SUPPLEMENTAL DIGITAL CONTENT
Protection of brain injury by amniotic mesenchymal stromal cells secreted metabolites Francesca Pischiutta, PhD1*; LauraBrunelli PhD2*; Pietro Romele, MS3; Antonietta Silini, PhD3;
Eliana Sammali, MS1,4; Lara Paracchini, MS5; Sergio Marchini, PhD5; Giorgio B. Boncoraglio, MD4; Laura Talamini, MS6; Paolo Bigini PhD6; Roberta Pastorelli, PhD2; Maria-Grazia De Simoni,
PhD1; Ornella Parolini, PhD3#and Elisa R. Zanier, MD1#
1Department of Neuroscience, IRCCS – Istituto di Ricerche Farmacologiche Mario Negri, Milan;
2Department of Environmental Health Sciences, IRCCS – Istituto di Ricerche Farmacologiche Mario Negri, Milan; 3Centro di Ricerca E. Menni, Fondazione Poliambulanza-Istituto Ospedaliero, Brescia, Italy; 4Department of Cerebrovascular Diseases, Fondazione IRCCS – Istituto Neurologico Carlo Besta, Milan; 5Department of Oncology, IRCCS – Istituto di Ricerche Farmacologiche Mario Negri, Milan, 6Department of Biochemistry, IRCCS – Istituto di Ricerche Farmacologiche Mario Negri, Milan, Italy.
* These authors contributed equally to this work
#Corresponding authors:
Elisa R Zanier
Unit of Cell Therapy and Acute Brain Injury
Laboratory of Inflammation and Nervous System Diseases IRCCS - Istituto di Ricerche Farmacologiche Mario Negri via Giuseppe La Masa, 19
20156 Milan Italy
Phone: +39 02 390 14 204 Fax: +39 02 390 01 916
email: [email protected] and
Ornella Parolini
Centro di Ricerca E. Menni, Fondazione Poliambulanza Istituto Ospedaliero, Via Bissolati, 57 I
25124 Brescia, Italy Phone: +390303518904 Fax: +390303518915
email: [email protected]
SUPPLEMENTARY METHODS hAMSC Phenotype
hAMSC at passage 0 were washed with FACS buffer [0.1% sodium azide (Sigma-Aldrich) and 0.1% fetal bovine serum (FBS) (Sigma-Aldrich) in PBS], and then incubated for 20 min at 4°C with anti-human fluorescein isothiocyanate (FITC), PE, or APC conjugated monoclonal antibodies, or isotype-matched controls specified below, together with 20 mg/ml polyglobin (Kiovig, Baxter, Illinois, USA), which was prepared in PBS with 1% BSA and added to block non-specific binding.
The clones and suppliers of the antibodies used in this study were as follows: monoclonal antibodies against CD90 FITC (clone 5E10), CD73 PE (AD2), CD14 PE (MΦP8), HLA-DR APC (TU36), CD34 FITC (581/CD34), CD11b PE (ICRF44 (44)), CD45 APC (2D1), CD19 PE (HIB19) (all purchased from BD Biosciences) and CD105 FITC (SN6), (purchased from Serotec, Oxford, UK).
hAMSC Differentiation
For both Osteogenic and Adipogenic differentiation, 3×104/cm2 hAMSC at passage 0 were seeded in 48-well plates in DMEM (Sigma) supplemented with 20% FBS, 100 U/ml penicillin, 100 μg/ml streptomycin, and 2mM L-glutamine. After 3 days, culture medium was replaced with medium from either STEMPRO® Osteogenesis (Life Technologies) and Bulletkit Adipogenic Differentiation Medium (Cambrex) according to the manufacturer’s instructions. The medium was subsequently replaced twice a week. Osteogenic differentiation was assessed after 14 days and calcium deposits were visualized by Alizarin Red. Cells were fixed in 2% formaldehyde and 2%
Alizarin Red pH 4.2 was added, left for 25 minutes, and afterwards cells were washed with distilled water. Adipogenic differentiation was assessed after 21 days. Cells were fixed in 2% formaldehyde, washed with 60% isopropanol, and lipid vacuoles were stained with Oil Red O (Sigma).
For Chondrogenic studies, 25×104 hAMSC at passage 0 were centrifuged at 300g for 10 min and culture medium was replaced with Chondro Diff Medium (Miltenyi Biotec), then replaced twice a week. Chondrogenic differentiation was assessed after 21 days. The presence of metachromatic matrix was demonstrated by toluidine blue (Sigma) Staining on formalin-fixed, paraffin embedded cell aggregates.
Animals
(PHS) Policy on Human Care and Use of Laboratory Animals has been recently reviewed (9/9/2014) and will expire on September 30, 2019 (Animal Welfare Assurance #A5023-01). All efforts were made to minimize animal suffering and to reduce the number of animals used.
Behavioral tests
Sensorimotor deficits were evaluated by neuroscore and beam walk tests at 1d (before hAMSC infusion, baseline) and weekly up to 5 was previously described (1, 2). Neuroscore ranges from 12 (normal) to 0 (severely impaired). Beam walk: number of foot-faults of a trained mouse walking twice on an elevated and narrow wooden beam (5mm wide and 100cm length) normalized on the baseline value. Values lower than 1 indicate improvement.
