Differentiation Of Precursor Cells Research Articles

Themis suppresses the effector function of CD8+ T cells in acute viral infection.

CD8+ T cells play a central role in antiviral immune responses. Upon infection, naive CD8+ T cells differentiate into effector cells to eliminate virus-infected cells, and some of these effector cells further differentiate into memory cells to provide long-term protection after infection is resolved. Although extensively investigated, the underlying mechanisms of CD8+ T-cell differentiation remain incompletely understood. Themis is a T-cell-specific protein that plays critical roles in T-cell development. Recent studies using Themis T-cell conditional knockout mice also demonstrated that Themis is required to promote mature CD8+ T-cell homeostasis, cytokine responsiveness, and antibacterial responses. In this study, we used LCMV Armstrong infection as a probe to explore the role of Themis in viral infection. We found that preexisting CD8+ T-cell homeostasis defects and cytokine hyporesponsiveness do not impair viral clearance in Themis T-cell conditional knockout mice. Further analyses showed that in the primary immune response, Themis deficiency promoted the differentiation of CD8+ effector cells and increased their TNF and IFNγ production. Moreover, Themis deficiency impaired memory precursor cell (MPEC) differentiation but promoted short-lived effector cell (SLEC) differentiation. Themis deficiency also enhanced effector cytokine production in memory CD8+ T cells while impairing central memory CD8+ T-cell formation. Mechanistically, we found that Themis mediates PD-1 expression and its signaling in effector CD8+ T cells, which explains the elevated cytokine production in these cells when Themis is disrupted.

P-15 promotes chondrocyte proliferation in osteoarthritis by regulating SFPQ to target the Akt-RUNX2 axis

BackgroundThe disruption of chondrocyte proliferation and differentiation is a critical event during the process of joint injury in osteoarthritis (OA). P-15 peptides could bind to integrin receptors on various precursor cells, promote cell adhesion, release growth factors, and promote the differentiation of osteoblast precursor cells. However, the role of P-15 in OA, particularly in chondrocyte proliferation, is not fully understood.MethodsThe activity of SFPQ and RUNX2 in the bone tissue of patients with osteoarthritis was analyzed using quantitative real-time polymerase chain reaction (qRT-PCR). Interleukin-1β (IL-1β) inducer was performed to establish an in vitro model of OA. Cell proliferation was measured by CCK-8 assay. The expressions of COL2a1, ACAN, COMP, SOX9, and BMP2 related to cartilage differentiation were detected using qRT-PCR. In addition, the expression levels of SFPQ, AKT, p-AKT, and RUNX2 were detected using Western blotting.ResultsThe results showed that the expression of SFPQ was significantly decreased and the expression of RUNX2 was significantly increased in osteoarthritis cartilage tissue. P-15 peptide reversed IL-1β-induced cell proliferation obstruction and alleviated chondrocyte damage. Furthermore, P-15 polypeptide increased the expression levels of cartilage differentiation genes COL2a1, ACAN, and BMP2, while decreasing the expression of COMP and SOX9 in an inverse dose-dependent manner. Then specific interfering RNA proved that P-15 maintains chondrocyte stability and is associated with the SFPQ gene. Finally, we confirmed that P-15 inhibited the Akt-RUNX2 pathway, which is regulated in the expression of SFPQ.ConclusionsP-15 can mitigate chondrocyte damage and osteoarthritis progression by inhibiting cell death and modulating SFPQ-Akt-RUNX2 pathway, offering an opportunity to develop new strategies for the treatment of osteoarthritis.

The progress in the relationship between trace elements and acute lymphoblastic leukemia.

Trace elements are very important substances with low content in the human body. If the content of some trace elements changes, they are also related to diseases. Acute lymphoblastic leukemia (ALL) is a malignant blood tumor, and its relationship with trace elements has also been a concern by scholars. Not only have the trace element levels in ALL patients changed, but the efficacy of different treatment methods has also been linked to the corresponding trace element changes. The characteristics of ALL may be related to the dysregulation of differentiation and proliferation of lymphoid precursor cells. Cell proliferation and differentiation are often affected by changes in DNA levels. However, trace elements are involved in DNA damage and repair mechanisms. In recent years, as an increasing number of studies believe that ALL is related to the abnormal metabolism of trace elements in the body, this paper intends to discuss the research progress on the relationship between trace elements and ALL to provide more information on trace elements for the diagnosis, treatment, and prevention of ALL.

