Chronological transitions of hepatocyte growth factor treatment effects in spinal cord injury tissue.

Inflammatory responses are known to suppress neural regeneration in patients receiving stem cell-based regenerative therapy for spinal cord injury (SCI). Consequently, pathways involved in neurogenesis and immunomodulation, such as the hepatocyte growth factor (HGF)/MET signaling cascade, have garnered significant attention. Notably, various studies, including our own, have highlighted the enhanced recovery of locomotor functions achieved in SCI animal models by combining HGF pretreatment and human induced stem cell-derived neural stem/progenitor cell (hiPSC-NS/PC) transplantation. However, these studies implicitly hypothesized that the functionality of HGF in SCI would be time consistent and did not elucidate its dynamics. In the present article, we investigated the time-course of the effect of HGF on SCI, aiming to uncover a more precise mechanism for HGF administration, which is indispensable for developing crystallizing protocols for combination therapy. To this end, we performed a detailed investigation of the temporal variation of HGF using the RNA-seq data we obtained in our most recent study. Leveraging the time-series design of the data, which we did not fully exploit previously, we identified three components in the effects of HGF that operate at different times: early effects, continuous effects, and delayed effects. Our findings suggested a concept where the three components together contribute to the acceleration of neurogenesis and immunomodulation, which reinforce the legitimacy of empirically fine-tuned protocols for HGF administration and advocate the novel possibility that the time-inconsistent effects of HGF progressively augment the efficacy of combined therapy.

Hepatocyte growth factor pretreatment boosts functional recovery after spinal cord injury through human iPSC-derived neural stem/progenitor cell transplantation.

BACKGROUND: Human induced pluripotent stem cell-derived neural stem/progenitor cell (hiPSC-NS/PC)-based cell transplantation has emerged as a groundbreaking method for replacing damaged neural cells and stimulating functional recovery, but its efficacy is strongly influenced by the state of the injured spinal microenvironment. This study evaluates the impact of a dual therapeutic intervention utilizing hepatocyte growth factor (HGF) and hiPSC-NS/PC transplantation on motor function restoration following spinal cord injury (SCI). METHODS: Severe contusive SCI was induced in immunocompromised rats, followed by continuous administration of recombinant human HGF protein into the subarachnoid space immediately after SCI for two weeks. Acute-phase histological and RNA sequencing analyses were conducted. Nine days after the injury, hiPSC-NS/PCs were transplanted into the lesion epicenter of the injured spinal cord, and the functional and histological outcomes were determined. RESULTS: The acute-phase HGF-treated group exhibited vascularization, diverse anti-inflammatory effects, and activation of endogenous neural stem cells after SCI, which collectively contributed to tissue preservation. Following cell transplantation into a favorable environment, the transplanted NS/PCs survived well, facilitating remyelination and neuronal regeneration in host tissues. These comprehensive effects led to substantial enhancements in motor function in the dual-therapy group compared to the single-treatment groups. CONCLUSIONS: We demonstrate that the combined therapeutic approach of HGF preconditioning and hiPSC-NS/PC transplantation enhances locomotor functional recovery post-SCI, highlighting a highly promising therapeutic strategy for acute to subacute SCI.

Gene therapy using genome-edited iPS cells for targeting malignant glioma.

Glioblastoma is characterized by diffuse infiltration into the normal brain. Invasive glioma stem cells (GSCs) are an underlying cause of treatment failure. Despite the use of multimodal therapies, the prognosis remains dismal. New therapeutic approach targeting invasive GSCs is required. Here, we show that neural stem cells (NSCs) derived from CRISRP/Cas9-edited human-induced pluripotent stem cell (hiPSC) expressing a suicide gene had higher tumor-trophic migratory capacity compared with mesenchymal stem cells (MSCs), leading to marked in vivo antitumor effects. High migratory capacity in iPSC-NSCs was related to self-repulsive action and pathotropism involved in EphB-ephrinB and CXCL12-CXCR4 signaling. The gene insertion to ACTB provided higher and stable transgene expression than other common insertion sites, such as GAPDH or AAVS1. Ferroptosis was associated with enhanced antitumor immune responses. The thymidylate synthase and dihydroprimidine dehydrogenase expressions predicted the treatment efficacy of therapeutic hiPSC-NSCs. Our results indicate the potential benefit of genome-edited iPS cells based gene therapy for invasive GSCs. Furthermore, the present research concept may become a platform to promote clinical studies using hiPSC.

