Transplanted oligodendrocyte progenitor cells improve neurological defects in a rat model of preterm white-matter injury during the sequela phase

Introduction

Preterm white-matter injury (PWMI) is one of the primary contributors to mortality and morbidity in preterm infants, and no specific or effective treatment for this condition has been developed (1). Despite advancements in neonatal intensive care, the incidence of complications such as white-matter injury (WMI) remains high (2). Approximately 25–50% surviving premature infants develop neurological impairment, including sensory disorders, cognitive dysfunction, and behavioral issues, which pose an increased risk of mental health disorder (3-6). Severe motor disabilities, such as cerebral palsy, affect 5–10% of these infants, and lead to cognitive deficit, reduced physical function, and psychological disorder (2).

The primary pathological feature of PWMI is disrupted myelination due to impaired differentiation and maturation of oligodendrocyte progenitor cells (OPCs) (7). Between the 23rd and 32nd gestational weeks, OPCs and premyelinating oligodendrocytes (pre-OLs) dominate the fetal white matter. Preterm infants exposed to inflammation or hypoxia during this period are at risk of oligodendrocyte (OL) differentiation defects and myelination failure, leading to persistent neurological dysfunction and progression to the sequela phase. Therefore, as transplanted cells can replace damaged OPCs and restore myelination, OPC transplantation may be a promising therapeutic strategy (8-10). Studies have demonstrated the therapeutic potential of OPCs in animal experimental models of white-matter dysplasia and spinal cord injury (8,11).

The research on PWMI treatment has primarily focused on the acute phase, with limited studies targeting the sequela phase (12,13). Currently, there is no effective treatment for PWMI sequelae with persistent neurological deficits. Cell therapy, particularly with OPCs, holds considerable promise for neurological disease treatment. In contrast to the neural stem cells (NSCs) and mesenchymal stem cells (MSCs) used in some WMI models (14,15), OPCs have a unique ability to self-renew and differentiate into myelin-generating OLs—a characteristic absent in other cell types (16,17). Moreover, PWMI sequela models better align with clinical needs, as acute-phase treatments are mostly symptomatic and do not typically include cell therapy (18-20). Traditional cell delivery methods, such as systemic administration and intraventricular injection, face challenges due to the blood-brain barrier (BBB). Systemic delivery often limits donor cell entry into the central nervous system (CNS), while intraventricular injection often provides limited cell migration to the target region (21). In contrast, intraparenchymal injection directly targets the damaged white matter by bypassing the BBB, thus enhancing therapeutic potential.

In this study, a rat model in the sequela stage of PWMI was optimized and established. The purpose of this study was to clarify the effect of the intracerebral parenchymal transplantation of human oligodendrocyte progenitor cells (hOPCs) on myelin regeneration and neurobehavioral outcomes in rats. Additionally, the patterns of migration and differentiation of hOPCs within the injured brains of rats were observed, with the ultimate aim of providing evidence-based support for future clinical research on hOPC transplantation. We present this article in accordance with the ARRIVE and MDAR reporting checklists (available at https://tp.amegroups.com/article/view/10.21037/tp-2025-175/rc).

Methods

Optimization and establishment of a rat model of PWMI sequelae

All animal experiments were performed under a project license (No. 221228-SWDWF-004) granted by the institutional Animal Ethics and Welfare Committee of the Beijing Center for Physical and Chemical Analysis, in compliance with national guidelines for the care and use of animals. Specific pathogen-free (SPF) Sprague-Dawley (SD) rats were procured from Sibeifu (Beijing) Biotechnology Co., Ltd. [Animal Use License No. SCXK(Jing) 2024-0001]. A protocol was prepared before the study without registration. Animal use and suffering were minimized. A modified Rice-Vannucci model was used to induce hypoxia-ischemia (HI) in 48 SD rats of both sexes, all of which were at postnatal day 3 (P3) (22). Briefly, pups were anesthetized on ice for a duration of 10–15 minutes. Subsequently, the right common carotid artery was separated and permanently blocked via electrocoagulation. After 90 minutes of recovery, the pups were exposed to 6% oxygen for 120 minutes at 37 ℃. The 32 surviving rats were equally randomized to a PWMI group and a PWMI + hOPC group. Additionally, 10 other animals that underwent sham operations, during which there was neither artery occlusion nor occurrence of hypoxia, were used as the control specimens.

PWMI was confirmed via pathological staining 3 weeks after HI and was consistent with PWMI sequelae.

