Comparative study of the efficacy of intra-arterial and intravenous transplantation of human induced pluripotent stem cells-derived neural progenitor cells in experimental stroke

Cell culture

iPSCs were derived according to the protocol described previously (Salikhova et al., 2020). Donor skin biopsy collection was approved by the Institutional Ethics Committee of the Research Centre for Medical Genetics (Protocol No. 2019-2/3 from October 13, 2020). All patients given written informed consent for using the tissue for research purposes. CTS CytoTune-iPS 2.1 Sendai reprogramming kit (Invitrogen, Carlsbad, CA, USA) were used for fibroblast reprogramming. Obtained iPSCs were cultivated in Essential E8 Medium (Gibco, Waltham, MA, USA) using Petri dishes covered by recombinant vitronectin (10 µg/mL, Gibco, Waltham, MA, USA). iPSCs were detached upon reaching a culture of 80% confluency by incubation in Versen solution (PanEco, Berg am Irchel, Switzerland) for 5 min at 37 °C. The cell suspension was centrifuged at 800 rpm for 5 min and the pellet was transferred to new Petri dishes with the addition of ROCK inhibitor (5 µM, Merck Millipore, Rahway, NJ, USA) to the culture medium for 24 h.

The pluripotent status of the iPSCs was confirmed, as previously described (Salikhova et al., 2021), by their ability to differentiate into cells belonging to all three germ layers, to form embryoid bodies, and to express the pluripotent markers Oct4, Nanog, SSEA4, TRA-1-81. iPSCs were transferred as colony fragments to 24-well Ultra Low Adhesion Plates (Corning, Corning, NY, USA) and cultured in DMEM/F12 medium supplemented 20% FBS, 2 mM glutamine, 1% mixture of essential amino acids, 100 mg/L penicillin-streptomycin (Paneko, Moscow, Russia) for formation of embryoid bodies. The formed embryoid bodies were transferred to Petri dishes coated with 0.1% gelatin (Paneko, Moscow, Russia) for observation of cell migration after 2–3 weeks of cultivation. The obtained extensive areas of differentiated cells were fixed and immunocytochemical analysis was performed for markers of three germ layers using primary antibodies against pan-cytokeratin (ectoderm marker, ab7753, Abcam, Boston, MA, USA), vimentin (mesoderm marker, ab92547, Abcam, Boston, MA, USA), α-fetoprotein (endodermal marker, ab3980, Abcam, Boston, MA, USA). To confirm the phenotype of iPSCs, the immunocytochemical analysis on markers of pluripotency was performed using primary antibodies to NANOG, SSEA4, OCT4, TRA-1-81 (StemLight Pluripotency Antibody Kit, Cell Signaling Technology, San Antonio, TX, USA).

iNPCs were derived from iPSCs using the mix of small molecules: 10 µM SB431542 (Tocris, USA), 2 µM Dorsomorphine (Sigma Algrich, St. Louis, MO, USA), and 200 nM LDN193189 (Sigma Algrich, St. Louis, MO, USA) which were TGF-β and BMP signaling inhibitors. The differentiation lasted for 2 weeks on Petri dishes covered by Matrigel (Corning, Corning, NY, USA). The formed radially organized cellular structures (also called neural rosette) were subcultured with Versen solution for 5 min at 37 °C. The cell suspension was centrifuged at 1,200 rpm for 5 min. The obtained pellet of the iNPCs was cultivated in the DMEM/F12 medium (PanEco, Berg am Irchel, Switzerland) supplemented by 2% of the B27 (Gibco, Waltham, MA, USA), 2 mM glutamine (PanEco, Berg am Irchel, Switzerland), 100 mg/L penicillin-streptomycin (PanEco, Berg am Irchel, Switzerland), and 10 ng/ml FGF-2 (ProSpec, UK) using Matrigel substrate. The phenotype of the obtained iNPCs confirmed immunocytochemically using primary antibodies against Nestin (ab105389, Abcam), PAX6 (ab5790, Abcam, Boston, MA, USA), and Sox2 (ab79351, Abcam, Boston, MA, USA).

