Silicate Microfiber Scaffolds Support the Formation and Expansion of the Cortical Neuronal Layer of Cerebral Organoids With a Sheet-Like Configuration.

Cerebral organoids (COs) are derived from human-induced pluripotent stem cells in vitro and mimic the features of the human fetal brain. The development of COs is largely dependent on "self-organization" mechanisms, in which differentiating cells committed to cortical cells autonomously organize into the cerebral cortex-like tissue. However, extrinsic manipulation of their morphology, including size and thickness, remains challenging. In this study, we discovered that silicate microfiber scaffolds could support the formation of cortical neuronal layers and successfully generated cortical neuronal layers, which are 9 times thicker than conventional COs, in 70 days. These cortical neurons in the silicate microfiber layer were differentiated in a fetal brain-like lamination pattern. While these cellular characteristics such as cortical neurons and neural stem/progenitor cells were like those of conventional COs, the cortical neuronal layers were greatly thickened in sheet-like configuration. Moreover, the cortical neurons in the scaffolds showed spontaneous electrical activity. We concluded that silicate microfiber scaffolds support the formation of the cortical neuronal layers of COs without disturbing self-organization-driven corticogenesis. The extrinsic manipulation of the formation of the cortical neuronal layers of COs may be useful for the research of developmental mechanisms or pathogenesis of the human cerebral cortex, particularly for the development of regenerative therapy and bioengineering.

Continuous Monitoring of Changes in Heart Rate during the Periprocedural Course of Carotid Artery Stenting Using a Wearable Device: A Prospective Observational Study.

This prospective observational study will evaluate the change in heart rate (HR) during the periprocedural course of carotid artery stenting (CAS) via continuous monitoring using a wearable device. The participants were recruited from our outpatient clinic between April 2020 and March 2023. They were instructed to continuously wear the device from the last outpatient visit before admission to the first outpatient visit after discharge. The changes in HR of interest throughout the periprocedural course of CAS were assessed. In addition, the Bland-Altman analysis was adopted to compare the HR measurement made by the wearable device during CAS with that made by the electrocardiogram (ECG). A total of 12 patients who underwent CAS were included in the final analysis. The time-series analysis revealed that a percentage change in HR decrease occurred on day 1 following CAS and that the most significant HR decrease rate was 12.1% on day 4 following CAS. In comparing the measurements made by the wearable device and ECG, the Bland-Altman analysis revealed the accuracy of the wearable device with a bias of -1.12 beats per minute (bpm) and a precision of 3.16 bpm. Continuous HR monitoring using the wearable device indicated that the decrease in HR following CAS could persist much longer than previously reported, providing us with unique insights into the physiology of carotid sinus baroreceptors.

Atlantoaxial Intradural Synovial Cyst Mimicking an Extradural Lesion Adjacent to a Retro-odontoid Pseudotumor: A Case Report.

Atlantoaxial synovial cysts can very rarely penetrate the dura mater into the thecal sac and cause direct neural compression. Several case reports have been available on "intradural synovial cysts" (IDSCs). In this study, we report on a case with an atlantoaxial IDSC mimicking an extradural lesion. A 90-year-old man was diagnosed with a cystic lesion located laterally to the atlantoaxial joint adjacent to the retro-odontoid pseudotumor (ROP) causing cervical spinal cord compression. Thus, surgical removal was planned. On preoperative examination, the cyst, which had a two-layer structure showing a T2-isointense small mass inside a T2-hyperintense lesion, was thought to be located in the extradural region. However, operative findings showed that the cyst was located inside the dura mater. Histopathological examination suggested a synovial cyst. No recurrence of the cyst was observed until the latest follow-up after 3 years, and the ROP decreased in size. Almost all IDSCs reported previously were observed in the medial site of the atlantoaxial joint. In our case, however, the cyst was observed adjacent to the posteromedial site of the right atlantoaxial joint and the ROP, mimicking an extradural lesion. We had no knowledge regarding the IDSC before the surgery and assumed an extradural lesion. Albeit rare, the existence of such a condition should be considered.

Case of Syringomyelia Associated with Ossified Yellow Ligament and Arachnoid Web.