Neuroprotective Effects of Genome-Edited Human iPS Cell-Derived Neural Stem/Progenitor Cells on Traumatic Brain Injury.

Despite developing neurosurgical procedures, few treatment options have achieved functional recovery from traumatic brain injury (TBI). Neural stem/progenitor cells (NS/PCs) may produce a long-term effect on neurological recovery. Although induced pluripotent stem cells (iPSCs) can overcome ethical and practical issues of human embryonic or fetal-derived tissues in clinical applications, the tumorigenicity of iPSC-derived populations remains an obstacle to their safe use in regenerative medicine. We herein established a novel treatment strategy for TBI using iPSCs expressing the enzyme-prodrug gene yeast cytosine deaminase-uracil phosphoribosyl transferase (yCD-UPRT). NS/PCs derived from human iPSCs displayed stable and high transgene expression of yCD-UPRT following CRISPR/Cas9-mediated genome editing. In vivo bioluminescent imaging and histopathological analysis demonstrated that NS/PCs concentrated around the damaged cortex of the TBI mouse model. During the subacute phase, performances in both beam walking test and accelerating rotarod test were significantly improved in the treatment group transplanted with genome-edited iPSC-derived NS/PCs compared with the control group. The injury area visualized by extravasation of Evans blue was smaller in the treatment group compared with the control group, suggesting the prevention of secondary brain injury. During the chronic phase, cerebral atrophy and ventricle enlargement were significantly less evident in the treatment group. Furthermore, after 5-fluorocytosine (5-FC) administration, 5-fluorouracil converted from 5-FC selectively eliminated undifferentiated NS/PCs while preserving the adjacent neuronal structures. NS/PCs expressing yCD-UPRT can be applied for safe regenerative medicine without the concern for tumorigenesis.

The original strain of SARS-CoV-2, the Delta variant, and the Omicron variant infect microglia efficiently, in contrast to their inability to infect neurons: Analysis using 2D and 3D cultures.

COVID-19 causes neurological damage, systemic inflammation, and immune cell abnormalities. COVID-19-induced neurological impairment may be caused by severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2), which directly infects cells of the central nervous system (CNS) and exerts toxic effects. Furthermore, SARS-CoV-2 mutations occur constantly, and it is not well understood how the infectivity of the virus to cells of the CNS changes as the virus mutates. Few studies have examined whether the infectivity of cells of CNS - neural stem/progenitor cells (NS/PCs), neurons, astrocytes, and microglia - varies among SARS-CoV-2 mutant strains. In this study, therefore, we investigated whether SARS-CoV-2 mutations increase infectivity to CNS cells, including microglia. Since it was essential to demonstrate the infectivity of the virus to CNS cells in vitro using human cells, we generated cortical neurons, astrocytes, and microglia from human induced pluripotent stem cells (hiPSCs). We added pseudotyped lentiviruses of SARS-CoV-2 to each type of cells, and then we examined their infectivity. We prepared three pseudotyped lentiviruses expressing the S protein of the original strain (the first SARS-CoV-2 discovered in the world), the Delta variant, and the Omicron variant on their envelopes and analyzed differences of their ability to infect CNS cells. We also generated brain organoids and investigated the infectivity of each virus. The viruses did not infect cortical neurons, astrocytes, or NS/PCs, but microglia were infected by the original, Delta, and Omicron pseudotyped viruses. In addition, DPP4 and CD147, potential core receptors of SARS-CoV-2, were highly expressed in the infected microglia, while DPP4 expression was deficient in cortical neurons, astrocytes, and NS/PCs. Our results suggest that DPP4, which is also a receptor for Middle East respiratory syndrome-coronavirus (MERS-CoV), may play an essential role in the CNS. Our study is applicable to the validation of the infectivity of viruses that cause various infectious diseases in CNS cells, which are difficult to sample from humans.

A set-theoretic definition of cell types with an algebraic structure on gene regulatory networks and application in annotation of RNA-seq data.

The emergence of single-cell RNA sequencing (RNA-seq) has radically changed the observation of cellular diversity. Although annotations of RNA-seq data require preserved properties among cells of an identity, annotations using conventional methods have not been able to capture universal characters of a cell type. Analysis of expression levels cannot be accurately annotated for cells because differences in transcription do not necessarily explain biological characteristics in terms of cellular functions and because the data themselves do not inform about the correct mapping between cell types and genes. Hence, in this study, we developed a new representation of cellular identities that can be compared over different datasets while preserving nontrivial biological semantics. To generalize the notion of cell types, we developed a new framework to manage cellular identities in terms of set theory. We provided further insights into cells by installing mathematical descriptions of cell biology. We also performed experiments that could correspond to practical applications in annotations of RNA-seq data.