Preparation of hOPCs

The hOPCs were prepared and furnished by the Pediatric Laboratory at The Sixth Medical Center of PLA General Hospital (Beijing, China). This study was conducted in accordance with the Declaration of Helsinki and its subsequent amendments. All studies involving human participants underwent a thorough review and received approval from the Ethics Committee of The Sixth Medical Center of PLA General Hospital (ethical approval No. 2015013). Prior to their participation, the patients or participants were provided with comprehensive information regarding the study and voluntarily submitted their written informed consent. In brief, human neural stem cells (hNSCs) were extracted from the cortex of human embryos that were between 10 and 13 weeks old. We carried out a karyotype analysis on the provided cells, and the result was XY. The cell line is derived from human cells, and our accession number is NSC-9-OPC. These hNSCs were then propagated in NSC medium. After being cultured for a period of 10 days, they formed neurospheres. Subsequently, these neurospheres were dissociated and induced to differentiate toward hOPCs (23). The OPC purity (80–90%) was confirmed via staining with PDGFR-α, A2B5, SOX10, NG2, and Olig2 (24). Prior to transplantation, hOPCs were redispersed in 4 µL of phosphate-buffered saline (PBS), with a cell density of 5×10⁵ cells.

Transplantation of hOPCs

On postinjury day 22, hOPCs were injected into the brain parenchyma of the rats in the PWMI sequelae + hOPC group. The animals were anesthetized through administration of isoflurane, with an induction concentration of 2.5% and a maintenance concentration of 1.5%. They were then fixed in a stereotactic frame, and an incision was made on the scalp, with the following coordinates (in relation to the bregma) being employed: an anteroposterior position of –0.8 mm, a mediolateral position of –2.8 mm, and a dorsoventral position of –2.5 mm (near the corpus callosum, ipsilateral to the carotid ligation). A dental drill was used to penetrate the skull at the marked site. A microinjector was inserted to deliver 5×10⁵ cells (4 µL) at a rate of 0.5 µL/min over 8 minutes. The injector remained in its position for another 5 minutes before it was gradually pulled out. The rat was positioned on a heating blanket so that it could recuperate and was later taken back to its cage. Meanwhile, the rats in the PWMI sequelae group were given a corresponding volume (4 µL) of PBS at the above-described coordinates. In both groups, all rats were administered intraperitoneal injections of Sandimmun (cyclosporine A; Novartis, Basel, Switzerland) at a rate of 10 mg/kg/day starting 3 days before transplantation, which was continued for 4 weeks post-surgery. To mitigate the risk of rejection, cyclosporine A was subsequently administered in drinking water at a strength of 100 µg/mL throughout the duration of the experiment.

Neurobehavioral test

At 9–10 weeks’ post-transplantation, the Morris water maze test, a frequently used test to assess an individual’s capacity for spatial learning and memory (12,25), was performed at the same time period in the morning to observe changes in behavior of the rats. The test was conducted by a well-experienced researcher and an assistant, neither of whom had any knowledge of the experimental plan. In brief, the test took place in a circular pool that measured 150 cm in diameter and 50 cm in height. The pool was filled with water to a depth of 30 cm, and the water temperature was maintained at 22±2 ℃. A submerged platform, which had a diameter of 12 cm, was positioned in the second quadrant of the pool. The rats were first acclimated to the test room 1 day prior to minimize the impact of stress on the results of behavioral tests. During the navigation test, each rat participated in 4 daily trials over a period of 4 days, starting from different positions. Throughout the experiment, it was observed that most rats could locate the platform by day 3, and consequently, the navigation test was reduced from 5 to 4 days. The trials ended when the rats remained on the platform for 3 seconds; if they had not located the platform after 60 seconds, they were guided to it. On day 5, a probe test lasting 60 seconds was carried out after the platform had been removed. Data were analyzed using Labmaze v3.0 software (Zhongsheng Beidong, Beijing, China). Parameters including escape latency, platform crossings, and entries into the target quadrant were recorded for analysis.

Tissue collection and processing

Thirteen weeks following the transplantation, the rats were thoroughly anesthetized with 10% chloral hydrate. Subsequently, they underwent perfusion, first with 0.9% saline and then with 4% paraformaldehyde (PFA). The excised brains were immersed in 4% PFA for a duration of 24 hours for post-fixation. Following this, they were cryoprotected successively in solutions of 15% and 30% sucrose. Finally, a CM1850 cryostat (Leica Biosystems, Nussloch, Germany) was employed to coronally section the brains at a thickness of 8 µm. Sections corresponding to Bregma levels –0.48, –1.44, –2.40, –3.36, and +0.48 mm were treated with the hematoxylin-eosin (HE) and Luxol fast blue (LFB) staining techniques (26).

