Cell culture and conditioned media preparation
hiPSCs were derived from human dermal fibroblasts by using CTS CytoTune-iPS 2.1 Sendai Reprogramming Kit (Invitrogen, USA). To obtain neural stem cells (NSCs) for subsequent differentiation into neuronal and glial lineages, hiPSCs were cultured in DMEM/F12 (Gibco, USA) supplemented with N-2 (to 1X, Gibco), 2 mM L-glutamine (PanEco, Russia), 100 mg/L penicillin-streptomycin (PanEco), 10 µM SB431542 (Stemcell Technologies, USA), 2 µM dorsomorphin (Stemcell Technologies) and 200 nM LDN193189 (Sigma-Aldrich, USA) [10].
To obtain NPСs, NSCs were cultured for 2 weeks in DMEM/F12 (PanEco) supplemented with B-27 (to 1X, Gibco), 2 mM L-glutamine, 100 mg/L penicillin-streptomycin, 10 ng/mL FGF-2 (ProSpec, UK) and 1 µM purmorphamine (Stemcell Technologies) [11].
To obtain GPСs, NSCs were cultured in DMEM/F12-based growth medium supplemented invariably with N-2, 2 mM L-glutamine, 100 mg/L penicillin-streptomycin and 1% fetal bovine serum (PanEco). The induction proceeded in 3 stages, 3 days each. For the first stage, the growth medium was additionally supplemented with 10 ng/mL FGF-2 and 20 ng/mL EGF (ProSpec). For the second stage, the growth medium was additionally supplemented with 10 ng/mL FGF-2, 20 ng/mL EGF, and 20 ng/mL CNTF (PeproTech, USA). For the third stage, the growth medium was additionally supplemented with 20 ng/mL EGF and 20 ng/mL CNTF [12,13].
To obtain the conditioned media (CM), NPС and GPС cultures were washed twice with PBS and incubated in DMEM/F12 (PanEco) for 12 h. The medium was collected and centrifuged for 5 min at 3000 rpm. The supernatant was passed through 0.2 µm syringe filter (PanEco) and used in the experiments.
Immunocytochemistry
The cells were fixed in 4% paraformaldehyde solution (PanEco) for 10 min at room temperature, washed with PBS, pre-incubated in 0.25% Triton X-100 and 1% BSA in PBS for 30 min, and further incubated with primary antibodies to βIII tubulin (ab182070, Abcam, UK), S100b (ab52642, Abcam), NANOG, SSEA4, OCT4, TRA-1-81, SOX2 (StemLight Pluripotency Antibody Kit, Cell Signaling Technology, USA), pancytokeratin (ab7753, Abcam), desmin (ab32362, Abcam), α-fetoprotein (ab3980, Abcam), Nestin (ab105389, Abcam) or PAX6 (ab5790, Abcam) at +4 °C overnight. Secondary antibodies (Alexa Fluor 555 or Alexa Fluor 488 conjugated, Invitrogen) were applied for 60 min in the dark. The nuclei were counterstained with DAPI solution (1 µg/mL in PBS). The signals were observed and images recorded with an Axio Observer.D1 inverted fluorescence microscope equipped with AxioCam HRc (Carl Zeiss, Germany).
Flow cytometry
The cells were detached with Versene solution (PanEco) and pelleted by centrifugation at 1800 rpm for 5 min, then washed with HBSS (PanEco), fixed in 4% paraformaldehyde for 10 min, washed with PBS, permeabilized with 70% methanol on ice for 10 min, washed twice with PBS and collected by centrifugation at 1800 rpm for 5 min. Fixed cells were incubated with primary antibodies to PAX6 (ab5790, Abcam), glial marker S100b (ab52642, Abcam) or neuronal marker βIII tubulin (ab182070, Abcam) at +4 °С for 12 h, then washed with PBS, collected by centrifugation at 1800 rpm for 5 min and incubated with secondary antibodies (Alexa Fluor 488 conjugated, Invitrogen) for 60 min in the dark. Fixed cells exposed to secondary antibodies only were used as a negative control for the flow cytometry-based quantification. Stained cells were analyzed on a CyFlow ML flow cytometer with FloMax software (Partec, Germany).
Proteomic analysis
Ultimate 3000 RSLCnano HPLC system (Thermo Scientific, USA) connected to Q-exactive HF mass spectrometer (Thermo Scientific) was used for the analysis. Mass spectra were recorded in the ion-positive mode with nanoelectrospray ionization. Identification of proteins in the spectra was carried out in SearchGUI v.3.3.16 software with X!Tandem, OMSSA and MS-GF+ algorithms (Vaudel M. et al., 2011). The alignments were made with Uniprot database ('human'-filtered). Structuring of the data by classes and functions of the proteins was carried out by using Uniprot and PANTHER databases [15].
