- Cultivation and identification of hOPCs
HOPCs were prepared by the Pediatric Laboratory of the Sixth Medical Center of the Chinese People's Liberation Army General Hospital using previously established methods for their cultivation and identification (Text S1). The study was not pre-registered at the start of the methods. Next, the hOPCs were identified by flow cytometry and cell morphology. HOPCs were surface-stained with PDGFR-α BV421 mouse anti-human (Cat. #562799, BD Biosciences, Franklin Lake, New Jersey, USA), A2B5 PE mouse anti-human (Cat. #130-093-581, Miltenyi Biotec, Bergisch-Gladbach, Germany), and NG2 APC mouse anti-human (Cat. #FAB2585A, R&D Systems, Minnesota, USA) antibodies for flow cytometry (FACSanto II, BD Biosciences, Franklin Lake, New Jersey, USA).
Sprague–Dawley (SD) rats and homozygous shiverer mice (The Jackson Laboratory, Maine) were maintained in a specific pathogen-free environment at the Sixth Medical Center of PLA General Hospital. The room temperature was set to 23 ± 2℃, the humidity to 60 ± 10%, with light and dark cycles of 12 h. Animals had ad libitum access to sterile food and water. In this study, 48 newborn SD rats were randomly selected from a pool of 60, and 24 newborn shiverer mice were randomly selected from a pool of 40. For the inclusion criteria, the difference between the weight of each animal and the average weight was set to < 1g, whereas otherwise excluded; no animals were excluded since all fit the inclusion criteria. The total number of one-day-old rats or mice in each the experimental and control groups was 12 (6 males and 6 females), and each cage contained four animals. During animal testing following transplantation, the tester was unaware of the animal grouping. Table S1 shows the initial weight of the animals at one day old. We estimated the sample size based on the initial weight of the animal using the following equation:
In the formula: α = 0.05, β = 0.9, Z0.05 = 1.96, Z0.9 = 1.28, σ= δ = X1 - X2. α refers to Type I error while β to Type II, and S1 and S2 refer to the standard deviation of the animals' weight, while Z1 and Z2 refer to the mean.
- Safety of hOPCs transplantation into the lateral ventricle of SD rats
In the acute toxicity experiments, the transplantation was performed in the rats on post-partum day 6. We measured weight, tibia length, righting reflex, and cliff avoidance reaction every 2 days over the 14 days following transplantation (21 days after birth). These tests were conducted at 6 pm on the day of the experiment. On day 14, the young rats were sacrificed by cervical dislocation. In the chronic toxicity experiment, a total of three transplants were performed, and the transplantation time points corresponded to post-partum days 6, 20, and 40. Following the initial transplantation, the body weight and tibial length of the mice were measured every 3 days until the end of the experiment (post-partum day 90). These tests were conducted at 6 pm on the day of the experiment. After the third transplantation, blood was taken from the tail vein every 2 weeks for routine and biochemical blood tests. The haematology test was divided into two days: The first day was the transplantation group, while the second day was the control. The daily experiment duration was from 10 am to 5 pm. After the third blood collection, blood immune factor detection, and blood immune cell detection were carried out simultaneously. Immunofluorescence was used to detect the residual hOPCs in the various tissues. Before being sacrificed, all transplanted rats were photographed. After sacrifice, the coefficients of the heart, liver, spleen, lung, kidney, brain, thymus, spinal cord, testes, and ovary organs were calculated using the formula OC = m / M%, whereby m represents the organ’s weight, and M represents the rat’s weight.