Immunohistochemistry and imaging

The cryosections underwent a blocking step for 1 hour using PBS supplemented with 2% bovine serum albumin and 0.3% Triton X-100. They were then left to incubate throughout the night at 4 ℃ with the primary antibodies including anti-stem121 (diluted 1:500; mouse monoclonal antibody; Takara Bio, Kusatsu, Japan) to detect transplanted human cells, anti-glial fibrillary acidic protein (GFAP) (diluted 1:1,000; rabbit polyclonal antibody; Abcam, Cambridge, UK) to detect astrocytes, anti-Olig2 (diluted 1:200; rabbit polyclonal antibody; MilliporeSigma, Burlington, MA, USA) to detect OLs, anti-myelin basic protein (MBP) (diluted 1:100; rabbit polyclonal antibody; Abcam) for myelin staining, and anti-NeuN (diluted 1:1,000; rabbit polyclonal antibody; MilliporeSigma) for neuronal differentiation. Following this, the sections were rinsed with PBS and then incubated for 1 hour at 37 ℃ with the corresponding Alexa Fluor-488- or Fluor-594-conjugated secondary antibodies. Immediately after the incubation, images of the stained sections were promptly captured using a fluorescence microscope (Olympus IX51, Olympus, Tokyo, Japan). Specifically, sections were obtained at Bregma levels −0.48, −1.44, −2.40, −3.36, and +0.48 mm. To measure the capacity of hOPCs to differentiate into Olig2-positive OLs, the percentage of cells that were positive for both stem121 and Olig2 was calculated. For each animal, images were obtained from three fields of view under a 40× objective lens for every section. To assess the extent of differentiation of hOPCs into MBP-positive OLs or GFAP-positive astrocytes, the proportion of area that cells were positive for both stem121 and MBP or positive for both stem121 and GFAP in relation to the total number of stem121-positive cells was quantified. This quantification was carried out using ImageJ software (US National Institutes of Health, Bethesda, MD, USA). For each animal, in accordance with the same criteria described above, images were obtained from nine fields of view under a 40× objective lens.

MBP immunostaining was performed using a rabbit monoclonal antibody (1:100; Abcam) to evaluate the intactness of the host’s myelin. The secondary antibody employed was conjugated goat anti-rabbit immunoglobulin G (IgG) (1:200; Abcam). In order to assess the intensity of MBP in the ipsilateral cingulate gyrus, region-specific images were obtained from the approximate bregma level of –0.48 mm in each animal. Subsequently, the fluorescence intensity of these images was analyzed via ImageJ software.

Transmission electron microscopy

The anterior transplant-site-adjacent corpus callosum (around 1 mm × 1 mm × 1 mm) was dissected and fixed at 4 ℃ within a solution consisting of 2.5% glutaraldehyde and 2% PFA. Subsequently, the samples underwent embedding in epoxy resin. An ultrathin microtome was used to slice the samples, which were then stained with uranium acetate and lead citrate. Imaging with a TEM (Hitachi, Tokyo, Japan) revealed detailed ultrastructural characteristics of ultrathin sections for further analysis. The g-ratio within the TEM images was calculated by dividing the axon diameter by the sum of the axon diameter and the thickness of its myelin sheath via ImageJ software. A lower g-ratio corresponds to a thicker myelin sheath. For quantification, 50 myelinated axons were randomly selected at 6,000× magnification per animal, with three animals per group.

Statistical analysis

Statistical analyses were performed using GraphPad Prism 8 (Dotmatics, Boston, MA, USA). Data were independently evaluated by blinded investigators unaware of experimental group assignments. The results are expressed as the mean ± standard deviation (SD). For Morris water maze continuous variables, repeated-measures two-way analysis of variance (ANOVA) with least significant difference (LSD) post hoc analysis was applied as appropriate. Meanwhile, one-way ANOVA was used for the other datasets. A P value <0.05 indicated statistical significance.