Cell tracking in mouse organs after iv or icv infusion
Frozen organs were cut in slices with a thickness of 1mm and the fluorescence was measured by an automated scanner (Typhoon, GE Healthcare, Fairfield, CT, USA) using the red laser (532 nm).
Images were processed by Image-J (NIH) with a grey-level scale as previously described (3, 4).
Tissue processing for histopathological analysis
At 5w mice were euthanized for histological analysis. Under deep anesthesia (ketamine 30 mg/medetomidine 0.3 mg), animals were transcardially perfused with 20 mL of phosphate buffer saline (PBS) 0.1 mol/L, pH 7.4, followed by 50 mL of chilled paraformaldehyde (4%) in PBS. The brains were carefully removed from the skull and post fixed for 6h at 4°C, and then transferred to 30% sucrose in 0.1 mol/L phosphate buffer for 24h until equilibration (5). The brains were frozen by immersion in isopentane at -45°C for 3 minutes before being sealed into vials and stored at -80°C until use. Twenty µm thick serial sections were cut using a cryostat from bregma +1mm to bregma -4mm.
Neuronal count
Cresyl Violet stained brain sections were used for neuronal count. Three coronal sections per mouse, located at -0.4, -1.6, -2.8 mm from bregma were chosen to assess the viability of neurons in the injured cortex. An OlympusBX-61-VSmicroscope, inter-faced with VS-ASW-FL software (Olympus Tokyo, Japan) was used to acquire the whole sections at 20x of magnification. The ipsilateral cortex was analyzed over an area of 1 mm depth from the edge of the contusion. A corresponding area of the contralateral side was analyzed.The degree of neuronal loss was calculated by pooling the number of stained neurons in each hemisphere and was expressed as a percentage of the contralateral hemisphere. Images were analyzed using the open source platform software Fiji (http://fiji.sc/Fiji) (6) and segmentation was used to discriminate neurons from glia on the basis of cell size(5, 7).
Immunohistochemistry
Immunohistochemistry was performed on 20 µm thick brain coronal sections using anti-DCX (1:200; Santa Cruz Biotechnology, Santa Cruz, CA, USA), anti-CD31 (1:100; BD), anti-mouse CD11b (1:50, Ab86860, Abcam), anti-mouse CD68 (1:200; Serotec, Kidlington, UK), to measure
The quantification of DCX expression and vessel density were quantified as previously described (1). For CD11b and CD68 three brain coronal sections per mouse (at 0.4, 1.6, and 2.8 mm posterior to bregma), were acquired at 20x by Olympus BX-61-VS microscope. The ipsilateral cortex was analyzed over an area of 0.6 mm depth from the edge of the contusion. Images were analyzed using Fiji software. The immunostained area for each marker is expressed as positive pixels/total assessed pixels, and reported as the percentage staining area for subsequent statistical analysis.
Organotypic cortical brain culture
The experimental design for in vitro experiments is shown in Fig.2A. Organotypic cortical brain slices (named cortical slices from now on) were obtained from prefrontal cortex of C57BL/6 mouse pups (P1-3) (9). Mouse pup brains were removed from the skull under sterile conditions and were immersed into a 3% agar solution. Tissue blocks containing mesencephalic and forebrain levels were dissected out, fixed onto a specimen stage of a vibratome (Leica, VT 1000S) with Super Attack glue, and placed in ice-cold (4°C) artificial cerebral spinal fluid (ACSF) solution (NaCl=87mM, NaHCO3=25mM, NaH2PO4=1.25mM, MgCl2=7mM, CaCl2=0.5mM, KCl=2.5mM, D-glucose=25mM, sucrose=75mM, Penicillin=50U/ml, Streptomicin=50μg/ml, equilibrated with 95% O2 and 5% CO2, pH=7.4). Prefrontal cortex coronal sections of 200μm thickness were cut.
Cortical slices were transferred into petri dishes filled with ice-cold ACSF. Only intact cortical slices were placed on membranes of tissue culture inserts (Millicell Culture insert, 0.4μm pore size, Merk-Millipore) with two slices per insert and placed in 6-well plates, each filled with 1ml of culture medium (MEM-Glutamax 25%, basal medium eagle 25% (Invitrogen), horse serum 25%
(Euroclone), glucose 0.6%, Penicillin 100U/ml, Streptomicin 100μg/mL (Euroclone); pH=7.2). All the cultures were maintained at 37°C in 5% CO2. After 2d, the incubation medium was changed with NB/B27 and replaced there after every 2d.