3.1 Transplantation of hOPCs into the lateral ventricle of SD rats
HOPCs were transplanted into the right lateral ventricle of the experimental group (n = 12), and saline was injected into the right lateral ventricle of the control group (n = 12). The subgroups were the following: 1) male cell (MC, n = 6), 2) female cell (FC, n = 6), 3) male saline (MS, n = 6), and 4) female saline (FS, n = 6). Animals were anaesthetised with isoflurane anaesthesia (3-4%) immediately prior to surgery to minimise animal suffering. The animal’s head was then placed into a stereotaxic apparatus mask (Stoelting, USA), and the concentration of isoflurane was maintained at 2–2.5%. The anesthetized with isoflurane rats were fixed on a rat brain stereotaxic apparatus (Stoelting, USA) and their heads were disinfected before the skin was incised to expose the skull. The anterior fontanelle was the zero point. The injection site (anterior and posterior, midline-lateral, and depth) was approached based on the zero point (Windrem et al. 2004). In the acute toxicity experiments, the lateral ventricle of each rat was injected with either 5 μL of hOPCs (1×106 cells) or 5 μL of saline; injection rate: 0.5 μL/min; the lateral ventricle coordinates were (AP: 0.5 mm, ML: 1 mm, DV: 2.0 mm). In the chronic toxicity experiments, each rat was transplanted three times; once every 2 weeks. On the first, fourth, and seventh week each rat was injected a volume of 5 μL, 10 μL, and 10 μL, respectively, of either hOPCs (1×106 cells, 2×106 cells, and 2×106 cells) or saline. The coordinates of the lateral ventricle corresponding to the three transplants were (AP: 0.5 mm, ML: 1 mm, DV: 2.0 mm), (AP: 0.5 mm, ML: 1 mm, DV: 1.0 mm), and (AP: 0.5 mm, ML: 1 mm, DV: 1.0 mm). After transplantation, the incision was sutured, the skin was disinfected, and the young rats were returned to their mothers for feeding and were closely observed for 48 h. After weaning, the female and male rats were reared in separate cages.
3.2 Neural reflex detection
As part of the acute toxicity experiments, the neural reflex test was performed. Righting reflex: After transplantation, whether the supine young rats could turn over and touch the ground within 2 s was observed and assessed. If all young rats were positive for three consecutive tests on the same day, the day was considered a standard day. Cliff avoidance reflex: Starting from the first day after transplantation, young rats were placed on the edge of a workbench with a height of 30 cm. If the rats retreated or turned around from the edge within 90 s, the reaction was considered positive. If all rats in the same cage were positive, the day was considered a standard day.
3.3 Blood routine and serum biochemistry
We collected three blood samples after the final transplantation in the chronic toxicity experiment at 2-week intervals (weeks 9, 11, and 13, after birth). The blood was transferred to an anticoagulant tube containing EDTA. The XT2000iv blood analyzer (SYSMEX, JAPAN) was used to determine the blood routine. Detection indicators included red blood cells, white blood cells, platelets, hematocrit, hemoglobin, and mean platelet volume. Approximately 1 mL of blood was centrifuged at 2,000 rpm for 10 minutes at 4 °C, and the serum was obtained for further biochemical testing. Using a biochemical analyzer (HITACHI 7020, JAPAN), we measured alanine aminotransferase, aspartate aminotransferase, albumin, creatinine, urea, glucose, total protein, total cholesterol, and triglycerides.
3.4 Detection of cytokines in peripheral blood
At the last blood collection in the chronic toxicity experiment (week 13 after birth), whole blood samples were placed at room temperature for 2 h, before being centrifuged at 2,000 rpm for 20 min at 4 °C, and the supernatant was collected for testing. An enzyme-linked immunosorbent assay (ELISA, R&D Systems, Minnesota, USA) was used to detect peripheral blood immune factors. Detection indicators included IFN-γ, TNF-α, IL-2, IL-4, IL-5, and IL-6. The assay was performed according to the manufacturer’s instructions.
3.5 Detection of peripheral blood immune cells
In the final blood collection of the chronic toxicity experiment (week 13 after birth), the fresh blood sample was mixed with an equivalent volume of normal saline and slowly added to the test tube containing lymphocyte separation solution (Cat. # abs930, Absin Bioscience Inc, Shanghai, China). Then the blood was centrifuged at 2,000 rpm for 20 min at 4 °C to be divided into its compartments: plasma, WBCs, and RBCs (top to bottom). A capillary tube was used to collect the WBC-rich cell suspension above the RBC layer, and this was transferred to another test tube. Hanks solution was added without Ca2+ and Mg2+ ions; it was then mixed and centrifuged at 2,000 rpm for 10 min at 4 °C. Finally, a cell suspension was made with RPMI 1640 medium. Cell suspensions were surface-stained with CD3 APC mouse anti-human (Cat. #562799, BD Biosciences, Franklin Lake, New Jersey, USA), CD4 APC-A750 (Cat. #130-093-581, Miltenyi Biotec, Bergisch-Gladbach, Germany), CD8 PC5.5 (Cat. #FAB2585A, R&D Systems, Minnesota, USA), and CD45RA PC7 mouse anti-human antibodies for flow cytometry (FACSanto II, BD Biosciences, Franklin Lake, New Jersey, USA).