Results

Successful establishment of the sequela phase model of PWMI in rats

Hypoxia-related manifestations including respiration, skin color, and limb mobility were evaluated immediately after modeling, and varying degrees of impairment were observed. Three weeks after modeling, four rats were selected randomly for HE staining before cell transplantation, and the result showed moderate-to-severe brain tissue abnormalities, as indicated by numerous neuronal cells in the cerebral cortex, extensive neuronal degeneration, darker staining, fewer Nissl bodies, and dense tissue structures with no edema or inflammatory cell infiltration (Figure 1). This suggested that the brain structure had progressed beyond the acute phase of inflammatory edema by 3 weeks after modeling, with only occasional microglial cells being present, a distinctive feature as compared to other studies. Further validation of the model was implemented via behavioral tests and tissue analysis.

Figure 1 Three weeks after model induction, hematoxylin-eosin staining under 20× magnification revealed significant brain tissue abnormalities. In the hippocampal region, neuronal cell arrangement was disrupted, showing clear signs of degeneration. Neuronal cell bodies appeared pycnotic and deeply stained, with Nissl bodies absent (as marked by red arrows). Despite these changes, the tissue structure remained compact, with no edema or inflammatory cell infiltration. The black arrow indicates microglia cells.

Transplanted hOPCs migrated within the rat brain during the sequela phase of PWMI

Thirteen weeks following transplantation, during the PWMI sequela phase in rats, stem121-positive cells were found to be extensively dispersed within the damaged white-matter area of the rat brain (Figure 2A). These cells migrated to the contralateral cingulate gyrus and external capsule via the corpus callosum, with scattered cells also observed in the subcortical layer and hippocampal umbrella-shaped gyrus (Figure 2B-2H). The highest density of stem121-positive cells was near the transplantation site, with more cells on the grafted side than on the contralateral side (Figure 2G). Notably, hOPCs displayed distinct morphologies depending on their location. Cells in the subcortical layer were round with radial processes corresponding to type I morphology (Figure 2G,2H). In contrast, cells localized to the corpus callosum, external capsule, and hippocampal regions exhibited fusiform morphology with extensive parallel projections, consistent with the characteristics of type II/III OPCs (Figure 2C-2E). Surviving cells in the brain, most migrating cells in the corpus callosum exhibited a spindle-shaped morphology (27) (Figure 2C).

Figure 2 Migration of transplanted hOPCs. (A) Schematic illustration of the migration location of hOPCs. The letter labels on (A) correspond one-to-one with the alphabetical order of the images. (B) Thirteen weeks after transplantation, stem121⁺ cells in rats with PWMI sequelae were extensively dispersed throughout the damaged white matter and exhibited migration toward the contralateral side of the brain through the corpus callosum. Scale bar: 200 µm. (C) The cells within the corpus callosum exhibited a spindle-shaped morphology, accompanied by long, parallel cellular processes. (D-F) The cells that migrated to the contralateral corpus callosum, external capsule, and hippocampal regions also showed a spindle-shaped morphology. (G,H) Stem121⁺ cells in the subcortical layer and around the transplantation site appeared round with extensive radial processes. Every coronal section was subjected to counterstaining using DAPI. Scale bar of magnified images: 50 µm (C-H). The staining in (B-H) shows stem121 immunofluorescence (mouse, TaKaRa, Y40410) with DAPI counterstaining. hOPCs, human oligodendrocyte progenitor cells; PWMI, preterm white-matter injury.

Transplanted hOPCs improved myelination by differentiating into mature OLs within the rat brain with PWMI sequelae

To assess the differentiation status of transplanted cells, double immunofluorescence staining was performed. Quantitative analysis revealed that 83.34%±3.81% of stem121-positive cells co-expressed OL progenitor marker Olig2. Moreover, the surface area ratio of stem121-positive or MBP-positive cells to total stem121-positive cells was 77.15%±3.06%, while that of stem121- and GFAP-positive or stem121-positive cells was only 1.05%±0.97% (Figure 3), indicating that most transplanted cells matured into OLs and produced MBP in the damaged white matter, with minimal differentiation into astrocytes. NeuN staining in some rat brains revealed no significant neuronal differentiation of stem121-positive cells, further confirming that the transplanted hOPCs primarily differentiated into OLs and contributed to improved myelination.