Case report: meningeal lymphangiogenesis around ependymoma forming along the dura matter.

Recently, there has been growing interest in the presence and function of meningeal lymphatic vessels, with no direct evidence linking these vessels to primary brain tumors. We report a unique case of recurrent ependymoma in the dura mater, showing histopathological signs of lymphatic proliferation at the tumor attachment site. The patient initially presented with a headache, and was diagnosed with ZFTA fusion-positive supratentorial ependymoma, central nervous system WHO Grade 3. Following multiple dura mater recurrences and surgery, the fifth procedure revealed numerous tumors contralateral to the original site, with genetic testing confirming ZFTA fusion positivity, indicating recurrent ependymoma. Immunohistochemical analysis showed D2-40+ lymphatic vessel proliferation around tumor attachment sites within the dura mater. Elevated expression of ZEB1, which is an epithelial-to-mesenchymal transition factor, was also observed, implicating potential involvement in the unique pathophysiology. The present case suggests a new process of metastasis through meningeal lymphatic vessels, although we were unable to visually confirm tumor cell infiltration into the lymphatic vessels. This case is the first report suggesting ependymoma metastasis through dural lymphatic vessels, underlining the need for further case accumulation and study to understand the mechanisms of this phenomenon.

Subarachnoid hemorrhage triggers neuroinflammation of the entire cerebral cortex, leading to neuronal cell death.

BACKGROUND: Subarachnoid hemorrhage (SAH) is a fatal disease, with early brain injury (EBI) occurring within 72 h of SAH injury contributes to its poor prognosis. EBI is a complicated phenomenon involving multiple mechanisms. Although neuroinflammation has been shown to be important prognosis factor of EBI, whether neuroinflammation spreads throughout the cerebrum and the extent of its depth in the cerebral cortex remain unknown. Knowing how inflammation spreads throughout the cerebrum is also important to determine if anti-inflammatory agents are a future therapeutic strategy for EBI. METHODS: In this study, we induced SAH in mice by injecting hematoma into prechiasmatic cistern and created models of mild to severe SAH. In sections of the mouse cerebrum, we investigated neuroinflammation and neuronal cell death in the cortex distal to the hematoma injection site, from anterior to posterior region 24 h after SAH injury. RESULTS: Neuroinflammation caused by SAH spread to all layers of the cerebral cortex from the anterior to the posterior part of the cerebrum via the invasion of activated microglia, and neuronal cell death increased in correlation with neuroinflammation. This trend increased with the severity of the disease. CONCLUSIONS: Neuroinflammation caused by SAH had spread throughout the cerebrum, causing neuronal cell death. Considering that the cerebral cortex is responsible for long-term memory and movement, suppressing neuroinflammation in all layers of the cerebral cortex may improve the prognosis of patients with SAH.

The GADD45G/p38 MAPK/CDC25B signaling pathway enhances neurite outgrowth by promoting microtubule polymerization.

GADD45G, one of the genes containing the human-specific conserved deletion enhancer-sequence (hCONDEL), has contributed to the evolution of the human cerebrum, but its function in human neurons has not been established. Here, we show that the GADD45G/p38 MAPK/CDC25B signaling pathway promotes neurite outgrowth in human neurons by facilitating microtubule polymerization. This pathway ultimately promotes dephosphorylation of phosphorylated CRMP2 which in turn promotes microtubule assembly. We also found that GADD45G was highly expressed in developing human cerebral specimens. In addition, RK-682, which is the inhibitor of a phosphatase of p38 MAPK and was found in Streptomyces sp., was shown to promote microtubule polymerization and neurite outgrowth by enhancing p38 MAPK/CDC25B signaling. These in vitro and in vivo results indicate that GADD45G/p38 MAPK/CDC25B enhances neurite outgrowth in human neurons.

Neurological pathogenesis of SARS-CoV-2 (COVID-19): from virological features to clinical symptoms.

Since the worldwide outbreak of coronavirus disease 2019 (COVID-19) in 2020, various research reports and case reports have been published. It has been found that COVID-19 causes not only respiratory disorders but also thrombosis and gastrointestinal disorders, central nervous system (CNS) disorders, and peripheral neuropathy. Compared to other disorders, there are low number of research reports and low number of summaries on COVID-19-related neural disorders. Therefore, focusing on neural disorders, we outline both basic research and clinical manifestations of COVID-19-related neural disorders.