Syringomyelia is often associated with Chiari malformation, trauma, infection, and spinal cord tumor. Although they are relatively rare diseases, arachnoid cysts and its related pathology, "arachnoid web" can sometimes lead to syrinx formation at the thoracic vertebral level. However, syrinx formation caused by degenerative spinal disorders, particularly at the thoracic vertebral levels, has rarely been reported. Herein, we present a case of syringomyelia with thoracic ossified yellow ligament (OYL) in a 79-year-old man, who underwent initial posterior decompression followed by arachnoid web removal. Posterior decompression via laminectomy of thoracic vertebra 2 and removal of the OYL improved the syrinx partially, but dorsal indentation of the spinal cord and a remnant syrinx were observed in post-operative magnetic resonance images, subsequent to the second surgery's successful removal of the arachnoid web, which had dorsally compressed the spinal cord. After the second operation, the syrinx shrunk further, and the patient could walk independently at 5 months after the operations. In our case, both the OYL and arachnoid web were responsible for syrinx formation. Therefore, the coincidence of degenerative vertebral diseases with a syrinx might indicate the coexistence of an underlying lesion. Furthermore, the arachnoid web in this case might have formed due to the denaturation of the arachnoid cyst triggered by the OYL.

Dysbiosis of Gut Microbiome Is Associated With Rupture of Cerebral Aneurysms.

BACKGROUND AND PURPOSE: Environmental factors are important with respect to the rupture of cerebral aneurysms. However, the relationship between the gut microbiome, an environmental factor, and aneurysm rupture is unclear. Therefore, we compared the gut microbiome in patients with unruptured intracranial aneurysms (UIAs) and ruptured aneurysms (RAs) to identify the specific bacteria causing the rupture of cerebral aneurysms. METHODS: A multicenter, prospective case-control study was conducted over one year from 2019 to 2020. The fecal samples of patients with stable UIAs and RAs immediately after onset were collected. Their gut microbiomes were analyzed using 16S rRNA sequencing. Subsequently, a phylogenetic tree was constructed, and polymerase chain reaction was performed to identify the specific species. RESULTS: A total of 28 RAs and 33 UIAs were included in this study. There was no difference in patient characteristics between RAs and UIAs: age, sex, hypertension, dyslipidemia, diabetes status, body mass index, and smoking. No difference was observed in alpha diversity; however, beta diversity was significantly different in the unweighted UniFrac distances. At the phylum level, the relative abundance of Campylobacter in the RA group was larger than that in the UIA group. Furthermore, the gut microbiome in the RA and UIA groups exhibited significantly different taxonomies. However, Campylobacter was focused on because it is widely known as pathogenic among these bacteria. Then, a phylogenetic tree of operational taxonomic units related to Campylobacter was constructed and 4 species were identified. Polymerase chain reaction for these species identified that the abundance of the genus Campylobacter and Campylobacter ureolyticus was significantly higher in the RA group. CONCLUSIONS: The gut microbiome profile of patients with stable UIAs and RAs were significantly different. The genus Campylobacter and Campylobacter ureolyticus may be associated with the rupture of cerebral aneurysms.

Successful neurosurgical separation of conjoined spinal cords in pygopagus twins: illustrative cases.

BACKGROUND: Conjoined twins represent a rare congenital malformation. Pygopagus twins are fused at the sacrum and perineum, with union of the spine. The authors report a successful separation of a unique case of pygopagus twins sharing a U-shaped spinal cord, which the authors identified through aberrant nerves by intraoperative physiological spinal root examination. OBSERVATIONS: The 6-month-old male pygopagus conjoined twins, who were diagnosed in the prenatal period, underwent separation. They had a single dural sac containing a U-shaped continuous spinal cord; their filum terminale appeared completely fused and the anatomical border of the spinal cord was not distinguishable. A triggered electromyogram (tEMG) was used on each nerve root to determine which belonged to one twin versus the other, to detect nerve cross, and to identify functional midline cleavage. Finally, the twins were separated after spinal division. Both twins recovered uneventfully with no lower limb neurological deficits or walking impairment for 16 months. LESSONS: Pygopagus twins with a conjoined spinal cord are very rare, but a good long-term functional prognosis can be expected with successful separation. Intraoperative tEMG is useful in spinal separation surgery for twins with a conjoined spinal cord.