Enzyme-linked immunosorbent assay (ELISA)
Concentrations of GDNF, BDNF, NGF and CNTF in CM were measured by ELISA (RnD systems, USA) in accordance with the manufacturer's protocol. The collected CM samples were concentrated 24-fold with 3 kDa Amicon Ultra filter units (Sigma-Aldrich) to 0.5 mg/mL total protein concentrations prior to the analysis. Optical densities (absorbance at 450 nm) were measured in a plate reader (PerkinElmer, USA).
PCR assay
For the reverse transcription polymerase chain reaction (RT-PCR) assay, cells or tissues were collected in RNAlater (Thermo Fisher Scientific, USA) for the preservation of RNA molecules during storage. Total RNA from the collected cell or tissue samples was isolated with RNeasy Mini Kit (Qiagen, USA) according to the manufacturer's protocols. cDNA synthesis was carried out with RevertAid First Strand cDNA Synthesis Kit (Thermo Fisher Scientific) according to the manufacturer's protocol. The real-time PCR mixtures were set up with qPCRmix-HS SYBR (Evrogen, Russia); the reactions were carried out in a Bio-Rad iQ thermal cycler as primary denaturation 95 °C for 5 min followed by 40 cycles of denaturation 95 °C for 20 seconds, annealing 55–63 °C for 20 seconds and elongation 72 °C for 20 seconds. Raw data for the genes of interest were normalized against constitutively expressed reference genes GAPDH (Gapdh, glyceraldehyde-3-phosphate dehydrogenase) and ACTB (Actb, beta-actin). Expression levels were calculated by 2-ΔΔC(T) approach. The oligonucleotide sequences are given in Supplementary Table 1.
In vitro and in vivo study design
In vitro experiments involved SHSY-5Y neuroblastoma cell line. CM were applied at 4 h after hypoxia modeling to the final total protein concentration of 5 µg/mL. At 24 h after hypoxia modeling, cell survival was assessed by MTT and LDH tests, vital staining with propidium iodide and Hoechst 33342, and the levels of apoptosis-related gene expression. In the longer incubation experiments, CM were added every other day to the same final protein concentration. Formation of axons and dendrites was assessed by expression markers of neuritogenesis MAP2 and GAP43 and alterations in cell morphology on days 1, 3 and 7 of the experiment.
In vivo experiments involved adult male Wistar rats of 250–300 g body weight (n=34) purchased from AlCondi Ltd., Russia. All manipulations with the animals were approved by the Ethical Committee at N.I. Pirogov Russian National Research Medical University. All in vivo experiments were carried out in accordance with EU Directive 2010/63/EU.
The experimental ischemic stroke was modeled by temporary occlusion of the right middle cerebral artery. At 24 h after the stroke, the animals (n=34) were randomized into 3 groups: control group (n=10) and CM recipients (n=12 each) to receive intra-arterial infusions of, respectively, the non-conditioned DMEM/F12-based medium, concentrated GPC-CM or NPC-CM to 50 µg/mL of total protein. Therapeutic effects were assessed the following parameters: survival, neurological deficit and the stroke volume immediately before the infusion (day 1) and on days 7, 14 and 30 post infusion (p/i). The animals were sacrificed at the end of the experiment (day 30 p/i). At the autopsy, the ischemized portions of the brain tissue were dissected into 3 fragments. One fragment was fixed in 10% formalin for paraffin sections, one was preserved in liquid nitrogen for cryosections, and one was preserved in RNAlater reagent for PCR analysis. Corresponding area from the unaffected contralateral hemisphere was dissected in the same way to be used as a control.
In vitro study of neuroprotective properties
Modeling of chemically induced hypoxia
SHSY-5Y cells were cultured in DMEM/F12 (PanEco) supplemented with 10% FBS (PanEco). One week prior to the addition of the mimetic, the cells were seeded in 12-well plates (Corning, USA) coated with collagen type I solution (Gibco) at 90 × 103 cells per well and incubated in Gibco Opti-MEM supplemented with 10 mM retinoic acid (Gibco) and 0.5% FBS (PanEco) for 2 days. The growth medium was subsequently replaced with DMEM/F12 (PanEco) supplemented with B-27 (to 1X, Gibco) and 50 ng/mL BDNF (PeproTech). At 72 prior to hypoxia, the medium was depleted of BDNF. The cells were exposed to 250 µM CoCl2 for 4 h, washed with HBSS (PanEco) and replated in DMEM/F12 (PanEco) supplemented with B-27 (Gibco) [16].
