, Nam S-H, Kim R, Song S, Park K, Park YJ, Kim J-I, Koh S-H, Chang M-S, Neuroplasticity-enhancing therapy using glia-like cells derived from human mesenchymal stem cells for the recovery of sequelae of cerebral infarction, Molecular Therapy (2024), doi: https://doi.org/10.1016/j.ymthe.2024.11.022. This is a PDF file of an article that has undergone enhancements after acceptance, such as the addition of a cover page and metadata, and formatting for readability, but it is not yet the definitive version of record. This version will undergo additional copyediting, typesetting and review before it is published in its final form, but we are providing this version to give early visibility of the article. Please note that, during the production process, errors may be discovered which could affect the content, and all legal disclaimers that apply to the journal pertain. © 2024 Published by Elsevier Inc. on behalf of The American Society of Gene and Cell Therapy. Abstract Despite a dramatic increase in ischemic stroke incidence worldwide, effective therapies for attenuating sequelae of cerebral infarction are lacking. This study investigates the use of human mesenchymal stem cells (hMSCs) induced toward glia-like cells (ghMSCs) toameliorate chronic sequelae resulting from cerebral infarction. Transcriptome analysis demonstrated that ghMSCs exhibited astrocytic characteristics, and assessments conducted ex vivo using organotypic brain slice cultures demonstrated that ghMSCs exhibited superior neuroregenerative and neuroprotective activity against ischemic damage compared to hMSCs. The observed beneficial effects of ghMSCs were diminished by pre-treatment with a CXCR2 antagonist, indicating a direct role for CXCR2 signaling. Studies conducted in rats subjected to cerebral infarction demonstrated that ghMSCs restored neurobehavioral functions and reduced chronic brain infarction in a dose-dependent manner when transplanted at the subacute-to- chronic phase. These beneficial impacts were also inhibited by a CXCR2 antagonist. Molecular analyses confirmed that increased neuroplasticity contributed to ghMSCs’ neuroregenerative effects. These data indicate that ghMSCs hold promise for treating refractory sequelae resulting from cerebral infarction by enhancing neuroplasticity and identify CXCR2 signaling as an important mediator of ghMSCs’ mechanism of action./ Introduction Stroke ranks as the second leading cause of mortality, following ischemic heart disease. The prevalence of stroke, including instances of young-onset stroke, is anticipated to rise significantly in the coming years.1,2 Cerebral infarction due to ischemia accounts for over 80% of strokes,3 and while thrombolytic therapies exist, approximately 50% of patients who receive such therapy following the onset of ischemic cerebral infarction fail to respond.4 Moreover, while oral antithrombotic drugs may prevent the recurrence of cerebral infarction, they are not treatments for already damaged brain areas. Since 50 to 80% of patients with cerebral infarction suffer from serious neurological aftereffects, which increase the social and economic burden on patients and their guardians, there is a critical need for novel therapies that preserve or restore the function of brain regions damaged by cerebral infarction.5 It is widely acknowledged that the adult brain has a limited capacity for regeneration after injuries, and that this capacity diminishes as humans age.6 Considering that most patients with cerebral infarction are elderly, it is unrealistic to expect that damage induced by cerebral infarction, which includes neuronal loss, synaptic loss, gliosis, and decreased blood supply, will recover spontaneously thereby reducing the incidence of long-lasting sequelae.7 Consequently, establishing approaches to enhance brain regeneration, plasticity, and blood supply in the damaged region would constitute a breakthrough in treating patients with sequelae. Over the past few decades, cell-based therapies have emerged as new therapeutic modalities for treating cerebral infarction.8 While numerous cell types have been evaluated as therapeutics, human mesenchymal stem cells (hMSCs) have gained favor due to their ready availability, amenability to large-scale expansion, and documented ability to secrete various paracrine acting factors with pro-angiogenic, neurotrophic, and anti-inflammatory activity. Recent studies have demonstrated the differentiation of hMSCs into neural phenotypes.9,10 Moreover, mouse bone marrow-derived MSCs have been differentiated into neurons, astrocytes, and oligodendrocytes.11 Our previous work has also demonstrated the potential of late-passage hMSCs, when induced to adopt a glia-like phenotype (ghMSCs), to exhibit neuroprotective effects in pre-clinical stroke models.12 The therapeutic benefits of ghMSCs are attributed to their secretion of neurotrophic factors and their induction into astrocyte-like cells.12-14