Figure 3 The differentiation of the transplanted hOPCs (immunofluorescence staining). Representative images captured at 13 weeks after transplantation indicated that approximately 80% of the transplanted cells expressed both Olig2 and MBP. In contrast, there was barely discernible containing of stem121+ cells with GFAP or NeuN (n=5 per group; for each rat brain, three sampling sites were randomly selected). The staining order is sequential: MBP (Rb, Abcam, ab124493), Olig2 (Rb, Millipore, AB9610), GFAP (Rb, Abcam, ab7260), and NeuN (Rb, Millipore, ABN78). They were all double-stained with stem121 (mouse, TaKaRa, Y40410). All panels are counterstained with DAPI for nuclear labeling. The white arrows denote the representative differentiation status of the transplanted cells within the rat brain. GFAP, glial fibrillary acidic protein; hOPCs, human oligodendrocyte progenitor cells; MBP, myelin basic protein; Rb, rabbit.

At 13 weeks after transplantation, MBP fluorescence intensity measurement showed that MBP intensity was reduced in both the cerebral hemispheres of rats with HI injury examined as compared to controls (Figure 4A). Quantitative analysis showed that the MBP intensity in the ipsilateral cingulate gyrus of HI-injured rats was significantly lower than that in the controls (P<0.001; LSD). It was found that the rats in the PWMI sequelae + hOPC group exhibited a significantly higher MBP intensity as compared to those in the PWMI group (P=0.002; LSD) but had a lower intensity as compared to controls (P=0.02; LSD) (Figure 4B).

Figure 4 Immunohistochemistry, LFB staining, and TEM analysis of myelin. (A,C,D) Representative images of MBP immunohistochemistry (scale bar: 200 µm) (A), LFB-stained myelin in the cingulate gyrus (20× magnification) (C), and TEM of the corpus callosum myelin (scale bar: 5 µm; inset: 500 nm) across groups 13 weeks after transplantation (D). (B) Quantified MBP fluorescence intensity in the cingulate gyrus (n=5 per group; 3 sections per rat. One-way ANOVA with LSD post hoc test was used). (E,F) Analysis of the g-ratio of myelinated axons within the corpus callosum (n=3 per group; one-way ANOVA with LSD post hoc test). Data are expressed as mean ± SEM. *, P<0.05; **, P<0.01; ***, P<0.001. ANOVA, analysis of variance; hOPCs, human oligodendrocyte progenitor cells; LSD, least significant difference; MBP, myelin basic protein; PWMI, premature white-matter injury; SEM, standard error of the mean; TEM, transmission electron microscopy.

LFB staining at 13 weeks post-transplantation revealed that myelin in PWMI rats was lighter, more swollen, and sparser as compared to that in controls. In contrast, PWMI rats receiving hOPC transplantation showed deeper staining and denser myelin morphology, suggesting a tendency toward myelin repair (Figure 4C).

TEM showed that the myelin sheaths in the corpus callosum of control animals were thick and compact, while those in rats with PWMI sequelae were sparse with thin and disrupted structures. After hOPC transplantation, myelin density and thickness were improved in rats with PWMI sequelae, although they remained inferior to those in controls (Figure 4D). These findings were supported by g-ratio quantification (control: 0.614±0.051; PWMI: 0.768±0.065; PWMI + hOPC: 0.632±0.049) (PWMI sequelae vs. control: P<0.001; PWMI sequelae + hOPC vs. PWMI sequelae: P<0.001; PWMI sequelae + hOPC vs. control: P=0.04; LSD) (Figure 4E,4F). Overall, these results indicate that the transplanted hOPCs managed to partly recover the myelin integrity in PWMI sequela rats.

Transplanted hOPCs alleviated the neurobehavioral defects in rats with PWMI sequelae

In the Morris water maze test, it was observed that the three groups of animals exhibited varying degrees of memory and learning abilities during the navigation test and spatial probe trials (Figure 5A). Starting from day 2 of the Morris water maze test, the PWMI sequela model rats required a significantly longer duration to locate the platform as compared to those in the control group (day 2: P<0.001; day 3: P<0.001; day 4: P<0.001), indicating spatial learning impairment. However, PWMI rats receiving hOPC transplantation showed near-normal platform-finding times (day 2: P=0.01; day 3: P=0.04; day 4: P=0.02; LSD; Figure 5B). As over 90% of the rats located the platform by day 3, the test duration was reduced from 5 to 4 days. The spatial probe test showed that PWMI model rats required significantly fewer attempts to cross the platform and platform quadrant than did the controls (platform crosses: P=0.004; quadrant crosses: P=0.003; LSD; Figure 5C,5D). hOPC transplantation increased the number of crossings the platform and platform quadrant in PWMI rats. These findings demonstrated that hOPC transplantation improved motor and cognitive deficits in rats with PWMI sequelae.