Composite Coatings Based on Recombinant Spidroins and Peptides with Motifs of the Extracellular Matrix Proteins Enhance Neuronal Differentiation of Neural Precursor Cells Derived from Human Induced Pluripotent Stem Cells

The production and transplantation of functionally active human neurons is a promising approach to cell therapy. Biocompatible and biodegradable matrices that effectively promote the growth and directed differentiation of neural precursor cells (NPCs) into the desired neuronal types are very important. The aim of this study was to evaluate the suitability of novel composite coatings (CCs) containing recombinant spidroins (RSs) rS1/9 and rS2/12 in combination with recombinant fused proteins (FP) carrying bioactive motifs (BAP) of the extracellular matrix (ECM) proteins for the growth of NPCs derived from human induced pluripotent stem cells (iPSC) and their differentiation into neurons. NPCs were produced by the directed differentiation of human iPSCs. The growth and differentiation of NPCs cultured on different CC variants were compared with a Matrigel (MG) coating using qPCR analysis, immunocytochemical staining, and ELISA. An investigation revealed that the use of CCs consisting of a mixture of two RSs and FPs with different peptide motifs of ECMs increased the efficiency of obtaining neurons differentiated from iPSCs compared to Matrigel. CC consisting of two RSs and FPs with Arg-Gly-Asp-Ser (RGDS) and heparin binding peptide (HBP) is the most effective for the support of NPCs and their neuronal differentiation.

Effect of Vancomycin Applied to the Surgical Site on Fracture Healing in a Diabetic Rat Model.

Prophylactic vancomycin treatment decreases the prevalence of surgical site and deep infections by >70% in diabetic patients undergoing reconstructive foot and ankle surgery. Thus, determining whether clinically relevant local vancomycin doses affect diabetic fracture healing is of medical interest. We hypothesized that application of vancomycin powder to the fracture site during surgery would not affect healing outcomes, but continuous exposure of vancomycin would inhibit differentiation of osteoblast precursor cells and their osteogenic activity in vitro. The vancomycin dose used to treat the diabetic rats was a modest increase to routine surgical site vancomycin application of 1 to 2 g for a 70-kg adult (21 mg/kg). After femur fracture in BB-Wistar type 1 diabetic rats, powdered vancomycin (25 mg/kg) was administered to the fracture site. Bone marrow and periosteal cells isolated from diabetic bones were cultured and treated with increasing levels of vancomycin (0, 5, 50, 500, or 5000 µg/mL). Radiographic scoring, micro-computed tomography (µCT) analysis, and torsion mechanical testing failed to identify any statistical difference between the vancomycin-treated and the untreated fractured femurs 6 weeks postfracture. Low to moderate levels of vancomycin treatment (5 and 50 µg/mL) did not impair cell viability, osteoblast differentiation, or calcium deposition in either the periosteum or bone marrow-derived cell cultures. In contrast, high doses of vancomycin (5000 µg/mL) did impair viability, differentiation, and calcium deposition. In this diabetic rodent fracture model, vancomycin powder application at clinically relevant doses did not affect fracture healing or osteogenesis.

TREM2 mediates physical exercise-promoted neural functional recovery in rats with ischemic stroke via microglia-promoted white matter repair