In vitro acute brain injury
After one week in culture, cortical slices were subjected to oxygen-glucose deprivation (OGD), an in vitro model of brain ischemia (10) that recapitulates critical features of secondary injury at the contusion border in TBI.The culture medium was removed, cortical slices were washed twice with PBS and transferred into a temperature‐controlled (37°±1°C) hypoxic chamber (InvivO2 400, Baker Ruskinn) at [O2]=0.1%, [CO2]=5%, [N2]=95%. Once in the hypoxic chamber, the PBS was replaced with deoxygenatedglucose‐free medium. After a 2h OGD period, cortical slices were returned to a normoxic incubator and medium was replaced with NB/B27. Control cortical slices (CTRL, not exposed to ischemic injury) were maintained in normoxic incubator with NB/B27.
fractions were obtained as follows: (step-1) 1ml of CM<2kDa was loaded onto column and the eluates were recovered by adding 2.4ml of water 3 times, collecting 3 sequential fractions (FrA, FrB, FrC), that were tested in the in vitro OGD model; (step-2) 1ml of CM<2kDa was loaded on a PD MiniTrap G-10 and the eluates were recovered by adding 1.2ml of water twice, collecting 2 sequential fractions (FrA1, FrA2) that were tested in the in vitro OGD model.These fractions were then further analyzed for “omics“ profiling; (step-3) 1ml of CM<2kDa was loaded on a PD MiniTrap G-10 and eluates were recovered by adding 0.6ml of water 4 times, collecting 4 sequential fractions (FrA1a, FrA1b, FrA2a, FrA2b). Fractions A2a and A2b were tested in the in vitro OGD model. The fractionation protocol was also applied to non-conditioned NB/B27 medium as control. All fractions were tested in the in vitro OGD model at the concentration of 50% in NB/B27.
Gene expression analysis
Forty-eight hours after injury, slices were collected and total RNA was extracted by miRNeasy mini kit (Qiagen). Samples were treated with DNase (Life Technologies) and reverse-transcribed with random hexamer primers using Multi-Scribe Reverse Transcriptase (Life Technologies). Real-time reverse transcription PCR was performed and relative gene expression determined by ΔΔCt method.
Data are expressed as log2 of the fold difference from the CTRL group. Genes and primer sequences are in Supplemental Digital Contents Table-1.
Peptidomics
CM FrA1 and FrA2 fractions and their control counterparts were subjected to solid phase extraction methods for peptide enrichment and concentration using Oasis HLB cartridges (Waters).
Samplewas first acidified at pH3 with 50% trifluoroacetic acid (TFA), centrifuged at 12500g and supernatant was then loaded on cartridge previously conditioned with acetonitrile and equilibrated with 0.1% TFA. The cartridge was washed with 0.1% TFA/H2O. The eluate was then dried, reconstituted in 10microL of 0.1M ammonium bicarbonate and analyzed on a high-resolution reversed-phase capillary LC system coupled with Thermo Fischer Scientific LTQ-Orbitrap as previously reported (11). The ten most abundant parent ions, excluding single charge state, were selected for MS/MS using both collision-induced dissociation and high-energy collision dissociation with a normalized collision energy setting of 30. A dynamic exclusion time of 30s was used. LC-MS/MS raw data were converted into dta files (Bioworks browser 3.3.1, Thermo Scientific). MS/MS spectra originating from the same precursor ion were grouped together using a tolerance of 2 ppm for MS/MS spectra with at least 100-count intensity and at least 10 ions, with automatic assignment of charge state.Dta files were merged, and submitted as an “mgf” file to the search engine Mascot (in-house 2.3.02, Matrix Science, Boston, MA). Mascot was set up to search both the forward and reversed sequences of the SwissProt_2015 database with no enzyme specification, fragment ion mass tolerance of 1.00 Da, parent ion tolerance of 2.0 ppm and Pyroglutamination, C-terminal amidation, N-terminal acetylation deamidation of asparagine and oxidation of methionine were set as variable modifications.Scaffold (version 4.4.3, Proteome Software Inc., Portland, OR, US) was used to validate MS/MS-based peptide and protein identifications. Peptide identifications were accepted if they could be established at greater than
parsimony. These filtering criteria established a false positive identification rate (FDR) of 0% for the human proteome dataset based on the reported decoy database search strategy.
Target Metabolomics
Targeted metabolomics analysis was performed using the BiocratesAbsoluteIDQTM p180 kit (Biocrates Life Science AG). This validated targeted assay allows for simultaneous detection and quantification of metabolites in a high-throughput manner. One ml of each fraction was concentrated to 200 μL under a stream of N2. Thirty μl of each concentrated fraction were processed following manufacturer instructions and analyzed on a triple-quadropole mass spectrometer (AB SCIEX triple-quad 5500) operating in the multiple reaction monitoring (MRM) mode. The assay is based on PITC (phenylisothiocyanate)-derivatization in the presence of internal standards for the analysis of aminoacids and biogenic amines resolved and quantified by LC-MS/MS using scheduled MRMs. Subsequent flow injection analysis tandem mass spectrometry (FIA-MS/MS) is performed to analyze acylcarnitines, glycerophospolipids, hexose. MRM detection is used for quantification applying spectra parsing algorithm integrated into the MetIQ software (Biocrates Life Science AG).
Concentration are calculated and evaluated by comparing measured analytes in a defined extracted ion count section to those of specific labelled internal standards or non-labelled, provided by the kit.
The measurements are made in a 96-well format. Seven calibration standards, four quality control samples, three zero samples (PBS) and one blank (solvents) are integrated into the plate.