Astrocytes can be classified into two major subtypes: A1 and A2. A1 astrocytes, often found in various human neurodegenerative diseases, are neurotoxic as they secrete neurotoxins and upregulate complement cascade genes that damage synapses. Conversely, A2 astrocytes promote neuronal survival and tissue repair, primarily through the upregulation of neurotrophic factors.15 We hypothesize that the A2 astrocyte-like properties of ghMSCs may underlie the neuroprotective effects observed in our previous studies.12,14 Recognizing the significance of A2 markers on ghMSCs, this study sought to clarify the role of A2 astrocyte-like properties in mediating neuroprotection and neuroregeneration. To achieve this, we first induced astrocytic induction in hMSCs and validated the induction efficacy using RNA-sequencing (RNA-seq) and protein analyses to confirm the A2 astrocyte- like phenotype of ghMSCs. We then tested the ability of these induced astrocytes to protect against oxygen-glucose deprivation (OGD)-induced cell death in rodent brain slices, an ex vivo model of cerebral infarction. We further investigated whether the neuroprotective effects might be mediated through the activation of the CXCR2 signaling pathway by exposing neonatal rat brain slices to a CXCR2 antagonist. Finally, we evaluated the therapeutic efficacy of ghMSC transplantation in vivo in an adult rat model of middle cerebral artery occlusion (MCAO). Our findings demonstrate that ghMSCs exhibit superior neuroprotective and neuroregenerative effects in these models compared to hMSCs and highlight the activation of CXCR2 signaling as a key mechanism driving these beneficial effects. Results Changes in cell morphology and gene expression support induction of an astrocytic phenotype in ghMSCs Consistent with our previous studies on ghMSCs derived from hMSCs,12-14 hMSCs initially exhibited a flattened, fibroblast-like morphology prior to induction (Day 0). However, at the end of the induction period (Day 12), ghMSCs adopted a morphology similar to astrocytes (Figure 1A). To determine if phenotypic differences reflect changes in cell function, we performed RNA-seq on hMSCs obtained from two different donors and their corresponding ghMSCs. Deposited data from three immortalized human astrocyte lines were also used as a reference. Unsupervised hierarchical clustering analysis of the most highly expressed 10,000 genes indicated that ghMSCs were more closely related to human astrocytes than to hMSCs (Figure 1B). This analysis also identified differentially expressed genes (DEGs, adjusted P- value < 0.05 and log2(fold change) > 1) between ghMSCs vs. hMSCs with 313 up- and 63 down-regulated (Figure 1C). Notably, genes encoding the cytokine ligands, such as CXCL1, CXCL3, and CXCL8, which all bind CXCR2,16 were significantly upregulated in ghMSCs vs. hMSCs. Included among the 313 DEGs were the astrocyte specific markers SLC16A6, SNAP25, SOX9, APOE, SLC16A4, DIO2, SLC13A (GLAST), and GJA1 (Table S1). Furthermore, the expression of PTGS2 and SPHK1 in ghMSCs indicates that the cells expressed an A2 astrocyte- like phenotype, which is associated with neuroprotection.15
Gene set enrichment analysis (GSEA) of these DEGs returned the Gene ontology (GO) terms cell communication, regulation of cell population proliferation, cell migration, tissue development, neuron differentiation, and cytokine activity (Figure 1D). We also conducted GSEA using a manually curated gene set comprising 65 astrocyte markers including 63 obtained from PangladDB with the addition of SOX2 and PAX6. GSEA results indicated an enrichment of astrocyte marker genes in ghMSCs with an adjusted P-value < 0.1 (Figure 1E). These results confirm previous studies indicating that ghMSCs exhibit glia-like characteristics.12
ghMSCs exhibit increased expression of astrocyte markers and release of cytokines and trophic factors Immunohistochemistry, flow cytometry and ELISA assays of condition media all show increases proteins associated with A2 astrocytes in ghMSCs, compared to hMSCs. Immunocytochemistry analysis demonstrated that expressed levels of astrocyte-specific markers, SRY-box transcription factor 9 (SOX9) and glutamate aspartate transporter 1 (GLAST), were significantly higher in ghMSCs than in hMSCs17,18 (Figure 1F). Additionally, flow cytometry analysis demonstrated that levels of glial fibrillary acidic protein (GFAP) and GLAST were also significantly higher in ghMSCs than in hMSCs (Figure 1G). Lastly, Western blot analysis demonstrated a substantial increase in expression of SOX9, an astrocyte-specific nuclear marker in adult brain,17 in ghMSCs vs. hMSCs (Figure 1H). Using a human cytokine and trophic factor array, we further showed that ghMSCs secreted significantly higher levels of interleukin-8 (IL-8, CXCL8), growth-related oncogene (GRO- α/β/γ, CXCL1, 2, 3), GRO-α (CXCL1), monocyte chemoattractant protein-1 (MCP-1, CCL2), vascular endothelial growth factor (VEGF), insulin-like growth factor binding protein-4 (IGFBP-4), and hepatocyte growth factor (HGF) compared to hMSCs (Figure 1I). Differences in expressed levels of IL-8, GRO-α/β/γ, and IGFBP-4 between ghMSCs and hMSCs were also confirmed by ELISA (Figure 1J).