BackgroundThe repair of white matter injury is of significant importance for functional recovery after ischemic stroke, and the up-regulation of triggering receptors expressed on myeloid cells 2 (TREM2) after ischemic stroke is neuroprotective and implicated in remyelination. However, the lack of effective therapies calls for the need to investigate the regenerative process of remyelination and the role of rehabilitation therapy. This study sought to investigate whether and how moderate physical exercise (PE) promotes oligodendrogenesis and remyelination in rats with transient middle cerebral artery occlusion (tMCAO).MethodsMale Sprague–Dawley rats (weighing 250–280 g) were subjected to tMCAO. AAV-shRNA was injected into the lateral ventricle to silence the Trem2 gene before the operation. The rats in the physical exercise group started electric running cage training at 48 h after the operation. The Morris water maze and novel object recognition test were used to evaluate cognitive function. Luxol fast blue staining, diffusion tensor imaging, and electron microscopy were used to observe myelin injury and repair. Immunofluorescence staining was applied to observe the proliferation and differentiation of oligodendrocyte precursor cells (OPCs). Expression of key molecules were detected using immunofluorescence staining, quantitative real-time polymerase chain reaction, Western blotting, and Enzyme-linked immunosorbent assay, respectively.ResultsPE exerted neuroprotective efects by modulating microglial state, promoting remyelination and recovery of neurological function of rats over 35 d after stroke, while silencing Trem2 expression in rats suppressed the aforementioned effects promoted by PE. In addition, by leveraging the activin-A neutralizing antibody, we found a direct beneficial effect of PE on microglia-derived activin-A and its subsequent role on oligodendrocyte differentiation and remyelination mediated by the activin-A/Acvr axis.ConclusionsThe present study reveals a novel regenerative role of PE in white matter injury after stroke, which is mediated by upregulation of TREM2 and microglia-derived factor for oligodendrocytes regeneration. PE is an effective therapeutic approach for improving white matter integrity and alleviating neurological function deficits after ischemic stroke.

Bicuculline and Bumetanide Attenuate Sevoflurane-Induced Impairment of Myelination and Cognition in Young Mice.

Sevoflurane (Sevo) is one of the most commonly used general anesthetics for infants and young children. We investigated whether Sevo impairs neurological functions, myelination, and cognition via the γ-aminobutyric acid A receptor (GABAAR) and Na+-K+-2Cl- cotransporter (NKCC1) in neonatal mice. On postnatal days 5-7, mice were exposed to 3% Sevo for 2 h. On postnatal day 14, mouse brains were dissected, and oligodendrocyte precursor cell line level lentivirus knockdown of GABRB3, immunofluorescence, and transwell migration assays were performed. Finally, behavioral tests were conducted. Multiple Sevo exposure groups exhibited increased neuronal apoptosis levels and decreased neurofilament protein levels in the mouse cortex compared with the control group. Sevo exposure inhibited the proliferation, differentiation, and migration of the oligodendrocyte precursor cells, thereby affecting their maturation process. Electron microscopy revealed that Sevo exposure reduced myelin sheath thickness. The behavioral tests showed that multiple Sevo exposures induced cognitive impairment. GABAAR and NKCC1 inhibition provided protection against Sevo-induced neurotoxicity and cognitive dysfunction. Thus, bicuculline and bumetanide can protect against Sevo-induced neuronal injury, myelination impairment, and cognitive dysfunction in neonatal mice. Furthermore, GABAAR and NKCC1 may be mediators of Sevo-induced myelination impairment and cognitive dysfunction.

Nerve growth factor promotes differentiation and protects the oligodendrocyte precursor cells from in vitro hypoxia/ischemia.

Nerve growth factor (NGF) is a pleiotropic molecule acting on different cell types in physiological and pathological conditions. However, the effect of NGF on the survival, differentiation and maturation of oligodendrocyte precursor cells (OPCs) and oligodendrocytes (OLs), the cells responsible for myelin formation, turnover, and repair in the central nervous system (CNS), is still poorly understood and heavily debated. Here we used mixed neural stem cell (NSC)-derived OPC/astrocyte cultures to clarify the role of NGF throughout the entire process of OL differentiation and investigate its putative role in OPC protection under pathological conditions. We first showed that the gene expression of all the neurotrophin receptors (TrkA, TrkB, TrkC, and p75NTR ) dynamically changes during the differentiation. However, only TrkA and p75NTR expression depends on T3-differentiation induction, as Ngf gene expression induction and protein secretion in the culture medium. Moreover, in the mixed culture, astrocytes are the main producer of NGF protein, and OPCs express both TrkA and p75NTR . NGF treatment increases the percentage of mature OLs, while NGF blocking by neutralizing antibody and TRKA antagonist impairs OPC differentiation. Moreover, both NGF exposure and astrocyte-conditioned medium protect OPCs exposed to oxygenglucose deprivation (OGD) from cell death and NGF induces an increase of AKT/pAKT levels in OPCs nuclei by TRKA activation. This study demonstrated that NGF is implicated in OPC differentiation, maturation, and protection in the presence of metabolic challenges, also suggesting implications for the treatment of demyelinating lesions and diseases.

Genesis of osteoclasts on calcium phosphate ceramics and their role in material-induced bone formation.