For glycerophospolipids the precise position of the double bonds and the distribution of the carbon atoms in different fatty acid side chains cannot be determined with this technology. Lipid side-chain composition is abbreviated as Cx:y, where x denotes the number of carbons in the side chain and y the number of double bonds. The nature of fatty acids linkage is expressed as aa for diacyl or ae for acyl-alkyl. For example, PCaaC32:1 denotes diacyl-phosphatidylcholine with 32 carbons in the two fatty acids side chains and a single double bond in one of them.
To ensure data quality, the following filtering criteria were applied: (i) metabolites measured with more than 20% missing data (no detectable peak) were excluded; (ii) metabolites for which the concentration was below the limit of detection (<LOD) in at least ≥ 50% of analyzed samples were excluded.
SUPPLEMENTARY RESULTS hAMSC characterization
As previously described by Parolini and colleagues (12), the consensus on placental MSC is based on that the Mesenchymal and Tissue Stem Cell Committee of the International Society for Cellular Therapy, which are the minimal criteria to define human MSC (13). These include the ability of MSC to differentiate toward mesodermal lineages and phenotype as defined as the expression of CD105, CD73 and CD90, as measured by flow cytometry, and lack of CD45, CD34, CD14 or CD11b, CD79a or CD19 and HLA class II (13). In accordance with our previously reported results which report the phenotype of hAMSC either at p0 (14–17), or p2 (18, 19) or at p4 (20), in Supp.Fig.1 we show that hAMSC at passage 2 express the typical MSC phenotype with expression of CD90, CD73, CD105, and lack of the hematopoietic cell marker CD45, and CD11b, CD19, CD14, HLA-DR class II histocompatibility antigens, and CD34. Moreover, hAMSC were able to differentiate towards the 3 mesodermal lineages (Supp.Fig.2).
PKH26-labelled hAMSCfate in TBI mice
Supp.Fig.3 shows the quantification of PKH26-labelled hAMSC signal in brain (A), liver (B), lungs (C) and spleen (D), at 2 days, 1 and 5 weeks after TBI and iv (light grey, left) or icv (dark grey, right) injection of PKH26-labelled hAMSC. Brain fluorescence intensity related to PKH26 was increased in icv treated mice 2 days after TBI. As expected, no migration of cells to the peripheral organs was found. This reflected previous findings from our group obtained in a model of amniotic lateral sclerosis and icv administration of mesenchymal cells (21). On the contrary, no fluorescence in the brain was detected at any time point considered in iv treated mice. The systemic administration of PKH26-labelled hAMSC led to a strong, fast, but deciduous migration in lungs (Supp.Fig.3C) and a slower and transient segregation towards liver parenchyma. No relevant increase of signal was observed in the spleen (Supp.Fig.3C) up to 1 week with a significant increase at 5 weeks. This delayed accumulation of fluorescent signal in the spleen deserves further investigation and may suggest a migration of circulating scavenger cells which efficiently incorporated fragments of dead hAMSC along the time.
SUPPLEMENTARY FIGURES Supplementary Figure 1
Supplementary Figure 1. hAMSC phenotype. Phenotype analysis with corresponding monoclonal antibodies (white histograms) or isotype-matched IgG controls (gray histograms) are shown. The histograms show one representative experiment (n=3), and the mean percentage of positive cells with standard deviation is indicated in each plot.
Supplementary Figure 2
Supplementary Figure 2. hAMSC differentiation. The images show hAMSC cultured in regular culture medium (left column) or cultured in adipogenic, osteogenic and chondrogenic medium (right column). Adipogenic differentiation was analyzed with Oil red solution, osteogenic differentiation with Alizarin red staining and chondrogenic differentiation with toluidine blue.
Supplementary Figure 3
Supplementary Figure 3. Organ distribution of fluorescent PKH26-labelled hAMSC.
Histograms show the amount of fluorescence in brain (A), liver (B), lungs (C) and spleen (D), at2 days, 1 and 5 weeks after TBI and iv (light grey, left) or icv (dark grey, right) injection of PKH26- labelled hAMSC. The values are normalized to the same regions of TBI PBS treated mice(n = 3).
** p < 0.01 compared to TBI PBS treated mice (unpaired t-test).
Supplementary Figure 4
Supplementary Figure 4. Microglia activation in brain slices. Forty-eight hours after OGD, CD11b expression (in red) was not increased compared to CTRL (A-B) and no expression of Ym1 (in green) was observed. hAMSC (C) and CM (D) treatments were associated with an increased microglial activation which displayed an hypertrophic morphology together with the polarization toward the M2 phenotype as shown by the co-expression with the Ym1 marker (C-D).
Supplementary Figure 5
Supplementary Figure 5. Heat treatment does not affect CM efficacy. Quantification of PI incorporation 48 h after OGD shows similar effects of unheated or heated CM. Data are mean + SD, n=6, one experiment. One-way ANOVA p<0.001, followed by Tukey post-hoc test.
Supplementary Figure 6
Supplementary Figure 6. BDNF production increases after treatment with CM and FrA2.
Confocal microscopy 48h after injury of CTRL (A), OGD (B), OGD CM (C), OGD CM FrA2 (D) cortical slices. Both CM and CM FrA2 increased the production of BDNF (red) expressed by
neuronal (yellow in the merge right panel) and non-neuronal (red in the merge right panel) populations. Bar = 20 μm.