Generation of Induced Pluripotent Stem Cells and Neural Stem/Progenitor Cells from Newborns with Spina Bifida Aperta.

STUDY DESIGN: We established induced pluripotent stem cells (iPSCs) and neural stem/progenitor cells (NSPCs) from three newborns with spina bifida aperta (SBa) using clinically practical methods. PURPOSE: We aimed to develop stem cell lines derived from newborns with SBa for future therapeutic use. OVERVIEW OF LITERATURE: SBa is a common congenital spinal cord abnormality that causes defects in neurological and urological functions. Stem cell transplantation therapies are predicted to provide beneficial effects for patients with SBa. However, the availability of appropriate cell sources is inadequate for clinical use because of their limited accessibility and expandability, as well as ethical issues. METHODS: Fibroblast cultures were established from small fragments of skin obtained from newborns with SBa during SBa repair surgery. The cultured cells were transfected with episomal plasmid vectors encoding reprogramming factors necessary for generating iPSCs. These cells were then differentiated into NSPCs by chemical compound treatment, and NSPCs were expanded using neurosphere technology. RESULTS: We successfully generated iPSC lines from the neonatal dermal fibroblasts of three newborns with SBa. We confirmed that these lines exhibited the characteristics of human pluripotent stem cells. We successfully generated NSPCs from all SBa newborn-derived iPSCs with a combination of neural induction and neurosphere technology. CONCLUSIONS: We successfully generated iPSCs and iPSC-NSPCs from surgical samples obtained from newborns with SBa with the goal of future clinical use in patients with SBa.

Visualization of migration of human cortical neurons generated from induced pluripotent stem cells.

BACKGROUND: Neuronal migration is considered a key process in human brain development. However, direct observation of migrating human cortical neurons in the fetal brain is accompanied by ethical concerns and is a major obstacle in investigating human cortical neuronal migration. NEW METHOD: We established a novel system that enables direct visualization of migrating cortical neurons generated from human induced pluripotent stem cells (hiPSCs). RESULTS: We observed the migration of cortical neurons generated from hiPSCs derived from a control and from a patient with lissencephaly. METHODS: Our system needs no viable brain tissue, which is usually used in slice culture. Migratory behavior of human cortical neuron can be observed more easily and more vividly by its fluorescence and glial scaffold than that by earlier methods. CONCLUSIONS: Our in vitro experimental system provides a new platform for investigating development of the human central nervous system and brain malformation.

In vitro characterization of neurite extension using induced pluripotent stem cells derived from lissencephaly patients with TUBA1A missense mutations.

BACKGROUND: Lissencephaly, or smooth brain, is a severe congenital brain malformation that is thought to be associated with impaired neuronal migration during corticogenesis. However, the exact etiology of lissencephaly in humans remains unknown. Research on congenital diseases is limited by the shortage of clinically derived resources, especially for rare pediatric diseases. The research on lissencephaly is further limited because gyration in humans is more evolved than that in model animals such as mice. To overcome these limitations, we generated induced pluripotent stem cells (iPSCs) from the umbilical cord and peripheral blood of two lissencephaly patients with different clinical severities carrying alpha tubulin (TUBA1A) missense mutations (Patient A, p.N329S; Patient B, p.R264C). RESULTS: Neural progenitor cells were generated from these iPSCs (iPSC-NPCs) using SMAD signaling inhibitors. These iPSC-NPCs expressed TUBA1A at much higher levels than undifferentiated iPSCs and, like fetal NPCs, readily differentiated into neurons. Using these lissencephaly iPSC-NPCs, we showed that the neurons derived from the iPSCs obtained from Patient A but not those obtained from Patient B showed abnormal neurite extension, which correlated with the pathological severity in the brains of the patients. CONCLUSION: We established iPSCs derived from lissencephaly patients and successfully modeled one aspect of the pathogenesis of lissencephaly in vitro using iPSC-NPCs and iPSC-derived neurons. The iPSCs from patients with brain malformation diseases helped us understand the mechanism underlying rare diseases and human corticogenesis without the use of postmortem brains.

Establishment of Human Neural Progenitor Cells from Human Induced Pluripotent Stem Cells with Diverse Tissue Origins.