Innate immune responses play important roles in material-induced bone formation and such roles were further explored in the current study with an emphasis on M2 macrophages and osteoclastogenesis. With the presence of M-CSF and RANKL, M0 macrophages from FVB mouse bone marrow-derived monocytes (BMMs) fused to osteoclasts with both M2 marker and osteoclast marker at day 5, and such osteoclast formation at day 5 was enhanced when the cells were treated with IL-4 at day 3. With IL-4 treatment alone for 24 h, M0 polarized into M2 macrophages. Conditioned medium of M2 macrophages enhanced osteogenic differentiation of MC3T3-E1 (pre-osteoblasts) while osteoclast conditioned medium enhanced osteogenic differentiation of CRL-12424 (osteogenic precursors). TCPs (a typical osteoinductive material) supported M2 macrophage polarization at day 4 and osteoclast formation at day 5, while TCPb (a typical non-osteoinductive material) was less effective. Moreover, osteoclasts formed on TCPs produced osteogenic factors including S1P, Wnt10B and BMP-6, resulting osteogenic differentiation of CRL-12424 cells. Similar to in vitro testing, TCPs favored M2 macrophage polarization followed by the formation of osteoclasts in vivo, as compared to TCPb. The overall data provided evidence of a coupling between M2 macrophages, osteoclasts and material-induced bone formation: osteoclasts formed from M2 macrophages secrete osteogenic cytokines to induce osteogenic differentiation of osteogenic precursor cells to finally form bone. The current findings outlined a biological mechanism of material-induced bone formation and further rationalized the use of osteoinductive materials for bone regeneration. STATEMENT OF SIGNIFICANCE: This paper provides evidence for finding out the relationship between M2 macrophages, osteoclasts and osteogenesis in material-induced bone formation. It suggested that osteoinductive materials enhanced macrophage polarization to M2 macrophages which fuses to osteoclasts, osteoclasts subsequently secret osteogenic cytokines to differentiate finally osteogenic precursors to form bone in osteoinductive materials. The data supports scientifically the superiority of osteoinductive materials for bone regeneration in clinics.

Evidence-Based Approach to Orthobiologics for Osteoarthritis and Other Joint Disorders.

Osteoarthritis and cartilage lesions are a major cause of functional limitations which is why the goal of biological treatment is to preserve the native joint to delay the onset of OA. As a result of improvements in surgical techniques and technology, treatment options are more and more available, allowing the treatment of a whole range of injuries, from minor to extensive lesions both acute and chronic. In chondral lesion treatment, restoring hyaline-like cartilage provides improved durability of repaired tissue and desirable wear characteristics. Biological cell-based cartilage restoration treatment was developed to address the need for the long-term viability of repaired tissue. These procedures provide a reliable source of chondrocytes, whether directly or through the differentiation of multipotent precursor cells, capable of producing hyaline-like cartilage, with the minimal formation of fibrocartilage tissue. However, if arthritic changes begin biological therapies offer possibilities to delay. This chapter aims to discuss and give insights into these regenerative, joint preservation techniques for cartilage treatment and possible biological treatment in OA.

Advances in osseointegration of biomimetic mineralized collagen and inorganic metal elements of natural bone for bone repair.

At this stage, bone defects caused by trauma, infection, tumor, or congenital diseases are generally filled with autologous bone or allogeneic bone transplantation, but this treatment method has limited sources, potential disease transmission and other problems. Ideal bone-graft materials remain continuously explored, and bone defect reconstruction remains a significant challenge. Mineralized collagen prepared by bionic mineralization combining organic polymer collagen with inorganic mineral calcium phosphate can effectively imitate the composition and hierarchical structure of natural bone and has good application value in bone repair materials. Magnesium, strontium, zinc and other inorganic components not only can activate relevant signaling pathways to induce differentiation of osteogenic precursor cells but also stimulate other core biological processes of bone tissue growth and play an important role in natural bone growth, and bone repair and reconstruction. This study reviewed the advances in hydroxyapatite/collagen composite scaffolds and osseointegration with natural bone inorganic components, such as magnesium, strontium and zinc.