Immunocytochemistry

iPSCs and their derivate cultures were fixed in 4% paraformaldehyde solution (PanEco, Berg am Irchel, Switzerland) 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 incubated with primary antibodies for 60 min in the dark. Secondary antibodies (anti-mouse IgG conjugated to Alexa Fluor 555 (A-21422, Invitrogen, Carlsbad, CA) or anti-rabbit IgG conjugated to Alexa Fluor 488 (A-11008, Invitrogen, Carlsbad, CA) was applied for 60 min in the dark. The nuclei was counterstained with DAPI (Sigma-Aldrich, , St. Louis, MO, USA) solution (1 µg/mL in PBS). The images were recorded with an Axio Observer.D1 inverted fluorescence microscope equipped with Axio-Cam HRc camera (Carl Zeiss, Jena, Germany).

Cell labeling

The transplanted iNPCs were pre-labeled with the GFP protein using the LVT-TagGFP lentiviral vector (Evrogen, Moscow, Russia) for visualization. The virus suspension was added to the culture medium with 4 µg/ml polybrene (Sigma-Aldrich, St. Louis, MO, USA) according to the manufacturer’s instructions. The cells were washed twice with the phosphate-buffered saline (PanEco, Moscow, Russia) after 12 h incubation and the maintenance culture medium was added. The iNPCs were additionally labeled with the PKH26 Red Fluorescent Cell Linker kit (Sigma-Aldrich, USA) following the instructions of the manufacturer. Briefly, the cells were harvested using Versen solution (PanEco, Moscow, Russia), centrifuged at 1,200 rpm for 5 min, washed with Hank’s Balanced Salt Solution (HBSS), and re-suspended in 1 ml of the Diluent C reagent. The cell suspension was mixed with an equal volume of the labelling solution (4 nM PKH26), and incubated at room temperature for 5 min. The staining process was stopped by the addition of 2 ml of fetal bovine serum. Cells were washed twice with HBSS, and re-suspended in the phosphate-buffered saline (PanEco, Moscow, Russia). The obtained suspension was analyzed 72 h post-transfection using a CyFlow ML flow cytometer (Partec, München Germany), and the number of labeled cells was counted using the FloMax software.

Animals

Adult male Wistar rats weighing 250–300 g (n = 69) were purchased from “SMK STESAR” (Vladimir, Russian Federation). The experimental animals were housed 4 to 5 per cage under standard housing conditions (12-h/12-h light/dark cycle, room temperature 22 ± 2 °C, humidity 45–65%) with free access to standard rodent chow and water. Enrichment based on no risk to the animals (i.e., cause injuries or excessive aggression), to the humans (i.e., jeopardize the health and safety of the animal staff), or to the experiments (i.e., cause undesirable interference or an excessive increase in the number of animals used). All animal experiments were carried out in accordance with the guidelines of the Declaration of Helsinki and the Directive 2010/63/EU on the protection of animals used for scientific purposes of the European Parliament and the Council of European Union dated 22 September 2010 and were approved by the Pirogov Russian National Research Medical University Animal Care and Use Commission (protocol code No 24/2021 from 10 December 2021). In vivo studies are reported according to the ARRIVE guidelines (v. 2.0). Surgery was performed under inhalation anesthesia with the mixture of 2,5–3% isoflurane (Aerrane, Baxter HealthCare Corporation, Deerfield, IL, USA) and of 97–98,5% atmospheric air using an animal anesthesia system (E-Z-7000 Classic System, E-Z-Anesthesia^® Systems, Palmer, PA, USA). After the stroke modeling using endovascular middle cerebral artery occlusion method, the animals were kept separately in a cage with a 37 °C heating pad to avoid additional injury and for better recovery after surgery. The animals were intraperitoneally administered with 2 ml of saline solution in order to avoid hypovolemia in the first hours after surgery, when the mobility of the animal would be limited due to the severity of the condition. At the end of the observation period (14 days after cell transplantation) and for histological examination (2 h, 3 and 7 days after transplantation) the experimental animals were euthanized by inhalation anesthesia with a lethal dose of isoflurane and by injection of a lethal dose of Zoletil.