Evaluation of cell survival
MTT ((3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide, PanEco) was added to the culture medium to a final concentration of 0.5 mg/mL. The measurements were carried out after 2 h incubation with MTT and subsequent dissolution of formazan crystals with DMSO (PanEco). Evaluation of necrosis rates by LDH tests was carried out by using LDH Activity Assay Kit (Sigma-Aldrich) according to the manufacturer's protocol. Optical densities (absorbance at 570/620 nm for MTT test or 450 nm for LDH test) were measured in a plate reader (PerkinElmer). Cell death rates were assessed by staining the cells with vital fluorescent dyes Hoechst 33342 (Sigma-Aldrich, 5 µg/mL for 30 min) and propidium iodide (Sigma-Aldrich, 1 µg/mL for 10 min) to visualize nuclear fragmentation and membrane integrity. The fluorescence patterns were observed and recorded with Axio Observer.D1 inverted fluorescence microscope equipped with AxioCam HRc (Carl Zeiss).
Assessment of neurite outgrowth
Formation of axons and dendrites was assessed by morphological examination combined with immunocytochemistry for βIII tubulin, and also by real-time PCR assay for the transcriptomic markers of neuritogenesis (MAP2, GAP43).
In vivo study of neuroprotective properties
Modeling of ischemic stroke
Transient occlusion of the middle cerebral artery in rats was performed by standard method (the procedure originally developed by Koizumi [17] and modified by Longa [18]). The animals were anesthesized with 2.5–3% isoflurane/97–97.5% air mixture. The right middle cerebral artery was temporary occluded with a silicone rubber-coated monofilament (diameter 0.19 mm, length 30 mm, diameter with coating 0.37±0.02 mm, coating length 3–4 mm; Doccol, USA) for 90 min. During the operation and until the emergence from anesthesia the body temperature was maintained at 37 °С with a warming pad.
Comparative assessment of the neurological deficit and functional recovery
Therapeutic effects of CM administration were determined by survival rates, infarct volume and neurological deficits as assessed by using the modified neurological severity scores (mNSS) for rodents [19] immediately before the infusion (day 1) and on days 7, 14 and 30 p/i.
Magnetic resonance imaging (MRI)
The imaging was accomplished in a 7T ClinScan small animal MRI system (Bruker BioSpin, USA). Intraoperative MRI (monitoring of the monofilament position, intracranial blood flow and hemorrhagic complications) was carried out as described previously [20]. For MRI evaluation of in vivo dynamics of the stroke volume, axial plane T2-weighted brain images (Т2-WI) were acquired immediately before the infusion (day 0) and on days 7, 14 and 30 p/i. Quantitative reconstruction of the infarct volume on the basis of T2-WI was accomplished in ImageJ software (Wayne Rasband, National Institute of Mental Health, Bethesda, MD, USA) by calculating V= (S1+...+Sn) × (h+d), where S values correspond to the infarct area measured in individual sections, h is slice thickness, and d is spacing between the slices.
Histology
The formalin-fixed tissues were dehydrated and embedded in paraffin and cut on a microtome. The 5–7 µm thick sections were positioned on gelatin-coated slides and dried at 37 °С for 24 h. The sections were deparafinized in xylene and rehydrated in 100°–70° ethanol series, stained with H&E, dehydrated and mounted. Pathohistological examination of the sections by light microscopy was carried out using Axio Observer.D1 equipped with AxioCam HRc (Carl Zeiss). Formation of new blood vessels at the site of injury was assessed in the images of the H&E stained histological sections at х400 magnification by using ImageJ software. Blood vessels were identified as hollow structures of characteristic morphology and red blood cells in the limina. The total number of blood vessels per sq mm of the section and the volume density of blood vessels (estimated as the total cross-sectional area of blood vessels per sq mm of the section) were evaluated.
Immunohistochemistry
The tissues cryopreserved in liquid nitrogen were cryosectioned at a 4–5 µm thickness. The sections were pre-blocked in PBS with 0.3% Triton X-100 and 2% BSA for 1 h, incubated with anti-CD68 primary antibodies (ab125212, Abcam) at +4 °C overnight, washed and incubated with secondary antibodies (Alexa Fluor 488 conjugated, Invitrogen) for 60 min in the dark. The nuclei were counterstained with DAPI solution (1 µg/mL in PBS). The images were recorded with Axio Observer.D1 inverted fluorescence microscope equipped with AxioCam HRc (Carl Zeiss). Total counts of CD68+ cells per sq mm of the section were determined with ImageJ software by counting the positively stained cells at x200 magnification.
Statistical analysis
The data were processed in SigmaPlot 12.5 software. Each single group included 3–5 independent experiments. Pairwise comparisons were carried out by t-test in the case of normal distribution or Mann Whitney test in the case of non-normal distribution. For multiple comparisons, one-way ANOVA with Holm-Sidak method was used for the cases of normal distribution, whereas ANOVA on ranks with Dunn's test was used for the cases of non-normal distribution. Differences at p≤0.05 were considered significant.