Whole-exome sequencing reveals that the induction of ghMSCs from hMSCs does not 18 superior therapeutic effects at lower concentrations than recombinant peptides, potentially reducing side effects. Synergistic effects among factors secreted by ghMSCs may also produce enhanced therapeutic impacts. Notably, results in this study are consistent with previous findings showing significantly higher IGFBP-4 secretion, which exhibited a neuroprotective effect in both in vitro and in vivo models of acute ischemia, by ghMSCs compared to hMSCs.12 IGFBP-4 has been shown to activate the AKT pathway, which promotes cell survival and proliferation.43,44 Activation of CXCR2, triggered by ligands, such as CXCL1/2/3 and CXCL8 also leads to the stimulation of the AKT pathway, promoting cell survival and proliferation.16 Both IGFBP-4 and CXCR2 pathways converge on the AKT pathway, suggesting potential synergistic effects in mediating the neuroprotective properties of ghMSCs, as observed in both our previous and current studies. We propose that the enhanced therapeutic effects of ghMSCs may result from synergistic interactions among multiple secreted factors. Furthermore, in our study, we utilized late-passage hMSCs rather than early-passage hMSCs. While the use of late-passage hMSCs raises concerns due to potential senescence, obtaining sufficient quantities of early-passage hMSCs for clinical applications can be challenging and expensive. Therefore, it is essential to develop efficient methods that allow the use of late-passage hMSCs while maintaining or even enhancing their therapeutic properties. Our approach aims to efficiently convert late-passage hMSCs into cells with A2 astrocyte-like properties. This strategy could potentially offer comparable or even superior efficacy to early- passage hMSCs, making it a more practical and scalable option for clinical use. The results of WES further confirmed that induction of ghMSCs from hMSCs did not elevate the risk of tumor development, ensuring the safety of these cells for potential clinical application. Despite the promising results, our study has several limitations. The in vivo results were conducted exclusively in male rats. In middle-aged humans, the incidence of ischemic stroke is higher in men compared to women, and clinical symptoms are more severe in men.45,46 19 Additionally, it is widely known that female sex hormones have the ability to protect against ischemic damage as evidenced in a previous study.47 Although the symptom severity is greater for male than female rats after MCAO surgery, the therapeutic efficacy of MSC transplantation is the same.48 Consequently, it will be important in future studies to extend our findings to female rats. Considering that cerebral infarction mainly occurs in aged individuals, it will also be important to repeat studies using rats of varying ages.49,50 Numerous investigations have analyzed the susceptibility to ischemic damage across different age groups. In these studies, older rats demonstrated a significantly elevated mortality rate of 43.5%, which is substantially higher compared to the 6.3% observed in younger cohorts. However, there was no statistically significant difference in the extent of cerebral infarction between the age groups up to 28 days post-MCAO surgery.50,51 Therefore, employing older rats in research may necessitate a greater number of animal sacrifices. It is recommended that further investigations be carried out following a more thorough examination of the impact of ischemic injury in older rats. Although our research does not entirely replicate the cerebral environment of an aged rat, we have assessed the efficacy of ghMSCs transplantation during the subacute to chronic phases of the stroke, employing a temporal phase post-surgery in 8-week-old rats, a model frequently utilized in similar studies.52-54 As mentioned above, we conducted the experiments under specific conditions in many aspects. Further studies in less restrictive environments are needed for future clinical applications, and although no adverse effects of treatment were observed during our experimental period, long-term toxicity of transplanted ghMSCs should also be explored. In this study, we employed organotypic brain slice culture to elucidate the underlying mechanisms of action of ghMSCs on cell survival and neuroregeneration in brain