Teriflunomide as a therapeutic means for myelin repair

BackgroundPromotion of myelin repair in the context of demyelinating diseases such as multiple sclerosis (MS) still represents a clinical unmet need, given that this disease is not only characterized by autoimmune activities but also by impaired regeneration processes. Hence, this relates to replacement of lost oligodendrocytes and myelin sheaths—the primary targets of autoimmune attacks. Endogenous remyelination is mainly mediated via activation and differentiation of resident oligodendroglial precursor cells (OPCs), whereas its efficiency remains limited and declines with disease progression and aging. Teriflunomide has been approved as a first-line treatment for relapsing remitting MS. Beyond its role in acting via inhibition of de novo pyrimidine synthesis leading to a cytostatic effect on proliferating lymphocyte subsets, this study aims to uncover its potential to foster myelin repair.MethodsWithin the cuprizone mediated de-/remyelination model teriflunomide dependent effects on oligodendroglial homeostasis and maturation, related to cellular processes important for myelin repair were analyzed in vivo. Teriflunomide administration was performed either as pulse or continuously and markers specific for oligodendroglial maturation and mitochondrial integrity were examined by means of gene expression and immunohistochemical analyses. In addition, axon myelination was determined using electron microscopy.ResultsBoth pulse and constant teriflunomide treatment efficiently boosted myelin repair activities in this model, leading to accelerated generation of oligodendrocytes and restoration of myelin sheaths. Moreover, teriflunomide restored mitochondrial integrity within oligodendroglial cells.ConclusionsThe link between de novo pyrimidine synthesis inhibition, oligodendroglial rescue, and maintenance of mitochondrial homeostasis appears as a key for successful myelin repair and hence for protection of axons from degeneration.

PPAR Gamma Agonist Leriglitazone Recovers Alterations Due to Pank2-Deficiency in hiPS-Derived Astrocytes

The novel brain-penetrant peroxisome proliferator-activated receptor gamma agonist leriglitazone, previously validated for other rare neurodegenerative diseases, is a small molecule that acts as a regulator of mitochondrial function and exerts neuroprotective, anti-oxidative and anti-inflammatory effects. Herein, we tested whether leriglitazone can be effective in ameliorating the mitochondrial defects that characterize an hiPS-derived model of Pantothenate kinase-2 associated Neurodegeneration (PKAN). PKAN is caused by a genetic alteration in the mitochondrial enzyme pantothenate kinase-2, whose function is to catalyze the first reaction of the CoA biosynthetic pathway, and for which no effective cure is available. The PKAN hiPS-derived astrocytes are characterized by mitochondrial dysfunction, cytosolic iron deposition, oxidative stress and neurotoxicity. We monitored the effect of leriglitazone in comparison with CoA on hiPS-derived astrocytes from three healthy subjects and three PKAN patients. The treatment with leriglitazone did not affect the differentiation of the neuronal precursor cells into astrocytes, and it improved the viability of PKAN cells and their respiratory activity, while diminishing the iron accumulation similarly or even better than CoA. The data suggest that leriglitazone is well tolerated in this cellular model and could be considered a beneficial therapeutic approach in the treatment of PKAN.

Inhibition of CXCR2 enhances CNS remyelination via modulating PDE10A/cAMP signaling pathway

CXC chemokine receptor 2 (CXCR2) plays an important role in demyelinating diseases, but the detailed mechanisms were not yet clarified. In the present study, we mainly investigated the critical function and the potential molecular mechanisms of CXCR2 on oligodendrocyte precursor cell (OPC) differentiation and remyelination. The present study demonstrated that inhibiting CXCR2 significantly enhanced OPC differentiation and remyelination in primary cultured OPCs and ethidium bromide (EB)-intoxicated rats by facilitating the formation of myelin proteins, including PDGFRα, MBP, MAG, MOG, and Caspr. Further investigation identified phosphodiesterase 10A (PDE10A) as a main downstream protein of CXCR2, interacting with the receptor to regulate OPC differentiation, in that inhibition of CXCR2 reduced PDE10A expression while suppression of PDE10A did not affect CXCR2. Furthermore, inhibition of PDE10A promoted OPC differentiation, whereas overexpression of PDE10A down-regulated OPC differentiation. Our data also revealed that inhibition of CXCR2/PDE10A activated the cAMP/ERK1/2 signaling pathway, and up-regulated the expression of key transcription factors, including SOX10, OLIG2, MYRF, and ZFP24, that ultimately promoted remyelination and myelin protein biosynthesis. In conclusion, our findings suggested that inhibition of CXCR2 promoted OPC differentiation and enhanced remyelination by regulating PDE10A/cAMP/ERK1/2 signaling pathway. The present data also highlighted that CXCR2 may serve as a potential target for the treatment of demyelination diseases.