Human neural progenitor cells (hNPCs) have previously been generated from limited numbers of human induced pluripotent stem cell (hiPSC) clones. Here, 21 hiPSC clones derived from human dermal fibroblasts, cord blood cells, and peripheral blood mononuclear cells were differentiated using two neural induction methods, an embryoid body (EB) formation-based method and an EB formation method using dual SMAD inhibitors (dSMADi). Our results showed that expandable hNPCs could be generated from hiPSC clones with diverse somatic tissue origins. The established hNPCs exhibited a mid/hindbrain-type neural identity and uniform expression of neural progenitor genes.

Differentiation, polarization, and migration of human induced pluripotent stem cell-derived neural progenitor cells co-cultured with a human glial cell line with radial glial-like characteristics.

Here we established a unique human glial cell line, GDC90, derived from a human glioma and demonstrated its utility as a glial scaffold for the polarization and differentiation of human induced pluripotent stem cell-derived neural progenitor cells (iPSC-NPCs). When co-cultured with GDC90 cells, iPSC-NPCs underwent rapid polarization and neurite extension along the radially spreading processes of the GDC90 cells, and showed migratory behavior. This method is potentially useful for detailed examination of neurites or for controlling neurites behavior for regenerative medicine.

Human Decidua-Derived Mesenchymal Cells Are a Promising Source for the Generation and Cell Banking of Human Induced Pluripotent Stem Cells.

Placental tissue is a biomaterial with remarkable potential for use in regenerative medicine. It has a three-layer structure derived from the fetus (amnion and chorion) and the mother (decidua), and it contains huge numbers of cells. Moreover, placental tissue can be collected without any physical danger to the donor and can be matched with a variety of HLA types. The decidua-derived mesenchymal cells (DMCs) are highly proliferative fibroblast-like cells that express a similar pattern of CD antigens as bone marrow-derived mesenchymal cells (BM-MSCs). Here we demonstrated that induced pluripotent stem (iPS) cells could be efficiently generated from DMCs by retroviral transfer of reprogramming factor genes. DMC-hiPS cells showed equivalent characteristics to human embryonic stem cells (hESCs) in colony morphology, global gene expression profile (including human pluripotent stem cell markers), DNA methylation status of the OCT3/4 and NANOG promoters, and ability to differentiate into components of the three germ layers in vitro and in vivo. The RNA expression of XIST and the methylation status of its promoter region suggested that DMC-iPSCs, when maintained undifferentiated and pluripotent, had three distinct states: (1) complete X-chromosome reactivation, (2) one inactive X-chromosome, or (3) an epigenetic aberration. Because DMCs are derived from the maternal portion of the placenta, they can be collected with the full consent of the adult donor and have considerable ethical advantages for cell banking and the subsequent generation of human iPS cells for regenerative applications.

Diagnosis, treatment, and long-term outcomes of fetal hydrocephalus.

This study analyzed 156 cases of fetal hydrocephalus treated at Osaka National Hospital from 1992 to 2011 to review current methods for diagnosing and treating fetal hydrocephalus, and for estimating its clinical outcome. This was a retrospective study of a single institute (Osaka National Hospital). Of 156 cases in total, 37% were diagnosed as isolated ventriculomegaly, 50% as another type of malformation (36 cases of myelomeningocele, six of holoprosencephaly, three of Dandy-Walker syndrome, one case of Joubert syndrome, 12 of arachnoid cyst, nine of encephalocele, three of atresia of Monro and eight of corpus callosum agenesis, and 13% as secondary hydrocephalus. Diagnoses were made between 13 and 40 weeks of gestation (average 27 weeks). Diagnosis was made before 21 weeks of gestation in 24% of cases, from the first day of 22 weeks to the sixth day of 27 weeks in 27%, and after the first day of 28 weeks in 49%. With the exclusion of 17 aborted cases and 40 cases in which the patients were too young to evaluate or lost during follow-up, the final outcome was analyzed for 90 cases. Of these, 17% of the patients died, 21% showed severe retardation, 13% moderate retardation, 26% mild retardation, and 23% showed a good outcome. The long-term outcome was mostly influenced by the basic disease and accompanying anomaly. The time of diagnosis showed no correlation with outcome. Hydrocephalus associated with arachnoid cyst, atresia of Monro, and corpus callosum agenesis, and hydrocephalus due to fetal intracranial hemorrhage, resulted in good outcomes. By contrast, holoprosencephaly, hydrocephalus associated with encephalocele, syndromic hydrocephalus, and hydrocephalus due to fetal virus infection led to poor outcomes. For accurate diagnosis and proper counseling, established protocols are important for the diagnosis and treatment of fetal hydrocephalus, including not only fetal sonography, fetal magnetic resonance imaging, and TORCH (toxoplasma, rubella, cytomegalovirus, herpes simplex) screening test, but also chromosomal and gene testing.