Supplementary Figure 7.
Supplementary Figure 7. CM-DERMA does not maintain the microenvironmental gene
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SUPPLEMENTARY TABLES Supplementary Table 1
Gen e
NCBI refSequenc
Forward Primer Reverse Primer β-
ACT
3 CGCGAGCACAGCTTCT
TT
GCAGCGATATCGTCATC RPL CAT
27
NM_01128 9.3
TCATGAAACCCGGGAA AGT
GAGGTGCCATCGTCAA B2 TGT
M
NM_00973 5.3
CTGACCGGCCTGTATG CTAT
TATGTTCGGCTTCCCAT MA TCT
P2
NM_00103 9934.1
TCAGCTGACAGAGAAA CAGCA
TTGTGTTGGGCTTCCTT CD3 CTC
1
NM_00103 2378
GTCGTCCATGTCCCGA GAA
GCACAGGACTCTCGCA GFA ATCC
P
NM_00113 1020
GAAACCGCATCACCAT TCC
TCGGATGGAGGTTGGA OLI GA
G2
NM_01696 7
CATTGTACAAAACGGC CACAA
GTGCAGGCAGGAAGT CD1 TCCA
1b
NM_01056 2.2
GAGCAGCACTGAGATC CTGTTTAA
ATACGACTCCTGCCCTG Ym1 NM_00989 GAA
2.2
TCTGGTGAAGGAAATG CGTAAA
GCAGCCTTGGAATGTC TTTCTC
TNF -α
NM_01369 3.2
AGACCCTCACACTCAG ATCATCTTC
TTGCTACGACGTGGGC BD TACA
NF
NM_00754 0.4
AGGCACTGGAACTCGC AATG
AAGGGCCCGAACATAC VEG GATT
F
NM_00950 5.4
CCTGCAAAAACACAGA CTCGC
CGTTTAACTCAAGCTG CCTCG
Supplementary Table 1 List of primers used for real-time reverse transcription polymerase chain reaction
Supplementary Table 2. Metabolites determined using the Biocrates Absolute IDQ p180 kit
METABOLITE CLASS NUMBER METABOLITE NAME OR ABBREVIATION BIOLOGICAL RELEVANCE
(SELECTED EXAMPLES) AMINO ACIDS 21 Alanine, arginine, aspartate, citrulline, glutamine, glutamate, glycine,
histidine, isoleucine, leucine, lysine, methionine, ornithine, phenylalanine, proline, serine, threonine, tryptophan, tyrosine, valine
Amino acid metabolism, urea cycle, activity of gluconeogenesis and glycolysis, insulin sensitivity, neurotransmitter metabolism, oxidative stress
CARNITINE 1 C0 Energy metabolism, fatty acid transport and
mitochondrial fatty acid oxidation, ketosis, oxidative stress, mitochondrial membrane damage
ACYLCARNITINE 39 C2, C3, C3:1, C3-OH, C4, C4:1, C4-OH, C5, C5:1, C5:1-DC, C5-DC, C5-M-DC, C5-OH, C6, C6:1, C7-DC, C8, C9, C10, C10:1, C10:2, C12, C12-DC, C14, C14:1, C14:1-OH, C14:2, C14:2-OH, C16, C16:1, C16:1-OH, C16:2, C16:2-OH, C16- OH, C18, C18:1, C18:1-OH, C18:2
BIOGENIC AMINES 19 Acetylornithine, asymmetric dimethylarginine, total dimethylarginine, alpha-aminoadipic acid, carnosine, creatinine, histamine, kynurenine, methionine sulfoxide, nitrotyrosine, hydroxyproline, phenylethylamine, putrescine, sarcosine, serotonin, spermidine, spermine, taurine
Neurological disorders, cell proliferation, cell cycle progression, DNA stability, oxidative stress
LYSO-PHOSPHATIDYLCHOLINES 14 lysoPC a
C14:0/C16:0/C16:1/C17:0/C18:0/C18:1/C18:2/C20:3/C20:4/C26:0/C26:1/C2 8:0/C28:1
Degradation of phospholipids, membrane damage, signaling cascades, fatty acid profile DIACYL-
PHOSPHATIDYLCHOLINES
38 PC aa
C24:0/C26:0/C28:1/C30:0/C30:2/C32:0/C32:1/C32:2/C32:3/C34:1/C32:2/C3 4:3/C32:4/C36:0/C36:1/C36:2/C36:3/C36:4/C36:5/C36:6/C38:0/C38:1/C38:
3/C38:4/C38:5/C38:6/C40:1/C40:2/C40:3/C40:4/C40:5/C40:6/C42:0/C42:1/
C42:2/C42:4/C42:5/C42:6
Dyslipidemia, membrane composition and damage, fatty acid profile, activity of desaturases
Supplementary Table 3. Gene expression values (log2 of fold change normalized over CTRL group) relative to graphs in Fig.3
CTRL OGD OGD hAMSC OGD CM
MAP2 0.00±0.27 -2.00±0.49 °°° -1.03±0.43 °°° *** -1.15±0.56 °°° **
CD31 0.00±0.74 -0.27±0.92 0.026±0.44 1.47±0.51 °° *** ##
GFAP -0.10±0.58 0.89±0.54 1.25±1.81 0.48±1.63
CD11b -0.02±0.27 0.24±0.66 1.70±0.41 °°° *** 1.50±0.30 °°° ***
Ym1 0.00±0.62 -0.48±1.00 2.11±0.42 °°° *** 1.43±0.95 °° ***
TNFα -0.12±0.63 1.02±0.64 °° 1.28±0.47 °°° 1.08±0.61 °°
BDNF 0.00±0.48 -2.95±0.50 °°° -1.839±0.31 °°° ** -1.49±0.95 °°° ***
VEGF -0.03±0.29 -2.47±0.16 °°° -2.06±0.30 °°° -1.83±0.42 °°° **
Data are presented as mean±SD, n=6-8, One way ANOVA p<0.001, followed by Tukey post-hoc test. °°p<0.01, °°°p<0.001 compared to CTRL; **p<0.01,