The Generation and Functional Characterization of Human Microglia-Like Cells Derived from iPS and Embryonic Stem Cells.

The following protocol describes the generation of microglia cells from human-induced pluripotent stem cells (hiPSCs) using commercially available kits by StemCell Technologies. This protocol consists of three major steps: (1) Differentiation of hematopoietic precursor cells, (2) Microglia differentiation, and (3) Microglia maturation. Assays are described to characterize hematopoietic precursor cells and mature microglia.

Optogenetic Stimulation of mPFC Alleviates White Matter Injury-Related Cognitive Decline after Chronic Ischemia through Adaptive Myelination.

White matter injury (WMI), which reflects myelin loss, contributes to cognitive decline or dementia caused by cerebral vascular diseases. However, because pharmacological agents specifically for WMI are lacking, novel therapeutic strategies need to be explored. It is recently found that adaptive myelination is required for homeostatic control of brain functions. In this study, adaptive myelination-related strategies are appliedto explore the treatment for ischemicWMI-related cognitive dysfunction. Here, bilateral carotid artery stenosis (BCAS) is used to model ischemic WMI-related cognitive impairment and uncover that optogenetic and chemogenetic activation of glutamatergic neurons in the medial prefrontal cortex (mPFC) promote the differentiation of oligodendrocyte precursor cells (OPCs) in the corpus callosum, leading to improvements in myelin repair and working memory. Mechanistically, these neuromodulatory techniques exert a therapeutic effect by inducing the secretion of Wnt2 from activated neuronal axons, which acts on oligodendrocyte precursor cells and drives oligodendrogenesis and myelination. Thus, this study suggests that neuromodulation is a promising strategy for directing myelin repair and cognitive recovery through adaptive myelination in the context of ischemic WMI.

Fine-tuning of mTOR signaling by the UBE4B-KLHL22 E3 ubiquitin ligase cascade in brain development.

Spatiotemporal regulation of the mechanistic target of rapamycin (mTOR) pathway is pivotal for establishment of brain architecture. Dysregulation of mTOR signaling is associated with a variety of neurodevelopmental disorders. Here, we demonstrate that the UBE4B-KLHL22 E3 ubiquitin ligase cascade regulates mTOR activity in neurodevelopment. In a mouse model with UBE4B conditionally deleted in the nervous system, animals display severe growth defects, spontaneous seizures and premature death. Loss of UBE4B in the brains of mutant mice results in depletion of neural precursor cells and impairment of neurogenesis. Mechanistically, UBE4B polyubiquitylates and degrades KLHL22, an E3 ligase previously shown to degrade the GATOR1 component DEPDC5. Deletion of UBE4B causes upregulation of KLHL22 and hyperactivation of mTOR, leading to defective proliferation and differentiation of neural precursor cells. Suppression of KLHL22 expression reverses the elevated activity of mTOR caused by acute local deletion of UBE4B. Prenatal treatment with the mTOR inhibitor rapamycin rescues neurogenesis defects in Ube4b mutant mice. Taken together, these findings demonstrate that UBE4B and KLHL22 are essential for maintenance and differentiation of the precursor pool through fine-tuning of mTOR activity.

PDGFRβ-positive cell-mediated post-stroke remodeling of fibronectin and laminin α2 for tissue repair and functional recovery