tissues. The rationale for selecting organotypic brain slice cultures stems from their extensive use in neuroscience research19,20 and the fact that they preserve cytoarchitecture and physiological features of the brain thereby enabling the study of the microenvironment, including cell-cell 20 interactions, neuronal networks, and synaptic organization, as well as impacts in specific brain regions.19,20 For our experiments, we selected rats at postnatal day 7 (P7) as opposed to 8-week- old rats, which were used in in vivo studies. This decision was based on the fact that slices from adult brains require meticulous optimization for long-term culturing, such as reducing thickness, given the limited cell survival and the lack of retention of cytoarchitectural organization over an extended period.19,20 Therefore, brain slices from early postnatal days are preferred because they show greater resistance to mechanical trauma during preparation, though it is important to note that the brain microenvironment at this developmental stage may differ from that of adults.19,20 Our results clearly indicate a significant increase in cell death in the cortex following OGD/R treatment compared to the normal group, with the cortex being the primary region affected. Importantly, at least 60% of the dead cells in the cortex were identified as neurons (Figure 2B). These findings underscore the vulnerability of the cortex to OGD/R-induced damage. This is consistent with the fact that the cortex is significantly affected in stroke patients and plays a crucial role in essential functions, such as cognition, motor skills, and sensory processing.55 Therefore, the ex vivo organotypic brain slice model used in our study serves as a relevant representation of the pathophysiological state observed in cerebral infarction patients, reinforcing the translational value of our findings. In conclusion, our results demonstrate that ghMSCs exhibit distinct gene expression signatures resembling astrocytes and possess superior therapeutic efficacy compared to hMSCs in treating sequelae resulting from chronic cerebral infarction, particularly motor sequelae. They also implicate activation of CXCR2 signaling in endogenous cells as a mode of action of ghMSCs. Given the superior neuroregenerative effects of ghMSCs over hMSCs, we postulate that ghMSCs represent a promising therapeutic alternative for treating patients with sequelae of cerebral infarction, deserving consideration for testing in clinical trials.458Journal Pre-proof21 Materials & methods Compliance with ethical standards for animal welfare and identification of source of human cells All studies involving animals were approved by the Institutional Animal Care and Use Committee (IACUC) of Seoul National University (SNU-221206-5) and Hanyang University (2022-0034A), Republic of Korea. Sprague-Dawley (SD) rats at postnatal day 7 (P7), used for the culture of organotypic brain slices, were obtained from Orient Bio (Gapyeong, Korea) and sacrificed on the same day for use in experiments. Male SD rats, aged 8-9 weeks and weighing 250–280 g, used for MCAO surgery, were procured from KOATECH (Pyeongtaek, Korea). These animals were housed in a controlled environment with constant humidity (50 ± 10%), temperature (22 ± 2℃), and a 12-hour light/dark cycle (lights on at 8 a.m.). Diet and water were provided ad libitum Adult hMSCs from the bone marrow of healthy donors were purchased from STEMCELL Technologies Inc. (70022, Vancouver, Canada) and Lonza (PT-2501, Basel, Switzerland). The in vivo experiments involving hMSCs and ghMSCs were reviewed and approved by the Institutional Review Board of Hanyang University (HYUIRB-202202-007). Cell culture Adult hMSCs were cultured in low-glucose Dulbecco’s modified Eagle’s medium (DMEM) supplemented with 10% fetal bovine serum (FBS; 16000044, Gibco, MA, USA) and 1% penicillin/streptomycin (P/S; 15140, Gibco). Previously published protocols were used to generate ghMSCs.12-14 Briefly, hMSCs were treated for 24 h with 1 mM β-mercaptoethanol (63689, Sigma-Aldrich, St Louis, MO, USA) followed by 0.28 μg/mL all-trans-retinoic acid (R2625, Sigma-Aldrich) for three days. Thereafter, cells were treated with a cocktail containing 22 10 ng/mL basic fibroblast growth factor (bFGF; Peprotech, Rocky Hill, NJ, USA), 5 ng/mL platelet-derived growth factor-AA (PDGF-AA; 