***p<0.001 compared to OGD; ##p<0.01 compared to OGD hAMSC.
Supplementary Table 4.Gene expression values (log2 of fold change normalized over CTRL group) relative to graphs in Fig.5
CTRL OGD OGD CM OGD CM FrA2
MAP2 0.00±0.29 -1.90±0.45 °°° -0.58±0.96 * 0.63±0.78 *** ^^
CD31 0.00±0.74 -0.05±0.94 1.98±0.58 °° ** 1.64±1.41 ° **
GFAP -0.07±0.50 0.63±0.72 0.33±1.88 -1.07±1.18 *
CD11b -0.01±0.23 0.31±0.50 1.24±0.39 °° * -1.38±1.16 °°° *** ^^^
Ym1 0.00±0.62 -0.65±1.34 1.79±1.00 °° *** 1.79±0.88 ° ***
TNFα -0.12±0.62 1.02±0.65 ° 0.86±0.72 ° 0.41±0.60
BDNF 0.00±0.48 -2.76±0.49 °°° -1.06±0.81 ° *** -1.50±0.64 °°° ***
VEGF -0.03±0.29 -2.48±0.13 °°° -1.72±0.35 °°° ** -0.75±0.66 °° *** ^^^
Data are presented as mean±SD, n=8, One way ANOVA p<0.001, followed by Tukey post-hoc test. °p<0.05, °°p<0.01, °°°p<0.001 compared to CTRL;
*p<0.05, **p<0.01, ***p<0.001 compared to OGD, ^^p<0.01, ^^^p<0.001 compared to OGD CM.
Supplementary Table 5. Gene expression values (log2 of fold change normalized over CTRL group) relative to graphs in Supplementary Fig.3
CTRL OGD OGD CM OGD CM DERMA
MAP2 0.00±0.32 -1.86±0.47 °° -0.48±1.10 * -1.72±0.54 °
CD31 0.00±0.69 0.63±0.57 2.33±0.52 °°° ** 0.20±0.72 ^^^
GFAP -0.05±0.51 0.62±0.71 0.34±1.85 1.01±0.40
CD11b 0.00±0.23 0.50±0.16 ° 0.97±0.35 °°° * 0.73±0.59 °°°
Ym1 0.00±0.42 1.51±0.95 ° 3.72±0.90 °°° ** 2.08±0.66 °° ^
TNFα 0.00±0.34 1.50±0.37 °° 0.86±0.29 1.76±0.74 °°°
BDNF 0.00±0.68 -2.40±0.65 °°° -1.24±0.89 ° * -2.41±0.39 °°° ^
VEGF 0.00±0.19 -1.69±0.43 °°° -1.12±0.18 °°° * -1.65±0.34 °°° ^
Data are presented as mean±SD, n=6, One way ANOVA p<0.001, followed by Tukey post-hoc test. °p<0.05, °°p<0.01, °°°p<0.001 compared to CTRL;
*p<0.05, **p<0.01, compared to OGD, ^p<0.05, ^^^p<0.001 compared to OGD CM.
Supplementary Table 6. Differential expression data (as normalised spectral counts) for each identified protein in CM FrA1 and CM FrA2 and non- conditioned NB/B27 medium-derived fractionations as control counterparts, NB-FrA1 and NB-FrA2, using relaxed criteria for protein identifications (peptide identification probability 80%, protein identification probability 80% with one peptide identified).