Analysis of the transcription factor cascade that induces endocrine and exocrine cell lineages from pancreatic progenitor cells using a polyoma-based episomal vector system.

UNLABELLED: Aims/Introduction: We recently established a strategy for isolating multipotential duct-like cells, called pdx-1-positive pancreatic cell-derived (PPPD) cells, from the pancreas. To analyze the molecular mechanisms of pancreatic cell differentiation, we introduced a polyoma-based episomal vector system into PPPD cells. MATERIALS AND METHODS: PPPD cells were stably transfected with a polyoma large T (PLT)-expressing plasmid vector, which included the polyoma origin of replication, to generate PLT-PPPD cells. Various cDNA for pancreas-related transcription factors were subcloned into the expression plasmid pPyCAG, which included the polyoma origin of replication. PLT-PPPD cells were stably transfected with the resulting plasmid vectors and then subjected to gene and protein expression analyses. RESULTS: The coexpression of Mafa, Neurod1 and Ipf1 induced Ins1 and Ins2 expression in PLT-PPPD cells. The forced expression of Pax6 alone induced the expression of glucagon. The coexpression of Neurod1 and Isl1 induced Ins2 and Sst expression. In contrast, the expression of Ptf1a and Foxa2 induced the expression of exocrine markers Cpa1 and Amy2. Transfections with multiple transcription factors showed that Isl1 is required for the differentiation of both insulin-positive cells and somatostatin-positive cells. In addition, Foxa2 induced the differentiation of glucagon-positive cells and inhibited the differentiation of insulin-positive and somatostatin-positive cells. PLT-PPPD cells allow episomal vector-based gene expression and should be useful for studying the transcription factor cascades involved in the differentiation of pancreatic cell types in vitro. CONCLUSIONS: Our coexpression study showed novel critical roles for Isl1 and Foxa2 in the differentiation of PPPD cells into endocrine cells. (J Diabetes Invest, doi: 10.1111/j.2040-1124.2011.00136.x, 2012).

Three-dimensional reconstructed computed tomography-magnetic resonance fusion image-based preoperative planning for surgical procedures for spinal lipoma or tethered spinal cord after myelomeningocele repair.

Surgical procedures for spinal lipoma or tethered spinal cord after myelomeningocele (MMC) repair are often difficult and complicated, because the anatomical structures can be deformed in complex and unpredictable ways. Imaging helps the surgeon understand the patient's spinal anatomy. Whereas two-dimensional images provide only limited information for surgical planning, three-dimensional (3D) reconstructed computed tomography (CT)-magnetic resonance (MR) fusion images produce clearer representations of the spinal regions. Here we describe simple and quick methods for obtaining 3D reconstructed CT-MR fusion images for preoperative planning of surgical procedures using the iPlan(®) cranial (BrainLAB AG, Feldkirchen, Germany) neuronavigation software. 3D CT images of the vertebral bone were combined with heavily T(2)-weighted MR images of the spinal cord, lipoma, cerebrospinal fluid (CSF) space, and nerve root through a process of fusion, segmentation, and reconstruction of the 3D images. We also used our procedure called "Image Overlay" to directly project the 3D reconstructed image onto the body surface using an LED projector. The final reconstructed 3D images took 10-30 minutes to obtain, and provided the surgeon with a representation of the individual pathological structures, so enabled the design of effective surgical plans, even in patients with bony deformity such as scoliosis. None of the 19 patients treated based on our 3D reconstruction method has had neurological complications, except for CSF leakage. This 3D reconstructed imaging method, combined with Image Overlay, improves the visual understanding of complicated surgical situations, and should improve surgical efficiency and outcome.