Figure 5 Assessment of learning and memory abilities. (A) Representative swimming paths in the Morris water maze test across groups. (B) In the navigation trial, rats with PWMI sequelae showed increased platform latency, which normalized after hOPC transplantation (control: n=10; PWMI sequelae and PWMI sequelae + hOPCs: n=13; repeated-measures ANOVA with LSD post hoc test). (C,D) In the spatial probe trial, PWMI sequelae rats made fewer crossings of the platform and its quadrant, but this increased post-transplantation (control: n=10; PWMI sequelae and PWMI sequelae + hOPCs: n=13; one-way ANOVA with LSD). Data are the mean ± SEM. *, P<0.05; **, P<0.01; ***, P<0.001. ANOVA, analysis of variance; hOPCs, human oligodendrocyte progenitor cells; LSD, least significant difference; PWMI, preterm white-matter injury; SEM, standard error of the mean.

Discussion

Building upon prior studies conducted by our research team, we optimized and established a rat model of PWMI sequelae and transplanted hOPCs into the brain parenchyma (12,28), where they differentiated into mature OLs, increasing myelinated nerve fibers and myelin thickness, thereby improving neurological deficits. Unlike previous studies that focused on the acute phase (12,13), we targeted the sequela phase by observing differentiation into myelin-forming OLs 13 weeks’ post-transplantation. Our findings constitute preliminary clinical evidence supporting hOPC transplantation in PWMI sequela treatment.

Globally, 15 million premature infants are born each year, with 5–10% experiencing severe motor dysfunction, cognitive impairment, or cerebral palsy, which are known to be the leading causes of neurodevelopmental disability. Premature infants under 32 gestational weeks or with very low birth weight account for 16% of these cases (29-31). Advances in perinatal medicine have increased survival rates but have also exacerbated PWMI issues. In humans, PWMI susceptibility peaks between 23 and 32 gestational weeks, when brain white matter consists primarily of late OPCs. These exhibit a high degree of sensitivity to oxidative stress. As a result, the primary characteristic of brain WMI in premature infants is the depletion of OPCs. This stage is equivalent to the P2–P5 period in rats, and we thus opted for P3 to conduct HI treatment as a means of mimicking PWMI (7,32).

Previous studies have showed that ischemia (induced by unilateral or bilateral carotid artery transection) accompanied with hypoxia (sustained for 0.5 to 3.5 hours under 5–8% oxygen concentration) in neonatal rats causes myelination defects and neurological impairments (32,33). HE staining in our study showed that 2-hour hypoxia could induce the desired injury level. The sequela stage, occurring beyond 3 weeks after injury, is characterized by demyelination and glial scar formation (34,35). These pathological findings align with the magnetic resonance imaging observations in our model at 3 weeks post-hypoxic-ischemic injury, which showed ventricular enlargement and cystic degeneration of white matter. Research involving TEM and immunohistochemistry has identified pronounced myelin degradation and pathological remodeling of axonal architecture during the observation phase. Unlike previous studies that predominantly focused on the acute phase (3–7 days’ postinjury) for cell transplantation therapy, our study observed the late phase in order to examine the potential benefits of inflammatory factor chemotaxis, endogenous stem cell mobilization, and cytokine paracrine effects, among others (8-12). The sequelae phase of PWMI represents a chronic stage following acute injury, characterized by impaired OL maturation, axonal damage, and glial scar formation. During this phase, acute inflammatory responses subside, but astrocyte activation and extracellular matrix remodeling may inhibit remyelination. Although pre-OLs survive in this stage, they fail to differentiate into mature OLs, leading to myelination deficits. This stagnation is associated with interactions between high-molecular-weight hyaluronic acid (HA) secreted by astrocytes and its receptor CD44. While the glial scar limits inflammatory spread, its secretion of inhibitory factors [e.g., abnormal bone morphogenetic protein (BMP) and Wnt/β-catenin signaling pathways] impedes pre-OL differentiation and axonal regeneration (36). Additionally, chronic WMI is often accompanied by axonal degeneration, further reducing targets for myelination. However, in clinical practice, cell transplantation therapy during the acute phase is rarely implemented due to limitations imposed by conventional treatment recommendations (7). Instead, the majority of exploratory treatments are administered during the sequela stage. Thus, conducting cell therapy research in the sequela stage would more closely align with actual clinical needs. Notably, the rodent model recapitulated two hallmark features of human PWMI sequelae—region-specific neuropathological alterations and persistent sensorimotor/cognitive deficits—establishing its construct validity for preclinical studies.