3.6 Detection of spleen immune cells
Following the final blood collection in the chronic toxicity experiments (week 13 after birth), rats were sacrificed by cervical dislocation. The spleen was removed under aseptic conditions and rinsed three times with normal saline. It was then cut and ground into a homogenate without tissue mass, filtered with a 40 μm cell sieve (Cat. #352340, BD Biosciences, Franklin Lake, New Jersey, USA), and diluted with saline to prepare a spleen cell suspension. The cell suspension was added to the centrifuge tube containing lymphocyte separation solution and centrifuged at 2,000 rpm for 20 min. A capillary tube was used to collect the WBC-rich cell suspension over the RBC layer, which was then transferred to a 50 mL centrifuge tube and mixed with physiological saline. Thereafter, this underwent centrifugation at 1,700 rpm for 7 min. Finally, an RPMI 1640 medium was used to prepare a cell suspension. Cell suspensions were then surface-stained with CD3 APC, CD4 APC-A750, and CD8 PC5.5 mouse anti-human antibodies for flow cytometry.
3.7 Histopathological analysis and biodistribution of transplanted hOPCs
After the chronic toxicity experiments (week 13 after birth), the main organs (heart, liver, spleen, kidney, thymus, spinal cord, testes, ovary, brain, and lung) were fixed in 10% formalin. The tissue sections were stained with hematoxylin and eosin, and an upright optical microscope (NIKON ECLIPSE CI, JAPAN) was used for observation, inspection, and evaluation of histopathological lesions. Immunofluorescence staining with STEM121 (mouse anti-human STEM121 antibody, Cat. # Y40410, 1:500, Takara Bio, Japan) was performed to label the residual hOPCs in each tissue.
- Study of the effectiveness of hOPCs-induced myelination
4.1 hOPCs transplantation in shiverer mice and tremor assessment
Newborn shiverer mice were transplanted within one day after birth. Animals were anaesthetised as described in section 3.1 at an isoflurane induction concentration of 2-3%, and a maintenance concentration of 1-1.5%. A mouse brain stereotaxic apparatus (Stoelting, USA) was used to inject hOPCs (1.5 μL; 2×105 cells) into the right corpus callosum of shiverer mice (n = 6). The transplant coordinates were as follows: AP, 0.5 mm; ML, 1 mm; and DV, 0.5 mm. The control group consisted of six randomly selected non-transplanted homozygotes. After the operation, the body weights were recorded at the same time every 7 days until the end of the experiment. At 30, 60, and 90 days after transplantation, the tremor was assessed. The indicators included the amplitude of the tremor and the duration of continuous tremor.
4.2 Electron microscopy and tissue immunofluorescence staining
Three months after transplantation, mouse brain samples were imaged using a transmission electron microscope (TEM, H7650-B, HITACHI, Tokyo, Japan). A field of view was randomly selected for each mouse, and the G-ratio was calculated (myelin inner diameter / myelin outer diameter) according to the amount of myelin in the field of view. The remaining mouse brain tissues were immunostained with myelin basic protein (MBP) (rat anti-MBP antibody, Cat. #Ab7349, Abcam, Cambridge, UK). A fluorescence microscope (IX-70, Olympus Corporation) was used to image the samples and the average optical density of MBP was calculated using Image Pro Plus 6.0 (Media Cybernetics).
- Statistical analysis
Statistical analyses were conducted using SPSS version 22.0. Data expressed as the mean ± standard error. The normality of the data was evaluated using a normal distribution graph. The mean of continuous data was analyzed using an analysis of variance. Two-way ANOVA was used to compare the groups. Values of p < 0.05 were considered indicative of statistically significant differences.