Myelin, the structure that surrounds and insulates neuronal axons, is an important component of the central nervous system. The visualization of the myelinated fibers in brain tissues can largely facilitate the diagnosis of myelin-related diseases and understand how the brain functions. However, the most widely used fluorescent probes for myelin visualization, such as Vybrant DiD and FluoroMyelin, have strong background staining, low-staining contrast, and low brightness. These drawbacks may originate from their self-quenching properties and greatly limit their applications in three-dimensional (3D) imaging and myelin tracing. Chemical probes for the fluorescence imaging of myelin in 3D, especially in optically cleared tissue, are highly desirable but rarely reported. We herein developed a near-infrared aggregation-induced emission (AIE)-active probe, PM-ML, for high-performance myelin imaging. PM-ML is plasma membrane targeting with good photostability. It could specifically label myelinated fibers in teased sciatic nerves and mouse brain tissues with a high–signal-to-background ratio. PM-ML could be used for 3D visualization of myelin sheaths, myelinated fibers, and fascicles with high-penetration depth. The staining is compatible with different brain tissue–clearing methods, such as Clear^T and Clear^T2. The utility of PM-ML staining in demyelinating disease studies was demonstrated using the mouse model of multiple sclerosis. Together, this work provides an important tool for high-quality myelin visualization across scales, which may greatly contribute to the study of myelin-related diseases. Significance The high spatial resolution of three-dimensional (3D) fluorescence imaging of myelinated fibers will greatly facilitate the understanding of 3D neural networks and the pathophysiology of demyelinating diseases. However, existing myelin probes are far from satisfactory because of their low–signal-to-background ratio and poor tissue permeability. We herein developed a near-infrared aggregation-induced emission-active probe, PM-ML, for high-performance myelin imaging. PM-ML could specifically image myelinated fibers in teased sciatic nerves and mouse brain tissues with high contrast, good photostability, and deep penetration depth. PM-ML staining is compatible with several tissue-clearing methods. Its application in assessing myelination for neuropathological studies was also demonstrated using a multiple sclerosis mouse model.

The in vitro derivation of Schwann cells from human bone marrow stromal cells (hBMSCs) opens avenues for autologous transplantation to achieve remyelination therapy for post-traumatic neural regeneration. Towards this end, we exploited human induced pluripotent stem-cell-derived sensory neurons to direct Schwann-cell-like cells derived from among the hBMSC-neurosphere cells into lineage-committed Schwann cells (hBMSC-dSCs). These cells were seeded into synthetic conduits for bridging critical gaps in a rat model of sciatic nerve injury. With improvement in gait by 12-week post-bridging, evoked signals were also detectable across the bridged nerve. Confocal microscopy revealed axially aligned axons in association with MBP-positive myelin layers across the bridge in contrast to null in non-seeded controls. Myelinating hBMSC-dSCs within the conduit were positive for both MBP and human nucleus marker HuN. We then implanted hBMSC-dSCs into the contused thoracic cord of rats. By 12-week post-implantation, significant improvement in hindlimb motor function was detectable if chondroitinase ABC was co-delivered to the injured site; such cord segments showed axons myelinated by hBMSC-dSCs. Results support translation into a protocol by which lineage-committed hBMSC-dSCs become available for motor function recovery after traumatic injury to both peripheral and central nervous systems.

/ SUMMARY: Oligodendrocytes (OLs) are the only myelinating glia in the central nervous system (CNS). In congenital myelin disorders, OL dysfunction or death results in loss of myelin. This causes progressive and irreversible impairment to motor and cognitive functions, and is amongst the most disabling neurological disorder. Neonatal engraftment by glial progenitor cells (GPCs) allows the robust myelination of congenitally dysmyelinated brain, thereby preserving brain function and quality of life of patients. However, endogenous sources of glial progenitors are hard to obtain without causing secondary injury, while use of exogenous sources such as embryonic stem cells and induced-pluripotent stem cells face considerable ethical and safety issues. To circumvent such hurdles, we asked whether NG2 ⁺ cells in the bone marrow could be a potential cell source for GPCs. We successfully generated glial progenitor cells (GPCs) from human bone marrow stromal cells (hBMSCs) from 3 donors using a 14- day induction protocol. The generated hBMSC-GPCs were highly enriched in OPC marker expression, including OLIG2, PDGFRα, NG2, SOX10 and O4, and showed efficient differentiation into myelinogenic oligodendrocytes when transplanted into postnatal day 7 (P7) myelin-deficient shiverer mice. Remyelination of the shiverer mouse brain significantly extended lifespan and improved motor function. The novel induction protocol described here provides a method for fast, simple and effective glial therapy for myelin disorders, overcoming existent hurdles of cell source restriction and time frame requirement. Graphical Highlights: - Human bone marrow stromal cells (BMSCs) can be programmed to myelinating glia (GPCs, glial progenitor cells), via a novel 14-day in vitro induction protocol - Transplantation of these hBM-GPCs robustly remyelinated myelin deficient shiverer mice. - hBM-GPC transplant significantly extended lifespan, increased body weight and improved motor function

The serotonin system modulates nearly all neural circuits across development and adulthood and is a major pharmacological target for mood and anxiety disorders. Forebrain-projecting serotonin neurons are organized into projection-defined subsystems that align with molecular heterogeneity, but how these subsystems assemble during development remains unclear. We combined whole-brain developmental projection maps with single-cell transcriptomics to define the spatiotemporal and molecular logic of postnatal serotonin innervation. Using a deep-learning pipeline, we generated four-dimensional whole-brain maps of postnatal serotonin axon density and uncovered two coupled modes of regulation: region-specific trajectories of axon growth and refinement, and an early-postnatal, brain-wide synchronization of innervation. To link these dynamics to cell-intrinsic programs, we profiled single-cell transcriptomes of forebrain-projecting serotonin neurons from embryonic through postnatal stages and reconstructed developmental trajectories. We found that serotonin neuron heterogeneity was established embryonically and that the subtype taxonomy was largely conserved between mice and humans. Across serotonin subtypes, we observed a coordinated surge in wiring-molecule expression at mouse postnatal day 4 that aligned with the subsequent global innervation wave. Tracing subtypes defined by developmentally conserved markers at birth revealed complementary projection territories and engagement of distinct postnatal wiring modules that matched region-specific terminal maturation. Together, these results show that brain-wide serotonin innervation is built through embryonic subtype specification and two layers of postnatal control: system-level synchronization and subtype-specific terminal development.