Animal study design

The experimental ischemic stroke was induced in rats (n = 69) using the model of transient middle cerebral artery occlusion (MCAO). The animals were tested using the Modified Neurological Severity Scores (mNSS) 24 h after stroke modeling, before cell administration, and MRI study was performed in order to assess the severity of the rats’ condition, the volume of brain damage, and the exclusion of complications of the model. Rats with hemorrhagic complications were removed from the experiment in order to objectify the evaluation of the data. Then animals were randomly divided into three groups: control group—rats without cell therapy (n = 21); iNPCs IA—rats with intra-arterial transplantation of iNPCs (n = 22, 11 for evaluation of the therapeutic efficiency and 11 for the study of cell distribution); iNPCs IV—rats with intravenous transplantation of iNPCs (n = 26, 15 for evaluation of the therapeutic efficiency and 11 for the study of cell distribution). Only the person responsible for the administration knew the classification of the groups.

The therapeutic efficiency of iNPCs transplantation was estimated by the survival rate, neurological deficit and stroke volume of animals within a 14 days period. The cells’ distribution was evaluated by histological examination of the rat brain. For histochemical studies, rats were sacrificed at 2 h, 3 and 7 days after iNPCs transplantation using the method described above. All manipulations (tests, cell administration and histological examination) were assessed for all groups at the same time of the day to minimize potential confounders, such as the order of treatments and measurements or animal/cage location and treatment groups.

Stroke modeling

The experimental ischemic stroke was induced using endovascular transient (90 min) MCAO developed by Koizumi (1986) and modified by Longa (Longa et al., 1989) as was described in detail previously (Gubskiy et al., 2018). The accuracy of the MCAO and possible complications were monitored by MRI guidance. The endovascular occlusion of the middle cerebral artery was carried out by a blunt-ended silicon coating monofilament (Doccol Corporation, Sharon, MA, USA; diameter of filament was 0.19 mm, length 30 mm, diameter of the tip 0.37+0.02 mm, length of the tip 3–4 mm). After removing the filament and suturing the surgical wound the animals were injected 2 ml of saline intraperitoneally and 0.2 ml of gentamicin intramuscularly and placed in a heated cage to recover from anesthesia.

Cell transplantation

IA and IV cell transplantation was performed 24 h after stroke modeling as previously described (Gubskiy et al., 2022). For the IA administration, the bifurcation of the right common carotid artery was isolated and the pterygopalatine artery was ligated. A microcatheter (rodent tail vein catheter with 1F (1/3 mm) diameter and 28 cm length, Braintree Scientific, Inc., Braintree, MA, USA) filled with saline was inserted through the stump of the external carotid artery into the lumen of the common carotid artery for 5–6 mm in the direction opposite to the blood flow. The transplantation of iNPCs was performed through the microcatheter with the use of microinjector (Leica Microsystems GmbH, Braintree, MA, Germany) at the speed of 100 µl/min. Importantly, the blood flow in the common and internal carotid arteries was maintained during the transplantation to avoid cerebral embolism. For the IV cell transplantation, the right femoral vein was exposed and a microcatheter (rodent tail vein catheter with 1F (1/3 mm) diameter and 28 cm length, Braintree Scientific, Inc., Braintree, MA, USA) was inserted. The transplantation of iNPCs was performed at the speed of 250 µl/min to avoid pulmonary embolism. The density of cell suspensions for IA and IV transplantations was 7 × 10⁵ and 2 × 10⁶ of iNPCs in 1 ml of phosphate-buffered saline respectively. For the rats from the groups for the evaluation of the therapeutic efficiency unlabeled iNPCs were administrated in order to avoid the potential influence of the labels to the cells’ effect. Labeled cells were transplanted only into the rats for the histological study for evaluation of cell distribution.

Neurological outcomes

The neurological deficit was estimated using the Modified Neurological Severity Scores (mNSS) for animals with stroke on the 1st, 7th and 14th day after transplantation. The mNSS combines a set of tests and allows to evaluate motor and sensory functions, reflexes and balance (Schaar, Brenneman & Savitz, 2010). In each test one point is given for failure and no points for success. The severity of stroke is estimated by the sum of points scored. According to the mNSS scale the maximum number of points is 18, which corresponds to the most severe neurological deficit, 12–18 points estimated as severe stroke, 8–12–moderate stroke, 1–7–mild stroke.