Expression of ACE2 and a viral virulence-regulating factor CCN family member 1 in human iPSC-derived neural cells: implications for COVID-19-related CNS disorders.

It has been reported that coronavirus disease 2019 (COVID-19) causes not only pneumonia but also systemic inflammations including central nervous system (CNS) disorders. However, little is known about the mechanism that triggers the COVID-19-associated CNS disorders, due to the lack of appropriate experimental systems. Our present study showed that angiotensin-converting enzyme-2 (ACE2), a cellular receptor for SARS-CoV-2, is expressed in human induced pluripotent stem cell (iPSC)-derived neural stem/progenitor cells (hiPSC-NS/PCs) and young neurons. Furthermore, together with database analysis, we found that a viral virulent factor CCN family member 1 (CCN1), which is known to be induced by SARS-CoV-2 infection, is expressed in these cells at basal levels. Considering the role of CCN1 which is known to be involved in viral toxicity and inflammation, hiPSC-NS/PCs could provide an excellent model for COVID-19-associated CNS disorders from the aspect of SARS-CoV-2 infection-ACE2-CCN1 axis. In addition, we identified compounds that reduce CCN1 expression. Collectively, our study using hiPSC-NS/PCs may aid in the development of a therapeutic target for COVID-19-related CNS disorders.

Current understanding of adult neurogenesis in the mammalian brain: how does adult neurogenesis decrease with age?

Adult neurogenesis occurs throughout life in restricted brain regions in mammals. However, the number of neural stem cells (NSCs) that generate new neurons steadily decreases with age, resulting in a decrease in neurogenesis. Transplantation of mesenchymal cells or cultured NSCs has been studied as a promising treatment in models of several brain injuries including cerebral infarction and cerebral contusion. Considering the problems of host-versus-graft reactions and the tumorigenicity of transplanted cells, the mobilization of endogenous adult NSCs should be more feasible for the treatment of these brain injuries. However, the number of adult NSCs in the adult brain is limited, and their mitotic potential is low. Here, we outline what we know to date about why the number of NSCs and adult neurogenesis decrease with age. We also discuss issues applicable to regenerative medicine.

Involvement of p38 in Age-Related Decline in Adult Neurogenesis via Modulation of Wnt Signaling.

Neurogenesis in specific brain regions in adult mammals decreases with age. Progressive reduction in the proliferation of neural stem and progenitor cells (NS/PCs) is a primary cause of this age-associated decline. However, the mechanism responsible for this reduction is poorly understood. We identify p38 MAPK as a key factor in the proliferation of neural progenitor cells (NPCs) in adult neurogenic niches. p38 expression in adult NS/PCs is downregulated during aging. Deletion of p38α in NS/PCs specifically reduces the proliferation of NPCs but not stem cells. Conversely, forced expression of p38α in NS/PCs in the aged mouse subventricular zone (SVZ) restores NPC proliferation and neurogenesis, and prevents age-dependent SVZ atrophy. We also found that p38 is necessary for suppressing the expression of Wnt antagonists DKK1 and SFRP3, which inhibit the proliferation of NPCs. Age-related reduction in p38 thus leads to decreased adult neurogenesis via downregulation of Wnt signaling.

Treatment with a Gamma-Secretase Inhibitor Promotes Functional Recovery in Human iPSC- Derived Transplants for Chronic Spinal Cord Injury.

Treatment involving regenerative medicine for chronic spinal cord injury (SCI) is difficult due to phase-dependent changes in the intraspinal environment. We previously reported that treatment with a gamma-secretase inhibitor (GSI), which inhibits Notch signaling, promotes the differentiation into mature neurons in human induced pluripotent stem cell-derived neural stem/progenitor cell (hiPSC-NS/PC) transplantation for subacute SCI. Here, we evaluated the efficacy of GSI-treated hiPSC-NS/PC transplantation in treating chronic SCI, which resulted in significantly enhanced axonal regrowth, remyelination, inhibitory synapse formation with the host neural circuitry, and reticulo spinal tract fiber formation. Interestingly, inhibiting Notch signaling with GSI caused phosphorylation of p38 MAPK, which is a key molecule required to promote axonal regeneration. These favorable outcomes contributed to motor function improvement. Therefore, treating cells with GSI provides a beneficial effect after transplantation, even in the chronic phase following SCI.