Post-stroke intra-infarct repair promotes peri-infarct neural reorganization leading to functional recovery. Herein, we examined the remodeling of extracellular matrix proteins (ECM) that constitute the intact basal membrane after permanent middle cerebral artery occlusion (pMCAO) in mice. Among ECM, collagen type IV remained localized on small vessel walls surrounding CD31-positive endothelial cells within infarct areas. Fibronectin was gradually deposited from peri-infarct areas to the ischemic core, in parallel with the accumulation of PDGFRβ-positive cells. Cultured PDGFRβ-positive pericytes produced fibronectin, which was enhanced by the treatment with PDGF-BB. Intra-infarct deposition of fibronectin was significantly attenuated in pericyte-deficient Pdgfrb+/− mice. Phagocytic activity of macrophages against myelin debris was significantly enhanced on fibronectin-coated dishes. In contrast, laminin α2, produced by GFAP- and aquaporin 4-positive astrocytes, accumulated strongly in the boundary of peri-infarct areas. Pericyte-conditioned medium increased the expression of laminin α2 in cultured astrocytes, partly through TGFβ1. Laminin α2 increased the differentiation of oligodendrocyte precursor cells into oligodendrocytes and the expression of myelin-associated proteins. Peri-infarct deposition of laminin α2 was significantly reduced in Pdgfrb+/− mice, with attenuated oligodendrogenesis in peri-infarct areas. Collectively, intra-infarct PDGFRβ-positive cells may orchestrate post-stroke remodeling of key ECM that create optimal environments promoting clearance of myelin debris and peri-infarct oligodendrogenesis.

TAT‐LBD‐Ngn2‐improved cognitive functions after global cerebral ischemia by enhancing neurogenesis

Stroke is the major cause of adult neurocognitive disorders (NCDs), and presents a significant burden on both of the families and society. To improve the cerebral injury, we generated a blood-brain barrier penetrating peptide TAT-LBD-Ngn2, in which Ngn2 (Neurogenin2) is a classical preneural gene that enhances neurogenesis, and neural precursor cells survival and differentiation. We previously demonstrated that it has a short-term protective effect against cerebral ischemia-reperfusion injury.However, it is uncertain if TAT-LBD-Ngn2 could promote neurogenesis to exhibit long-term therapeutic impact. In present study, TAT-LBD-Ngn2 was administered for 14 or 28 days following bilateral common carotid arteries occlusion (BCCAO). After confirming that TAT-LBD-Ngn2 could cross the brain blood barrier and aggregate in the hippocampus, we conducted open field test, Morris water maze and contextual fear conditioning to examine the long-term effect of TAT-LBD-Ngn2 on cognition. We discovered that TAT-LBD-Ngn2 significantly improved the spatial and contextual learning and memory on both days 14 and 28 after BCCAO, while TAT-LBD-Ngn2 exhibited anxiolytic effect only on day 14, but had no effect on locomotion. Using western blot and immunofluorescence, TAT-LBD-Ngn2 was also shown to promote neurogenesis, as evidenced by increased BrdU+ and DCX+ neurons in dentate gyrus. Meanwhile, TAT-LBD-Ngn2 elevated the expression of brain derived neurotrophic factor rather than nerve growth factor compared to the control group. Our findings revealed that TAT-LBD-Ngn2 could dramatically promote learning and memory in long term by facilitating neurogenesis in the hippocampus after global cerebral ischemia, indicating that TAT-LBD-Ngn2 may be an appealing candidate for treating poststroke NCD.

STEM-21. REPURPOSING CLEMASTINE TO SUPPRESS GLIOBLASTOMA STEM CELLS

Abstract Glioblastoma stem cells (GSCs), also known as brain tumor initiating cells (BTICs), drive tumor progression, heterogeneity, and resistance to treatments, posing formidable challenges to advancing effective treatments against glioblastoma (GBM). We postulated that inducing BTIC differentiation can serve as a solution to diminishing their stem-like features. Here, we report that clemastine, an over-the-counter oral medication for allergy relief, attenuates stemness and proliferation of BTICs. These effects of clemastine were accompanied by altered transcriptional programs suggestive of a shift from maintaining stem cell identity to differentiation, resonating with the ability of clemastine to promote oligodendrocyte precursor cell (OPC) differentiation to mature oligodendrocytes. Genetic perturbation and small-molecule inhibition of putative pharmacological targets of clemastine revealed that Emopamil-binding protein (EBP), an enzyme in the sterol biosynthesis pathway, played a pivotal role in mediating the differentiating and anti-tumor effects of clemastine. Notably, loss-of-function assays showed that EBP expression was indispensable for BTIC propagation. In contrast, overexpression of EBP stimulated BTIC proliferation, thus uncovering a previously unknown role of sterol metabolism in BTIC maintenance. Finally, we demonstrated that a mouse neural stem cell-derived glioma model was similarly susceptible to clemastine treatment. Taken together, our study identifies pathways essential for the perpetuation of stemness in GBM, and implicates a non-oncology drug with a well-established safety profile that can be repurposed to mitigate the stem-like properties of GBM.