Transplanting OPCs is a promising strategy for treating PWMI neurological deficits. OPCs excel in migration and differentiation, making them ideal for myelination. Other cell types, including NSCs, exhibit a low degree of differentiation into OLs and myelin formation. Furthermore, MSCs and other types of cells are limited in their ability to generate myelin in the CNS and thus do not exert particularly strong cell replacement effects when transplanted into the brain (37). A study has shown that exogenous OPCs can integrate into the recipient’s brain tissue and further mature into myelinating OLs, encapsulating axons to restore the structure of white matter (38). Research has also demonstrated that in rat models of WMI, lateral ventricle injection of hOPCs can restore the white-matter structure, increase myelin thickness, and improve learning and memory abilities (8,11). In our study, hOPCs were able to survive in the brain, where they differentiated into mature OLs and alleviated white-matter damage in rats with PWMI sequelae. We used water maze tests to assess memory and learning abilities (39) and found them to be improved post-transplantation. We propose that the observed functional deficits stem not merely from PWMI but also reflect concurrent gray-matter pathology within the corticomotor network. Notably, our PWMI animal model exhibited microstructural alterations in both axonal tracts and cortical layer, suggesting a dual-mechanism pathogenesis underlying these neurological manifestations.

Our research provides the first evidence of sustained hOPC survival (>13 weeks’ post-transplantation) in a rat model of sequelae, in which the cells migrated to injury areas via fiber tracts of the corpus callosum. hOPCs were extensively distributed throughout the brain white-matter regions, including the corpus callosum, external capsule, striatum, and deep subcortical areas. The observed migration of hOPCs may be attributed to the chemotactic effect produced by the damaged white-matter tissue (12). Dual immunofluorescence staining showed that hOPCs differentiated into MBP-expressing mature OLs, while TEM and LFB staining revealed myelin repair. Webber et al. implanted rat-derived OPCs in animals with periventricular leukomalacia and found a relatively low proportion of these cells differentiating into mature Ols (40). Li et al. reported that the ability of rat-derived OPCs to generate OLs in a middle cerebral artery occlusion model was quite limited (41). Compared to these in previous studies (40,41), our hOPCs showed longer survival and higher differentiation rates, probably due to the cell source and transplantation timing. We used hOPCs induced from human fetal NSCs, which differ from embryonic stem cells or OPCs of murine origin. Notably, nearly 90% of hOPCs in our study had sustained expression of PDGFR-α, A2B5, and O4, which corresponds to the phenotype exhibited by late OPCs. In contrast, Webber et al. utilized early OPCs expressing NG2, Olig2, and A2B5 (40). Transplanting at the sequela stage, when inflammation is minimal, may enhance the ability of hOPCs to overcome microenvironment constraints and also avoid the inhibitory effect of the microenvironment in the WMI area on OPC differentiation (42). Additionally, unlike a previous study on cellular therapy for PWMI that typically selected treatment time points within 3 days or even earlier after injury—which corresponds to the neonatal stage in humans (43)—the therapeutic time window chosen in our study was 25 days after birth for rats. At this stage, SD rats have reached sexual maturity, equivalent to near adulthood in humans, and are therefore more prone to immune rejection. Furthermore, we administered cyclosporine starting 3 days prior to transplantation and continued until the study’s conclusion to guarantee the survival of exogenous cells. Other research has also proposed that cyclosporine therapy can mitigate xenogeneic cell rejection and extend the survival duration of transplanted cells (44).