MRI

The magnetic resonance imaging (MRI) was carried out using a 7T ClinScan system for small animals (Bruker BioSpin, USA) under isoflurane inhalation anesthesia (3.5–4% isoflurane mixed with pure oxygen). To perform stroke volume morphometry, T2-weighted imaging was carried out at the 1st, 7th and 14th day after transplantation. MRI morphometry was performed using ImageJ software (Rasband, W.S., ImageJ, National Institutes of Health, Bethesda, Maryland, USA). The volume of the stroke (V) was analyzed based on T2-weighted images by the summation of volumes measured in adjacent cross sections according to the following formula: V = (S1 + …+ Sn) × (h + d), where S1, …,Sn is area measured on slice, h is the slice thickness, and d is the gap interval between slices.

Immunohistochemistry

Animals were sacrificed at 2 h, 3 and 7 days after the IA or IV transplantation of iNPCs by inhalation anesthesia with a lethal dose of isoflurane and additional injection of a lethal dose of Zoletil. After transcardial perfusion using 4% paraformaldehyde (PFA), the brains were removed and post-fixed at 4 °C overnight in the same fixative, washed with PBS and cryoprotected in the 30% sucrose solution. The coronal sections (40 µm thick) were obtained at a cryostat microtome (Leica CM1900, Munich, Germany). The sections were collected and stored for subsequent immunohistochemistry. The sections were incubated in a mixture of 5% normal goat serum (Sigma-Aldrich, St. Louis, MO, USA), 0.3% Triton x-100 (Sigma-Aldrich, St. Louis, MO, USA) and primary antibodies anti-RECA (rat endothelial cell of the blood–brain barrier antibody) (1:200, ab264524, Abcam, Cambridge, UK) or anti-EBA (endothelial cells of the blood–brain barrier antibody) (1:100, Biolegend, San Diego, USA) in 0.01 M PBS (pH 7.4) at 4 °C overnight. Then sections were rinsed with PBS and incubated with secondary antibodies (1:500, anti-mouse IgG Alexa fluor 647, Invitrogen, USA) for 2 h at room temperature. Nuclei were counterstained with DAPI solution (2 µg/mL, Sigma-Aldrich, St. Louis, MO, USA). Fluorescence confocal micrographs were taken with the Nikon A1R MP laser scanning confocal microscope (Nikon Instruments Inc., Tokyo, Japan). For the group of rats with IA iNPCs administration the morphometric analysis of obtained images was carried out. The percentage of double-labeled cells was calculated on the assumption of the total number of cells (cell nuclei) in the histological section. The percentage of GFP+ and PKH26+ cells was calculated according to the following formula: (labeled cells/total number of cells) × 100%.

Statistical analysis

Statistical analysis and data visualization were performed using Python 3.10 (Van Rossum & Drake, 2009) in Jupyter Notebook (Kluyver et al., 2016). The normality of distribution was evaluated by Shapiro–Wilk’s test. Animal survival was assessed by the Kaplan–Meier method using the Log Rank test (Davidson-Pilon, 2019). Two-way ANOVA was used to evaluate the dynamics changes of the neurological deficit and stroke volume of experimental animals. The values of mNSS score and stroke volume obtained on day 7 and 14 were normalized based on the data obtained on day 1. The pairwise comparison of the groups with the FDR correction with Benjamini–Hochberg method was performed. The final results are presented after the pairwise comparison. The significance level was set at 0.05.

The sample size for the animal experiments was estimated based on the results of our previous study of the therapeutic effects (mNSS) of neural precursor cells after IA transplantation in MCAO rats (Namestnikova et al., 2021). The sample size was initially estimated on the assumption of power = 0.8 and significance level p = 0.05 for three groups with three measurements, and the calculations showed that the number of rats in each group should be at least n = 6. Originally, the number of rats in each group was planned to be n = 12 (two times more than calculated sample size), however, considering the high mortality of rats from the control group (rats with experimental stroke without any treatment) and the necessity to withdrawal rats for histological examination from the groups with iNPCs transplantation, the number of rats in each group was increased and was 21, 22 and 26 for the control group, IA and IV group respectively.