100-13A, Peprotech), 10 μM forskolin (F6886, Sigma-Aldrich), and 200 ng/mL recombinant human heregulin-β1 (HRG-β1; 396-HB, R&D Systems, Minneapolis, MN, USA) for 8 days and then harvested for experiments. RNA extraction, library generation, and RNA Quant-sequencing RNA was extracted from cells using the Qiagen RNeasy kit according to the manufacturer's instructions (74004, Qiagen, Hilden, Germany). RNA quality was assessed with an Agilent 2100 bioanalyzer using an RNA 6000 nano chip (5067-1511, Agilent Technologies, Santa Clara, CA, USA). Libraries were constructed using the QuantSeq 3’-mRNA-Seq Library Prep Kit (113.96, Lexogen Inc., Vienna, Austria) according to the manufacturer’s instructions at the Genomic Medicine Institute Research Service Center. The quality of the libraries was checked with an Agilent 2100 bioanalyzer using a High Sensitivity DNA Chip and sequenced paired-end (2 × 150 bp read length) on the Hiseq platform (Illumina, San Diego, CA, USA) to produce 2 Gb of data per sample. Raw data quality control including per base sequence quality and GC content was performed using FastQC (v0.11.9). Each read’s unique molecular index (UMI) was extracted with the ‘extract’ function from UMI-tools (v1.1.2).56 Using Illumina sequencing adapters and polyA fasta as reference files, sequencing reads were trimmed using the bbduk.sh script from the BBMap software (38.87). After trimming, reads were then aligned to human reference genome (GRCh38/hg38) using STAR aligner (2.7.9a)57 together with Samtools (1.13+htslib-1.13).58 PCR duplicates were removed using UMI-tools dedup function. HTSeq (v0.13.5)59 htseq-count function was used to quantify gene expression. Data analysis of RNA-seq The read counts and counts per million (CPM) values of the generated RNA-seq data 23 (ghMSCs and hMSCs) were imported into R. For human astrocytes, RNA-seq raw data were retrieved from a previously published study.60 Deposited data from three immortalized human astrocyte lines (SRR2557092, SRR2557093 and SRR2557094) were utilized as a reference.61 These fastq files were aligned to the human reference genome (GRCh38/hg38) specifically generated for RSEM (1.3.3), using rsem-calculate-expression function, together with STAR aligner (2.7.9a). Subsequently, the count data from publicly available astrocytes, combined with those from ghMSCs and hMSCs, underwent normalization and batch correction through the limma package within the DESeq2 tool.60 The most variable 10,000 genes across all samples were then selected for unsupervised hierarchical clustering, and a heatmap was generated using the pheatmap package. Differential expression analysis was then performed using DESeq2 package, and volcano plots of DEG were visualized using the Enhanced Volcano package. Base-mean and fold changes were calculated within the DESeq2 algorithm with statistical significance assumed at Padj < 0.05. Then, significant DEG genes were identified with an adjusted P-value < 0.05 and log2(fold change) > 1. In the DEG list, we applied the scoring system of log2(fold change) value multiplied by negative logP value to rank the significant genes. A web-based tool, the g:profiler, was then used to evaluate the ontology to identify the underlying pathways of the genes identified. GO terms were plotted by https://www.bioinformatics.com.cn/en, a free online data analysis and visualization platform. The GO analysis considered P adjust values below 0.05. GSEA was performed using astrocyte markers downloaded from PanglaoDB.62 A total of 18,640 genes were ranked according to the stat value calculated by DESeq2 package, with upregulation observed in ghMSCs compared to hMSCs. The analysis utilized an adjusted P- value of 0.0962. WES and data processing 24 Genomic DNA was extracted from frozen cells with the DNeasy Blood and Tissue Kit (69504, Qiagen). Agilent SureSelectXT Low Input Target Enrichment System (Agilent Technology Inc.) was used for DNA library preparation. All experiments were performed according to the manufacturer’s instructions. Exome libraries for WES were sequenced on an Illumina platform. Most bioinformatic analyses of sequencing data were performed using the computing server at the Genomic Medicine Institute Research Service Center. We aligned the DNA sequence reads to the human reference genome (GRCh38) using Burrows–Wheeler Aligner (BWA).63 Thereafter, we