CM NB
FR A1 FR A2 FR A1 FR A2
Identified Proteins Accession Number MWa) NSCb) Covc) NSCb) Covc) NSCb) Covc) NSCb) Covc) Phosphoribosyl pyrophosphate synthase-associated protein 2 KPRB_HUMAN 41 0 0 11 2.17% 0 0 26 2.17%
Cirhin CIR1A_HUMAN 77 0 0 0 0 0 0 2 1.46%
RNA-binding protein 27 RBM27_HUMAN 119 0 0 0 0 0 0 1 1.04%
MFS-type transporter SLC18B1 S18B1_HUMAN 49 0 0 0 0 0 0 1 2.63%
Ankyrin-1 ANK1_HUMAN 206 0 0 0 0 0 0 4 0.64%
Bromodomain and WD repeat-containing protein 3 BRWD3_HUMAN 204 0 0 0 0 3 0.39% 0 0
Coiled-coil domain-containing protein 108 CC108_HUMAN 217 0 0 0 0 0 0 4 0.62%
Bromodomain-containing protein 3 BRD3_HUMAN 80 0 0 0 0 2 1.65% 0 0
Transcription initiation factor TFIID subunit 5 TAF5_HUMAN 87 0 0 0 0 3 1.37% 0 0
Multidrug resistance-associated protein 7 MRP7_HUMAN 162 0 0 0 0 1 0.94% 0 0
E3 ubiquitin-protein ligase HUWE1 HUWE1_HUMAN 482 0 0 0 0 1 0.25% 0 0
Zinc finger CCCH domain-containing protein 4 ZC3H4_HUMAN 140 0 0 3 3.61% 0 0 4 5.91%
Centromere protein H CENPH_HUMAN 28 0 0 0 0 0 0 1 3.64%
Alpha-parvin PARVA_HUMAN 42 0 0 0 0 0 0 1 3.76%
SH3 and multiple ankyrin repeat domains protein 1 SHAN1_HUMAN 225 0 0 0 0 0 0 2 0.46%
Alpha-sarcoglycan SGCA_HUMAN 43 0 0 0 0 0 0 1 2.58%
Unconventional myosin-Ie MYO1E_HUMAN 127 0 0 0 0 0 0 1 0.72%
Taste receptor type 2 member 10 T2R10_HUMAN (+1) 35 0 0 1 3.91% 0 0 0 0
Receptor-type tyrosine-protein phosphatase H PTPRH_HUMAN 122 0 0 0 0 0 0 1 0.90%
Coiled-coil domain-containing protein 24 CCD24_HUMAN 34 0 0 0 0 0 0 1 4.56%
Ras-like protein family member 11B RSLBB_HUMAN 28 0 0 0 0 0 0 1 4.84%
Histone acetyltransferase KAT6B KAT6B_HUMAN 231 0 0 0 0 0 0 1 0.72%
Rho-associated protein kinase 2 ROCK2_HUMAN 161 0 0 0 0 0 0 1 0.58%
Coiled-coil domain-containing protein 81 CCD81_HUMAN 76 0 0 0 0 0 0 1 1.23%
Zinc finger MIZ domain-containing protein 1 ZMIZ1_HUMAN 115 0 0 1 3.75% 0 0 0 0
Ribosyldihydronicotinamide dehydrogenase [quinone] NQO2_HUMAN 26 0 0 0 0 0 0 1 3.90%
Pecanex-like protein 2 PCX2_HUMAN 237 0 0 0 0 0 0 1 0.98%
Vesicular acetylcholine transporter VACHT_HUMAN 57 0 0 0 0 0 0 1 1.88%
FACT complex subunit SSRP1 SSRP1_HUMAN 81 0 0 0 0 0 0 1 1.41%
Developmental pluripotency-associated protein 2 DPPA2_HUMAN 34 0 0 1 5.37% 0 0 0 0
Transmembrane protein 52 TMM52_HUMAN 22 0 0 1 12.40% 0 0 0 0
Transcription factor TFIIIB component B'' homolog BDP1_HUMAN 294 0 0 1 0.95% 0 0 0 0
Chromodomain-helicase-DNA-binding protein 3 CHD3_HUMAN 227 0 0 0 0 0 0 1 1.35%
cAMP and cAMP-inhibited cGMP 3',5'-cyclic
phosphodiesterase 10A PDE10_HUMAN 88 0 0 0 0 2 1.03% 0 0
Putative olfactory receptor 10AC1 O10AC_HUMAN 35 0 0 0 0 0 0 1 3.69%
Ras-related protein Rab-38 RAB38_HUMAN 24 0 0 0 0 0 0 1 3.79%
Blood group Rh(CE) polypeptide RHCE_HUMAN 46 0 0 0 0 0 0 1 2.88%
ATP synthase subunit beta, mitochondrial ATPB_HUMAN 57 0 0 0 0 0 0 1 1.70%
Transmembrane channel-like protein 2 TMC2_HUMAN 103 0 0 0 0 0 0 1 0.88%
Prominin-2 PROM2_HUMAN 92 0 0 1 2.16% 0 0 0 0
Histone H1.0 H10_HUMAN 21 0 0 1 15.50% 0 0 0 0
Unconventional myosin-XVIIIa MY18A_HUMAN 233 0 0 0 0 0 0 1 0.68%
Retinoic acid-induced protein 1 RAI1_HUMAN 203 0 0 0 0 0 0 1 0.53%
Round spermatid basic protein 1-like protein RSBNL_HUMAN 95 0 0 0 0 0 0 1 1.42%
Magnesium transporter NIPA3 NIPA3_HUMAN 45 0 0 0 0 0 0 1 7.32%
Protein SCAF8 SCAF8_HUMAN 141 0 0 0 0 0 0 1 0.71%