Cell culture characterization

Human iPSCs formed smooth edged colonies morphologically similar to embryonic stem cells (ESCs). The colonies consisted of tightly packed cells with high nucleus-cytoplasm ratio, immunopositive for the following pluripotency markers: TRA-1-81, SSEA4, Oct4, Nanog (Fig. 1A). The cells had the ability to form embryoid bodies, and spontaneously differentiate into the derivatives of three germ layers –mesoderm (vimentin+ cells), ectoderm (pan-cytokeratin+ cells), and endoderm (α-fetoprotein+ cells) (Fig. 1B). Neural differentiation of iPSCs was induced by dual SMAD inhibition that led to formation of neural rosettes. The obtained iNPCs were immunopositive for neural markers Pax6, Nestin, and Sox2 (Fig. 1C). For the in vivo cell tracking, iNPCs were transduced by lentiviral vector expressing the GFP and additionally stained by the lipophilic dye PKH26. The efficiency of transduction and cell labeling were determined by flow cytometry. The fluorescence intensity of transduced (GFP+) cells presented on the abscissa (FL1 channel), and PKH26+ labeled cells on the ordinate (FL2 channel). Accordingly, the efficiency of iNPCs transduction was 79.3 ± 4% (gates QA2 and QA4), and efficiency of PKH26+ labeling cells was 91 ± 6% (gates QA1 and QA2). The percentage of double-labeled cells was 75.3 ± 5% (gate QA2) (Fig. 1D).

Therapeutic efficiency of the iNPCs transplantation

The survival of animals after iNPCs transplantation was estimated over the period of 14 days. The Kaplan–Meier survival curves are presented at Fig. 2A. According to the results of statistical analysis, the survival rate of animals from therapy groups was significantly higher compared to the control group. No significant differences were found between groups with different routes of iNPCs transplantation. Both IA and IV administration of iNPCs improved animal survival rate (p ≤ 0.05) compared to the control group.

The neurological outcomes were evaluated using the mNSS on the 1st, 7th and 14th day post-transplantation. As seen from Fig. 2B, the dynamics of changes of the neurological deficit in rats getting the IA or IV iNPCs transplantation differed significantly from that in the control group (p ≤ 0.05). However, no significant difference was revealed between groups with different ways of iNPCs transplantation. Therefore, both routes of the systemic administration of iNPCs seem equally effective.

The stroke volume was evaluated by MRI on the 1st, 7th and 14th day after transplantation of iNPCs or vehicle (Fig. 2C). Only IA iNPCs transplantation led to significantly faster and pronounced reduction of the stroke volume compared to the control group during the observation period (p ≤ 0.05). No significant difference in the dynamics of stroke volume changes was found between the group with IV iNPCs administration and the control group.

Cell distribution

In case of the IA administration of iNPCs, labeled cells were detected inside cerebral blood vessels 2 h after transplantation (Fig. 3). Transplanted cells were distributed in the right hemisphere in the infarct area and the peri-infarct zone.

Transplanted cells were found 2 h after IA administration in all experimental animals (n = 3). The percentage of such cells was 2.2 ± 0.3% per histological section of the rat brain. Subsequently, 72 h after IA administration just single cells remained inside the cerebral blood vessels and were detected only in one rat from the experimental group (the total number n = 4). Therefore, it can be concluded that the number of transplanted labeled cells decreased over time (the example is given in Fig. 4). As can be seen from Fig. 4A 2 h after IA administration the transplanted iNPCs were detected in several blood vessels, whereas after 72 h (Fig. 4B) only single labelled cells were observed. Remarkably, within 72 h after cell administration human iNPCs were visualized inside cerebral blood vessels in contact with their inner wall (vessel were immunostained for the endothelial barrier antigen (EBA)) and the migration of transplanted cells through the structure of the blood–brain barrier into the brain parenchyma were not observed. After 7 days post-transplantation labeled iNPCs were not detected in the brain in all experimental animals (n = 4).

In the case of IV administration of iNPCs, labeled cells (GFP+ and PKH26+) were not detected in the brain 2 h (n = 4) after injection, as well as on the 3rd (n = 4) and 7th day (n = 3) after transplantation (Fig. 5).