performed preprocessing procedures for BAM files, including local realignment around insertions/deletions (INDELs) and base recalibration, according to the Genome Analysis Toolkit (GATK) best practices document.64,65 Somatic single nucleotide polymorphism (SNPs) and INDELs specific to ghMSCs were called with GATK Mutect2 using hMSCs as matching normal sample and gnomAD project allele frequency information for filtering of likely germline variation. GATK FilterMutectCalls was performed to filter outputs from Mutect2 calling with tumor segmentation file from GetPileupSummaries and contamination table from CalculateContamination on minimum allele fraction of 0.001 and minimum reads per strand 1. All mutations were annotated with OncoKB-Annotator. For SNP and INDELs specific to ghMSCs, we measured the tumorigenicity by mutations annotated as ‘Likely Oncogenic’ or ‘Oncogenic’ by the OncoKB-Annotator. All the analyses used the computing server at Genomic Medicine Institute Research Service Center. Immunocytochemistry staining Immunocytochemistry was performed to investigate expression levels of astrocyte markers, SOX9 and GLAST, in cells. Cells were seeded at 1 × 104 cells per well onto 12-mm cover glass (Marienfeld, Lauda-Königshofen, Germany), fixed with 4% PFA, washed with phosphate- buffered saline (PBS), and permeabilized with 1% bovine serum albumin (BSA) in PBS containing 0.1% Triton X-100 (TX-100) for 20 min. Blocking was performed with 5% normal goat serum (NGS) in PBS containing 0.1% TX-100 for 1 h. Cells were incubated overnight at 4°C with primary antibodies diluted in blocking solution (5% NGS in PBS containing 0.1% TX-100), then washed and treated for 1 h at room temperature (RT) with secondary antibodies diluted in PBS. After washing, cells were stained with 4',6-diamidino-2-phenylindole (DAPI) (P36931, Invitrogen, MA, USA) for 20 min at RT in the dark. The cells were washed again with PBS and mounted with a fluorescence mounting medium (S302380-2, Dako Agilent, Santa Clara, CA, USA). Stained cells were imaged using inverted fluorescence microscopy (DM6B, Leica, Wetzlar, Germany). All DAPI-positive cells were counted as total cells, and the proportion of cells stained with each primary antibody was expressed as a percentage of total cells. Relative fluorescence intensities were quantified using ImageJ software (National Institutes of Health, NIH, Bethesda, MD, USA). Fluorescence-activated cell sorter analysis Cultured ghMSCs and hMSCs were detached with TrypLE Select (Gibco) and centrifuged at 1,200 RPM for 5 min. The pellets were resuspended in 5% FBS/PBS (FACS buffer). Cells were washed with FACS buffer, fixed with 4% paraformaldehyde (PFA) for 20 min, and permeabilized for 15 min with 0.4% TX-100 at RT. Cells were incubated with the fluorescence- conjugated primary antibodies against human anti-GFAP-fluorescein isothiocyanate (FITC, 1:250; 561449, BD Biosciences, Franklin Lakes, NJ, USA) and GLAST-phycoerythrin (PE, 1:200, 130-118-483, Miltenyi Biotec., Bergisch Gladbach, Germany) for 1 h at RT in the dark. The cells without antibody binding were used as controls. Cells were washed with FACS buffer twice and strained with a 40 μm strainer. Then, cells were transferred to a polystyrene round bottom tube. Percentages of cells expressing each marker were calculated based on 10,000 gated cell events. FACS performance was acquired on a BD LSRFortessa™ X-20 flow cytometer and analyzed with BD FACSDiva™ software (BD Biosciences). Assays of human cytokines and growth factors To prepare conditioned medium (CM) from cultured hMSCs and ghMSCs, cells (4 × 103 cells/cm2) were rinsed four times with PBS and incubated in serum-free Neurobasal-A medium (NB; 10888022, Gibco) for 18 h. The CM from ghMSCs or hMSCs was analyzed using a Human Cytokine Array C1000 (AAH-CYT-1000-2, RayBiotech, Norcross, GA, USA). Briefly, array membranes were blocked with a blocking buffer and incubated with 1 mL of CM at 4℃ overnight. Array membranes were washed three times with wash buffer I and twice with wash buffer II. Membranes were then incubated at 37℃ with 1 mL of biotinylated antibody cocktail for 2 h with horseradish peroxidase (HRP)-streptavidin, and visualized by chemiluminescence (ChemiDoc MP, Bio-Rad, Hercules, CA, USA).
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Li Zhou†, 
Jiani Wang
Jiagui Huang
Qin Yang