The effect of hOPCs in WMI areas likely involves multiple mechanisms. The initial goal of cell therapy is to replace the damaged tissue through cellular substitution. In our study, immunofluorescence staining, LFB staining (for gross morphological analysis), and TEM revealed that transplanted hOPCs differentiated into mature OLs and directly participated in myelin repair and remyelination. Additionally, objective behavioral assessments indicated that hOPC transplantation significantly improved neurobehavioral performance in rats with PWMI sequelae. Myelin repair promotes neural conduction efficiency, while white-matter regeneration facilitates synaptic reorganization. Although the myelin thickness in transplanted rats showed improvement compared to the model group, it still fell short of normal levels. We recognize that the quality of these myelin sheaths may critically influence neurological function in the rats. In our follow-up studies, we plan to optimize transplantation protocols (e.g., adjusting cell dosage) and increase sample sizes to rigorously evaluate both the degree of myelin restoration and its correlation with functional outcomes. These refinements will help clarify the therapeutic potential and limitations of this approach. Collectively, these processes contribute to the recovery of neurological functions. In addition to cellular substitution, hOPCs may mobilize endogenous stem cells and exert paracrine effects. Kishida et al. discovered that OPCs secreted angiogenic factors, including vascular endothelial growth factor (VEGF) and angiopoietin-1, when exposed to hypoxic conditions (45). VEGF, a major regulator of angiogenesis, can nourish neuronal cells, induce revascularization, and improve blood flow to the brain tissue. Yue et al. (13) transplanted hOPCs within 12 hours after hypoxic injury in a fetal sheep model of PWMI and found that HI conditions decreased brain tissue levels of glial cell line-derived neurotrophic factor (GDNF) and brain-derived neurotrophic factor (BDNF), while hOPC transplantation increased these levels. Based on these findings, we speculate that the transplanted hOPCs may prevent myelin loss and promote endogenous myelin formation by exerting a paracrine effect in the local environment.

At 4 weeks’ post-transplantation, we randomly selected the brain tissue surrounding the corpus callosum from three rats in each group for transcriptome sequencing analysis. We found that the myelin-related genes in hOPC transplantation group were upregulated as compared those in the other groups. These genes included Myrf, Olig2, Plp1, Mag, Pmp22, Pllp, Sox10, Sema4d, Wnt3a, Epha8, Inpp5f, and Gata3. It was also found that upregulation of myelin-related genes in rats with hOPC transplantation enriched OL differentiation and myelination pathways. The enriched Kyoto Encyclopedia of Genes and Genomes (KEGG) pathways included GABAergic synapse, axon protection, TGF-β signaling, Notch signaling, VEGF signaling, and regulation of stem cell pluripotency.

Indeed, there remain critical safety concerns regarding OPC therapy for cerebral white matter injury in preterm infants that require special attention before clinical translation. Members of our research team have conducted extensive safety validation in these areas, including toxicology studies (acute and chronic toxicology tests) of transplanted cells, delivery routes, post-transplantation biodistribution in vivo, tumorigenic potential, and biosafety evaluations (46). All of these preclinical safety-related work demand our rigorous scrutiny and meticulous attention.

Although our study produced promising findings, due to limitations such as sample size, further validation has not yet been conducted. Meanwhile, additional confounding factors such as body weight and gender should be incorporated into the analysis to enhance the credibility and robustness of the study. Future research will focus on elucidating the mechanisms by which hOPC transplantation promotes myelin repair in PWMI sequelae and identifying other potential therapeutic targets.

Conclusions

PWMI is one of the most severe diseases affecting the prognosis of premature infants, but there is currently no effective clinical treatment in the sequela phase. In this study, we observed that exogenous hOPCs were able to differentiate into mature OLs in rats during the sequela stage of PWMI. This finding attests to the regenerative capacity of hOPCs in rats with PWMI sequelae. Consequently, hOPC transplantation may be a promising therapeutic approach for managing the sequelae of PWMI in children.

Acknowledgments

None.

Footnote

Reporting Checklist: The authors have completed the ARRIVE and MDAR reporting checklists. Available at https://tp.amegroups.com/article/view/10.21037/tp-2025-175/rc

Data Sharing Statement: Available at https://tp.amegroups.com/article/view/10.21037/tp-2025-175/dss

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Funding: This work received support from the National Key R & D Program of China (No. 2017YFA0104203) and was funded by the Ministry of Science and Technology of the People’s Republic of China.

Conflicts of Interest: All authors have completed the ICMJE uniform disclosure form (available at https://tp.amegroups.com/article/view/10.21037/tp-2025-175/coif). The authors have no conflicts of interest to declare.

Ethical Statement: The authors are accountable for all aspects of the work in ensuring that questions related to the accuracy or integrity of any part of the work are appropriately investigated and resolved. The study was conducted in accordance with the Declaration of Helsinki and its subsequent amendments. All studies involving human participants underwent a thorough review and received approval from the Ethics Committee of The Sixth Medical Center of PLA General Hospital (ethical approval No. 2015013). Prior to their participation, the patients or participants were provided with comprehensive information regarding the study and voluntarily submitted their written informed consent. All animal experiments were performed under a project license (No. 221228-SWDWF-004) granted by the institutional Animal Ethics and Welfare Committee of the Beijing Center for Physical and Chemical Analysis, in compliance with national guidelines for the care and use of animals.

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