Ras GTPase-activating protein SynGAP SYGP1_HUMAN 148 0 0 0 0 0 0 1 0.67%
Probable E3 ubiquitin-protein ligase HERC4 HERC4_HUMAN 119 0 0 0 0 0 0 1 1.23%
Prostate stem cell antigen PSCA_HUMAN 13 0 0 0 0 1 10.60% 0 0
Docking protein 1 DOK1_HUMAN 52 0 0 0 0 0 0 1 1.66%
Plasma membrane calcium-transporting ATPase 3 AT2B3_HUMAN 134 0 0 1 2.54% 0 0 0 0
Mothers against decapentaplegic homolog 5 SMAD5_HUMAN 52 0 0 0 0 0 0 1 6.67%
Phosphofurin acidic cluster sorting protein 2 PACS2_HUMAN (+1) 98 0 0 0 0 0 0 1 0.90%
Ras association domain-containing protein 4 RASF4_HUMAN 37 0 0 0 0 0 0 1 2.80%
Probable cation-transporting ATPase 13A1 AT131_HUMAN 133 0 0 0 0 0 0 1 0.66%
Toll-like receptor 8 TLR8_HUMAN 120 k 0 0 0 0 0 0 1 0.77%
Inositol 1,4,5-trisphosphate receptor type 3 ITPR3_HUMAN 304 0 0 0 0 0 0 1 0.34%
Neuropathy target esterase PLPL6_HUMAN 150 0 0 1 2.27% 0 0 0 0
GPI inositol-deacylase PGAP1_HUMAN 105 0 0 0 0 0 0 1 1.08%
Protein FAM126B F126B_HUMAN 59 0 0 0 0 0 0 1 1.89%
Protein patched homolog 2 PTC2_HUMAN 131 0 0 1 2.49% 0 0 0 0
Bile salt sulfotransferase ST2A1_HUMAN 34 0 0 0 0 0 0 1 2.81%
N-arachidonyl glycine receptor GPR18_HUMAN 38 0 0 1 7.25% 0 0 0 0
NKG2D ligand 4 N2DL4_HUMAN 30 0 0 0 0 0 0 1 3.04%
a) molecular weight express as kDa (MW), b) number of normalized spectral counts (NSC), c) percent of protein sequence coverage (cov)
Supplementary Table 7. Concentration (µM) of quantified metabolites in conditioned medium –derived active fractions (CM) and their non- conditioned medium-derived fractions as controls (NB)
METABOLITE NB<2KDA CM<2KD NB FrA1 CM FrA1 NB FrA2 CM FrA2
LYSOPC A C16:1 0.218 0.219 0.048 0.046 0.121 0.069
LYSOPC A C17:0 0.122 0.097 0.042 0.040 0.101 0.063
LYSOPC A C18:2 0.266 0.662 0.087 0.133 0.385 0.682
LYSOPC A C20:3 0.184 0.106 0.046 0.049 0.189 0.151
LYSOPC A C20:4 0.741 0.323 0.045 0.025 0.371 0.136
LYSOPC A C24:0 0.135 0.093 0.053 0.050 0.165 0.124
LYSOPC A C26:0 0.244 0.159 0.414 0.366 0.144 0.217
LYSOPC A C26:1 0.078 0.074 0.035 0.034 0.157 0.127
LYSOPC A C28:1 0.065 0.037 0.031 0.018 0.133 0.057
PC AA C24:0 0.121 0.062 0.025 0.027 0.076 0.048
PC AA C30:2 0.029 0.006 0.001 0.002 0.021 0.022
PC AA C32:0 2.374 1.286 0.411 0.524 1.029 0.625
PC AA C32:1 1.963 1.272 0.289 0.268 0.763 0.273
PC AA C32:2 0.298 0.194 0.088 0.060 0.261 0.133
PC AA C32:3 0.317 0.150 0.034 0.046 0.288 0.123
PC AA C34:1 6.164 3.137 0.844 0.792 1.800 0.934
PC AA C34:2 3.367 1.508 0.379 0.375 0.782 0.416
PC AA C34:3 0.761 0.272 0.439 0.409 0.478 0.277
PC AA C34:4 0.218 0.150 0.047 0.027 0.216 0.093
PC AA C36:0 0.651 0.293 0.183 0.176 0.324 0.182
PC AA C36:1 1.129 0.410 0.142 0.143 0.334 0.140
PC AA C36:2 1.849 1.189 0.325 0.318 0.667 0.410
PC AA C36:3 1.400 0.552 0.162 0.142 0.427 0.164
PC AA C36:4 0.738 0.551 0.104 0.111 0.208 0.116
PC AA C36:5 0.449 0.333 0.099 0.093 0.190 0.102
PC AA C36:6 0.245 0.038 0.057 0.046 0.118 0.041
PC AA C38:0 0.354 0.246 0.056 0.078 0.140 0.058
PC AA C38:1 0.133 0.115 0.050 0.057 0.116 0.032
PC AA C38:3 0.310 0.307 0.093 0.088 0.109 0.051
PC AA C38:4 0.731 0.361 0.118 0.094 0.414 0.107
PC AA C38:5 0.859 0.433 0.158 0.199 0.150 0.171
PC AA C38:6 0.442 0.140 0.064 0.057 0.117 0.081
PC AA C40:1 0.623 0.422 0.598 0.464 0.380 0.403
PC AA C40:2 0.542 0.404 0.184 0.144 0.189 0.084
PC AA C40:3 0.248 0.122 0.040 0.033 0.068 0.021